Whole Genome Sequencing of High-Risk Families to Identify New Mutational Mechanisms of Breast Cancer Predisposition

DTIC Science & Technology

2014-10-01

INTRODUCTION: Despite tremendous advances in mutation detection with gene panels and exome sequencing the majority of high risk breast...2a. Align reads to the reference sequence (months 4-10) 2b. Identify SNPs, indels, CNVs and rearrangements by bioinformatic tools (months 4-10) 2c

MTHFR Functional Polymorphism C677T and Genomic Instability in the Etiology of Idiopathic Autism in Simplex Families

DTIC Science & Technology

2014-12-01

facts that de novo CNVs rates are consistently high in SPX ASD (5.8%-10.2%) versus familial ASD (2-3%), we hypothesize that low-activity MTHFR 677T...allele leads to increase global DNA hypomethylation and consequently results in increased generation of de novo CNVs bringing about a higher risk for...developing sporadic cases of autism. We proposed to test 1) the association of MTHFR 677T allele with rate of ASD related de novo CNVs ; 2) the

MTHFR Functional Polymorphism C677T and Genomic Instability in the Etiology of Idiopathic Autism in Simplex Families. Revision

DTIC Science & Technology

2013-10-01

that de novo CNVs rates are consistently high in SPX ASD (5.8%-10.2%) versus familial ASD (2-3%), we hypothesize that low-activity MTHFR 677T allele...leads to increase global DNA hypomethylation and consequently results in increased generation of de novo CNVs bringing about a higher risk for...developing sporadic cases of autism. We proposed to test 1) the association of MTHFR 677T allele with rate of ASD related de novo CNVs ; 2) the

Clinical relevance of small copy-number variants in chromosomal microarray clinical testing.

PubMed

Hollenbeck, Dana; Williams, Crescenda L; Drazba, Kathryn; Descartes, Maria; Korf, Bruce R; Rutledge, S Lane; Lose, Edward J; Robin, Nathaniel H; Carroll, Andrew J; Mikhail, Fady M

2017-04-01

The 2010 consensus statement on diagnostic chromosomal microarray (CMA) testing recommended an array resolution ≥400 kb throughout the genome as a balance of analytical and clinical sensitivity. In spite of the clear evidence for pathogenicity of large copy-number variants (CNVs) in neurodevelopmental disorders and/or congenital anomalies, the significance of small, nonrecurrent CNVs (<500 kb) has not been well established in a clinical setting. We investigated the clinical significance of all nonpolymorphic small, nonrecurrent CNVs (<500 kb) in patients referred for CMA clinical testing over a period of 6 years, from 2009 to 2014 (a total of 4,417 patients). We excluded from our study patients with benign or likely benign CNVs and patients with only recurrent microdeletions/microduplications <500 kb. In total, 383 patients (8.67%) were found to carry at least one small, nonrecurrent CNV, of whom 176 patients (3.98%) had one small CNV classified as a variant of uncertain significance (VUS), 45 (1.02%) had two or more small VUS CNVs, 20 (0.45%) had one small VUS CNV and a recurrent CNV, 113 (2.56%) had one small pathogenic or likely pathogenic CNV, 17 (0.38%) had two or more small pathogenic or likely pathogenic CNVs, and 12 (0.27%) had one small pathogenic or likely pathogenic CNV and a recurrent CNV. Within the pathogenic group, 80 of 142 patients (56% of all small pathogenic CNV cases) were found to have a single whole-gene or exonic deletion. The themes that emerged from our study are presented in the Discussion section. Our study demonstrates the diagnostic clinical relevance of small, nonrecurrent CNVs <500 kb during CMA clinical testing and underscores the need for careful clinical interpretation of these CNVs.Genet Med 19 4, 377-385.

Increased Frequency of De Novo Copy Number Variations in Congenital Heart Disease by Integrative Analysis of SNP Array and Exome Sequence Data

PubMed Central

Rodriguez-Murillo, Laura; Fromer, Menachem; Mazaika, Erica; Vardarajan, Badri; Italia, Michael; Leipzig, Jeremy; DePalma, Steven R.; Golhar, Ryan; Sanders, Stephan J.; Yamrom, Boris; Ronemus, Michael; Iossifov, Ivan; Willsey, A. Jeremy; State, Matthew W.; Kaltman, Jonathan R.; White, Peter S.; Shen, Yufeng; Warburton, Dorothy; Brueckner, Martina; Seidman, Christine; Goldmuntz, Elizabeth; Gelb, Bruce D.; Lifton, Richard; Seidman, Jonathan; Hakonarson, Hakon; Chung, Wendy K.

2014-01-01

Rationale Congenital heart disease (CHD) is among the most common birth defects. Most cases are of unknown etiology. Objective To determine the contribution of de novo copy number variants (CNVs) in the etiology of sporadic CHD. Methods and Results We studied 538 CHD trios using genome-wide dense single nucleotide polymorphism (SNP) arrays and/or whole exome sequencing (WES). Results were experimentally validated using digital droplet PCR. We compared validated CNVs in CHD cases to CNVs in 1,301 healthy control trios. The two complementary high-resolution technologies identified 63 validated de novo CNVs in 51 CHD cases. A significant increase in CNV burden was observed when comparing CHD trios with healthy trios, using either SNP array (p=7x10−5, Odds Ratio (OR)=4.6) or WES data (p=6x10−4, OR=3.5) and remained after removing 16% of de novo CNV loci previously reported as pathogenic (p=0.02, OR=2.7). We observed recurrent de novo CNVs on 15q11.2 encompassing CYFIP1, NIPA1, and NIPA2 and single de novo CNVs encompassing DUSP1, JUN, JUP, MED15, MED9, PTPRE SREBF1, TOP2A, and ZEB2, genes that interact with established CHD proteins NKX2-5 and GATA4. Integrating de novo variants in WES and CNV data suggests that ETS1 is the pathogenic gene altered by 11q24.2-q25 deletions in Jacobsen syndrome and that CTBP2 is the pathogenic gene in 10q sub-telomeric deletions. Conclusions We demonstrate a significantly increased frequency of rare de novo CNVs in CHD patients compared with healthy controls and suggest several novel genetic loci for CHD. PMID:25205790

Increased frequency of de novo copy number variants in congenital heart disease by integrative analysis of single nucleotide polymorphism array and exome sequence data.

PubMed

Glessner, Joseph T; Bick, Alexander G; Ito, Kaoru; Homsy, Jason; Rodriguez-Murillo, Laura; Fromer, Menachem; Mazaika, Erica; Vardarajan, Badri; Italia, Michael; Leipzig, Jeremy; DePalma, Steven R; Golhar, Ryan; Sanders, Stephan J; Yamrom, Boris; Ronemus, Michael; Iossifov, Ivan; Willsey, A Jeremy; State, Matthew W; Kaltman, Jonathan R; White, Peter S; Shen, Yufeng; Warburton, Dorothy; Brueckner, Martina; Seidman, Christine; Goldmuntz, Elizabeth; Gelb, Bruce D; Lifton, Richard; Seidman, Jonathan; Hakonarson, Hakon; Chung, Wendy K

2014-10-24

Congenital heart disease (CHD) is among the most common birth defects. Most cases are of unknown pathogenesis. To determine the contribution of de novo copy number variants (CNVs) in the pathogenesis of sporadic CHD. We studied 538 CHD trios using genome-wide dense single nucleotide polymorphism arrays and whole exome sequencing. Results were experimentally validated using digital droplet polymerase chain reaction. We compared validated CNVs in CHD cases with CNVs in 1301 healthy control trios. The 2 complementary high-resolution technologies identified 63 validated de novo CNVs in 51 CHD cases. A significant increase in CNV burden was observed when comparing CHD trios with healthy trios, using either single nucleotide polymorphism array (P=7×10(-5); odds ratio, 4.6) or whole exome sequencing data (P=6×10(-4); odds ratio, 3.5) and remained after removing 16% of de novo CNV loci previously reported as pathogenic (P=0.02; odds ratio, 2.7). We observed recurrent de novo CNVs on 15q11.2 encompassing CYFIP1, NIPA1, and NIPA2 and single de novo CNVs encompassing DUSP1, JUN, JUP, MED15, MED9, PTPRE SREBF1, TOP2A, and ZEB2, genes that interact with established CHD proteins NKX2-5 and GATA4. Integrating de novo variants in whole exome sequencing and CNV data suggests that ETS1 is the pathogenic gene altered by 11q24.2-q25 deletions in Jacobsen syndrome and that CTBP2 is the pathogenic gene in 10q subtelomeric deletions. We demonstrate a significantly increased frequency of rare de novo CNVs in CHD patients compared with healthy controls and suggest several novel genetic loci for CHD. © 2014 American Heart Association, Inc.

Investigation of 15q11-q13, 16p11.2 and 22q13 CNVs in Autism Spectrum Disorder Brazilian Individuals with and without Epilepsy

PubMed Central

Moreira, Danielle P.; Griesi-Oliveira, Karina; Bossolani-Martins, Ana L.; Lourenço, Naila C. V.; Takahashi, Vanessa N. O.; da Rocha, Kátia M.; Moreira, Eloisa S.; Vadasz, Estevão; Meira, Joanna Goes Castro; Bertola, Debora; Halloran, Eoghan O’; Magalhães, Tiago R.; Fett-Conte, Agnes C.; Passos-Bueno, Maria Rita

2014-01-01

Copy number variations (CNVs) are an important cause of ASD and those located at 15q11-q13, 16p11.2 and 22q13 have been reported as the most frequent. These CNVs exhibit variable clinical expressivity and those at 15q11-q13 and 16p11.2 also show incomplete penetrance. In the present work, through multiplex ligation-dependent probe amplification (MLPA) analysis of 531 ethnically admixed ASD-affected Brazilian individuals, we found that the combined prevalence of the 15q11-q13, 16p11.2 and 22q13 CNVs is 2.1% (11/531). Parental origin could be determined in 8 of the affected individuals, and revealed that 4 of the CNVs represent de novo events. Based on CNV prediction analysis from genome-wide SNP arrays, the size of those CNVs ranged from 206 kb to 2.27 Mb and those at 15q11-q13 were limited to the 15q13.3 region. In addition, this analysis also revealed 6 additional CNVs in 5 out of 11 affected individuals. Finally, we observed that the combined prevalence of CNVs at 15q13.3 and 22q13 in ASD-affected individuals with epilepsy (6.4%) was higher than that in ASD-affected individuals without epilepsy (1.3%; p<0.014). Therefore, our data show that the prevalence of CNVs at 15q13.3, 16p11.2 and 22q13 in Brazilian ASD-affected individuals is comparable to that estimated for ASD-affected individuals of pure or predominant European ancestry. Also, it suggests that the likelihood of a greater number of positive MLPA results might be found for the 15q13.3 and 22q13 regions by prioritizing ASD-affected individuals with epilepsy. PMID:25255310

Investigation of 15q11-q13, 16p11.2 and 22q13 CNVs in autism spectrum disorder Brazilian individuals with and without epilepsy.

PubMed

Moreira, Danielle P; Griesi-Oliveira, Karina; Bossolani-Martins, Ana L; Lourenço, Naila C V; Takahashi, Vanessa N O; da Rocha, Kátia M; Moreira, Eloisa S; Vadasz, Estevão; Meira, Joanna Goes Castro; Bertola, Debora; O'Halloran, Eoghan; Magalhães, Tiago R; Fett-Conte, Agnes C; Passos-Bueno, Maria Rita

2014-01-01

Copy number variations (CNVs) are an important cause of ASD and those located at 15q11-q13, 16p11.2 and 22q13 have been reported as the most frequent. These CNVs exhibit variable clinical expressivity and those at 15q11-q13 and 16p11.2 also show incomplete penetrance. In the present work, through multiplex ligation-dependent probe amplification (MLPA) analysis of 531 ethnically admixed ASD-affected Brazilian individuals, we found that the combined prevalence of the 15q11-q13, 16p11.2 and 22q13 CNVs is 2.1% (11/531). Parental origin could be determined in 8 of the affected individuals, and revealed that 4 of the CNVs represent de novo events. Based on CNV prediction analysis from genome-wide SNP arrays, the size of those CNVs ranged from 206 kb to 2.27 Mb and those at 15q11-q13 were limited to the 15q13.3 region. In addition, this analysis also revealed 6 additional CNVs in 5 out of 11 affected individuals. Finally, we observed that the combined prevalence of CNVs at 15q13.3 and 22q13 in ASD-affected individuals with epilepsy (6.4%) was higher than that in ASD-affected individuals without epilepsy (1.3%; p<0.014). Therefore, our data show that the prevalence of CNVs at 15q13.3, 16p11.2 and 22q13 in Brazilian ASD-affected individuals is comparable to that estimated for ASD-affected individuals of pure or predominant European ancestry. Also, it suggests that the likelihood of a greater number of positive MLPA results might be found for the 15q13.3 and 22q13 regions by prioritizing ASD-affected individuals with epilepsy.

Copy number variants in patients with short stature

PubMed Central

van Duyvenvoorde, Hermine A; Lui, Julian C; Kant, Sarina G; Oostdijk, Wilma; Gijsbers, Antoinet CJ; Hoffer, Mariëtte JV; Karperien, Marcel; Walenkamp, Marie JE; Noordam, Cees; Voorhoeve, Paul G; Mericq, Verónica; Pereira, Alberto M; Claahsen-van de Grinten, Hedi L; van Gool, Sandy A; Breuning, Martijn H; Losekoot, Monique; Baron, Jeffrey; Ruivenkamp, Claudia AL; Wit, Jan M

2014-01-01

Height is a highly heritable and classic polygenic trait. Recent genome-wide association studies (GWAS) have revealed that at least 180 genetic variants influence adult height. However, these variants explain only about 10% of the phenotypic variation in height. Genetic analysis of short individuals can lead to the discovery of novel rare gene defects with a large effect on growth. In an effort to identify novel genes associated with short stature, genome-wide analysis for copy number variants (CNVs), using single-nucleotide polymorphism arrays, in 162 patients (149 families) with short stature was performed. Segregation analysis was performed if possible, and genes in CNVs were compared with information from GWAS, gene expression in rodents' growth plates and published information. CNVs were detected in 40 families. In six families, a known cause of short stature was found (SHOX deletion or duplication, IGF1R deletion), in two combined with a de novo potentially pathogenic CNV. Thirty-three families had one or more potentially pathogenic CNVs (n=40). In 24 of these families, segregation analysis could be performed, identifying three de novo CNVs and nine CNVs segregating with short stature. Four were located near loci associated with height in GWAS (ADAMTS17, TULP4, PRKG2/BMP3 and PAPPA). Besides six CNVs known to be causative for short stature, 40 CNVs with possible pathogenicity were identified. Segregation studies and bioinformatics analysis suggested various potential candidate genes. PMID:24065112

Increasing the Yield in Targeted Next-Generation Sequencing by Implicating CNV Analysis, Non-Coding Exons and the Overall Variant Load: The Example of Retinal Dystrophies

PubMed Central

Eisenberger, Tobias; Neuhaus, Christine; Khan, Arif O.; Decker, Christian; Preising, Markus N.; Friedburg, Christoph; Bieg, Anika; Gliem, Martin; Issa, Peter Charbel; Holz, Frank G.; Baig, Shahid M.; Hellenbroich, Yorck; Galvez, Alberto; Platzer, Konrad; Wollnik, Bernd; Laddach, Nadja; Ghaffari, Saeed Reza; Rafati, Maryam; Botzenhart, Elke; Tinschert, Sigrid; Börger, Doris; Bohring, Axel; Schreml, Julia; Körtge-Jung, Stefani; Schell-Apacik, Chayim; Bakur, Khadijah; Al-Aama, Jumana Y.; Neuhann, Teresa; Herkenrath, Peter; Nürnberg, Gudrun; Nürnberg, Peter; Davis, John S.; Gal, Andreas; Bergmann, Carsten; Lorenz, Birgit; Bolz, Hanno J.

2013-01-01

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover “hidden mutations” such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5′ exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5′-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading. PMID:24265693

Increasing the yield in targeted next-generation sequencing by implicating CNV analysis, non-coding exons and the overall variant load: the example of retinal dystrophies.

PubMed

Eisenberger, Tobias; Neuhaus, Christine; Khan, Arif O; Decker, Christian; Preising, Markus N; Friedburg, Christoph; Bieg, Anika; Gliem, Martin; Charbel Issa, Peter; Holz, Frank G; Baig, Shahid M; Hellenbroich, Yorck; Galvez, Alberto; Platzer, Konrad; Wollnik, Bernd; Laddach, Nadja; Ghaffari, Saeed Reza; Rafati, Maryam; Botzenhart, Elke; Tinschert, Sigrid; Börger, Doris; Bohring, Axel; Schreml, Julia; Körtge-Jung, Stefani; Schell-Apacik, Chayim; Bakur, Khadijah; Al-Aama, Jumana Y; Neuhann, Teresa; Herkenrath, Peter; Nürnberg, Gudrun; Nürnberg, Peter; Davis, John S; Gal, Andreas; Bergmann, Carsten; Lorenz, Birgit; Bolz, Hanno J

2013-01-01

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover "hidden mutations" such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5' exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5'-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.

The clinical significance of small copy number variants in neurodevelopmental disorders.

PubMed

Asadollahi, Reza; Oneda, Beatrice; Joset, Pascal; Azzarello-Burri, Silvia; Bartholdi, Deborah; Steindl, Katharina; Vincent, Marie; Cobilanschi, Joana; Sticht, Heinrich; Baldinger, Rosa; Reissmann, Regina; Sudholt, Irene; Thiel, Christian T; Ekici, Arif B; Reis, André; Bijlsma, Emilia K; Andrieux, Joris; Dieux, Anne; FitzPatrick, David; Ritter, Susanne; Baumer, Alessandra; Latal, Beatrice; Plecko, Barbara; Jenni, Oskar G; Rauch, Anita

2014-10-01

Despite abundant evidence for pathogenicity of large copy number variants (CNVs) in neurodevelopmental disorders (NDDs), the individual significance of genome-wide rare CNVs <500 kb has not been well elucidated in a clinical context. By high-resolution chromosomal microarray analysis, we investigated the clinical significance of all rare non-polymorphic exonic CNVs sizing 1-500 kb in a cohort of 714 patients with undiagnosed NDDs. We detected 96 rare CNVs <500 kb affecting coding regions, of which 58 (60.4%) were confirmed. 6 of 14 confirmed de novo, one of two homozygous and four heterozygous inherited CNVs affected the known microdeletion regions 17q21.31, 16p11.2 and 2p21 or OMIM morbid genes (CASK, CREBBP, PAFAH1B1, SATB2; AUTS2, NRXN3, GRM8). Two further de novo CNVs affecting single genes (MED13L, CTNND2) were instrumental in delineating novel recurrent conditions. For the first time, we here report exonic deletions of CTNND2 causing low normal IQ with learning difficulties with or without autism spectrum disorder. Additionally, we discovered a homozygous out-of-frame deletion of ACOT7 associated with features comparable to the published mouse model. In total, 24.1% of the confirmed small CNVs were categorised as pathogenic or likely pathogenic (median size 130 kb), 17.2% as likely benign, 3.4% represented incidental findings and 55.2% remained unclear. These results verify the diagnostic relevance of genome-wide rare CNVs <500 kb, which were found pathogenic in ∼2% (14/714) of cases (1.1% de novo, 0.3% homozygous, 0.6% inherited) and highlight their inherent potential for discovery of new conditions. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Copy number variations at the Prader-Willi syndrome region on chromosome 15 and associations with obesity in whites.

PubMed

Chen, Yuan; Liu, Yong-Jun; Pei, Yu-Fang; Yang, Tie-Lin; Deng, Fei-Yan; Liu, Xiao-Gang; Li, Ding-You; Deng, Hong-Wen

2011-06-01

Obesity is a serious health problem with strong genetic determination. Copy number variation (CNV) is a common type of genomic variant associated with some complex human diseases. However, it is not clear how CNVs contribute to the etiology of obesity. In this study, we examined 1,000 unrelated US whites to search for CNVs that may predispose to obesity. We focused our analyses on the Prader-Willi syndrome (PWS) critical region (chromosome 15q11-q13), because the PWS region is a hotspot for CNV generation and obesity is one of the major clinical manifestations for chromosome abnormalities at this region. We constructed a map containing 39 CNVs at the PWS critical region with CNV occurrence rates higher than 1%. Among them, three CNVs were significantly associated with body fat mass (P < 0.05), with a higher copy number (CN) associated with an increase of 5.08-9.77 kg in body fat mass. These three CNVs are close to two known PWS genes, NDN (necdin homolog) and C15orf2 (chromosome 15 open reading frame 2), and partially overlap with another obesity gene PWRN1 (Prader-Willi region nonprotein-coding RNA 1). Interestingly, our recently published whole genome association scan study using the same sample by examining single-nucleotide polymorphisms (SNPs) did not find any significant associations at these CNV regions, suggesting the importance of examining both CNVs and SNPs for better understanding of genetic basis of obesity. Further studies are warranted to validate these CNVs and their importance to obesity.

Pathogenic copy number variants in patients with congenital hypopituitarism associated with complex phenotypes.

PubMed

Correa, Fernanda A; Jorge, Alexander Al; Nakaguma, Marilena; Canton, Ana Pm; Costa, Silvia S; Funari, Mariana F; Lerario, Antonio M; Franca, Marcela M; Carvalho, Luciani R; Krepischi, Ana Cv; Arnhold, Ivo Jp; Rosenberg, Carla; Mendonca, Berenice B

2018-03-01

The aetiology of congenital hypopituitarism (CH) is unknown in most patients. Rare copy number variants (CNVs) have been implicated as the cause of genetic syndromes with previously unknown aetiology. Our aim was to study the presence of CNVs and their pathogenicity in patients with idiopathic CH associated with complex phenotypes. We selected 39 patients with syndromic CH for array-based comparative genomic hybridization (aCGH). Patients with pathogenic CNVs were also evaluated by whole exome sequencing. Twenty rare CNVs were detected in 19 patients. Among the identified rare CNVs, six were classified as benign, eleven as variants of uncertain clinical significance (VUS) and four as pathogenic. The three patients with pathogenic CNVs had combined pituitary hormone deficiencies, and the associated complex phenotypes were intellectual disabilities: trichorhinophalangeal type I syndrome (TRPS1) and developmental delay/intellectual disability with cardiac malformation, respectively. Patient one has a de novo 1.6-Mb deletion located at chromosome 3q13.31q13.32, which overlaps with the region of the 3q13.31 deletion syndrome. Patient two has a 10.5-Mb de novo deletion at 8q23.1q24.11, encompassing the TRPS1 gene; his phenotype is compatible with TRPS1. Patient three carries a chromosome translocation t(2p24.3;4q35.1) resulting in two terminal alterations: a 2p25.3p24.3 duplication of 14.7 Mb and a 4-Mb deletion at 4q35.1q35.2. Copy number variants explained the phenotype in 8% of patients with hypopituitarism and additional complex phenotypes. This suggests that chromosomal alterations are an important contributor to syndromic hypopituitarism. © 2017 John Wiley & Sons Ltd.

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma

PubMed Central

Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

2017-01-01

Objective This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Methods Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Results Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification (P=0.009) or deletion (P=0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly (P=1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Conclusion Chromosomal CNVs may contribute to their transcript expression in cervical cancer. PMID:29312578

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma.

PubMed

Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

2017-12-12

This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification ( P =0.009) or deletion ( P =0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly ( P =1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Chromosomal CNVs may contribute to their transcript expression in cervical cancer.

Subtelomeric Copy Number Variations: The Importance of 4p/4q Deletions in Patients with Congenital Anomalies and Developmental Disability.

PubMed

Novo-Filho, Gil M; Montenegro, Marília M; Zanardo, Évelin A; Dutra, Roberta L; Dias, Alexandre T; Piazzon, Flavia B; Costa, Taís V M M; Nascimento, Amom M; Honjo, Rachel S; Kim, Chong A; Kulikowski, Leslie D

2016-01-01

The most prevalent structural variations in the human genome are copy number variations (CNVs), which appear predominantly in the subtelomeric regions. Variable sizes of 4p/4q CNVs have been associated with several different psychiatric findings and developmental disability (DD). We analyzed 105 patients with congenital anomalies (CA) and developmental and/or intellectual disabilities (DD/ID) using MLPA subtelomeric specific kits (P036 /P070) and 4 of them using microarrays. We found abnormal subtelomeric CNVs in 15 patients (14.3%), including 8 patients with subtelomeric deletions at 4p/4q (53.3%). Additional genomic changes were observed at 1p36, 2q37.3, 5p15.3, 5q35.3, 8p23.3, 13q11, 14q32.3, 15q11.2, and Xq28/Yq12. This indicates the prevalence of independent deletions at 4p/4q, involving PIGG, TRIML2, and FRG1. Furthermore, we identified 15 genes with changes in copy number that contribute to neurological development and/or function, among them CRMP1, SORCS2, SLC25A4, and HELT. Our results highlight the association of genes with changes in copy number at 4p and 4q subtelomeric regions and the DD phenotype. Cytogenomic characterization of additional cases with distal deletions should help clarifying the role of subtelomeric CNVs in neurological diseases. © 2016 S. Karger AG, Basel.

Genome-wide analysis of CNV (copy number variation) and their associations with narcolepsy in a Japanese population.

PubMed

Yamasaki, Maria; Miyagawa, Taku; Toyoda, Hiromi; Khor, Seik-Soon; Koike, Asako; Nitta, Aino; Akiyama, Kumi; Sasaki, Tsukasa; Honda, Yutaka; Honda, Makoto; Tokunaga, Katsushi

2014-05-01

In humans, narcolepsy with cataplexy (narcolepsy) is a sleep disorder that is characterized by sleepiness, cataplexy and rapid eye movement (REM) sleep abnormalities. Narcolepsy is caused by a reduction in the number of neurons that produce hypocretin (orexin) neuropeptide. Both genetic and environmental factors contribute to the development of narcolepsy.Rare and large copy number variations (CNVs) reportedly play a role in the etiology of a number of neuropsychiatric disorders. Narcolepsy is considered a neurological disorder; therefore, we sought to investigate any possible association between rare and large CNVs and human narcolepsy. We used DNA microarray data and a CNV detection software application, PennCNV-Affy, to detect CNVs in 426 Japanese narcoleptic patients and 562 healthy individuals. Overall, we found a significant enrichment of rare and large CNVs (frequency ≤1%, size ≥100 kb) in the patients (case-control ratio of CNV count=1.54, P=5.00 × 10(-4)). Next, we extended a region-based association analysis by including CNVs with its size ≥30 kb. Rare and large CNVs in PARK2 region showed a significant association with narcolepsy. Four patients were assessed to carry duplications of the gene region, whereas no controls carried the duplication, which was further confirmed by quantitative PCR assay. This duplication was also found in 2 essential hypersomnia (EHS) patients out of 171 patients. Furthermore, a pathway analysis revealed enrichments of gene disruptions by rare and large CNVs in immune response, acetyltransferase activity, cell cycle regulation and regulation of cell development. This study constitutes the first report on the risk association between multiple rare and large CNVs and the pathogenesis of narcolepsy. In the future, replication studies are needed to confirm the associations.

Pseudoautosomal abnormalities in terminal AZFb+c deletions are associated with isochromosomes Yp and may lead to abnormal growth and neuropsychiatric function.

PubMed

Castro, A; Rodríguez, F; Flórez, M; López, P; Curotto, B; Martínez, D; Maturana, A; Lardone, M C; Palma, C; Mericq, V; Ebensperger, M; Cassorla, F

2017-02-01

Are copy number variations (CNVs) in the pseudoautosomal regions (PARs) frequent in subjects with Y-chromosome microdeletions and can they lead to abnormal stature and/or neuropsychiatric disorders? Only subjects diagnosed with azoospermia factor (AZF)b+c deletions spanning to the end of the Y chromosome (i.e. terminal deletions) harbor Y isochromosomes and/or cells 45,X that lead to pseudoautosomal gene CNVs, which were associated with abnormal stature and/or neuropsychiatric disorders. The microdeletions in the long arm of the Y chromosome (Yq) that include the loss of one to three AZF regions, referred to as Yq microdeletions, constitute the most important known etiological factor for primary spermatogenic failure. Recently, controversy has arisen about whether Yq microdeletions are associated with gain or loss of PAR genes, which are implicated in skeletal development and neuropsychiatric function. We studied a cohort of 42 Chilean patients with complete AZF deletions (4 AZFa, 4 AZFb, 23 AZFc, 11 AZFb+c) from a university medical center, diagnosed over a period of 15 years. The subjects underwent complete medical examinations with special attention to their stature and neuropsychiatric function. All subjects were characterized for Yq breakpoints by PCR, and for CNVs in PARs by multiplex ligation-dependent probe amplification (MLPA), followed by qPCR analysis for genes in PAR1 (SHOX and ZBED1), PAR2 (IL9R) and two single copy genes (SRY and DDX3Y, respectively located in Yp11.3 and AZFa). In addition, karyotypes revision and fluorescence in situ hybridization (FISH) for SRY and centromeric probes for X (DXZ1) and Y (DYZ3) chromosomes were performed in males affected with CNVs. We did not detect CNVs in any of the 35 AZF-deleted men with interstitial deletions (AZFa, AZFb, AZFc or AZFb+c). However, six of the seven patients with terminal AZFb+c deletions showed CNVs: two patients showed a loss and four patients showed a gain of PAR1 genes, with the expected loss of VAMP-7 in PAR2. In these patients, the Yq breakpoints localized to the palindromes P8, P5 or P4. In the four cases with gain of PAR1, qPCR analysis showed duplicated signals for SRY and DDX3Y and one copy of IL9R, indicating isodicentric Yp chromosomes [idic(Y)] with breakpoint in Yq11.22. The two patients who had loss of PAR1, as shown by MLPA, had an additional reduction for SRY and DDX3Y, as shown by qPCR, associated with a high proportion of 45,X cells, as determined by FISH and karyotype. In agreement with the karyotype analysis, we detected DYZ3++ and DYZ3+ cells by FISH in the six patients, confirming idic(Y) and revealing additional monocentric Y chromosome [i(Y)]. Five patients had a history of major depressive disorders or bipolar disorder, and three had language impairment, whereas two patients showed severe short stature (Z score: -2.75 and -2.62), while a man with bipolar disorder was very tall (Z score: +2.56). N/A. The number of males studied with Y-chromosome microdeletions and normozoospermic controls with normal karyotypes may not be enough to rule out an association between AZF deletions and PAR abnormalities. The prevalence of Y isochromosomes and/or 45,X cells detected in peripheral blood does not necessarily reflect the variations of PAR genes in target tissues. This study shows that CNVs in PARs were present exclusively in patients with terminal AZFb+c deletions associated with the presence of Y isochromosomes and 45,X cells, and may lead to neuropsychiatric and growth disorders. In contrast, we show that men with interstitial Yq microdeletions with normal karyotypes do not have an increased risk of PAR abnormalities and of phenotypical consequences. Moreover, our results highlight the importance of performing molecular studies, which are not considered in the usual screening for patients with Yq microdeletions. This work was supported by the National Fund for Scientific and Technological Development of Chile (FONDECYT), grant no. 1120176 (A.C.). The authors declare that no conflicting interests exist. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Copy number variation in ALOX5 and PTGER1 is associated with NSAIDs-induced urticaria and/or angioedema.

PubMed

Plaza-Serón, María Del Carmen; Ayuso, Pedro; Pérez-Sánchez, Natalia; Doña, Inmaculada; Blanca-Lopez, Natalia; Flores, Carlos; Galindo, Luisa; Molina, Ana; Perkins, James R; Cornejo-García, Jose A; Agúndez, Jose A; García-Martín, Elena; Campo, Paloma; Canto, Gabriela; Blanca, Miguel

2016-06-01

Cross-intolerance to NSAIDs is a class of drug hypersensitivity reaction, of which NSAIDs-induced urticaria and/or angioedema (NIUA) are the most frequent clinical entities. They are considered to involve dysregulation of the arachidonic acid pathway; however, this mechanism has not been confirmed for NIUA. In this work, we assessed copy number variations (CNVs) in eight of the main genes involved in the arachidonic acid pathway and their possible genetic association with NIUA. CNVs in ALOX5, LTC4S, PTGS1, PTGS2, PTGER1, PTGER2, PTGER3, and PTGER4 were analyzed using TaqMan copy number assays. Genotyping was carried out by real-time quantitative PCR. Individual genotypes were assigned using the CopyCaller Software. Statistical analysis was carried out using GraphPad prism 5, PLINK, EPIDAT, and R version 3.1.2. A total of 151 cases and 139 controls were analyzed during the discovery phase and 148 cases and 140 controls were used for replication. CNVs in open reading frames were found for ALOX5, PTGER1, PTGER3, and PTGER4. Statistically significant differences in the CNV frequency between NIUA and controls were found for ALOX5 (Pc=0.017) and PTGER1 (Pc=1.22E-04). This study represents the first analysis showing an association between CNVs in exonic regions of ALOX5 and PTGER1 and NIUA. This suggests a role of CNVs in this pathology that should be explored further.

Copy Number Variation in the Horse Genome

PubMed Central

Ghosh, Sharmila; Qu, Zhipeng; Das, Pranab J.; Fang, Erica; Juras, Rytis; Cothran, E. Gus; McDonell, Sue; Kenney, Daniel G.; Lear, Teri L.; Adelson, David L.; Chowdhary, Bhanu P.; Raudsepp, Terje

2014-01-01

We constructed a 400K WG tiling oligoarray for the horse and applied it for the discovery of copy number variations (CNVs) in 38 normal horses of 16 diverse breeds, and the Przewalski horse. Probes on the array represented 18,763 autosomal and X-linked genes, and intergenic, sub-telomeric and chrY sequences. We identified 258 CNV regions (CNVRs) across all autosomes, chrX and chrUn, but not in chrY. CNVs comprised 1.3% of the horse genome with chr12 being most enriched. American Miniature horses had the highest and American Quarter Horses the lowest number of CNVs in relation to Thoroughbred reference. The Przewalski horse was similar to native ponies and draft breeds. The majority of CNVRs involved genes, while 20% were located in intergenic regions. Similar to previous studies in horses and other mammals, molecular functions of CNV-associated genes were predominantly in sensory perception, immunity and reproduction. The findings were integrated with previous studies to generate a composite genome-wide dataset of 1476 CNVRs. Of these, 301 CNVRs were shared between studies, while 1174 were novel and require further validation. Integrated data revealed that to date, 41 out of over 400 breeds of the domestic horse have been analyzed for CNVs, of which 11 new breeds were added in this study. Finally, the composite CNV dataset was applied in a pilot study for the discovery of CNVs in 6 horses with XY disorders of sexual development. A homozygous deletion involving AKR1C gene cluster in chr29 in two affected horses was considered possibly causative because of the known role of AKR1C genes in testicular androgen synthesis and sexual development. While the findings improve and integrate the knowledge of CNVs in horses, they also show that for effective discovery of variants of biomedical importance, more breeds and individuals need to be analyzed using comparable methodological approaches. PMID:25340504

Association between salivary amylase (AMY1) gene copy numbers and insulin resistance in asymptomatic Korean men.

PubMed

Choi, Y-J; Nam, Y-S; Yun, J M; Park, J H; Cho, B L; Son, H-Y; Kim, J I; Yun, J W

2015-12-01

Salivary amylase gene (AMY1) copy number variations (CNVs) correlate directly with salivary amylase activity and serum amylase levels. Previously, individuals with high AMY1 CNVs exhibited low postprandial glucose levels and postprandial early insulin surge, suggesting that high AMY1 gene copy numbers may play a role in lowering the risk of insulin resistance. We verified the relationship between AMY1 CNVs and homeostatic model assessment-insulin resistance (HOMA-IR) in a cohort of 1257 Korean men aged 20-65 years who visited two medical centres for regular health check-ups, and in subgroups of current smokers and regular alcohol drinkers. Individuals with fasting plasma glucose levels > 10.0 mmol/l, HbA1c ≥ 64 mmol/mol (8.0%) or who used oral hypoglycaemic agents or insulin were excluded. AMY1 CNVs correlated negatively with HOMA-IR even after adjusting for covariates (e.g. BMI, systolic blood pressure, triacylglycerol, alcohol consumption, smoking and physical activity). When the participants were divided according to current smoking and alcohol consumption habits, negative correlations between AMY1 CNVs and HOMA-IR were more evident among non-smokers and regular drinkers and were non-significant among smokers and non-regular drinkers. Low AMY1 CNVs correlated with high insulin resistance in asymptomatic Korean men, and such a relationship presented differently according to the status of smoking and alcohol consumption. © 2015 The Authors. Diabetic Medicine © 2015 Diabetes UK.

The contribution of de novo and rare inherited copy number changes to congenital heart disease in an unselected sample of children with conotruncal defects or hypoplastic left heart disease

PubMed Central

Ronemus, Michael; Kline, Jennie; Jobanputra, Vaidehi; Williams, Ismee; Anyane-Yeboa, Kwame; Chung, Wendy; Yu, Lan; Wong, Nancy; Awad, Danielle; Yu, Chih-yu; Leotta, Anthony; Kendall, Jude; Yamrom, Boris; Lee, Yoon-ha; Wigler, Michael; Levy, Dan

2013-01-01

Congenital heart disease (CHD) is the most common congenital malformation, with evidence of a strong genetic component. We analyzed data from 223 consecutively ascertained families, each consisting of at least one child affected by a conotruncal defect (CNT) or hypoplastic left heart disease (HLHS) and both parents. The NimbleGen HD2-2.1 comparative genomic hybridization platform was used to identify de novo and rare inherited copy number variants (CNVs). Excluding 10 cases with 22q11.2 DiGeorge deletions, we validated de novo CNVs in 8 % of 148 probands with CNTs, 12.7 % of 71 probands with HLHS and none in 4 probands with both. Only 2 % of control families showed a de novo CNV. We also identified a group of ultra-rare inherited CNVs that occurred de novo in our sample, contained a candidate gene for CHD, recurred in our sample or were present in an affected sibling. We confirmed the contribution to CHD of copy number changes in genes such as GATA4 and NODAL and identified several genes in novel recurrent CNVs that may point to novel CHD candidate loci. We also found CNVs previously associated with highly variable pheno-types and reduced penetrance, such as dup 1q21.1, dup 16p13.11, dup 15q11.2-13, dup 22q11.2, and del 2q23.1. We found that the presence of extra-cardiac anomalies was not related to the frequency of CNVs, and that there was no significant difference in CNV frequency or specificity between the probands with CNT and HLHS. In agreement with other series, we identified likely causal CNVs in 5.6 % of our total sample, half of which were de novo. PMID:23979609

The contribution of de novo and rare inherited copy number changes to congenital heart disease in an unselected sample of children with conotruncal defects or hypoplastic left heart disease.

PubMed

Warburton, Dorothy; Ronemus, Michael; Kline, Jennie; Jobanputra, Vaidehi; Williams, Ismee; Anyane-Yeboa, Kwame; Chung, Wendy; Yu, Lan; Wong, Nancy; Awad, Danielle; Yu, Chih-Yu; Leotta, Anthony; Kendall, Jude; Yamrom, Boris; Lee, Yoon-Ha; Wigler, Michael; Levy, Dan

2014-01-01

Congenital heart disease (CHD) is the most common congenital malformation, with evidence of a strong genetic component. We analyzed data from 223 consecutively ascertained families, each consisting of at least one child affected by a conotruncal defect (CNT) or hypoplastic left heart disease (HLHS) and both parents. The NimbleGen HD2-2.1 comparative genomic hybridization platform was used to identify de novo and rare inherited copy number variants (CNVs). Excluding 10 cases with 22q11.2 DiGeorge deletions, we validated de novo CNVs in 8 % of 148 probands with CNTs, 12.7 % of 71 probands with HLHS and none in 4 probands with both. Only 2 % of control families showed a de novo CNV. We also identified a group of ultra-rare inherited CNVs that occurred de novo in our sample, contained a candidate gene for CHD, recurred in our sample or were present in an affected sibling. We confirmed the contribution to CHD of copy number changes in genes such as GATA4 and NODAL and identified several genes in novel recurrent CNVs that may point to novel CHD candidate loci. We also found CNVs previously associated with highly variable phenotypes and reduced penetrance, such as dup 1q21.1, dup 16p13.11, dup 15q11.2-13, dup 22q11.2, and del 2q23.1. We found that the presence of extra-cardiac anomalies was not related to the frequency of CNVs, and that there was no significant difference in CNV frequency or specificity between the probands with CNT and HLHS. In agreement with other series, we identified likely causal CNVs in 5.6 % of our total sample, half of which were de novo.

Novel origins of copy number variation in the dog genome

PubMed Central

2012-01-01

Background Copy number variants (CNVs) account for substantial variation between genomes and are a major source of normal and pathogenic phenotypic differences. The dog is an ideal model to investigate mutational mechanisms that generate CNVs as its genome lacks a functional ortholog of the PRDM9 gene implicated in recombination and CNV formation in humans. Here we comprehensively assay CNVs using high-density array comparative genomic hybridization in 50 dogs from 17 dog breeds and 3 gray wolves. Results We use a stringent new method to identify a total of 430 high-confidence CNV loci, which range in size from 9 kb to 1.6 Mb and span 26.4 Mb, or 1.08%, of the assayed dog genome, overlapping 413 annotated genes. Of CNVs observed in each breed, 98% are also observed in multiple breeds. CNVs predicted to disrupt gene function are significantly less common than expected by chance. We identify a significant overrepresentation of peaks of GC content, previously shown to be enriched in dog recombination hotspots, in the vicinity of CNV breakpoints. Conclusions A number of the CNVs identified by this study are candidates for generating breed-specific phenotypes. Purifying selection seems to be a major factor shaping structural variation in the dog genome, suggesting that many CNVs are deleterious. Localized peaks of GC content appear to be novel sites of CNV formation in the dog genome by non-allelic homologous recombination, potentially activated by the loss of PRDM9. These sequence features may have driven genome instability and chromosomal rearrangements throughout canid evolution. PMID:22916802

Human Y chromosome copy number variation in the next generation sequencing era and beyond.

PubMed

Massaia, Andrea; Xue, Yali

2017-05-01

The human Y chromosome provides a fertile ground for structural rearrangements owing to its haploidy and high content of repeated sequences. The methodologies used for copy number variation (CNV) studies have developed over the years. Low-throughput techniques based on direct observation of rearrangements were developed early on, and are still used, often to complement array-based or sequencing approaches which have limited power in regions with high repeat content and specifically in the presence of long, identical repeats, such as those found in human sex chromosomes. Some specific rearrangements have been investigated for decades; because of their effects on fertility, or their outstanding evolutionary features, the interest in these has not diminished. However, following the flourishing of large-scale genomics, several studies have investigated CNVs across the whole chromosome. These studies sometimes employ data generated within large genomic projects such as the DDD study or the 1000 Genomes Project, and often survey large samples of healthy individuals without any prior selection. Novel technologies based on sequencing long molecules and combinations of technologies, promise to stimulate the study of Y-CNVs in the immediate future.

Analysis of structural diversity in wolf-like canids reveals post-domestication variants.

PubMed

Ramirez, Oscar; Olalde, Iñigo; Berglund, Jonas; Lorente-Galdos, Belen; Hernandez-Rodriguez, Jessica; Quilez, Javier; Webster, Matthew T; Wayne, Robert K; Lalueza-Fox, Carles; Vilà, Carles; Marques-Bonet, Tomas

2014-06-12

Although a variety of genetic changes have been implicated in causing phenotypic differences among dogs, the role of copy number variants (CNVs) and their impact on phenotypic variation is still poorly understood. Further, very limited knowledge exists on structural variation in the gray wolf, the ancestor of the dog, or other closely related wild canids. Documenting CNVs variation in wild canids is essential to identify ancestral states and variation that may have appeared after domestication. In this work, we genotyped 1,611 dog CNVs in 23 wolf-like canids (4 purebred dogs, one dingo, 15 gray wolves, one red wolf, one coyote and one golden jackal) to identify CNVs that may have arisen after domestication. We have found an increase in GC-rich regions close to the breakpoints and around 1 kb away from them suggesting that some common motifs might be associated with the formation of CNVs. Among the CNV regions that showed the largest differentiation between dogs and wild canids we found 12 genes, nine of which are related to two known functions associated with dog domestication; growth (PDE4D, CRTC3 and NEB) and neurological function (PDE4D, EML5, ZNF500, SLC6A11, ELAVL2, RGS7 and CTSB). Our results provide insight into the evolution of structural variation in canines, where recombination is not regulated by PRDM9 due to the inactivation of this gene. We also identified genes within the most differentiated CNV regions between dogs and wolves, which could reflect selection during the domestication process.

Noncoding copy-number variations are associated with congenital limb malformation.

PubMed

Flöttmann, Ricarda; Kragesteen, Bjørt K; Geuer, Sinje; Socha, Magdalena; Allou, Lila; Sowińska-Seidler, Anna; Bosquillon de Jarcy, Laure; Wagner, Johannes; Jamsheer, Aleksander; Oehl-Jaschkowitz, Barbara; Wittler, Lars; de Silva, Deepthi; Kurth, Ingo; Maya, Idit; Santos-Simarro, Fernando; Hülsemann, Wiebke; Klopocki, Eva; Mountford, Roger; Fryer, Alan; Borck, Guntram; Horn, Denise; Lapunzina, Pablo; Wilson, Meredith; Mascrez, Bénédicte; Duboule, Denis; Mundlos, Stefan; Spielmann, Malte

2017-10-12

PurposeCopy-number variants (CNVs) are generally interpreted by linking the effects of gene dosage with phenotypes. The clinical interpretation of noncoding CNVs remains challenging. We investigated the percentage of disease-associated CNVs in patients with congenital limb malformations that affect noncoding cis-regulatory sequences versus genes sensitive to gene dosage effects.MethodsWe applied high-resolution copy-number analysis to 340 unrelated individuals with isolated limb malformation. To investigate novel candidate CNVs, we re-engineered human CNVs in mice using clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing.ResultsOf the individuals studied, 10% harbored CNVs segregating with the phenotype in the affected families. We identified 31 CNVs previously associated with congenital limb malformations and four novel candidate CNVs. Most of the disease-associated CNVs (57%) affected the noncoding cis-regulatory genome, while only 43% included a known disease gene and were likely to result from gene dosage effects. In transgenic mice harboring four novel candidate CNVs, we observed altered gene expression in all cases, indicating that the CNVs had a regulatory effect either by changing the enhancer dosage or altering the topological associating domain architecture of the genome.ConclusionOur findings suggest that CNVs affecting noncoding regulatory elements are a major cause of congenital limb malformations.Genetics in Medicine advance online publication, 12 October 2017; doi:10.1038/gim.2017.154.

Complement activation on the surface of cell-derived microparticles during cardiac surgery with cardiopulmonary bypass - is retransfusion of pericardial blood harmful?

PubMed

Biró, E; van den Goor, J M; de Mol, B A; Schaap, M C; Ko, L-Y; Sturk, A; Hack, C E; Nieuwland, R

2011-01-01

To investigate whether cell-derived microparticles play a role in complement activation in pericardial blood of patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and whether microparticles in pericardial blood contribute to systemic complement activation upon retransfusion. Pericardial blood of 13 patients was retransfused in 9 and discarded in 4 cases. Microparticles were isolated from systemic blood collected before anesthesia (T1) and at the end of CPB (T2), and from pericardial blood. The microparticles were analyzed by flow cytometry for bound complement components C1q, C4 and C3, and bound complement activator molecules C-reactive protein (CRP), serum amyloid P-component (SAP), immunoglobulin (Ig)M and IgG. Fluid-phase complement activation products (C4b/c, C3b/c) and activator molecules were determined by ELISA. Compared with systemic T1 blood, pericardial blood contained increased C4b/c and C3b/c, and increased levels of microparticles with bound complement components. In systemic T1 samples, microparticle-bound CRP, whereas in pericardial blood, microparticle-bound SAP and IgM were associated with complement activation. At the end of CPB, increased C3b/c (but not C4b/c) was present in systemic T2 blood compared with T1, while concentrations of microparticles binding complement components and of those binding complement activator molecules were similar. Concentrations of fluid-phase complement activation products and microparticles were similar in patients whether or not retransfused with pericardial blood. In pericardial blood of patients undergoing cardiac surgery with CPB, microparticles contribute to activation of the complement system via bound SAP and IgM. Retransfusion of pericardial blood, however, does not contribute to systemic complement activation.

Chromosomal microarray analysis of consecutive individuals with autism spectrum disorders or learning disability presenting for genetic services.

PubMed

Roberts, Jennifer L; Hovanes, Karine; Dasouki, Majed; Manzardo, Ann M; Butler, Merlin G

2014-02-01

Chromosomal microarray analysis is now commonly used in clinical practice to identify copy number variants (CNVs) in the human genome. We report our experience with the use of the 105 K and 180K oligonucleotide microarrays in 215 consecutive patients referred with either autism or autism spectrum disorders (ASD) or developmental delay/learning disability for genetic services at the University of Kansas Medical Center during the past 4 years (2009-2012). Of the 215 patients [140 males and 75 females (male/female ratio=1.87); 65 with ASD and 150 with learning disability], abnormal microarray results were seen in 45 individuals (21%) with a total of 49 CNVs. Of these findings, 32 represented a known diagnostic CNV contributing to the clinical presentation and 17 represented non-diagnostic CNVs (variants of unknown significance). Thirteen patients with ASD had a total of 14 CNVs, 6 CNVs recognized as diagnostic and 8 as non-diagnostic. The most common chromosome involved in the ASD group was chromosome 15. For those with a learning disability, 32 patients had a total of 35 CNVs. Twenty-six of the 35 CNVs were classified as a known diagnostic CNV, usually a deletion (n=20). Nine CNVs were classified as an unknown non-diagnostic CNV, usually a duplication (n=8). For the learning disability subgroup, chromosomes 2 and 22 were most involved. Thirteen out of 65 patients (20%) with ASD had a CNV compared with 32 out of 150 patients (21%) with a learning disability. The frequency of chromosomal microarray abnormalities compared by subject group or gender was not statistically different. A higher percentage of individuals with a learning disability had clinical findings of seizures, dysmorphic features and microcephaly, but not statistically significant. While both groups contained more males than females, a significantly higher percentage of males were present in the ASD group. © 2013 Elsevier B.V. All rights reserved.

CNVs affecting cancer predisposing genes (CPGs) detected as incidental findings in routine germline diagnostic chromosomal microarray (CMA) testing.

PubMed

Innes, Josie; Reali, Lisa; Clayton-Smith, Jill; Hall, Georgina; Lim, Derek Hk; Burghel, George J; French, Kim; Khan, Unzela; Walker, Daniel; Lalloo, Fiona; Evans, D Gareth R; McMullan, Dominic; Maher, Eamonn R; Woodward, Emma R

2018-02-01

Identification of CNVs through chromosomal microarray (CMA) testing is the first-line investigation in individuals with learning difficulties/congenital abnormalities. Although recognised that CMA testing may identify CNVs encompassing a cancer predisposition gene (CPG), limited information is available on the frequency and nature of such results. We investigated CNV gains and losses affecting 39 CPGs in 3366 pilot index case individuals undergoing CMA testing, and then studied an extended cohort (n=10 454) for CNV losses at 105 CPGs and CNV gains at 9 proto-oncogenes implicated in inherited cancer susceptibility. In the pilot cohort, 31/3366 (0.92%) individuals had a CNV involving one or more of 16/39 CPGs. 30/31 CNVs involved a tumour suppressor gene (TSG), and 1/30 a proto-oncogene (gain of MET ). BMPR1A , TSC2 and TMEM127 were affected in multiple cases. In the second stage analysis, 49/10 454 (0.47%) individuals in the extended cohort had 50 CNVs involving 24/105 CPGs. 43/50 CNVs involved a TSG and 7/50 a proto-oncogene (4 gains, 3 deletions). The most frequently involved genes, FLCN (n=10) and SDHA (n=7), map to the Smith-Magenis and cri-du-chat regions, respectively. Incidental identification of a CNV involving a CPG is not rare and poses challenges for future cancer risk estimation. Prospective data collection from CPG-CNV cohorts ascertained incidentally and through syndromic presentations is required to determine the risks posed by specific CNVs. In particular, ascertainment and investigation of adults with CPG-CNVs and adults with learning disability and cancer, could provide important information to guide clinical management and surveillance. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

C4B gene influences intestinal microbiota through complement activation in patients with paediatric-onset inflammatory bowel disease.

PubMed

Nissilä, E; Korpela, K; Lokki, A I; Paakkanen, R; Jokiranta, S; de Vos, W M; Lokki, M-L; Kolho, K-L; Meri, S

2017-12-01

Complement C4 genes are linked to paediatric inflammatory bowel disease (PIBD), but the mechanisms have remained unclear. We examined the influence of C4B gene number on intestinal microbiota and in-vitro serum complement activation by intestinal microbes in PIBD patients. Complement C4A and C4B gene numbers were determined by genomic reverse transcription-polymerase chain reaction (RT-PCR) from 64 patients with PIBD (Crohn's disease or ulcerative colitis). The severity of the disease course was determined from faecal calprotectin levels. Intestinal microbiota was assessed using the HITChip microarray. Complement reactivity in patients was analysed by incubating their sera with Yersinia pseudotuberculosis and Akkermansia muciniphila and determining the levels of C3a and soluble terminal complement complex (SC5b-9) using enzyme immunoassays. The microbiota diversity was wider in patients with no C4B genes than in those with one or two C4B genes, irrespective of intestinal inflammation. C4B and total C4 gene numbers correlated positively with soluble terminal complement complex (TCC, SC5b-9) levels when patient serum samples were stimulated with bacteria. Our results suggest that the C4B gene number associates positively with inflammation in patients with PIBD. Multiple copies of the C4B gene may thus aggravate the IBD-associated dysbiosis through escalated complement reactivity towards the microbiota. © 2017 British Society for Immunology.

Rare Inherited and De Novo CNVs Reveal Complex Contributions to ASD Risk in Multiplex Families.

PubMed

Leppa, Virpi M; Kravitz, Stephanie N; Martin, Christa Lese; Andrieux, Joris; Le Caignec, Cedric; Martin-Coignard, Dominique; DyBuncio, Christina; Sanders, Stephan J; Lowe, Jennifer K; Cantor, Rita M; Geschwind, Daniel H

2016-09-01

Rare mutations, including copy-number variants (CNVs), contribute significantly to autism spectrum disorder (ASD) risk. Although their importance has been established in families with only one affected child (simplex families), the contribution of both de novo and inherited CNVs to ASD in families with multiple affected individuals (multiplex families) is less well understood. We analyzed 1,532 families from the Autism Genetic Resource Exchange (AGRE) to assess the impact of de novo and rare CNVs on ASD risk in multiplex families. We observed a higher burden of large, rare CNVs, including inherited events, in individuals with ASD than in their unaffected siblings (odds ratio [OR] = 1.7), but the rate of de novo events was significantly lower than in simplex families. In previously characterized ASD risk loci, we identified 49 CNVs, comprising 24 inherited events, 19 de novo events, and 6 events of unknown inheritance, a significant enrichment in affected versus control individuals (OR = 3.3). In 21 of the 30 families (71%) in whom at least one affected sibling harbored an established ASD major risk CNV, including five families harboring inherited CNVs, the CNV was not shared by all affected siblings, indicating that other risk factors are contributing. We also identified a rare risk locus for ASD and language delay at chromosomal region 2q24 (implicating NR4A2) and another lower-penetrance locus involving inherited deletions and duplications of WWOX. The genetic architecture in multiplex families differs from that in simplex families and is complex, warranting more complete genetic characterization of larger multiplex ASD cohorts. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Genomic Variation by Whole-Genome SNP Mapping Arrays Predicts Time-to-Event Outcome in Patients with Chronic Lymphocytic Leukemia

PubMed Central

Schweighofer, Carmen D.; Coombes, Kevin R.; Majewski, Tadeusz; Barron, Lynn L.; Lerner, Susan; Sargent, Rachel L.; O'Brien, Susan; Ferrajoli, Alessandra; Wierda, William G.; Czerniak, Bogdan A.; Medeiros, L. Jeffrey; Keating, Michael J.; Abruzzo, Lynne V.

2013-01-01

Genomic abnormalities, such as deletions in 11q22 or 17p13, are associated with poorer prognosis in patients with chronic lymphocytic leukemia (CLL). We hypothesized that unknown regions of copy number variation (CNV) affect clinical outcome and can be detected by array-based single-nucleotide polymorphism (SNP) genotyping. We compared SNP genotypes from 168 untreated patients with CLL with genotypes from 73 white HapMap controls. We identified 322 regions of recurrent CNV, 82 of which occurred significantly more often in CLL than in HapMap (CLL-specific CNV), including regions typically aberrant in CLL: deletions in 6q21, 11q22, 13q14, and 17p13 and trisomy 12. In univariate analyses, 35 of total and 11 of CLL-specific CNVs were associated with unfavorable time-to-event outcomes, including gains or losses in chromosomes 2p, 4p, 4q, 6p, 6q, 7q, 11p, 11q, and 17p. In multivariate analyses, six CNVs (ie, CLL-specific variations in 11p15.1-15.4 or 6q27) predicted time-to-treatment or overall survival independently of established markers of prognosis. Moreover, genotypic complexity (ie, the number of independent CNVs per patient) significantly predicted prognosis, with a median time-to-treatment of 64 months versus 23 months in patients with zero to one versus two or more CNVs, respectively (P = 3.3 × 10−8). In summary, a comparison of SNP genotypes from patients with CLL with HapMap controls allowed us to identify known and unknown recurrent CNVs and to determine regions and rates of CNV that predict poorer prognosis in patients with CLL. PMID:23273604

Clinical application of 2.7M Cytogenetics array for CNV detection in subjects with idiopathic autism and/or intellectual disability.

PubMed

Qiao, Y; Tyson, C; Hrynchak, M; Lopez-Rangel, E; Hildebrand, J; Martell, S; Fawcett, C; Kasmara, L; Calli, K; Harvard, C; Liu, X; Holden, J J A; Lewis, S M E; Rajcan-Separovic, E

2013-02-01

Higher resolution whole-genome arrays facilitate the identification of smaller copy number variations (CNVs) and their integral genes contributing to autism and/or intellectual disability (ASD/ID). Our study describes the use of one of the highest resolution arrays, the Affymetrix(®) Cytogenetics 2.7M array, coupled with quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF) for detection and validation of small CNVs. We studied 82 subjects with ASD and ID in total (30 in the validation and 52 in the application cohort) and detected putatively pathogenic CNVs in 6/52 cases from the application cohort. This included a 130-kb maternal duplication spanning exons 64-79 of the DMD gene which was found in a 3-year-old boy manifesting autism and mild neuromotor delays. Other pathogenic CNVs involved 4p14, 12q24.31, 14q32.31, 15q13.2-13.3, and 17p13.3. We established the optimal experimental conditions which, when applied to select small CNVs for QMPSF confirmation, reduced the false positive rate from 60% to 25%. Our work suggests that selection of small CNVs based on the function of integral genes, followed by review of array experimental parameters resulting in highest confirmation rate using multiplex PCR, may enhance the usefulness of higher resolution platforms for ASD and ID gene discovery. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Systematic Prioritization and Integrative Analysis of Copy Number Variations in Schizophrenia Reveal Key Schizophrenia Susceptibility Genes

PubMed Central

Luo, Xiongjian; Huang, Liang; Han, Leng; Luo, Zhenwu; Hu, Fang; Tieu, Roger; Gan, Lin

2014-01-01

Schizophrenia is a common mental disorder with high heritability and strong genetic heterogeneity. Common disease-common variants hypothesis predicts that schizophrenia is attributable in part to common genetic variants. However, recent studies have clearly demonstrated that copy number variations (CNVs) also play pivotal roles in schizophrenia susceptibility and explain a proportion of missing heritability. Though numerous CNVs have been identified, many of the regions affected by CNVs show poor overlapping among different studies, and it is not known whether the genes disrupted by CNVs contribute to the risk of schizophrenia. By using cumulative scoring, we systematically prioritized the genes affected by CNVs in schizophrenia. We identified 8 top genes that are frequently disrupted by CNVs, including NRXN1, CHRNA7, BCL9, CYFIP1, GJA8, NDE1, SNAP29, and GJA5. Integration of genes affected by CNVs with known schizophrenia susceptibility genes (from previous genetic linkage and association studies) reveals that many genes disrupted by CNVs are also associated with schizophrenia. Further protein-protein interaction (PPI) analysis indicates that protein products of genes affected by CNVs frequently interact with known schizophrenia-associated proteins. Finally, systematic integration of CNVs prioritization data with genetic association and PPI data identifies key schizophrenia candidate genes. Our results provide a global overview of genes impacted by CNVs in schizophrenia and reveal a densely interconnected molecular network of de novo CNVs in schizophrenia. Though the prioritized top genes represent promising schizophrenia risk genes, further work with different prioritization methods and independent samples is needed to confirm these findings. Nevertheless, the identified key candidate genes may have important roles in the pathogenesis of schizophrenia, and further functional characterization of these genes may provide pivotal targets for future therapeutics and diagnostics. PMID:24664977

Accuracy of CNV Detection from GWAS Data.

PubMed

Zhang, Dandan; Qian, Yudong; Akula, Nirmala; Alliey-Rodriguez, Ney; Tang, Jinsong; Gershon, Elliot S; Liu, Chunyu

2011-01-13

Several computer programs are available for detecting copy number variants (CNVs) using genome-wide SNP arrays. We evaluated the performance of four CNV detection software suites--Birdsuite, Partek, HelixTree, and PennCNV-Affy--in the identification of both rare and common CNVs. Each program's performance was assessed in two ways. The first was its recovery rate, i.e., its ability to call 893 CNVs previously identified in eight HapMap samples by paired-end sequencing of whole-genome fosmid clones, and 51,440 CNVs identified by array Comparative Genome Hybridization (aCGH) followed by validation procedures, in 90 HapMap CEU samples. The second evaluation was program performance calling rare and common CNVs in the Bipolar Genome Study (BiGS) data set (1001 bipolar cases and 1033 controls, all of European ancestry) as measured by the Affymetrix SNP 6.0 array. Accuracy in calling rare CNVs was assessed by positive predictive value, based on the proportion of rare CNVs validated by quantitative real-time PCR (qPCR), while accuracy in calling common CNVs was assessed by false positive/false negative rates based on qPCR validation results from a subset of common CNVs. Birdsuite recovered the highest percentages of known HapMap CNVs containing >20 markers in two reference CNV datasets. The recovery rate increased with decreased CNV frequency. In the tested rare CNV data, Birdsuite and Partek had higher positive predictive values than the other software suites. In a test of three common CNVs in the BiGS dataset, Birdsuite's call was 98.8% consistent with qPCR quantification in one CNV region, but the other two regions showed an unacceptable degree of accuracy. We found relatively poor consistency between the two "gold standards," the sequence data of Kidd et al., and aCGH data of Conrad et al. Algorithms for calling CNVs especially common ones need substantial improvement, and a "gold standard" for detection of CNVs remains to be established.

Chromosomal abnormalities and copy number variations in fetal left-sided congenital heart defects.

PubMed

Jansen, Fenna A R; Hoffer, Mariette J V; van Velzen, Christine L; Plati, Stephani Klingeman; Rijlaarsdam, Marry E B; Clur, Sally-Ann B; Blom, Nico A; Pajkrt, Eva; Bhola, Shama L; Knegt, Alida C; de Boer, Marion A; Haak, Monique C

2016-02-01

To demonstrate the spectrum of copy number variants (CNVs) in fetuses with isolated left-sided congenital heart defects (CHDs), and analyse genetic content. Between 2003 and 2012, 200 fetuses were identified with left-sided CHD. Exclusion criteria were chromosomal rearrangements, 22q11.2 microdeletion and/or extra-cardiac malformations (n = 64). We included cases with additional minor anomalies (n = 39), such as single umbilical artery. In 54 of 136 eligible cases, stored material was available for array analysis. CNVs were categorized as either (likely) benign, (likely) pathogenic or of unknown significance. In 18 of the 54 isolated left-sided CHDs we found 28 rare CNVs (prevalence 33%, average 1.6 CNV per person, size 10.6 kb-2.2 Mb). Our interpretation yielded clinically significant CNVs in two of 54 cases (4%) and variants of unknown significance in three other cases (6%). In left-sided CHDs that appear isolated, with normal chromosome analysis and 22q11.2 FISH analysis, array analysis detects clinically significant CNVs. When counselling parents of a fetus with a left-sided CHD it must be taken into consideration that aside from the cardiac characteristics, the presence of extra-cardiac malformations and chromosomal abnormalities influence the treatment plan and prognosis. © 2015 John Wiley & Sons, Ltd.

Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis.

PubMed

Tüzün, Erdem; Scott, Benjamin G; Goluszko, Elzbieta; Higgs, Stephen; Christadoss, Premkumar

2003-10-01

Abs to acetylcholine receptor (AChR) and complement are the major constituents of pathogenic events causing neuromuscular junction destruction in both myasthenia gravis (MG) and experimental autoimmune MG (EAMG). To analyze the differential roles of the classical vs alternative complement pathways in EAMG induction, we immunized C3(-/-), C4(-/-), C3(+/-), and C4(+/-) mice and their control littermates (C3(+/+) and C4(+/+) mice) with AChR in CFA. C3(-/-) and C4(-/-) mice were resistant to disease, whereas mice heterozygous for C3 or C4 displayed intermediate susceptibility. Although C3(-/-) and C4(-/-) mice had anti-AChR Abs in their sera, anti-AChR IgG production by C3(-/-) mice was significantly suppressed. Both C3(-/-) and C4(-/-) mice had reduced levels of B cells and increased expression of apoptotis inducers (Fas ligand, CD69) and apoptotic cells in lymph nodes. Immunofluorescence studies showed that the neuromuscular junction of C3(-/-) and C4(-/-) mice lacked C3 or membrane attack complex deposits, despite having IgG deposits, thus providing in vivo evidence for the incapacity of anti-AChR IgGs to induce full-blown EAMG without the aid of complements. The data provide the first direct genetic evidence for the classical complement pathway in the induction of EAMG induced by AChR immunization. Accordingly, severe MG and other Ab- and complement-mediated diseases could be effectively treated by inhibiting C4, thus leaving the alternative complement pathway intact.

Complement Evasion by Pathogenic Leptospira.

PubMed

Fraga, Tatiana Rodrigues; Isaac, Lourdes; Barbosa, Angela Silva

2016-01-01

Leptospirosis is a neglected infectious disease caused by spirochetes from the genus Leptospira . Pathogenic microorganisms, notably those which reach the blood circulation such as Leptospira , have evolved multiple strategies to escape the host complement system, which is important for innate and acquired immunity. Leptospira avoid complement-mediated killing through: (i) recruitment of host complement regulators; (ii) acquisition of host proteases that cleave complement proteins on the bacterial surface; and, (iii) secretion of proteases that inactivate complement proteins in the Leptospira surroundings. The recruitment of host soluble complement regulatory proteins includes the acquisition of Factor H (FH) and FH-like-1 (alternative pathway), C4b-binding protein (C4BP) (classical and lectin pathways), and vitronectin (Vn) (terminal pathway). Once bound to the leptospiral surface, FH and C4BP retain cofactor activity of Factor I in the cleavage of C3b and C4b, respectively. Vn acquisition by leptospires may result in terminal pathway inhibition by blocking C9 polymerization. The second evasion mechanism lies in plasminogen (PLG) binding to the leptospiral surface. In the presence of host activators, PLG is converted to enzymatically active plasmin, which is able to degrade C3b, C4b, and C5 at the surface of the pathogen. A third strategy used by leptospires to escape from complement system is the active secretion of proteases. Pathogenic, but not saprophytic leptospires, are able to secrete metalloproteases that cleave C3 (central complement molecule), Factor B (alternative pathway), and C4 and C2 (classical and lectin pathways). The purpose of this review is to fully explore these complement evasion mechanisms, which act together to favor Leptospira survival and multiplication in the host.

Complement Evasion by Pathogenic Leptospira

PubMed Central

Fraga, Tatiana Rodrigues; Isaac, Lourdes; Barbosa, Angela Silva

2016-01-01

Leptospirosis is a neglected infectious disease caused by spirochetes from the genus Leptospira. Pathogenic microorganisms, notably those which reach the blood circulation such as Leptospira, have evolved multiple strategies to escape the host complement system, which is important for innate and acquired immunity. Leptospira avoid complement-mediated killing through: (i) recruitment of host complement regulators; (ii) acquisition of host proteases that cleave complement proteins on the bacterial surface; and, (iii) secretion of proteases that inactivate complement proteins in the Leptospira surroundings. The recruitment of host soluble complement regulatory proteins includes the acquisition of Factor H (FH) and FH-like-1 (alternative pathway), C4b-binding protein (C4BP) (classical and lectin pathways), and vitronectin (Vn) (terminal pathway). Once bound to the leptospiral surface, FH and C4BP retain cofactor activity of Factor I in the cleavage of C3b and C4b, respectively. Vn acquisition by leptospires may result in terminal pathway inhibition by blocking C9 polymerization. The second evasion mechanism lies in plasminogen (PLG) binding to the leptospiral surface. In the presence of host activators, PLG is converted to enzymatically active plasmin, which is able to degrade C3b, C4b, and C5 at the surface of the pathogen. A third strategy used by leptospires to escape from complement system is the active secretion of proteases. Pathogenic, but not saprophytic leptospires, are able to secrete metalloproteases that cleave C3 (central complement molecule), Factor B (alternative pathway), and C4 and C2 (classical and lectin pathways). The purpose of this review is to fully explore these complement evasion mechanisms, which act together to favor Leptospira survival and multiplication in the host. PMID:28066433

Accurate clinical detection of exon copy number variants in a targeted NGS panel using DECoN.

PubMed

Fowler, Anna; Mahamdallie, Shazia; Ruark, Elise; Seal, Sheila; Ramsay, Emma; Clarke, Matthew; Uddin, Imran; Wylie, Harriet; Strydom, Ann; Lunter, Gerton; Rahman, Nazneen

2016-11-25

Background: Targeted next generation sequencing (NGS) panels are increasingly being used in clinical genomics to increase capacity, throughput and affordability of gene testing. Identifying whole exon deletions or duplications (termed exon copy number variants, 'exon CNVs') in exon-targeted NGS panels has proved challenging, particularly for single exon CNVs.  Methods: We developed a tool for the Detection of Exon Copy Number variants (DECoN), which is optimised for analysis of exon-targeted NGS panels in the clinical setting. We evaluated DECoN performance using 96 samples with independently validated exon CNV data. We performed simulations to evaluate DECoN detection performance of single exon CNVs and to evaluate performance using different coverage levels and sample numbers. Finally, we implemented DECoN in a clinical laboratory that tests BRCA1 and BRCA2 with the TruSight Cancer Panel (TSCP). We used DECoN to analyse 1,919 samples, validating exon CNV detections by multiplex ligation-dependent probe amplification (MLPA).  Results: In the evaluation set, DECoN achieved 100% sensitivity and 99% specificity for BRCA exon CNVs, including identification of 8 single exon CNVs. DECoN also identified 14/15 exon CNVs in 8 other genes. Simulations of all possible BRCA single exon CNVs gave a mean sensitivity of 98% for deletions and 95% for duplications. DECoN performance remained excellent with different levels of coverage and sample numbers; sensitivity and specificity was >98% with the typical NGS run parameters. In the clinical pipeline, DECoN automatically analyses pools of 48 samples at a time, taking 24 minutes per pool, on average. DECoN detected 24 BRCA exon CNVs, of which 23 were confirmed by MLPA, giving a false discovery rate of 4%. Specificity was 99.7%.  Conclusions: DECoN is a fast, accurate, exon CNV detection tool readily implementable in research and clinical NGS pipelines. It has high sensitivity and specificity and acceptable false discovery rate. DECoN is freely available at www.icr.ac.uk/decon.

Genome-wide copy number variation analysis identified deletions in SFMBT1 associated with fasting plasma glucose in a Han Chinese population.

PubMed

Chung, Ren-Hua; Chiu, Yen-Feng; Hung, Yi-Jen; Lee, Wen-Jane; Wu, Kwan-Dun; Chen, Hui-Ling; Lin, Ming-Wei; Chen, Yii-Der I; Quertermous, Thomas; Hsiung, Chao A

2017-08-08

Fasting glucose and fasting insulin are glycemic traits closely related to diabetes, and understanding the role of genetic factors in these traits can help reveal the etiology of type 2 diabetes. Although single nucleotide polymorphisms (SNPs) in several candidate genes have been found to be associated with fasting glucose and fasting insulin, copy number variations (CNVs), which have been reported to be associated with several complex traits, have not been reported for association with these two traits. We aimed to identify CNVs associated with fasting glucose and fasting insulin. We conducted a genome-wide CNV association analysis for fasting plasma glucose (FPG) and fasting plasma insulin (FPI) using a family-based genome-wide association study sample from a Han Chinese population in Taiwan. A family-based CNV association test was developed in this study to identify common CNVs (i.e., CNVs with frequencies ≥ 5%), and a generalized estimating equation approach was used to test the associations between the traits and counts of global rare CNVs (i.e., CNVs with frequencies <5%). We found a significant genome-wide association for common deletions with a frequency of 5.2% in the Scm-like with four mbt domains 1 (SFMBT1) gene with FPG (association p-value = 2×10 -4 and an adjusted p-value = 0.0478 for multiple testing). No significant association was observed between global rare CNVs and FPG or FPI. The deletions in 20 individuals with DNA samples available were successfully validated using PCR-based amplification. The association of the deletions in SFMBT1 with FPG was further evaluated using an independent population-based replication sample obtained from the Taiwan Biobank. An association p-value of 0.065, which was close to the significance level of 0.05, for FPG was obtained by testing 9 individuals with CNVs in the SFMBT1 gene region and 11,692 individuals with normal copies in the replication cohort. Previous studies have found that SNPs in SFMBT1 are associated with blood pressure and serum urate concentration, suggesting that SFMBT1 may have functional implications in some metabolic-related traits.

The role of complement in myasthenia gravis: serological evidence of complement consumption in vivo.

PubMed

Romi, Fredrik; Kristoffersen, Einar K; Aarli, Johan A; Gilhus, Nils Erik

2005-01-01

Antibodies to the acetylcholine receptor (AChR) titin and the ryanodine receptor (RyR) occur in myasthenia gravis (MG). These antibodies are capable of complement activation in vitro. The involvement of the complement system should cause consumption of complement components such as C3 and C4 in vivo. Complement components C3 and C4 were assayed in sera from 78 AChR antibody-positive MG patients and 52 healthy controls. Forty-eight of the patient sera contained titin antibodies as well, and 20 were also RyR antibody-positive. MG patients with AChR antibody concentrations above the median (11.2 nmol/l) had significantly lower mean C3 and C4 concentrations in serum compared to those with AChR antibody concentrations below the median. Titin antibody-positive MG patients, titin antibody-negative early-onset MG patients, titin antibody-negative late-onset MG patients, and controls had similar C3 and C4 concentrations. Nor did mean C3 and C4 concentrations differ in MG patients with RyR antibodies. Patients with severe MG (grades 4 and 5) had similar C3 and similar C4 levels compared to those with mild MG (grades 1 and 2). An increased in vivo complement consumption was detected in MG patients with high AChR antibody concentrations, unrelated to MG severity and non-AChR muscle antibodies.

Comparison of Constitutional and Replication Stress-Induced Genome Structural Variation by SNP Array and Mate-Pair Sequencing

PubMed Central

Arlt, Martin F.; Ozdemir, Alev Cagla; Birkeland, Shanda R.; Lyons, Robert H.; Glover, Thomas W.; Wilson, Thomas E.

2011-01-01

Copy-number variants (CNVs) are a major source of genetic variation in human health and disease. Previous studies have implicated replication stress as a causative factor in CNV formation. However, existing data are technically limited in the quality of comparisons that can be made between human CNVs and experimentally induced variants. Here, we used two high-resolution strategies—single nucleotide polymorphism (SNP) arrays and mate-pair sequencing—to compare CNVs that occur constitutionally to those that arise following aphidicolin-induced DNA replication stress in the same human cells. Although the optimized methods provided complementary information, sequencing was more sensitive to small variants and provided superior structural descriptions. The majority of constitutional and all aphidicolin-induced CNVs appear to be formed via homology-independent mechanisms, while aphidicolin-induced CNVs were of a larger median size than constitutional events even when mate-pair data were considered. Aphidicolin thus appears to stimulate formation of CNVs that closely resemble human pathogenic CNVs and the subset of larger nonhomologous constitutional CNVs. PMID:21212237

Abnormal Dosage of Ultraconserved Elements Is Highly Disfavored in Healthy Cells but Not Cancer Cells

PubMed Central

McCole, Ruth B.; Fonseka, Chamith Y.; Koren, Amnon; Wu, C.-ting

2014-01-01

Ultraconserved elements (UCEs) are strongly depleted from segmental duplications and copy number variations (CNVs) in the human genome, suggesting that deletion or duplication of a UCE can be deleterious to the mammalian cell. Here we address the process by which CNVs become depleted of UCEs. We begin by showing that depletion for UCEs characterizes the most recent large-scale human CNV datasets and then find that even newly formed de novo CNVs, which have passed through meiosis at most once, are significantly depleted for UCEs. In striking contrast, CNVs arising specifically in cancer cells are, as a rule, not depleted for UCEs and can even become significantly enriched. This observation raises the possibility that CNVs that arise somatically and are relatively newly formed are less likely to have established a CNV profile that is depleted for UCEs. Alternatively, lack of depletion for UCEs from cancer CNVs may reflect the diseased state. In support of this latter explanation, somatic CNVs that are not associated with disease are depleted for UCEs. Finally, we show that it is possible to observe the CNVs of induced pluripotent stem (iPS) cells become depleted of UCEs over time, suggesting that depletion may be established through selection against UCE-disrupting CNVs without the requirement for meiotic divisions. PMID:25340765

Clinical significance of rare copy number variations in epilepsy: a case-control survey using microarray-based comparative genomic hybridization.

PubMed

Striano, Pasquale; Coppola, Antonietta; Paravidino, Roberta; Malacarne, Michela; Gimelli, Stefania; Robbiano, Angela; Traverso, Monica; Pezzella, Marianna; Belcastro, Vincenzo; Bianchi, Amedeo; Elia, Maurizio; Falace, Antonio; Gazzerro, Elisabetta; Ferlazzo, Edoardo; Freri, Elena; Galasso, Roberta; Gobbi, Giuseppe; Molinatto, Cristina; Cavani, Simona; Zuffardi, Orsetta; Striano, Salvatore; Ferrero, Giovanni Battista; Silengo, Margherita; Cavaliere, Maria Luigia; Benelli, Matteo; Magi, Alberto; Piccione, Maria; Dagna Bricarelli, Franca; Coviello, Domenico A; Fichera, Marco; Minetti, Carlo; Zara, Federico

2012-03-01

To perform an extensive search for genomic rearrangements by microarray-based comparative genomic hybridization in patients with epilepsy. Prospective cohort study. Epilepsy centers in Italy. Two hundred seventy-nine patients with unexplained epilepsy, 265 individuals with nonsyndromic mental retardation but no epilepsy, and 246 healthy control subjects were screened by microarray-based comparative genomic hybridization. Identification of copy number variations (CNVs) and gene enrichment. Rare CNVs occurred in 26 patients (9.3%) and 16 healthy control subjects (6.5%) (P = .26). The CNVs identified in patients were larger (P = .03) and showed higher gene content (P = .02) than those in control subjects. The CNVs larger than 1 megabase (P = .002) and including more than 10 genes (P = .005) occurred more frequently in patients than in control subjects. Nine patients (34.6%) among those harboring rare CNVs showed rearrangements associated with emerging microdeletion or microduplication syndromes. Mental retardation and neuropsychiatric features were associated with rare CNVs (P = .004), whereas epilepsy type was not. The CNV rate in patients with epilepsy and mental retardation or neuropsychiatric features is not different from that observed in patients with mental retardation only. Moreover, significant enrichment of genes involved in ion transport was observed within CNVs identified in patients with epilepsy. Patients with epilepsy show a significantly increased burden of large, rare, gene-rich CNVs, particularly when associated with mental retardation and neuropsychiatric features. The limited overlap between CNVs observed in the epilepsy group and those observed in the group with mental retardation only as well as the involvement of specific (ion channel) genes indicate a specific association between the identified CNVs and epilepsy. Screening for CNVs should be performed for diagnostic purposes preferentially in patients with epilepsy and mental retardation or neuropsychiatric features.

The landscape of inherited and de novo copy number variants in a plasmodium falciparum genetic cross

PubMed Central

2011-01-01

Background Copy number is a major source of genome variation with important evolutionary implications. Consequently, it is essential to determine copy number variant (CNV) behavior, distributions and frequencies across genomes to understand their origins in both evolutionary and generational time frames. We use comparative genomic hybridization (CGH) microarray and the resolution provided by a segregating population of cloned progeny lines of the malaria parasite, Plasmodium falciparum, to identify and analyze the inheritance of 170 genome-wide CNVs. Results We describe CNVs in progeny clones derived from both Mendelian (i.e. inherited) and non-Mendelian mechanisms. Forty-five CNVs were present in the parent lines and segregated in the progeny population. Furthermore, extensive variation that did not conform to strict Mendelian inheritance patterns was observed. 124 CNVs were called in one or more progeny but in neither parent: we observed CNVs in more than one progeny clone that were not identified in either parent, located more frequently in the telomeric-subtelomeric regions of chromosomes and singleton de novo CNVs distributed evenly throughout the genome. Linkage analysis of CNVs revealed dynamic copy number fluctuations and suggested mechanisms that could have generated them. Five of 12 previously identified expression quantitative trait loci (eQTL) hotspots coincide with CNVs, demonstrating the potential for broad influence of CNV on the transcriptional program and phenotypic variation. Conclusions CNVs are a significant source of segregating and de novo genome variation involving hundreds of genes. Examination of progeny genome segments provides a framework to assess the extent and possible origins of CNVs. This segregating genetic system reveals the breadth, distribution and dynamics of CNVs in a surprisingly plastic parasite genome, providing a new perspective on the sources of diversity in parasite populations. PMID:21936954

In vitro C3 Deposition on Cryptococcus Capsule Occurs Via Multiple Complement Activation Pathways

PubMed Central

Mershon-Shier, Kileen L.; Vasuthasawat, Alex; Takahashi, Kazue; Morrison, Sherie L.; Beenhouwer, David O.

2011-01-01

Complement can be activated via three pathways: classical, alternative, and lectin. Cryptococcus gattii and C. neoformans are closely related fungal pathogens possessing a polysaccharide capsule composed mainly of glucuronoxylomannan (GXM), which serves as a site for complement activation and deposition of complement components. We determined C3 deposition on Cryptococcus spp. by flow cytometry and confocal microscopy after incubation with serum from C57BL/6J mice as well as mice deficient in complement components C4, C3, factor B, and mannose binding lectin (MBL). C. gattii and C. neoformans activate complement in EGTA-treated serum indicating that they can activate the alternative pathway. However, complement activation was seen with factor B−/− serum suggesting activation could also take place in the absence of a functional alternative pathway. Furthermore, we uncovered a role for C4 in the alternative pathway activation by Cryptococcus spp. We also identified an unexpected and complex role for MBL in complement activation by Cryptococcus spp. No complement activation occurred in the absence of MBL-A and -C proteins although activation took place when the lectin binding activity of MBL was disrupted by calcium chelation. In addition, alternative pathway activation by C. neoformans required both MBL-A and -C, while either MBL-A or -C was sufficient for alternative pathway activation by C. gattii. Thus, complement activation by Cryptococcus spp. can take place through multiple pathways and complement activation via the alternative pathway requires the presence of C4 and MBL proteins. PMID:21723612

A method for rapid, targeted CNV genotyping identifies rare variants associated with neurocognitive disease.

PubMed

Mefford, Heather C; Cooper, Gregory M; Zerr, Troy; Smith, Joshua D; Baker, Carl; Shafer, Neil; Thorland, Erik C; Skinner, Cindy; Schwartz, Charles E; Nickerson, Deborah A; Eichler, Evan E

2009-09-01

Copy-number variants (CNVs) are substantial contributors to human disease. A central challenge in CNV-disease association studies is to characterize the pathogenicity of rare and possibly incompletely penetrant events, which requires the accurate detection of rare CNVs in large numbers of individuals. Cost and throughput issues limit our ability to perform these studies. We have adapted the Illumina BeadXpress SNP genotyping assay and developed an algorithm, SNP-Conditional OUTlier detection (SCOUT), to rapidly and accurately detect both rare and common CNVs in large cohorts. This approach is customizable, cost effective, highly parallelized, and largely automated. We applied this method to screen 69 loci in 1105 children with unexplained intellectual disability, identifying pathogenic variants in 3.1% of these individuals and potentially pathogenic variants in an additional 2.3%. We identified seven individuals (0.7%) with a deletion of 16p11.2, which has been previously associated with autism. Our results widen the phenotypic spectrum of these deletions to include intellectual disability without autism. We also detected 1.65-3.4 Mbp duplications at 16p13.11 in 1.1% of affected individuals and 350 kbp deletions at 15q11.2, near the Prader-Willi/Angelman syndrome critical region, in 0.8% of affected individuals. Compared to published CNVs in controls they are significantly (P = 4.7 x 10(-5) and 0.003, respectively) enriched in these children, supporting previously published hypotheses that they are neurocognitive disease risk factors. More generally, this approach offers a previously unavailable balance between customization, cost, and throughput for analysis of CNVs and should prove valuable for targeted CNV detection in both research and diagnostic settings.

A physical map of the human regulator of complement activation gene cluster linking the complement genes CR1, CR2, DAF, and C4BP

PubMed Central

1988-01-01

We report the organization of the human genes encoding the complement components C4-binding protein (C4BP), C3b/C4b receptor (CR1), decay accelerating factor (DAF), and C3dg receptor (CR2) within the regulator of complement activation (RCA) gene cluster. Using pulsed field gel electrophoresis analysis these genes have been physically linked and aligned as CR1-CR2-DAF-C4BP in an 800-kb DNA segment. The very tight linkage between the CR1 and the C4BP loci, contrasted with the relative long DNA distance between these genes, suggests the existence of mechanisms interfering with recombination within the RCA gene cluster. PMID:2450163

Systematic analysis of copy number variation associated with congenital diaphragmatic hernia.

PubMed

Zhu, Qihui; High, Frances A; Zhang, Chengsheng; Cerveira, Eliza; Russell, Meaghan K; Longoni, Mauro; Joy, Maliackal P; Ryan, Mallory; Mil-Homens, Adam; Bellfy, Lauren; Coletti, Caroline M; Bhayani, Pooja; Hila, Regis; Wilson, Jay M; Donahoe, Patricia K; Lee, Charles

2018-05-15

Congenital diaphragmatic hernia (CDH), characterized by malformation of the diaphragm and hypoplasia of the lungs, is one of the most common and severe birth defects, and is associated with high morbidity and mortality rates. There is growing evidence demonstrating that genetic factors contribute to CDH, although the pathogenesis remains largely elusive. Single-nucleotide polymorphisms have been studied in recent whole-exome sequencing efforts, but larger copy number variants (CNVs) have not yet been studied on a large scale in a case control study. To capture CNVs within CDH candidate regions, we developed and tested a targeted array comparative genomic hybridization platform to identify CNVs within 140 regions in 196 patients and 987 healthy controls, and identified six significant CNVs that were either unique to patients or enriched in patients compared with controls. These CDH-associated CNVs reveal high-priority candidate genes including HLX , LHX1 , and HNF1B We also discuss CNVs that are present in only one patient in the cohort but have additional evidence of pathogenicity, including extremely rare large and/or de novo CNVs. The candidate genes within these predicted disease-causing CNVs form functional networks with other known CDH genes and play putative roles in DNA binding/transcription regulation and embryonic development. These data substantiate the importance of CNVs in the etiology of CDH, identify CDH candidate genes and pathways, and highlight the importance of ongoing analysis of CNVs in the study of CDH and other structural birth defects. Copyright © 2018 the Author(s). Published by PNAS.

Genome-Wide Mapping of Copy Number Variation in Humans: Comparative Analysis of High Resolution Array Platforms

PubMed Central

Haraksingh, Rajini R.; Abyzov, Alexej; Gerstein, Mark; Urban, Alexander E.; Snyder, Michael

2011-01-01

Accurate and efficient genome-wide detection of copy number variants (CNVs) is essential for understanding human genomic variation, genome-wide CNV association type studies, cytogenetics research and diagnostics, and independent validation of CNVs identified from sequencing based technologies. Numerous, array-based platforms for CNV detection exist utilizing array Comparative Genome Hybridization (aCGH), Single Nucleotide Polymorphism (SNP) genotyping or both. We have quantitatively assessed the abilities of twelve leading genome-wide CNV detection platforms to accurately detect Gold Standard sets of CNVs in the genome of HapMap CEU sample NA12878, and found significant differences in performance. The technologies analyzed were the NimbleGen 4.2 M, 2.1 M and 3×720 K Whole Genome and CNV focused arrays, the Agilent 1×1 M CGH and High Resolution and 2×400 K CNV and SNP+CGH arrays, the Illumina Human Omni1Quad array and the Affymetrix SNP 6.0 array. The Gold Standards used were a 1000 Genomes Project sequencing-based set of 3997 validated CNVs and an ultra high-resolution aCGH-based set of 756 validated CNVs. We found that sensitivity, total number, size range and breakpoint resolution of CNV calls were highest for CNV focused arrays. Our results are important for cost effective CNV detection and validation for both basic and clinical applications. PMID:22140474

Analysis of chromosome 22q11 copy number variations by multiplex ligation-dependent probe amplification for prenatal diagnosis of congenital heart defect.

PubMed

Zhang, Jingjing; Ma, Dingyuan; Wang, Yan; Cao, Li; Wu, Yun; Qiao, Fengchang; Liu, An; Li, Li; Lin, Ying; Liu, Gang; Liu, Cuiyun; Hu, Ping; Xu, Zhengfeng

2015-01-01

Congenital heart defects (CHD) represent one of the most common birth defects. This study aimed to evaluate the value of multiplex ligation-dependent probe amplification (MLPA) as a tool to detect the copy number variations (CNVs) of 22q11 in fetuses with CHD. A large cohort of 225 fetuses with CHD was screened by fetal echocardiography. Once common chromosome abnormalities in 30 fetuses were screened out by conventional G-banding analysis, the CNVs of chromosome 22q11 in the remaining 195 fetuses were determined by MLPA for prenatal genetic counseling. In 195 CHD fetuses with normal karyotype, 11 cases had pathological CNVs, including 22q11.2 deletion (seven cases), the deletion of 22q11 cat eye syndrome (CES) region (one case), 22q11.2 duplication (one case), 22q13.3 deletion (one case) and 17p13.3 deletion (one case). In total, our findings from MLPA screening represented 4.9 % in our cohort. Among these, three cases were inherited CNVs, and eight cases were de novo. These CNVs were further verified by single nucleotide polymorphism (SNP)-array analysis, and their chromosomal location was refined. This study indicated that MLPA could serve as an effective test for routine prenatal diagnosis of 22q11 in fetuses with CHD.

O1.6. INCREASED COMPLEMENT FACTORS C3 AND C4 IN SCHIZOPHRENIA AND THE EARLY STAGES OF PSYCHOSIS: IMPLICATIONS FOR CLINICAL SYMPTOMATOLOGY AND CORTICAL THICKNESS

PubMed Central

Cropley, Vanessa; Laskaris, Liliana; Zalesky, Andrew; Weickert, Cynthia Shannon; Biase, Maria Di; Chana, Gursharan; Baune, Bernhard; Bousman, Chad; Nelson, Barnaby; McGorry, Patrick D; Everall, Ian; Pantelis, Christos

2018-01-01

Abstract Background The complement system - a key component of the innate immune system, has been proposed to contribute to the pathogenesis of schizophrenia. Recently, complement C4 was associated with increased risk of schizophrenia, and in a mouse model, developmentally-timed synaptic pruning. These observations have led to proposals that abnormal activation of the complement system might contribute to the development of schizophrenia by disrupting synaptic pruning during key developmental periods. However, despite renewed interest in the complement system in schizophrenia it remains unclear whether peripheral complement levels differ in cases compared to controls, change over the course of illness and whether they are associated with current symptomatology and brain cortical thickness. This study aimed to: i) investigate whether peripheral complement protein levels are altered at different stages of illness, and ii) identify patterns among complement protein levels that predict clinical symptoms and grey matter thickness across the cortex. Methods Complement factors C1q, C3 and C4 were quantified in 183 participants [n=83 Healthy Controls (HC), n=10 Ultra-High Risk (UHR) for psychosis, n=40 First Episode Psychosis (FEP), n=50 Chronic schizophrenia] using Multiplex ELISA. Permutation-based t-tests were used to assess between-group differences in complement protein levels at each of the three illness stages, relative to age- and gender-matched healthy controls. Canonical correlation analysis was used to identify patterns of complement protein levels that correlated with clinical symptoms and regional thickness across the cortex. Results C3 and C4 were significantly increased in FEP and UHR patients, whereas only C4 was significantly increased in chronic patients. A molecular pattern of increased C4 and decreased C3 was associated with positive and negative symptom severity in the pooled patient sample. Increased C4 levels alone, or decreased C3 levels alone, did not correlate with symptom severity as strongly as the pattern of increased C4 in combination with decreased C3. Preliminary canonical correlation analyses revealed that, in healthy controls, a molecular pattern characterised by increased C3 and decreased C4 was associated with relatively thinner paracentral, inferior parietal and inferior temporal cortices, but relatively thicker insular, in the left hemisphere. In the pooled patient group, a trend for increased C3 in combination with decreased C1q was associated with relatively thinner left lateral occipital cortex and pars orbitalis but relatively thicker pars opercularis and precuneus. Discussion Our findings indicate that peripheral complement concentration is particularly increased early and preceding psychosis and its imbalance may be associated with symptom severity and variation in regional grey matter thickness across the cortex.

Differences in aggressive behavior and DNA copy number variants between BALB/cJ and BALB/cByJ substrains.

PubMed

Velez, Lady; Sokoloff, Greta; Miczek, Klaus A; Palmer, Abraham A; Dulawa, Stephanie C

2010-03-01

Some BALB/c substrains exhibit different levels of aggression. We compared aggression levels between male BALB/cJ and BALB/cByJ substrains using the resident intruder paradigm. These substrains were also assessed in other tests of emotionality and information processing including the open field, forced swim, fear conditioning, and prepulse inhibition tests. We also evaluated single nucleotide polymorphisms (SNPs) previously reported between these BALB/c substrains. Finally, we compared BALB/cJ and BALB/cByJ mice for genomic deletions or duplications, collectively termed copy number variants (CNVs), to identify candidate genes that might underlie the observed behavioral differences. BALB/cJ mice showed substantially higher aggression levels than BALB/cByJ mice; however, only minor differences in other behaviors were observed. None of the previously reported SNPs were verified. Eleven CNV regions were identified between the two BALB/c substrains. Our findings identify a robust difference in aggressive behavior between BALB/cJ and BALB/cByJ substrains, which could be the result of the identified CNVs.

Preeclampsia in autologous and oocyte donation pregnancy: is there a different pathophysiology?

PubMed

Lashley, Lisa E E L O; Buurma, Aletta; Swings, Godelieve M J S; Eikmans, Michael; Anholts, Jacqueline D H; Bakker, Jaap A; Claas, Frans H J

2015-06-01

Oocyte donation (OD) is a specific method of artificial reproductive technology that is accompanied by a higher risk of preeclampsia during pregnancy. The pathophysiological mechanism underlying preeclampsia in OD pregnancies is thought to differ from preeclampsia in autologous pregnancies. As preeclampsia in autologous pregnancies is suggested to be associated with complement activation, we studied C4d deposition, circulating complement components and placental complement regulatory proteins in preeclamptic OD pregnancies. Women with uncomplicated and preeclamptic pregnancies after OD or spontaneous conception were selected. We stained the placentas for C4d, marker for complement activation, measured complement factors C1q, C3 and C4 in maternal sera and quantified the placental mRNA expression of complement regulatory proteins CD46, CD55 and CD59. A significantly (p < 0.03) higher incidence of C4d deposition was observed in placentas from women with preeclampsia compared with uncomplicated pregnancies, both OD and autologous. The level of complement factors in serum did not differ between the groups. Children born in the autologous preeclampsia group were significantly lower in birth weight (p < 10th percentile) compared with the preeclamptic OD group. In addition, the placental mRNA expression level of complement regulatory proteins was significantly lower in uncomplicated and preeclamptic OD compared with the autologous pregnancies. In line with autologous preeclampsia pregnancies, there is excessive activation of complement in preeclamptic OD pregnancies. However, in contrast to autologous pregnancies this is not associated with counterbalancing upregulation of complement regulatory proteins. Furthermore, C4d deposition in OD pregnancies is not related to the severity of preeclampsia, suggesting another trigger or regulatory mechanism of placental C4d deposition in preeclamptic OD pregnancies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Rare copy number variants in a population-based investigation of hypoplastic right heart syndrome.

PubMed

Dimopoulos, Aggeliki; Sicko, Robert J; Kay, Denise M; Rigler, Shannon L; Druschel, Charlotte M; Caggana, Michele; Browne, Marilyn L; Fan, Ruzong; Romitti, Paul A; Brody, Lawrence C; Mills, James L

2017-01-20

Hypoplastic right heart syndrome (HRHS) is a rare congenital defect characterized by underdevelopment of the right heart structures commonly accompanied by an atrial septal defect. Familial HRHS reports suggest genetic factor involvement. We examined the role of copy number variants (CNVs) in HRHS. We genotyped 32 HRHS cases identified from all New York State live births (1998-2005) using Illumina HumanOmni2.5 microarrays. CNVs were called with PennCNV and prioritized if they were ≥20 Kb, contained ≥10 SNPs and had minimal overlap with CNVs from in-house controls, the Database of Genomic Variants, HapMap3, and Childrens Hospital of Philadelphia database. We identified 28 CNVs in 17 cases; several encompassed genes important for right heart development. One case had a 2p16-2p23 duplication spanning LBH, a limb and heart development transcription factor. Lbh mis-expression results in right ventricular hypoplasia and pulmonary valve defects. This duplication also encompassed SOS1, a factor associated with pulmonary valve stenosis in Noonan syndrome. Sos1 -/- mice display thin and poorly trabeculated ventricles. In another case, we identified a 1.5 Mb deletion associated with Williams-Beuren syndrome, a disorder that includes valvular malformations. A third case had a 24 Kb deletion upstream of the TGFβ ligand ITGB8. Embryos genetically null for Itgb8, and its intracellular interactant Band 4.1B, display lethal cardiac phenotypes. To our knowledge, this is the first study of CNVs in HRHS. We identified several rare CNVs that overlap genes related to right ventricular wall and valve development, suggesting that genetics plays a role in HRHS and providing clues for further investigation. Birth Defects Research 109:16-26, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Rare Copy Number Variants in a Population Based Investigation of Hypoplastic Right Heart Syndrome

PubMed Central

Dimopoulos, Aggeliki; Sicko, Robert J.; Kay, Denise M.; Rigler, Shannon L.; Druschel, Charlotte M.; Caggana, Michele; Browne, Marilyn L.; Fan, Ruzong; Romitti, Paul A.; Brody, Lawrence C.; Mills, James L.

2016-01-01

Background Hypoplastic right heart syndrome (HRHS) is a rare congenital defect characterized by underdevelopment of the right heart structures commonly accompanied by an atrial septal defect. Familial HRHS reports suggest genetic factor involvement. We examined the role of copy number variants (CNVs) in HRHS. Methods We genotyped 32 HRHS cases identified from all New York State live births (1998–2005) using Illumina HumanOmni2.5 microarrays. CNVs were called with PennCNV and prioritized if they were ≥20Kb, contained ≥10 SNPs and had minimal overlap with CNVs from in-house controls, the Database of Genomic Variants, HapMap3 and CHOP database. Results We identified 28 CNVs in 17 cases; several encompassed genes important for right heart development. One case had a 2p16–2p23 duplication spanning LBH, a limb and heart development transcription factor. Lbh mis-expression results in right ventricular hypoplasia and pulmonary valve defects. This duplication also encompassed SOS1, a factor associated with pulmonary valve stenosis in Noonan syndrome. Sos1−/− mice display thin and poorly trabeculated ventricles. In another case, we identified a 1.5Mb deletion associated with Williams Beuren syndrome, a disorder that includes valvular malformations. A third case had a 24Kb deletion upstream of the TGFβ ligand ITGB8. Embryos genetically null for Itgb8, and its intracellular interactant Band 4.1B, display lethal cardiac phenotypes. Conclusions To our knowledge, this is the first study of CNVs in HRHS. We identified several rare CNVs that overlap genes related to right ventricular wall and valve development, suggesting that genetics plays a role in HRHS and providing clues for further investigation. PMID:28009100

The effect of algorithms on copy number variant detection.

PubMed

Tsuang, Debby W; Millard, Steven P; Ely, Benjamin; Chi, Peter; Wang, Kenneth; Raskind, Wendy H; Kim, Sulgi; Brkanac, Zoran; Yu, Chang-En

2010-12-30

The detection of copy number variants (CNVs) and the results of CNV-disease association studies rely on how CNVs are defined, and because array-based technologies can only infer CNVs, CNV-calling algorithms can produce vastly different findings. Several authors have noted the large-scale variability between CNV-detection methods, as well as the substantial false positive and false negative rates associated with those methods. In this study, we use variations of four common algorithms for CNV detection (PennCNV, QuantiSNP, HMMSeg, and cnvPartition) and two definitions of overlap (any overlap and an overlap of at least 40% of the smaller CNV) to illustrate the effects of varying algorithms and definitions of overlap on CNV discovery. We used a 56 K Illumina genotyping array enriched for CNV regions to generate hybridization intensities and allele frequencies for 48 Caucasian schizophrenia cases and 48 age-, ethnicity-, and gender-matched control subjects. No algorithm found a difference in CNV burden between the two groups. However, the total number of CNVs called ranged from 102 to 3,765 across algorithms. The mean CNV size ranged from 46 kb to 787 kb, and the average number of CNVs per subject ranged from 1 to 39. The number of novel CNVs not previously reported in normal subjects ranged from 0 to 212. Motivated by the availability of multiple publicly available genome-wide SNP arrays, investigators are conducting numerous analyses to identify putative additional CNVs in complex genetic disorders. However, the number of CNVs identified in array-based studies, and whether these CNVs are novel or valid, will depend on the algorithm(s) used. Thus, given the variety of methods used, there will be many false positives and false negatives. Both guidelines for the identification of CNVs inferred from high-density arrays and the establishment of a gold standard for validation of CNVs are needed.

Complement system studies in systemic lupus erythematosus (SLE)

PubMed

Teisberg, P

1975-01-01

Complement system involvement has been studied in 16 patients with systemic lupus erythematosus (SLE). Circulating conversion products of C3 were observed in 4 cases. Low mean values of C4 and C3 were found, while C3 proactivator (properdin factor B) levels were low in only a few of the patients. The levels of C4, C3 and C3 proactivator were not lower in the 4 patients in whom C3 conversion products could be demonstrated than in the others. It is concluded that the low complement values found in SLE may be caused mainly by deficient synthesis. Signs of complement activation are in this patient material demonstrated early in the disease, and chiefly in patients not receiving immunosuppressive therapy.

CoDE-seq, an augmented whole-exome sequencing, enables the accurate detection of CNVs and mutations in Mendelian obesity and intellectual disability.

PubMed

Montagne, Louise; Derhourhi, Mehdi; Piton, Amélie; Toussaint, Bénédicte; Durand, Emmanuelle; Vaillant, Emmanuel; Thuillier, Dorothée; Gaget, Stefan; De Graeve, Franck; Rabearivelo, Iandry; Lansiaux, Amélie; Lenne, Bruno; Sukno, Sylvie; Desailloud, Rachel; Cnop, Miriam; Nicolescu, Ramona; Cohen, Lior; Zagury, Jean-François; Amouyal, Mélanie; Weill, Jacques; Muller, Jean; Sand, Olivier; Delobel, Bruno; Froguel, Philippe; Bonnefond, Amélie

2018-05-16

The molecular diagnosis of extreme forms of obesity, in which accurate detection of both copy number variations (CNVs) and point mutations, is crucial for an optimal care of the patients and genetic counseling for their families. Whole-exome sequencing (WES) has benefited considerably this molecular diagnosis, but its poor ability to detect CNVs remains a major limitation. We aimed to develop a method (CoDE-seq) enabling the accurate detection of both CNVs and point mutations in one step. CoDE-seq is based on an augmented WES method, using probes distributed uniformly throughout the genome. CoDE-seq was validated in 40 patients for whom chromosomal DNA microarray was available. CNVs and mutations were assessed in 82 children/young adults with suspected Mendelian obesity and/or intellectual disability and in their parents when available (n total  = 145). CoDE-seq not only detected all of the 97 CNVs identified by chromosomal DNA microarrays but also found 84 additional CNVs, due to a better resolution. When compared to CoDE-seq and chromosomal DNA microarrays, WES failed to detect 37% and 14% of CNVs, respectively. In the 82 patients, a likely molecular diagnosis was achieved in >30% of the patients. Half of the genetic diagnoses were explained by CNVs while the other half by mutations. CoDE-seq has proven cost-efficient and highly effective as it avoids the sequential genetic screening approaches currently used in clinical practice for the accurate detection of CNVs and point mutations. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

Transmission disequilibrium of small CNVs in simplex autism.

PubMed

Krumm, Niklas; O'Roak, Brian J; Karakoc, Emre; Mohajeri, Kiana; Nelson, Ben; Vives, Laura; Jacquemont, Sebastien; Munson, Jeff; Bernier, Raphe; Eichler, Evan E

2013-10-03

We searched for disruptive, genic rare copy-number variants (CNVs) among 411 families affected by sporadic autism spectrum disorder (ASD) from the Simons Simplex Collection by using available exome sequence data and CoNIFER (Copy Number Inference from Exome Reads). Compared to high-density SNP microarrays, our approach yielded ∼2× more smaller genic rare CNVs. We found that affected probands inherited more CNVs than did their siblings (453 versus 394, p = 0.004; odds ratio [OR] = 1.19) and that the probands' CNVs affected more genes (921 versus 726, p = 0.02; OR = 1.30). These smaller CNVs (median size 18 kb) were transmitted preferentially from the mother (136 maternal versus 100 paternal, p = 0.02), although this bias occurred irrespective of affected status. The excess burden of inherited CNVs among probands was driven primarily by sibling pairs with discordant social-behavior phenotypes (p < 0.0002, measured by Social Responsiveness Scale [SRS] score), which contrasts with families where the phenotypes were more closely matched or less extreme (p > 0.5). Finally, we found enrichment of brain-expressed genes unique to probands, especially in the SRS-discordant group (p = 0.0035). In a combined model, our inherited CNVs, de novo CNVs, and de novo single-nucleotide variants all independently contributed to the risk of autism (p < 0.05). Taken together, these results suggest that small transmitted rare CNVs play a role in the etiology of simplex autism. Importantly, the small size of these variants aids in the identification of specific genes as additional risk factors associated with ASD. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Genome-wide copy number variant analysis for congenital ventricular septal defects in Chinese Han population.

PubMed

An, Yu; Duan, Wenyuan; Huang, Guoying; Chen, Xiaoli; Li, Li; Nie, Chenxia; Hou, Jia; Gui, Yonghao; Wu, Yiming; Zhang, Feng; Shen, Yiping; Wu, Bailin; Wang, Hongyan

2016-01-08

Ventricular septal defects (VSDs) constitute the most prevalent congenital heart disease (CHD), occurs either in isolation (isolated VSD) or in combination with other cardiac defects (complex VSD). Copy number variation (CNV) has been highlighted as a possible contributing factor to the etiology of many congenital diseases. However, little is known concerning the involvement of CNVs in either isolated or complex VSDs. We analyzed 154 unrelated Chinese individuals with VSD by chromosomal microarray analysis. The subjects were recruited from four hospitals across China. Each case underwent clinical assessment to define the type of VSD, either isolated or complex VSD. CNVs detected were categorized into syndrom related CNVs, recurrent CNVs and rare CNVs. Genes encompassed by the CNVs were analyzed using enrichment and pathway analysis. Among 154 probands, we identified 29 rare CNVs in 26 VSD patients (16.9 %, 26/154) and 8 syndrome-related CNVs in 8 VSD patients (5.2 %, 8/154). 12 of the detected 29 rare CNVs (41.3 %) were recurrently reported in DECIPHER or ISCA database as associated with either VSD or general heart disease. Fifteen genes (5 %, 15/285) within CNVs were associated with a broad spectrum of complicated CHD. Among these15 genes, 7 genes were in "abnormal interventricular septum morphology" derived from the MGI (mouse genome informatics) database, and nine genes were associated with cardiovascular system development (GO:0072538).We also found that these VSD-related candidate genes are enriched in chromatin binding and transcription regulation, which are the biological processes underlying heart development. Our study demonstrates the potential clinical diagnostic utility of genomic imbalance profiling in VSD patients. Additionally, gene enrichment and pathway analysis helped us to implicate VSD related candidate genes.

Classical Complement Pathway Activation in the Kidneys of Women With Preeclampsia.

PubMed

Penning, Marlies; Chua, Jamie S; van Kooten, Cees; Zandbergen, Malu; Buurma, Aletta; Schutte, Joke; Bruijn, Jan Anthonie; Khankin, Eliyahu V; Bloemenkamp, Kitty; Karumanchi, S Ananth; Baelde, Hans

2015-07-01

A growing body of evidence suggests that complement dysregulation plays a role in the pathogenesis of preeclampsia. The kidney is one of the major organs affected in preeclampsia. Because the kidney is highly susceptible to complement activation, we hypothesized that preeclampsia is associated with renal complement activation. We performed a nationwide search for renal autopsy material in the Netherlands using a computerized database (PALGA). Renal tissue was obtained from 11 women with preeclampsia, 25 pregnant controls, and 14 nonpregnant controls with hypertension. The samples were immunostained for C4d, C1q, mannose-binding lectin, properdin, C3d, C5b-9, IgA, IgG, and IgM. Preeclampsia was significantly associated with renal C4d-a stable marker of complement activation-and the classical pathway marker C1q. In addition, the prevalence of IgM was significantly higher in the kidneys of the preeclamptic women. No other complement markers studied differed between the groups. Our findings in human samples were validated using a soluble fms-like tyrosine kinase 1 mouse model of preeclampsia. The kidneys in the soluble fms-like tyrosine kinase 1-injected mice had significantly more C4 deposits than the control mice. The association between preeclampsia and renal C4d, C1q, and IgM levels suggests that the classical complement pathway is involved in the renal injury in preeclampsia. Moreover, our finding that soluble fms-like tyrosine kinase 1-injected mice develop excess C4 deposits indicates that angiogenic dysregulation may play a role in complement activation within the kidney. We suggest that inhibiting complement activation may be beneficial for preventing the renal manifestations of preeclampsia. © 2015 American Heart Association, Inc.

Copy Number Variations in Tilapia Genomes.

PubMed

Li, Bi Jun; Li, Hong Lian; Meng, Zining; Zhang, Yong; Lin, Haoran; Yue, Gen Hua; Xia, Jun Hong

2017-02-01

Discovering the nature and pattern of genome variation is fundamental in understanding phenotypic diversity among populations. Although several millions of single nucleotide polymorphisms (SNPs) have been discovered in tilapia, the genome-wide characterization of larger structural variants, such as copy number variation (CNV) regions has not been carried out yet. We conducted a genome-wide scan for CNVs in 47 individuals from three tilapia populations. Based on 254 Gb of high-quality paired-end sequencing reads, we identified 4642 distinct high-confidence CNVs. These CNVs account for 1.9% (12.411 Mb) of the used Nile tilapia reference genome. A total of 1100 predicted CNVs were found overlapping with exon regions of protein genes. Further association analysis based on linear model regression found 85 CNVs ranging between 300 and 27,000 base pairs significantly associated to population types (R 2  > 0.9 and P > 0.001). Our study sheds first insights on genome-wide CNVs in tilapia. These CNVs among and within tilapia populations may have functional effects on phenotypes and specific adaptation to particular environments.

Modeling read counts for CNV detection in exome sequencing data.

PubMed

Love, Michael I; Myšičková, Alena; Sun, Ruping; Kalscheuer, Vera; Vingron, Martin; Haas, Stefan A

2011-11-08

Varying depth of high-throughput sequencing reads along a chromosome makes it possible to observe copy number variants (CNVs) in a sample relative to a reference. In exome and other targeted sequencing projects, technical factors increase variation in read depth while reducing the number of observed locations, adding difficulty to the problem of identifying CNVs. We present a hidden Markov model for detecting CNVs from raw read count data, using background read depth from a control set as well as other positional covariates such as GC-content. The model, exomeCopy, is applied to a large chromosome X exome sequencing project identifying a list of large unique CNVs. CNVs predicted by the model and experimentally validated are then recovered using a cross-platform control set from publicly available exome sequencing data. Simulations show high sensitivity for detecting heterozygous and homozygous CNVs, outperforming normalization and state-of-the-art segmentation methods.

Novel Scabies Mite Serpins Inhibit the Three Pathways of the Human Complement System

PubMed Central

Mika, Angela; Reynolds, Simone L.; Mohlin, Frida C.; Willis, Charlene; Swe, Pearl M.; Pickering, Darren A.; Halilovic, Vanja; Wijeyewickrema, Lakshmi C.; Pike, Robert N.; Blom, Anna M.; Kemp, David J.; Fischer, Katja

2012-01-01

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage. PMID:22792350

Comprehensive analysis of Stargardt macular dystrophy patients reveals new genotype-phenotype correlations and unexpected diagnostic revisions

PubMed Central

Zaneveld, Jacques; Siddiqui, Sorath; Li, Huajin; Wang, Xia; Wang, Hui; Wang, Keqing; Li, Hui; Ren, Huanan; Lopez, Irma; Dorfman, Allison; Khan, Ayesha; Wang, Feng; Salvo, Jason; Gelowani, Violet; Li, Yumei; Sui, Ruifang; Koenekoop, Robert; Chen, Rui

2014-01-01

Purpose Stargardt macular dystrophy (STGD) results in early central vision loss. We sought to explain the genetic cause of STGD in a cohort of 88 patients from three different cultural backgrounds. Methods Next Generation Sequencing using a novel capture panel was used to search for disease causing mutations. Unsolved patients were clinically re-examined and tested for copy number variations (CNVs) as well as intronic mutations. Results We determined the cause of disease in 67% of our patients. Our analysis identified 35 novel ABCA4 alleles. Eleven patients had mutations in genes not previously reported to cause STGD. Finally, 45% of our unsolved patients had single deleterious mutations in ABCA4, a recessive disease gene. No likely pathogenic CNVs were identified. Conclusions This study expands our knowledge of STGD by identifying dozens of novel STGD causing alleles. The frequency of patients with single mutations in ABCA4 is higher than controls, indicating these mutations contribute to disease. Eleven patients were explained by mutations outside ABCA4 underlining the need to genotype all retinal disease genes to maximize genetic diagnostic rates. Few ABCA4 mutations were observed in our French Canadian patients. This population may contain an unidentified founder mutation. Our results indicate that CNVs are unlikely to be a major cause of STGD. PMID:25474345

Population clustering based on copy number variations detected from next generation sequencing data.

PubMed

Duan, Junbo; Zhang, Ji-Gang; Wan, Mingxi; Deng, Hong-Wen; Wang, Yu-Ping

2014-08-01

Copy number variations (CNVs) can be used as significant bio-markers and next generation sequencing (NGS) provides a high resolution detection of these CNVs. But how to extract features from CNVs and further apply them to genomic studies such as population clustering have become a big challenge. In this paper, we propose a novel method for population clustering based on CNVs from NGS. First, CNVs are extracted from each sample to form a feature matrix. Then, this feature matrix is decomposed into the source matrix and weight matrix with non-negative matrix factorization (NMF). The source matrix consists of common CNVs that are shared by all the samples from the same group, and the weight matrix indicates the corresponding level of CNVs from each sample. Therefore, using NMF of CNVs one can differentiate samples from different ethnic groups, i.e. population clustering. To validate the approach, we applied it to the analysis of both simulation data and two real data set from the 1000 Genomes Project. The results on simulation data demonstrate that the proposed method can recover the true common CNVs with high quality. The results on the first real data analysis show that the proposed method can cluster two family trio with different ancestries into two ethnic groups and the results on the second real data analysis show that the proposed method can be applied to the whole-genome with large sample size consisting of multiple groups. Both results demonstrate the potential of the proposed method for population clustering.

High-resolution copy number variation analysis of schizophrenia in Japan.

PubMed

Kushima, I; Aleksic, B; Nakatochi, M; Shimamura, T; Shiino, T; Yoshimi, A; Kimura, H; Takasaki, Y; Wang, C; Xing, J; Ishizuka, K; Oya-Ito, T; Nakamura, Y; Arioka, Y; Maeda, T; Yamamoto, M; Yoshida, M; Noma, H; Hamada, S; Morikawa, M; Uno, Y; Okada, T; Iidaka, T; Iritani, S; Yamamoto, T; Miyashita, M; Kobori, A; Arai, M; Itokawa, M; Cheng, M-C; Chuang, Y-A; Chen, C-H; Suzuki, M; Takahashi, T; Hashimoto, R; Yamamori, H; Yasuda, Y; Watanabe, Y; Nunokawa, A; Someya, T; Ikeda, M; Toyota, T; Yoshikawa, T; Numata, S; Ohmori, T; Kunimoto, S; Mori, D; Iwata, N; Ozaki, N

2017-03-01

Recent schizophrenia (SCZ) studies have reported an increased burden of de novo copy number variants (CNVs) and identified specific high-risk CNVs, although with variable phenotype expressivity. However, the pathogenesis of SCZ has not been fully elucidated. Using array comparative genomic hybridization, we performed a high-resolution genome-wide CNV analysis on a mainly (92%) Japanese population (1699 SCZ cases and 824 controls) and identified 7066 rare CNVs, 70.0% of which were small (<100 kb). Clinically significant CNVs were significantly more frequent in cases than in controls (odds ratio=3.04, P=9.3 × 10 -9 , 9.0% of cases). We confirmed a significant association of X-chromosome aneuploidies with SCZ and identified 11 de novo CNVs (e.g., MBD5 deletion) in cases. In patients with clinically significant CNVs, 41.7% had a history of congenital/developmental phenotypes, and the rate of treatment resistance was significantly higher (odds ratio=2.79, P=0.0036). We found more severe clinical manifestations in patients with two clinically significant CNVs. Gene set analysis replicated previous findings (e.g., synapse, calcium signaling) and identified novel biological pathways including oxidative stress response, genomic integrity, kinase and small GTPase signaling. Furthermore, involvement of multiple SCZ candidate genes and biological pathways in the pathogenesis of SCZ was suggested in established SCZ-associated CNV loci. Our study shows the high genetic heterogeneity of SCZ and its clinical features and raises the possibility that genomic instability is involved in its pathogenesis, which may be related to the increased burden of de novo CNVs and variable expressivity of CNVs.

Convergence of Genes and Cellular Pathways Dysregulated in Autism Spectrum Disorders

PubMed Central

Pinto, Dalila; Delaby, Elsa; Merico, Daniele; Barbosa, Mafalda; Merikangas, Alison; Klei, Lambertus; Thiruvahindrapuram, Bhooma; Xu, Xiao; Ziman, Robert; Wang, Zhuozhi; Vorstman, Jacob A.S.; Thompson, Ann; Regan, Regina; Pilorge, Marion; Pellecchia, Giovanna; Pagnamenta, Alistair T.; Oliveira, Bárbara; Marshall, Christian R.; Magalhaes, Tiago R.; Lowe, Jennifer K.; Howe, Jennifer L.; Griswold, Anthony J.; Gilbert, John; Duketis, Eftichia; Dombroski, Beth A.; De Jonge, Maretha V.; Cuccaro, Michael; Crawford, Emily L.; Correia, Catarina T.; Conroy, Judith; Conceição, Inês C.; Chiocchetti, Andreas G.; Casey, Jillian P.; Cai, Guiqing; Cabrol, Christelle; Bolshakova, Nadia; Bacchelli, Elena; Anney, Richard; Gallinger, Steven; Cotterchio, Michelle; Casey, Graham; Zwaigenbaum, Lonnie; Wittemeyer, Kerstin; Wing, Kirsty; Wallace, Simon; van Engeland, Herman; Tryfon, Ana; Thomson, Susanne; Soorya, Latha; Rogé, Bernadette; Roberts, Wendy; Poustka, Fritz; Mouga, Susana; Minshew, Nancy; McInnes, L. Alison; McGrew, Susan G.; Lord, Catherine; Leboyer, Marion; Le Couteur, Ann S.; Kolevzon, Alexander; Jiménez González, Patricia; Jacob, Suma; Holt, Richard; Guter, Stephen; Green, Jonathan; Green, Andrew; Gillberg, Christopher; Fernandez, Bridget A.; Duque, Frederico; Delorme, Richard; Dawson, Geraldine; Chaste, Pauline; Café, Cátia; Brennan, Sean; Bourgeron, Thomas; Bolton, Patrick F.; Bölte, Sven; Bernier, Raphael; Baird, Gillian; Bailey, Anthony J.; Anagnostou, Evdokia; Almeida, Joana; Wijsman, Ellen M.; Vieland, Veronica J.; Vicente, Astrid M.; Schellenberg, Gerard D.; Pericak-Vance, Margaret; Paterson, Andrew D.; Parr, Jeremy R.; Oliveira, Guiomar; Nurnberger, John I.; Monaco, Anthony P.; Maestrini, Elena; Klauck, Sabine M.; Hakonarson, Hakon; Haines, Jonathan L.; Geschwind, Daniel H.; Freitag, Christine M.; Folstein, Susan E.; Ennis, Sean; Coon, Hilary; Battaglia, Agatino; Szatmari, Peter; Sutcliffe, James S.; Hallmayer, Joachim; Gill, Michael; Cook, Edwin H.; Buxbaum, Joseph D.; Devlin, Bernie; Gallagher, Louise; Betancur, Catalina; Scherer, Stephen W.

2014-01-01

Rare copy-number variation (CNV) is an important source of risk for autism spectrum disorders (ASDs). We analyzed 2,446 ASD-affected families and confirmed an excess of genic deletions and duplications in affected versus control groups (1.41-fold, p = 1.0 × 10−5) and an increase in affected subjects carrying exonic pathogenic CNVs overlapping known loci associated with dominant or X-linked ASD and intellectual disability (odds ratio = 12.62, p = 2.7 × 10−15, ∼3% of ASD subjects). Pathogenic CNVs, often showing variable expressivity, included rare de novo and inherited events at 36 loci, implicating ASD-associated genes (CHD2, HDAC4, and GDI1) previously linked to other neurodevelopmental disorders, as well as other genes such as SETD5, MIR137, and HDAC9. Consistent with hypothesized gender-specific modulators, females with ASD were more likely to have highly penetrant CNVs (p = 0.017) and were also overrepresented among subjects with fragile X syndrome protein targets (p = 0.02). Genes affected by de novo CNVs and/or loss-of-function single-nucleotide variants converged on networks related to neuronal signaling and development, synapse function, and chromatin regulation. PMID:24768552

CNVcaller: highly efficient and widely applicable software for detecting copy number variations in large populations.

PubMed

Wang, Xihong; Zheng, Zhuqing; Cai, Yudong; Chen, Ting; Li, Chao; Fu, Weiwei; Jiang, Yu

2017-12-01

The increasing amount of sequencing data available for a wide variety of species can be theoretically used for detecting copy number variations (CNVs) at the population level. However, the growing sample sizes and the divergent complexity of nonhuman genomes challenge the efficiency and robustness of current human-oriented CNV detection methods. Here, we present CNVcaller, a read-depth method for discovering CNVs in population sequencing data. The computational speed of CNVcaller was 1-2 orders of magnitude faster than CNVnator and Genome STRiP for complex genomes with thousands of unmapped scaffolds. CNV detection of 232 goats required only 1.4 days on a single compute node. Additionally, the Mendelian consistency of sheep trios indicated that CNVcaller mitigated the influence of high proportions of gaps and misassembled duplications in the nonhuman reference genome assembly. Furthermore, multiple evaluations using real sheep and human data indicated that CNVcaller achieved the best accuracy and sensitivity for detecting duplications. The fast generalized detection algorithms included in CNVcaller overcome prior computational barriers for detecting CNVs in large-scale sequencing data with complex genomic structures. Therefore, CNVcaller promotes population genetic analyses of functional CNVs in more species. © The Authors 2017. Published by Oxford University Press.

CNVcaller: highly efficient and widely applicable software for detecting copy number variations in large populations

PubMed Central

Wang, Xihong; Zheng, Zhuqing; Cai, Yudong; Chen, Ting; Li, Chao; Fu, Weiwei

2017-01-01

Abstract Background The increasing amount of sequencing data available for a wide variety of species can be theoretically used for detecting copy number variations (CNVs) at the population level. However, the growing sample sizes and the divergent complexity of nonhuman genomes challenge the efficiency and robustness of current human-oriented CNV detection methods. Results Here, we present CNVcaller, a read-depth method for discovering CNVs in population sequencing data. The computational speed of CNVcaller was 1–2 orders of magnitude faster than CNVnator and Genome STRiP for complex genomes with thousands of unmapped scaffolds. CNV detection of 232 goats required only 1.4 days on a single compute node. Additionally, the Mendelian consistency of sheep trios indicated that CNVcaller mitigated the influence of high proportions of gaps and misassembled duplications in the nonhuman reference genome assembly. Furthermore, multiple evaluations using real sheep and human data indicated that CNVcaller achieved the best accuracy and sensitivity for detecting duplications. Conclusions The fast generalized detection algorithms included in CNVcaller overcome prior computational barriers for detecting CNVs in large-scale sequencing data with complex genomic structures. Therefore, CNVcaller promotes population genetic analyses of functional CNVs in more species. PMID:29220491

Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls.

PubMed

Craddock, Nick; Hurles, Matthew E; Cardin, Niall; Pearson, Richard D; Plagnol, Vincent; Robson, Samuel; Vukcevic, Damjan; Barnes, Chris; Conrad, Donald F; Giannoulatou, Eleni; Holmes, Chris; Marchini, Jonathan L; Stirrups, Kathy; Tobin, Martin D; Wain, Louise V; Yau, Chris; Aerts, Jan; Ahmad, Tariq; Andrews, T Daniel; Arbury, Hazel; Attwood, Anthony; Auton, Adam; Ball, Stephen G; Balmforth, Anthony J; Barrett, Jeffrey C; Barroso, Inês; Barton, Anne; Bennett, Amanda J; Bhaskar, Sanjeev; Blaszczyk, Katarzyna; Bowes, John; Brand, Oliver J; Braund, Peter S; Bredin, Francesca; Breen, Gerome; Brown, Morris J; Bruce, Ian N; Bull, Jaswinder; Burren, Oliver S; Burton, John; Byrnes, Jake; Caesar, Sian; Clee, Chris M; Coffey, Alison J; Connell, John M C; Cooper, Jason D; Dominiczak, Anna F; Downes, Kate; Drummond, Hazel E; Dudakia, Darshna; Dunham, Andrew; Ebbs, Bernadette; Eccles, Diana; Edkins, Sarah; Edwards, Cathryn; Elliot, Anna; Emery, Paul; Evans, David M; Evans, Gareth; Eyre, Steve; Farmer, Anne; Ferrier, I Nicol; Feuk, Lars; Fitzgerald, Tomas; Flynn, Edward; Forbes, Alistair; Forty, Liz; Franklyn, Jayne A; Freathy, Rachel M; Gibbs, Polly; Gilbert, Paul; Gokumen, Omer; Gordon-Smith, Katherine; Gray, Emma; Green, Elaine; Groves, Chris J; Grozeva, Detelina; Gwilliam, Rhian; Hall, Anita; Hammond, Naomi; Hardy, Matt; Harrison, Pile; Hassanali, Neelam; Hebaishi, Husam; Hines, Sarah; Hinks, Anne; Hitman, Graham A; Hocking, Lynne; Howard, Eleanor; Howard, Philip; Howson, Joanna M M; Hughes, Debbie; Hunt, Sarah; Isaacs, John D; Jain, Mahim; Jewell, Derek P; Johnson, Toby; Jolley, Jennifer D; Jones, Ian R; Jones, Lisa A; Kirov, George; Langford, Cordelia F; Lango-Allen, Hana; Lathrop, G Mark; Lee, James; Lee, Kate L; Lees, Charlie; Lewis, Kevin; Lindgren, Cecilia M; Maisuria-Armer, Meeta; Maller, Julian; Mansfield, John; Martin, Paul; Massey, Dunecan C O; McArdle, Wendy L; McGuffin, Peter; McLay, Kirsten E; Mentzer, Alex; Mimmack, Michael L; Morgan, Ann E; Morris, Andrew P; Mowat, Craig; Myers, Simon; Newman, William; Nimmo, Elaine R; O'Donovan, Michael C; Onipinla, Abiodun; Onyiah, Ifejinelo; Ovington, Nigel R; Owen, Michael J; Palin, Kimmo; Parnell, Kirstie; Pernet, David; Perry, John R B; Phillips, Anne; Pinto, Dalila; Prescott, Natalie J; Prokopenko, Inga; Quail, Michael A; Rafelt, Suzanne; Rayner, Nigel W; Redon, Richard; Reid, David M; Renwick; Ring, Susan M; Robertson, Neil; Russell, Ellie; St Clair, David; Sambrook, Jennifer G; Sanderson, Jeremy D; Schuilenburg, Helen; Scott, Carol E; Scott, Richard; Seal, Sheila; Shaw-Hawkins, Sue; Shields, Beverley M; Simmonds, Matthew J; Smyth, Debbie J; Somaskantharajah, Elilan; Spanova, Katarina; Steer, Sophia; Stephens, Jonathan; Stevens, Helen E; Stone, Millicent A; Su, Zhan; Symmons, Deborah P M; Thompson, John R; Thomson, Wendy; Travers, Mary E; Turnbull, Clare; Valsesia, Armand; Walker, Mark; Walker, Neil M; Wallace, Chris; Warren-Perry, Margaret; Watkins, Nicholas A; Webster, John; Weedon, Michael N; Wilson, Anthony G; Woodburn, Matthew; Wordsworth, B Paul; Young, Allan H; Zeggini, Eleftheria; Carter, Nigel P; Frayling, Timothy M; Lee, Charles; McVean, Gil; Munroe, Patricia B; Palotie, Aarno; Sawcer, Stephen J; Scherer, Stephen W; Strachan, David P; Tyler-Smith, Chris; Brown, Matthew A; Burton, Paul R; Caulfield, Mark J; Compston, Alastair; Farrall, Martin; Gough, Stephen C L; Hall, Alistair S; Hattersley, Andrew T; Hill, Adrian V S; Mathew, Christopher G; Pembrey, Marcus; Satsangi, Jack; Stratton, Michael R; Worthington, Jane; Deloukas, Panos; Duncanson, Audrey; Kwiatkowski, Dominic P; McCarthy, Mark I; Ouwehand, Willem; Parkes, Miles; Rahman, Nazneen; Todd, John A; Samani, Nilesh J; Donnelly, Peter

2010-04-01

Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed approximately 19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated approximately 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with disease-IRGM for Crohn's disease, HLA for Crohn's disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetes-although in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases.

The phenotypic manifestations of rare CNVs in schizophrenia.

PubMed

Merikangas, Alison K; Segurado, Ricardo; Cormican, Paul; Heron, Elizabeth A; Anney, Richard J L; Moore, Susan; Kelleher, Eric; Hargreaves, April; Anderson-Schmidt, Heike; Gill, Michael; Gallagher, Louise; Corvin, Aiden

2014-09-01

There is compelling evidence for the role of copy number variants (CNVs) in schizophrenia susceptibility, and it has been estimated that up to 2-3% of schizophrenia cases may carry rare CNVs. Despite evidence that these events are associated with an increased risk across categorical neurodevelopmental disorders, there is limited understanding of the impact of CNVs on the core features of disorders like schizophrenia. Our objective was to evaluate associations between rare CNVs in differentially brain expressed (BE) genes and the core features and clinical correlates of schizophrenia. The sample included 386 cases of Irish ancestry with a diagnosis of schizophrenia, at least one rare CNV impacting any gene, and a core set of phenotypic measures. Statistically significant associations between deletions in differentially BE genes were found for family history of mental illness (decreased prevalence of all CNVs and deletions, unadjusted and adjusted) and for paternal age (increase in deletions only, unadjusted, among those with later ages at birth of patient). The strong effect of a lack of a family history on BE genes suggests that CNVs may comprise one pathway to schizophrenia, whereas a positive family history could index other genetic mechanisms that increase schizophrenia vulnerability. To our knowledge, this is the first investigation of the association between genome-wide CNVs and risk factors and sub-phenotypic features of schizophrenia beyond cognitive function. Copyright © 2014 Elsevier B.V. All rights reserved.

Complement C4a inhibits the secretion of hepatitis B virus screened by surface-enhanced laser desorption ionization time-flight mass spectrometry-based ProteinChip analysis.

PubMed

Song, Ya-Nan; Zhang, Gui-Biao; Hu, Xue-Qing; Lu, Yi-Yu; Zhao, Yu; Yang, Yang; Yang, Yi-Fu; Zhang, Yong-Yu; Hu, Yi-Yang; Su, Shi-Bing

2015-12-01

Chronic hepatitis B (CHB) is a kind of chronic liver disease caused by persistent hepatitis B virus (HBV) infection. The study aims to seek the factors of host resistance to HBV and investigate their roles. Protein profiles of 58 healthy controls and 121 CHB patients were obtained by SELDI-TOF/MS. Predicted protein was validated by ELISA. Protein expression was evaluated by Western blot in the persistently HBV expressing cell line HepG2.2.15 and non-HBV expressing cell line HepG2. The level of HBV DNA was subsequently detected by quantitative real-time PCR in HepG2.2.15 cells with complement C4a treatment. Significantly altered protein peaks were found through statistical analysis, and m/z 4300 was predicted by databases and successfully matched with the fragment of complement C4a. According to ELISA, serum complement C4a was found to be significantly lower in CHB patients compared with healthy controls (p < 0.001) and the area under receiver operating characteristics curve is 0.78. Furthermore, complement C4a showed lower expression in HepG2.2.5 cells and the secretion of HBV DNA was inhibited by complement C4a. The present study implied the important role of complement C4a in inhibiting the HBV DNA secretion in CHB. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Growing Importance of CNVs: New Insights for Detection and Clinical Interpretation

PubMed Central

Valsesia, Armand; Macé, Aurélien; Jacquemont, Sébastien; Beckmann, Jacques S.; Kutalik, Zoltán

2013-01-01

Differences between genomes can be due to single nucleotide variants, translocations, inversions, and copy number variants (CNVs, gain or loss of DNA). The latter can range from sub-microscopic events to complete chromosomal aneuploidies. Small CNVs are often benign but those larger than 500 kb are strongly associated with morbid consequences such as developmental disorders and cancer. Detecting CNVs within and between populations is essential to better understand the plasticity of our genome and to elucidate its possible contribution to disease. Hence there is a need for better-tailored and more robust tools for the detection and genome-wide analyses of CNVs. While a link between a given CNV and a disease may have often been established, the relative CNV contribution to disease progression and impact on drug response is not necessarily understood. In this review we discuss the progress, challenges, and limitations that occur at different stages of CNV analysis from the detection (using DNA microarrays and next-generation sequencing) and identification of recurrent CNVs to the association with phenotypes. We emphasize the importance of germline CNVs and propose strategies to aid clinicians to better interpret structural variations and assess their clinical implications. PMID:23750167

The phenotypic manifestations of rare genic CNVs in autism spectrum disorder

PubMed Central

Merikangas, A K; Segurado, R; Heron, E A; Anney, R J L; Paterson, A D; Cook, E H; Pinto, D; Scherer, S W; Szatmari, P; Gill, M; Corvin, A P; Gallagher, L

2015-01-01

Significant evidence exists for the association between copy number variants (CNVs) and Autism Spectrum Disorder (ASD); however, most of this work has focused solely on the diagnosis of ASD. There is limited understanding of the impact of CNVs on the ‘sub-phenotypes' of ASD. The objective of this paper is to evaluate associations between CNVs in differentially brain expressed (DBE) genes or genes previously implicated in ASD/intellectual disability (ASD/ID) and specific sub-phenotypes of ASD. The sample consisted of 1590 cases of European ancestry from the Autism Genome Project (AGP) with a diagnosis of an ASD and at least one rare CNV impacting any gene and a core set of phenotypic measures, including symptom severity, language impairments, seizures, gait disturbances, intelligence quotient (IQ) and adaptive function, as well as paternal and maternal age. Classification analyses using a non-parametric recursive partitioning method (random forests) were employed to define sets of phenotypic characteristics that best classify the CNV-defined groups. There was substantial variation in the classification accuracy of the two sets of genes. The best variables for classification were verbal IQ for the ASD/ID genes, paternal age at birth for the DBE genes and adaptive function for de novo CNVs. CNVs in the ASD/ID list were primarily associated with communication and language domains, whereas CNVs in DBE genes were related to broader manifestations of adaptive function. To our knowledge, this is the first study to examine the associations between sub-phenotypes and CNVs genome-wide in ASD. This work highlights the importance of examining the diverse sub-phenotypic manifestations of CNVs in ASD, including the specific features, comorbid conditions and clinical correlates of ASD that comprise underlying characteristics of the disorder. PMID:25421404

The phenotypic manifestations of rare genic CNVs in autism spectrum disorder.

PubMed

Merikangas, A K; Segurado, R; Heron, E A; Anney, R J L; Paterson, A D; Cook, E H; Pinto, D; Scherer, S W; Szatmari, P; Gill, M; Corvin, A P; Gallagher, L

2015-11-01

Significant evidence exists for the association between copy number variants (CNVs) and Autism Spectrum Disorder (ASD); however, most of this work has focused solely on the diagnosis of ASD. There is limited understanding of the impact of CNVs on the 'sub-phenotypes' of ASD. The objective of this paper is to evaluate associations between CNVs in differentially brain expressed (DBE) genes or genes previously implicated in ASD/intellectual disability (ASD/ID) and specific sub-phenotypes of ASD. The sample consisted of 1590 cases of European ancestry from the Autism Genome Project (AGP) with a diagnosis of an ASD and at least one rare CNV impacting any gene and a core set of phenotypic measures, including symptom severity, language impairments, seizures, gait disturbances, intelligence quotient (IQ) and adaptive function, as well as paternal and maternal age. Classification analyses using a non-parametric recursive partitioning method (random forests) were employed to define sets of phenotypic characteristics that best classify the CNV-defined groups. There was substantial variation in the classification accuracy of the two sets of genes. The best variables for classification were verbal IQ for the ASD/ID genes, paternal age at birth for the DBE genes and adaptive function for de novo CNVs. CNVs in the ASD/ID list were primarily associated with communication and language domains, whereas CNVs in DBE genes were related to broader manifestations of adaptive function. To our knowledge, this is the first study to examine the associations between sub-phenotypes and CNVs genome-wide in ASD. This work highlights the importance of examining the diverse sub-phenotypic manifestations of CNVs in ASD, including the specific features, comorbid conditions and clinical correlates of ASD that comprise underlying characteristics of the disorder.

Genome-wide copy number variation (CNV) detection in Nelore cattle reveals highly frequent variants in genome regions harboring QTLs affecting production traits.

PubMed

da Silva, Joaquim Manoel; Giachetto, Poliana Fernanda; da Silva, Luiz Otávio; Cintra, Leandro Carrijo; Paiva, Samuel Rezende; Yamagishi, Michel Eduardo Beleza; Caetano, Alexandre Rodrigues

2016-06-13

Copy number variations (CNVs) have been shown to account for substantial portions of observed genomic variation and have been associated with qualitative and quantitative traits and the onset of disease in a number of species. Information from high-resolution studies to detect, characterize and estimate population-specific variant frequencies will facilitate the incorporation of CNVs in genomic studies to identify genes affecting traits of importance. Genome-wide CNVs were detected in high-density single nucleotide polymorphism (SNP) genotyping data from 1,717 Nelore (Bos indicus) cattle, and in NGS data from eight key ancestral bulls. A total of 68,007 and 12,786 distinct CNVs were observed, respectively. Cross-comparisons of results obtained for the eight resequenced animals revealed that 92 % of the CNVs were observed in both datasets, while 62 % of all detected CNVs were observed to overlap with previously validated cattle copy number variant regions (CNVRs). Observed CNVs were used for obtaining breed-specific CNV frequencies and identification of CNVRs, which were subsequently used for gene annotation. A total of 688 of the detected CNVRs were observed to overlap with 286 non-redundant QTLs associated with important production traits in cattle. All of 34 CNVs previously reported to be associated with milk production traits in Holsteins were also observed in Nelore cattle. Comparisons of estimated frequencies of these CNVs in the two breeds revealed 14, 13, 6 and 14 regions in high (>20 %), low (<20 %) and divergent (NEL > HOL, NEL < HOL) frequencies, respectively. Obtained results significantly enriched the bovine CNV map and enabled the identification of variants that are potentially associated with traits under selection in Nelore cattle, particularly in genome regions harboring QTLs affecting production traits.

Burden of potentially pathologic copy number variants is higher in children with isolated congenital heart disease and significantly impairs covariate-adjusted transplant-free survival.

PubMed

Kim, Daniel Seung; Kim, Jerry H; Burt, Amber A; Crosslin, David R; Burnham, Nancy; Kim, Cecilia E; McDonald-McGinn, Donna M; Zackai, Elaine H; Nicolson, Susan C; Spray, Thomas L; Stanaway, Ian B; Nickerson, Deborah A; Heagerty, Patrick J; Hakonarson, Hakon; Gaynor, J William; Jarvik, Gail P

2016-04-01

Copy number variants (CNVs) are duplications or deletions of genomic regions. Large CNVs are potentially pathogenic and are overrepresented in children with congenital heart disease (CHD). We sought to determine the frequency of large CNVs in children with isolated CHD, and to evaluate the relationship of these potentially pathogenic CNVs with transplant-free survival. These cases are derived from a prospective cohort of patients with nonsyndromic CHD (n = 422) identified before first surgery. Healthy pediatric controls (n = 500) were obtained from the electronic Medical Records and Genetic Epidemiology Network, and CNV frequency was contrasted for CHD cases and controls. CNVs were determined algorithmically; subsequently screened for >95% overlap between 2 methods, size (>300 kb), quality score, overlap with a gene, and novelty (absent from databases of known, benign CNVs); and separately validated by quantitative polymerase chain reaction. Survival likelihoods for cases were calculated using Cox proportional hazards modeling to evaluate the joint effect of CNV burden and known confounders on transplant-free survival. Children with nonsyndromic CHD had a higher burden of potentially pathogenic CNVs compared with pediatric controls (12.1% vs 5.0%; P = .00016). Presence of a CNV was associated with significantly decreased transplant-free survival after surgery (hazard ratio, 3.42; 95% confidence interval, 1.66-7.09; P = .00090) with confounder adjustment. We confirm that children with isolated CHD have a greater burden of rare/large CNVs. We report a novel finding that these CNVs are associated with an adjusted 2.55-fold increased risk of death or transplant. These data suggest that CNV burden is an important modifier of survival after surgery for CHD. Copyright © 2016 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

[Combined G-banded karyotyping and multiplex ligation-dependent probe amplification for the detection of chromosomal abnormalities in fetuses with congenital heart defects].

PubMed

Liu, Yang; Xie, Jiansheng; Geng, Qian; Xu, Zhiyong; Wu, Weiqin; Luo, Fuwei; Li, Suli; Wang, Qin; Chen, Wubin; Tan, Hongxi; Zhang, Hu

2017-02-10

To assess the value of G-banded karyotyping in combination with multiplex ligation-dependent probe amplification (MLPA) as a tool for the detection of chromosomal abnormalities in fetuses with congenital heart defects. The combined method was used to analyze 104 fetuses with heart malformations identified by ultrasonography. Abnormal findings were confirmed with chromosomal microarray analysis (CMA). Nineteen (18%) fetuses were found to harbor chromosomal aberrations by G-banded karyotyping and MLPA. For 93 cases, CMA has detected abnormalities in 14 cases including 10 pathogenic copy number variations (CNVs) and 4 CNVs of uncertain significance (VOUS). MLPA was able to detect all of the pathogenic CNVs and 1 VOUS CNV. Combined use of G-banded karyotyping and MLPA is a rapid, low-cost and effective method to detect chromosomal abnormalities in fetuses with various heart malformations.

Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP▿

PubMed Central

Barbosa, Angela S.; Monaris, Denize; Silva, Ludmila B.; Morais, Zenaide M.; Vasconcellos, Sílvio A.; Cianciarullo, Aurora M.; Isaac, Lourdes; Abreu, Patricia A. E.

2010-01-01

We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis. PMID:20404075

Acquisition of Complement Inhibitor Serine Protease Factor I and Its Cofactors C4b-Binding Protein and Factor H by Prevotella intermedia

PubMed Central

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M.

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with 125I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases. PMID:22514678

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia.

PubMed

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

CNV-RF Is a Random Forest-Based Copy Number Variation Detection Method Using Next-Generation Sequencing.

PubMed

Onsongo, Getiria; Baughn, Linda B; Bower, Matthew; Henzler, Christine; Schomaker, Matthew; Silverstein, Kevin A T; Thyagarajan, Bharat

2016-11-01

Simultaneous detection of small copy number variations (CNVs) (<0.5 kb) and single-nucleotide variants in clinically significant genes is of great interest for clinical laboratories. The analytical variability in next-generation sequencing (NGS) and artifacts in coverage data because of issues with mappability along with lack of robust bioinformatics tools for CNV detection have limited the utility of targeted NGS data to identify CNVs. We describe the development and implementation of a bioinformatics algorithm, copy number variation-random forest (CNV-RF), that incorporates a machine learning component to identify CNVs from targeted NGS data. Using CNV-RF, we identified 12 of 13 deletions in samples with known CNVs, two cases with duplications, and identified novel deletions in 22 additional cases. Furthermore, no CNVs were identified among 60 genes in 14 cases with normal copy number and no CNVs were identified in another 104 patients with clinical suspicion of CNVs. All positive deletions and duplications were confirmed using a quantitative PCR method. CNV-RF also detected heterozygous deletions and duplications with a specificity of 50% across 4813 genes. The ability of CNV-RF to detect clinically relevant CNVs with a high degree of sensitivity along with confirmation using a low-cost quantitative PCR method provides a framework for providing comprehensive NGS-based CNV/single-nucleotide variant detection in a clinical molecular diagnostics laboratory. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Identification of new risk factors for rolandic epilepsy: CNV at Xp22.31 and alterations at cholinergic synapses.

PubMed

Addis, Laura; Sproviero, William; Thomas, Sanjeev V; Caraballo, Roberto H; Newhouse, Stephen J; Gomez, Kumudini; Hughes, Elaine; Kinali, Maria; McCormick, David; Hannan, Siobhan; Cossu, Silvia; Taylor, Jacqueline; Akman, Cigdem I; Wolf, Steven M; Mandelbaum, David E; Gupta, Rajesh; van der Spek, Rick A; Pruna, Dario; Pal, Deb K

2018-05-22

Rolandic epilepsy (RE) is the most common genetic childhood epilepsy, consisting of focal, nocturnal seizures and frequent neurodevelopmental impairments in speech, language, literacy and attention. A complex genetic aetiology is presumed in most, with monogenic mutations in GRIN2A accounting for >5% of cases. To identify rare, causal CNV in patients with RE. We used high-density SNP arrays to analyse the presence of rare CNVs in 186 patients with RE from the UK, the USA, Sardinia, Argentina and Kerala, India. We identified 84 patients with one or more rare CNVs, and, within this group, 14 (7.5%) with recurrent risk factor CNVs and 15 (8.0%) with likely pathogenic CNVs. Nine patients carried recurrent hotspot CNVs including at 16p13.11 and 1p36, with the most striking finding that four individuals (three from Sardinia) carried a duplication, and one a deletion, at Xp22.31. Five patients with RE carried a rare CNV that disrupted genes associated with other epilepsies ( KCTD7 , ARHGEF15 , CACNA2D1, GRIN2A and ARHGEF4 ), and 17 cases carried CNVs that disrupted genes associated with other neurological conditions or that are involved in neuronal signalling/development. Network analysis of disrupted genes with high brain expression identified significant enrichment in pathways of the cholinergic synapse, guanine-exchange factor activation and the mammalian target of rapamycin. Our results provide a CNV profile of an ethnically diverse cohort of patients with RE, uncovering new areas of research focus, and emphasise the importance of studying non-western European populations in oligogenic disorders to uncover a full picture of risk variation. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

A high-resolution cattle CNV map by population-scale genome sequencing

USDA-ARS?s Scientific Manuscript database

Copy Number Variations (CNVs) are common genomic structural variations that have been linked to human diseases and phenotypic traits. CNVs represent an important type of genetic variation among cattle breeds and even individual animals; however, only low-resolution maps of cattle CNVs currently exis...

Nebulized C1-Esterase Inhibitor does not Reduce Pulmonary Complement Activation in Rats with Severe Streptococcus Pneumoniae Pneumonia.

PubMed

de Beer, Friso; Lagrand, Wim; Glas, Gerie J; Beurskens, Charlotte J P; van Mierlo, Gerard; Wouters, Diana; Zeerleder, Sacha; Roelofs, Joris J T H; Juffermans, Nicole P; Horn, Janneke; Schultz, Marcus J

2016-12-01

Complement activation plays an important role in the pathogenesis of pneumonia. We hypothesized that inhibition of the complement system in the lungs by repeated treatment with nebulized plasma-derived human C1-esterase inhibitor reduces pulmonary complement activation and subsequently attenuates lung injury and lung inflammation. This was investigated in a rat model of severe Streptococcus pneumoniae pneumonia. Rats were intra-tracheally challenged with S. pneumoniae to induce pneumonia. Nebulized C1-esterase inhibitor or saline (control animals) was repeatedly administered to rats, 30 min before induction of pneumonia and every 6 h thereafter. Rats were sacrificed 20 or 40 h after inoculation with bacteria. Brochoalveolar lavage fluid and lung tissue were obtained for measuring levels of complement activation (C4b/c), lung injury and inflammation. Induction of pneumonia was associated with pulmonary complement activation (C4b/c at 20 h 1.24 % [0.56-2.59] and at 40 h 2.08 % [0.98-5.12], compared to 0.50 % [0.07-0.59] and 0.03 % [0.03-0.03] in the healthy control animals). The functional fraction of C1-INH was detectable in BALF, but no effect was found on pulmonary complement activation (C4b/c at 20 h 0.73 % [0.16-1.93] and at 40 h 2.38 % [0.54-4.19]). Twenty hours after inoculation, nebulized C1-esterase inhibitor treatment reduced total histology score, but this effect was no longer seen at 40 h. Nebulized C1-esterase inhibitor did not affect other markers of lung injury or lung inflammation. In this negative experimental animal study, severe S. pneumoniae pneumonia in rats is associated with pulmonary complement activation. Repeated treatment with nebulized C1-esterase inhibitor, although successfully delivered to the lungs, does not affect pulmonary complement activation, lung inflammation or lung injury.

Contribution of Global Rare Copy-Number Variants to the Risk of Sporadic Congenital Heart Disease

PubMed Central

Soemedi, Rachel; Wilson, Ian J.; Bentham, Jamie; Darlay, Rebecca; Töpf, Ana; Zelenika, Diana; Cosgrove, Catherine; Setchfield, Kerry; Thornborough, Chris; Granados-Riveron, Javier; Blue, Gillian M.; Breckpot, Jeroen; Hellens, Stephen; Zwolinkski, Simon; Glen, Elise; Mamasoula, Chrysovalanto; Rahman, Thahira J.; Hall, Darroch; Rauch, Anita; Devriendt, Koenraad; Gewillig, Marc; O’ Sullivan, John; Winlaw, David S.; Bu’Lock, Frances; Brook, J. David; Bhattacharya, Shoumo; Lathrop, Mark; Santibanez-Koref, Mauro; Cordell, Heather J.; Goodship, Judith A.; Keavney, Bernard D.

2012-01-01

Previous studies have shown that copy-number variants (CNVs) contribute to the risk of complex developmental phenotypes. However, the contribution of global CNV burden to the risk of sporadic congenital heart disease (CHD) remains incompletely defined. We generated genome-wide CNV data by using Illumina 660W-Quad SNP arrays in 2,256 individuals with CHD, 283 trio CHD-affected families, and 1,538 controls. We found association of rare genic deletions with CHD risk (odds ratio [OR] = 1.8, p = 0.0008). Rare deletions in study participants with CHD had higher gene content (p = 0.001) with higher haploinsufficiency scores (p = 0.03) than they did in controls, and they were enriched with Wnt-signaling genes (p = 1 × 10−5). Recurrent 15q11.2 deletions were associated with CHD risk (OR = 8.2, p = 0.02). Rare de novo CNVs were observed in ∼5% of CHD trios; 10 out of 11 occurred on the paternally transmitted chromosome (p = 0.01). Some of the rare de novo CNVs spanned genes known to be involved in heart development (e.g., HAND2 and GJA5). Rare genic deletions contribute ∼4% of the population-attributable risk of sporadic CHD. Second to previously described CNVs at 1q21.1, deletions at 15q11.2 and those implicating Wnt signaling are the most significant contributors to the risk of sporadic CHD. Rare de novo CNVs identified in CHD trios exhibit paternal origin bias. PMID:22939634

STUDIES ON THE ANTIGENIC PROPERTIES OF COMPLEMENT

PubMed Central

Klein, Paul G.; Burkholder, Peter M.

1960-01-01

Sheep erythrocytes sensitized with amboceptor and persensitized thereafter with guinea pig complement are agglutinated by rabbit anti-guinea pig globulin and by immune sera obtained by injection of rabbits with fixed complement. In this agglutination neither C'1 nor C'2 takes part. Fixed C'4 acts as an agglutinogen. An additional agglutinogen, distinct from C'4, was found on persensitized cells. This additional agglutinogen appears to be distinct from hemolytically active C'3. PMID:14409703

A genome-wide assessment of rare copy number variants in colorectal cancer.

PubMed

Li, Zhenli; Yu, Dan; Gan, Meifu; Shan, Qiaonan; Yin, Xiaoyang; Tang, Shunli; Zhang, Shuai; Shi, Yongyong; Zhu, Yimin; Lai, Maode; Zhang, Dandan

2015-09-22

Colorectal cancer (CRC) is a complex disease with an estimated heritability of approximately 35%. However, known CRC-related common single nucleotide polymorphisms (SNPs) can only explain ~0.65% of the heritability. This "missing heritability" may be explained partially by rare copy number variants (CNVs). In this study, we performed a genome-wide scan using Illumina Human-Omni Express BeadChip, 694 sporadic CRC cases and 1641 controls were eventually included in our analysis after quality control. The global burden analysis revealed a 1.53-fold excess of rare CNVs in CRC cases compared with controls (P < 1 × 10(-6)), and the difference being more pronounced for genic rare CNVs and CNVs overlapped with coding regions (1.65-fold and 1.84-fold, respectively, both P < 1 × 10(-6)). Interestingly, both the cases in the lowest and middle tertile of age carried a higher burden of rare CNVs comparing to the highest tertile. Furthermore, 639 CNV-disrupted genes exclusive to CRC cases were found to be significantly enriched in gene ontology (GO) terms concerning nucleosome assembly and olfactory receptor activity. Our study was the first to evaluate the burden of rare CNVs in sporadic CRC and suggested that rare CNVs contributed to the missing heritability of CRC.

Copy number variation in metabolic phenotypes.

PubMed

Lanktree, M; Hegele, R A

2008-01-01

Despite successes in identifying genetic contributors to common metabolic phenotypes, only part of the heritable component of these traits has thus far been explained. Copy number variation (CNV) is likely to be responsible for some of the unexplained variation. As observed with single nucleotide changes, it is probable that both rare and common CNVs will contribute to susceptibility to metabolic disease. For instance, CNVs in the LDLR gene underlie a substantial portion of disease in patients with heterozygous familial hypercholesterolemia. As well, a common CNV in LPA encoding apolipoprotein(a) is the primary determinant of plasma lipoprotein(a) concentrations, a risk factor for atherosclerosis. Recent efforts to map CNVs in control populations have defined their size, frequency and distribution. Many of the identified CNVs overlap genes with important functions in metabolic pathways. The overlap of CNVs that were found in control datasets with functional candidate genes or genes with previous evidence of association with metabolic syndrome presents an important subset for future CNV association studies. Finally, we describe an approach to search for CNVs in a rare high-penetrance metabolic disorder, namely lipodystrophy. As methods to identify CNVs increase in precision and accuracy, the prospect of identifying their role in both rare Mendelian and common complex metabolic phenotypes will become a reality. Copyright 2009 S. Karger AG, Basel.

Prediction of inflammatory responses induced by biomaterials in contact with human blood using protein fingerprint from plasma.

PubMed

Engberg, Anna E; Nilsson, Per H; Huang, Shan; Fromell, Karin; Hamad, Osama A; Mollnes, Tom Eirik; Rosengren-Holmberg, Jenny P; Sandholm, Kerstin; Teramura, Yuji; Nicholls, Ian A; Nilsson, Bo; Ekdahl, Kristina N

2015-01-01

Inappropriate complement activation is often responsible for incompatibility reactions that occur when biomaterials are used. Complement activation is therefore a criterion included in legislation regarding biomaterials testing. However, no consensus is yet available regarding appropriate complement-activation-related test parameters. We examined protein adsorption in plasma and complement activation/cytokine release in whole blood incubated with well-characterized polymers. Strong correlations were found between the ratio of C4 to its inhibitor C4BP and generation of 10 (mainly pro-inflammatory) cytokines, including IL-17, IFN-γ, and IL-6. The levels of complement activation products correlated weakly (C3a) or not at all (C5a, sC5b-9), confirming their poor predictive values. We have demonstrated a direct correlation between downstream biological effects and the proteins initially adhering to an artificial surface after contact with blood. Consequently, we propose the C4/C4BP ratio as a robust, predictor of biocompatibility with superior specificity and sensitivity over the current gold standard. Copyright © 2014 Elsevier Ltd. All rights reserved.

Type 2 diabetes mellitus disease risk genes identified by genome wide copy number variation scan in normal populations.

PubMed

Prabhanjan, Manasa; Suresh, Raviraj V; Murthy, Megha N; Ramachandra, Nallur B

2016-03-01

To identify the role of copy number variations (CNVs) on disease risk genes and its effect on disease phenotypes in type 2 diabetes mellitus (T2DM) in 12 random populations using high throughput arrays. CNV analysis was carried out on a total of 1715 individuals from 12 populations, from ArrayExpress Archive of the European Bioinformatics Institute along with our subjects using Affymetrix Genome Wide SNP 6.0 array. CNV effect on T2DM genes were analyzed using several bioinformatics tools and a molecular protein interaction network was constructed to identify the disease mechanism altered by the CNVs. Analysis showed 34.4% of the total population to be under CNV burden for T2DM, with 83 disease causal and associated genes being under CNV influence. Hotspots were identified on chromosomes 22, 12, 6, 19 and 11.Overlap studies with case cohorts revealed significant disease risk genes such as EGFR, E2F1, PPP1R3A, HLA and TSPAN8. CNVs play a significant role in predisposing T2DM in normal cohorts and contribute to the phenotypic effects. Thus, CNVs should be considered as one of the major contributors in predisposition of the disease. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Convergence of genes and cellular pathways dysregulated in autism spectrum disorders.

PubMed

Pinto, Dalila; Delaby, Elsa; Merico, Daniele; Barbosa, Mafalda; Merikangas, Alison; Klei, Lambertus; Thiruvahindrapuram, Bhooma; Xu, Xiao; Ziman, Robert; Wang, Zhuozhi; Vorstman, Jacob A S; Thompson, Ann; Regan, Regina; Pilorge, Marion; Pellecchia, Giovanna; Pagnamenta, Alistair T; Oliveira, Bárbara; Marshall, Christian R; Magalhaes, Tiago R; Lowe, Jennifer K; Howe, Jennifer L; Griswold, Anthony J; Gilbert, John; Duketis, Eftichia; Dombroski, Beth A; De Jonge, Maretha V; Cuccaro, Michael; Crawford, Emily L; Correia, Catarina T; Conroy, Judith; Conceição, Inês C; Chiocchetti, Andreas G; Casey, Jillian P; Cai, Guiqing; Cabrol, Christelle; Bolshakova, Nadia; Bacchelli, Elena; Anney, Richard; Gallinger, Steven; Cotterchio, Michelle; Casey, Graham; Zwaigenbaum, Lonnie; Wittemeyer, Kerstin; Wing, Kirsty; Wallace, Simon; van Engeland, Herman; Tryfon, Ana; Thomson, Susanne; Soorya, Latha; Rogé, Bernadette; Roberts, Wendy; Poustka, Fritz; Mouga, Susana; Minshew, Nancy; McInnes, L Alison; McGrew, Susan G; Lord, Catherine; Leboyer, Marion; Le Couteur, Ann S; Kolevzon, Alexander; Jiménez González, Patricia; Jacob, Suma; Holt, Richard; Guter, Stephen; Green, Jonathan; Green, Andrew; Gillberg, Christopher; Fernandez, Bridget A; Duque, Frederico; Delorme, Richard; Dawson, Geraldine; Chaste, Pauline; Café, Cátia; Brennan, Sean; Bourgeron, Thomas; Bolton, Patrick F; Bölte, Sven; Bernier, Raphael; Baird, Gillian; Bailey, Anthony J; Anagnostou, Evdokia; Almeida, Joana; Wijsman, Ellen M; Vieland, Veronica J; Vicente, Astrid M; Schellenberg, Gerard D; Pericak-Vance, Margaret; Paterson, Andrew D; Parr, Jeremy R; Oliveira, Guiomar; Nurnberger, John I; Monaco, Anthony P; Maestrini, Elena; Klauck, Sabine M; Hakonarson, Hakon; Haines, Jonathan L; Geschwind, Daniel H; Freitag, Christine M; Folstein, Susan E; Ennis, Sean; Coon, Hilary; Battaglia, Agatino; Szatmari, Peter; Sutcliffe, James S; Hallmayer, Joachim; Gill, Michael; Cook, Edwin H; Buxbaum, Joseph D; Devlin, Bernie; Gallagher, Louise; Betancur, Catalina; Scherer, Stephen W

2014-05-01

Rare copy-number variation (CNV) is an important source of risk for autism spectrum disorders (ASDs). We analyzed 2,446 ASD-affected families and confirmed an excess of genic deletions and duplications in affected versus control groups (1.41-fold, p = 1.0 × 10(-5)) and an increase in affected subjects carrying exonic pathogenic CNVs overlapping known loci associated with dominant or X-linked ASD and intellectual disability (odds ratio = 12.62, p = 2.7 × 10(-15), ∼3% of ASD subjects). Pathogenic CNVs, often showing variable expressivity, included rare de novo and inherited events at 36 loci, implicating ASD-associated genes (CHD2, HDAC4, and GDI1) previously linked to other neurodevelopmental disorders, as well as other genes such as SETD5, MIR137, and HDAC9. Consistent with hypothesized gender-specific modulators, females with ASD were more likely to have highly penetrant CNVs (p = 0.017) and were also overrepresented among subjects with fragile X syndrome protein targets (p = 0.02). Genes affected by de novo CNVs and/or loss-of-function single-nucleotide variants converged on networks related to neuronal signaling and development, synapse function, and chromatin regulation. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Deletion of Crry and DAF on murine platelets stimulates thrombopoiesis and increases factor H-dependent resistance of peripheral platelets to complement attack.

PubMed

Barata, Lidia; Miwa, Takashi; Sato, Sayaka; Kim, David; Mohammed, Imran; Song, Wen-Chao

2013-03-15

Complement receptor 1-related gene/protein y (Crry) and decay-accelerating factor (DAF) are two murine membrane C3 complement regulators with overlapping functions. Crry deletion is embryonically lethal whereas DAF-deficient mice are generally healthy. Crry(-/-)DAF(-/-) mice were viable on a C3(-/-) background, but platelets from such mice were rapidly destroyed when transfused into C3-sufficient mice. In this study, we used the cre-lox system to delete platelet Crry in DAF(-/-) mice and studied Crry/DAF-deficient platelet development in vivo. Rather than displaying thrombocytopenia, Pf4-Cre(+)-Crry(flox/flox) mice had normal platelet counts and their peripheral platelets were resistant to complement attack. However, chimera mice generated with Pf4-Cre(+)-Crry(flox/flox) bone marrows showed platelets from C3(-/-) but not C3(+/+) recipients to be sensitive to complement activation, suggesting that circulating platelets in Pf4-Cre(+)-Crry(flox/flox) mice were naturally selected in a complement-sufficient environment. Notably, Pf4-Cre(+)-Crry(flox/flox) mouse platelets became complement susceptible when factor H function was blocked. Examination of Pf4-Cre(+)-Crry(flox/flox) mouse bone marrows revealed exceedingly active thrombopoiesis. Thus, under in vivo conditions, Crry/DAF deficiency on platelets led to abnormal platelet turnover, but peripheral platelet count was compensated for by increased thrombopoiesis. Selective survival of Crry/DAF-deficient platelets aided by factor H protection and compensatory thrombopoiesis demonstrates the cooperation between membrane and fluid phase complement inhibitors and the body's ability to adaptively respond to complement regulator deficiencies.

Copy number variation signature to predict human ancestry

PubMed Central

2012-01-01

Background Copy number variations (CNVs) are genomic structural variants that are found in healthy populations and have been observed to be associated with disease susceptibility. Existing methods for CNV detection are often performed on a sample-by-sample basis, which is not ideal for large datasets where common CNVs must be estimated by comparing the frequency of CNVs in the individual samples. Here we describe a simple and novel approach to locate genome-wide CNVs common to a specific population, using human ancestry as the phenotype. Results We utilized our previously published Genome Alteration Detection Analysis (GADA) algorithm to identify common ancestry CNVs (caCNVs) and built a caCNV model to predict population structure. We identified a 73 caCNV signature using a training set of 225 healthy individuals from European, Asian, and African ancestry. The signature was validated on an independent test set of 300 individuals with similar ancestral background. The error rate in predicting ancestry in this test set was 2% using the 73 caCNV signature. Among the caCNVs identified, several were previously confirmed experimentally to vary by ancestry. Our signature also contains a caCNV region with a single microRNA (MIR270), which represents the first reported variation of microRNA by ancestry. Conclusions We developed a new methodology to identify common CNVs and demonstrated its performance by building a caCNV signature to predict human ancestry with high accuracy. The utility of our approach could be extended to large case–control studies to identify CNV signatures for other phenotypes such as disease susceptibility and drug response. PMID:23270563

A scabies mite serpin interferes with complement-mediated neutrophil functions and promotes staphylococcal growth.

PubMed

Swe, Pearl M; Fischer, Katja

2014-06-01

Scabies is a contagious skin disease caused by the parasitic mite Sarcoptes scabiei. The disease is highly prevalent worldwide and known to predispose to secondary bacterial infections, in particular by Streptococcus pyogenes and Staphylococcus aureus. Reports of scabies patients co-infected with methicillin resistant S. aureus (MRSA) pose a major concern for serious down-stream complications. We previously reported that a range of complement inhibitors secreted by the mites promoted the growth of S. pyogenes. Here, we show that a recently characterized mite serine protease inhibitor (SMSB4) inhibits the complement-mediated blood killing of S. aureus. Blood killing of S. aureus was measured in whole blood bactericidal assays, counting viable bacteria recovered after treatment in fresh blood containing active complement and phagocytes, treated with recombinant SMSB4. SMSB4 inhibited the blood killing of various strains of S. aureus including methicillin-resistant and methicillin-sensitive isolates. Staphylococcal growth was promoted in a dose-dependent manner. We investigated the effect of SMSB4 on the complement-mediated neutrophil functions, namely phagocytosis, opsonization and anaphylatoxin release, by flow cytometry and in enzyme linked immuno sorbent assays (ELISA). SMSB4 reduced phagocytosis of S. aureus by neutrophils. It inhibited the deposition of C3b, C4b and properdin on the bacteria surface, but did not affect the depositions of C1q and MBL. SMSB4 also inhibited C5 cleavage as indicated by a reduced C5b-9 deposition. We postulate that SMSB4 interferes with the activation of all three complement pathways by reducing the amount of C3 convertase formed. We conclude that SMSB4 interferes with the complement-dependent killing function of neutrophils, thereby reducing opsonization, phagocytosis and further recruitment of neutrophils to the site of infection. As a consequence secreted scabies mites complement inhibitors, such as SMSB4, provide favorable conditions for the onset of S. aureus co-infection in the scabies-infected microenvironment by suppressing the immediate host immune response.

Detection Copy Number Variants from NGS with Sparse and Smooth Constraints.

PubMed

Zhang, Yue; Cheung, Yiu-Ming; Xu, Bo; Su, Weifeng

2017-01-01

It is known that copy number variations (CNVs) are associated with complex diseases and particular tumor types, thus reliable identification of CNVs is of great potential value. Recent advances in next generation sequencing (NGS) data analysis have helped manifest the richness of CNV information. However, the performances of these methods are not consistent. Reliably finding CNVs in NGS data in an efficient way remains a challenging topic, worthy of further investigation. Accordingly, we tackle the problem by formulating CNVs identification into a quadratic optimization problem involving two constraints. By imposing the constraints of sparsity and smoothness, the reconstructed read depth signal from NGS is anticipated to fit the CNVs patterns more accurately. An efficient numerical solution tailored from alternating direction minimization (ADM) framework is elaborated. We demonstrate the advantages of the proposed method, namely ADM-CNV, by comparing it with six popular CNV detection methods using synthetic, simulated, and empirical sequencing data. It is shown that the proposed approach can successfully reconstruct CNV patterns from raw data, and achieve superior or comparable performance in detection of the CNVs compared to the existing counterparts.

No evidence for mosaic pathogenic copy number variations in cardiac tissue from patients with congenital heart malformations.

PubMed

Winberg, Johanna; Berggren, Håkan; Malm, Torsten; Johansson, Sune; Johansson Ramgren, Jens; Nilsson, Boris; Liedén, Agne; Nordenskjöld, Agneta; Gustavsson, Peter; Nordgren, Ann

2015-03-01

The aim of this study was to investigate if pathogenic copy number variations (CNVs) are present in mosaic form in patients with congenital heart malformations. We have collected cardiac tissue and blood samples from 23 patients with congenital heart malformations that underwent cardiac surgery and screened for mosaic gene dose alterations restricted to cardiac tissue using array comparative genomic hybridization (array CGH). We did not find evidence of CNVs in mosaic form after array CGH analysis. Pathogenic CNVs that were present in both cardiac tissue and blood were detected in 2/23 patients (9%), and in addition we found several constitutional CNVs of unclear clinical significance. This is the first study investigating mosaicism for CNVs in heart tissue compared to peripheral blood and the results do not indicate that pathogenic mosaic copy number changes are common in patients with heart malformations. Importantly, in line with previous studies, our results show that constitutional pathogenic CNVs are important factors contributing to congenital heart malformations. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

21 CFR 866.5240 - Complement components immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

.... 866.5240 Section 866.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... complement components C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9, in serum, other body fluids, and tissues. Complement is a group of serum proteins which destroy infectious agents. Measurements of these...

21 CFR 866.5240 - Complement components immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

.... 866.5240 Section 866.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... complement components C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9, in serum, other body fluids, and tissues. Complement is a group of serum proteins which destroy infectious agents. Measurements of these...

21 CFR 866.5240 - Complement components immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

.... 866.5240 Section 866.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... complement components C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9, in serum, other body fluids, and tissues. Complement is a group of serum proteins which destroy infectious agents. Measurements of these...

21 CFR 866.5240 - Complement components immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

.... 866.5240 Section 866.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... complement components C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9, in serum, other body fluids, and tissues. Complement is a group of serum proteins which destroy infectious agents. Measurements of these...

Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface

PubMed Central

Hovingh, Elise S.; Kuipers, Betsy; Pinelli, Elena; Rooijakkers, Suzan H. M.

2017-01-01

Whooping cough, or pertussis, is a contagious disease of the respiratory tract that is re-emerging worldwide despite high vaccination coverage. The causative agent of this disease is the Gram-negative Bordetella pertussis. Knowledge on complement evasion strategies of this pathogen is limited. However, this is of great importance for future vaccine development as it has become apparent that a novel pertussis vaccine is needed. Here, we unravel the effect of Virulence associated gene 8 (Vag8) of B. pertussis on the human complement system at the molecular level. We show that both recombinant and endogenously secreted Vag8 inhibit complement deposition on the bacterial surface at the level of C4b. We reveal that Vag8 binding to human C1-inhibitor (C1-inh) interferes with the binding of C1-inh to C1s, C1r and MASP-2, resulting in the release of active proteases that subsequently cleave C2 and C4 away from the bacterial surface. We demonstrate that the depletion of these complement components in the bacterial surrounding and subsequent decreased deposition on B. pertussis leads to less complement-mediated bacterial killing. Vag8 is the first protein described that specifically prevents C1s, C1r and MASP-2 binding to C1-inh and thereby mediates complement consumption away from the bacterial surface. Unravelling the mechanism of this unique complement evasion strategy of B. pertussis is one of the first steps towards understanding the interactions between the first line of defense complement and B. pertussis. PMID:28742139

Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface.

PubMed

Hovingh, Elise S; van den Broek, Bryan; Kuipers, Betsy; Pinelli, Elena; Rooijakkers, Suzan H M; Jongerius, Ilse

2017-07-01

Whooping cough, or pertussis, is a contagious disease of the respiratory tract that is re-emerging worldwide despite high vaccination coverage. The causative agent of this disease is the Gram-negative Bordetella pertussis. Knowledge on complement evasion strategies of this pathogen is limited. However, this is of great importance for future vaccine development as it has become apparent that a novel pertussis vaccine is needed. Here, we unravel the effect of Virulence associated gene 8 (Vag8) of B. pertussis on the human complement system at the molecular level. We show that both recombinant and endogenously secreted Vag8 inhibit complement deposition on the bacterial surface at the level of C4b. We reveal that Vag8 binding to human C1-inhibitor (C1-inh) interferes with the binding of C1-inh to C1s, C1r and MASP-2, resulting in the release of active proteases that subsequently cleave C2 and C4 away from the bacterial surface. We demonstrate that the depletion of these complement components in the bacterial surrounding and subsequent decreased deposition on B. pertussis leads to less complement-mediated bacterial killing. Vag8 is the first protein described that specifically prevents C1s, C1r and MASP-2 binding to C1-inh and thereby mediates complement consumption away from the bacterial surface. Unravelling the mechanism of this unique complement evasion strategy of B. pertussis is one of the first steps towards understanding the interactions between the first line of defense complement and B. pertussis.

Analysis of copy number variations at 15 schizophrenia-associated loci.

PubMed

Rees, Elliott; Walters, James T R; Georgieva, Lyudmila; Isles, Anthony R; Chambert, Kimberly D; Richards, Alexander L; Mahoney-Davies, Gerwyn; Legge, Sophie E; Moran, Jennifer L; McCarroll, Steven A; O'Donovan, Michael C; Owen, Michael J; Kirov, George

2014-02-01

A number of copy number variants (CNVs) have been suggested as susceptibility factors for schizophrenia. For some of these the data remain equivocal, and the frequency in individuals with schizophrenia is uncertain. To determine the contribution of CNVs at 15 schizophrenia-associated loci (a) using a large new data-set of patients with schizophrenia (n = 6882) and controls (n = 6316), and (b) combining our results with those from previous studies. We used Illumina microarrays to analyse our data. Analyses were restricted to 520 766 probes common to all arrays used in the different data-sets. We found higher rates in participants with schizophrenia than in controls for 13 of the 15 previously implicated CNVs. Six were nominally significantly associated (P<0.05) in this new data-set: deletions at 1q21.1, NRXN1, 15q11.2 and 22q11.2 and duplications at 16p11.2 and the Angelman/Prader-Willi Syndrome (AS/PWS) region. All eight AS/PWS duplications in patients were of maternal origin. When combined with published data, 11 of the 15 loci showed highly significant evidence for association with schizophrenia (P<4.1×10(-4)). We strengthen the support for the majority of the previously implicated CNVs in schizophrenia. About 2.5% of patients with schizophrenia and 0.9% of controls carry a large, detectable CNV at one of these loci. Routine CNV screening may be clinically appropriate given the high rate of known deleterious mutations in the disorder and the comorbidity associated with these heritable mutations.

Definition of a putative pathological region in PARK2 associated with autism spectrum disorder through in silico analysis of its functional structure.

PubMed

Conceição, Inês C; Rama, Maria M; Oliveira, Bárbara; Café, Cátia; Almeida, Joana; Mouga, Susana; Duque, Frederico; Oliveira, Guiomar; Vicente, Astrid M

2017-04-01

The PARK2 gene encodes Parkin, a component of a multiprotein E3 ubiquitin ligase complex that targets substrate proteins for proteasomal degradation. PARK2 mutations are frequently associated with Parkinson's disease, but structural alterations have also been described in patients with neurodevelopmental disorders (NDD), suggesting a pathological effect ubiquitous to neurodevelopmental and neurodegenerative brain processes. The present study aimed to define the critical regions for NDD within PARK2. To clarify PARK2 involvement in NDDs, we examined the frequency and location of copy number variants (CNVs) identified in patients from our sample and reported in the literature and relevant databases, and compared with control populations. Overall, the frequency of PARK2 CNVs was higher in controls than in NDD cases. However, closer inspection of the CNV location in PARK2 showed that the frequency of CNVs targeting the Parkin C-terminal, corresponding to the ring-between-ring (RBR) domain responsible for Parkin activity, is significantly higher in NDD cases than in controls. In contrast, CNVs targeting the N-terminal of Parkin, including domains that regulate ubiquitination activity, are very common both in cases and in controls. Although PARK2 may be a pathological factor for NDDs, likely not all variants are pathogenic, and a conclusive assessment of PARK2 variant pathogenicity requires an accurate analysis of their location within the coding region and encoded functional domains.

CONAN: copy number variation analysis software for genome-wide association studies

PubMed Central

2010-01-01

Background Genome-wide association studies (GWAS) based on single nucleotide polymorphisms (SNPs) revolutionized our perception of the genetic regulation of complex traits and diseases. Copy number variations (CNVs) promise to shed additional light on the genetic basis of monogenic as well as complex diseases and phenotypes. Indeed, the number of detected associations between CNVs and certain phenotypes are constantly increasing. However, while several software packages support the determination of CNVs from SNP chip data, the downstream statistical inference of CNV-phenotype associations is still subject to complicated and inefficient in-house solutions, thus strongly limiting the performance of GWAS based on CNVs. Results CONAN is a freely available client-server software solution which provides an intuitive graphical user interface for categorizing, analyzing and associating CNVs with phenotypes. Moreover, CONAN assists the evaluation process by visualizing detected associations via Manhattan plots in order to enable a rapid identification of genome-wide significant CNV regions. Various file formats including the information on CNVs in population samples are supported as input data. Conclusions CONAN facilitates the performance of GWAS based on CNVs and the visual analysis of calculated results. CONAN provides a rapid, valid and straightforward software solution to identify genetic variation underlying the 'missing' heritability for complex traits that remains unexplained by recent GWAS. The freely available software can be downloaded at http://genepi-conan.i-med.ac.at. PMID:20546565

DNA methylation and copy number variation analyses of human embryonic stem cell-derived neuroprogenitors after low-dose decabromodiphenyl ether and/or bisphenol A exposure.

PubMed

Du, L; Sun, W; Li, X M; Li, X Y; Liu, W; Chen, D

2018-05-01

The polybrominated diphenyl ether flame retardants decabromodiphenyl ether (BDE-209) and bisphenol A (BPA) are environmental contaminants that can cross the placenta and exert toxicity in the developing fetal nervous system. Copy number variants (CNVs) play a role in a number of genetic disorders and may be implicated in BDE-209/BPA teratogenicity. In this study, we found that BDE-209 and/or BPA exposure decreased neural differentiation efficiency of human embryonic stem cells (hESCs), although there was a >90% induction of neuronal progenitor cells (NPCs) from exposed hESCs. However, the mean of CNV numbers in the NPCs with BDE-209 + BPA treatment was significantly higher compared to the other groups, whereas DNA methylation was lower and DNA methyltransferase(DNMT1 and DNMT3A) expression were significantly decreased in all of the BDE-209 and/or BPA treatment groups compared with the control groups. The number of CNVs in chromosomes 3, 4, 11, 22, and X in NPCs with BDE-209 and/or BPA exposure was higher compared to the control group. In addition, CNVs in chromosomes 7, 8, 14, and 16 were stable in hESCs and hESCs-derived NPCs irrespective of BDE-209/BPA exposure, and CNVs in chromosomes 20 q11.21 and 16 p13.11 might be induced by neural differentiation. Thus, BDE-209/BPA exposure emerges as a potential source of CNVs distinct from neural differentiation by itself. BDE-209 and/or BPA exposure may cause genomic instability in cultured stem cells via reduced activity of DNA methyltransferase, suggesting a new mechanism of human embryonic neurodevelopmental toxicity caused by this class of environmental toxins.

Application of chromosomal microarrays in the evaluation of intellectual disability/global developmental delay patients - A study from a tertiary care genetic centre in India.

PubMed

Sharma, Pankaj; Gupta, Neerja; Chowdhury, Madhumita Roy; Sapra, Savita; Ghosh, Manju; Gulati, Sheffali; Kabra, Madhulika

2016-09-15

Intellectual disability (ID)/Global developmental delay (GDD) is a diverse group of disorders in terms of cognitive and non-cognitive functions and can occur with or without associated co-morbidities. It affects 1-3% of individuals globally and in at least 30-50% of cases the etiology remains unexplained. The widespread use of chromosomal microarray analysis (CMA) in a clinical setting has allowed the identification of submicroscopic copy number variations (CNVs), throughout the genome, associated with neurodevelopmental phenotypes including ID/GDD. In this study we investigated the utility of CMA in the detection of CNVs in 106 patients with unexplained ID/DD, dysmorphism with or without multiple congenital anomalies (MCA). CMA study was carried out using Agilent 8×60K chips and Illumina Human CytoSNP-12 chips. Pathogenic CNVs were found in 15 (14.2%) patients. In these patients, CNVs on single chromosome were detected in 10 patients while 5 patients showed co-occurrence CNVs on two chromosomes. The size of these CNVs ranged between 322kb to 13Mb. The yield of pathogenic CNVs was similar for both mild and severe ID/GDD cases. One patient described in this paper is considered to harbour a likely pathogenic CNV with deletion in 17q22 region. Only few cases have been described in literature for 17q22 deletion and patient reported here was found to have an atypical deletion in 17q22 region (Case 90). This study re-affirms the view point that CMA is a powerful diagnostic tool in the evaluation of idiopathic ID/GDD patients irrespective of the degree of severity. Identifying pathogenic CNVs helps in counseling and prenatal diagnosis if desired. Copyright © 2016 Elsevier B.V. All rights reserved.

Copy number variants are frequent in genetic generalized epilepsy with intellectual disability

PubMed Central

Mullen, Saul A.; Carvill, Gemma L.; Bellows, Susannah; Bayly, Marta A.; Berkovic, Samuel F.; Dibbens, Leanne M.

2013-01-01

Objective: We examined whether copy number variants (CNVs) were more common in those with a combination of intellectual disability (ID) and genetic generalized epilepsy (GGE) than in those with either phenotype alone via a case-control study. Methods: CNVs contribute to the genetics of multiple neurodevelopmental disorders with complex inheritance, including GGE and ID. Three hundred fifty-nine probands with GGE and 60 probands with ID-GGE were screened for GGE-associated recurrent microdeletions at 15q13.3, 15q11.2, and 16p13.11 via quantitative PCR or loss of heterozygosity. Deletions were confirmed by comparative genomic hybridization (CGH). ID-GGE probands also had genome-wide CGH. Results: ID-GGE probands showed a significantly higher rate of CNVs compared with probands with GGE alone, with 17 of 60 (28%) ID-GGE probands having one or more potentially causative CNVs. The patients with ID-GGE had a 3-fold-higher rate of the 3 GGE-associated recurrent microdeletions than probands with GGE alone (10% vs 3%, p = 0.02). They also showed a high rate (13/60, 22%) of rare CNVs identified using genome-wide CGH. Conclusions: This study shows that CNVs are common in those with ID-GGE with recurrent deletions at 15q13.3, 15q11.2, and 16p13.11, particularly enriched compared with individuals with GGE or ID alone. Recurrent CNVs are likely to act as risk factors for multiple phenotypes not just at the population level, but also in any given individual. Testing for CNVs in ID-GGE will have a high diagnostic yield in a clinical setting and will inform genetic counseling. PMID:24068782

CNVs leading to fusion transcripts in individuals with autism spectrum disorder

PubMed Central

Holt, Richard; Sykes, Nuala H; Conceição, Inês C; Cazier, Jean-Baptiste; Anney, Richard JL; Oliveira, Guiomar; Gallagher, Louise; Vicente, Astrid; Monaco, Anthony P; Pagnamenta, Alistair T

2012-01-01

There is strong evidence that rare copy number variants (CNVs) have a role in susceptibility to autism spectrum disorders (ASDs). Much research has focused on how CNVs mediate a phenotypic effect by altering gene expression levels. We investigated an alternative mechanism whereby CNVs combine the 5′ and 3′ ends of two genes, creating a ‘fusion gene'. Any resulting mRNA with an open reading frame could potentially alter the phenotype via a gain-of-function mechanism. We examined 2382 and 3096 rare CNVs from 996 individuals with ASD and 1287 controls, respectively, for potential to generate fusion transcripts. There was no increased burden in individuals with ASD; 122/996 cases harbored at least one rare CNV of this type, compared with 179/1287 controls (P=0.89). There was also no difference in the overall frequency distribution between cases and controls. We examined specific examples of such CNVs nominated by case–control analysis and a candidate approach. Accordingly, a duplication involving REEP1-POLR1A (found in 3/996 cases and 0/1287 controls) and a single occurrence CNV involving KIAA0319-TDP2 were tested. However, no fusion transcripts were detected by RT-PCR. Analysis of additional samples based on cell line availability resulted in validation of a MAPKAPK5-ACAD10 fusion transcript in two probands. However, this variant was present in controls at a similar rate and is unlikely to influence ASD susceptibility. In summary, although we find no evidence that fusion-gene generating CNVs lead to ASD susceptibility, discovery of a MAPKAPK5-ACAD10 transcript with an estimated frequency of ∼1/200 suggests that gain-of-function mechanisms should be considered in future CNVs studies. PMID:22549408

Genomic imbalances in syndromic congenital heart disease.

PubMed

Molck, Miriam Coelho; Simioni, Milena; Paiva Vieira, Társis; Sgardioli, Ilária Cristina; Paoli Monteiro, Fabíola; Souza, Josiane; Fett-Conte, Agnes Cristina; Félix, Têmis Maria; Lopes Monlléo, Isabella; Gil-da-Silva-Lopes, Vera Lúcia

To identify pathogenic genomic imbalances in patients presenting congenital heart disease (CHD) with extra cardiac anomalies and exclusion of 22q11.2 deletion syndrome (22q11.2 DS). 78 patients negative for the 22q11.2 deletion, previously screened by fluorescence in situ hybridization (FISH) and/or multiplex ligation probe amplification (MLPA) were tested by chromosomal microarray analysis (CMA). Clinically significant copy number variations (CNVs ≥300kb) were identified in 10% (8/78) of cases. In addition, potentially relevant CNVs were detected in two cases (993kb duplication in 15q21.1 and 706kb duplication in 2p22.3). Genes inside the CNV regions found in this study, such as IRX4, BMPR1A, SORBS2, ID2, ROCK2, E2F6, GATA4, SOX7, SEMAD6D, FBN1, and LTPB1 are known to participate in cardiac development and could be candidate genes for CHD. These data showed that patients presenting CHD with extra cardiac anomalies and exclusion of 22q11.2 DS should be investigated by CMA. The present study emphasizes the possible role of CNVs in CHD. Copyright © 2017 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Ectromelia virus inhibitor of complement enzymes protects intracellular mature virus and infected cells from mouse complement.

PubMed

Moulton, Elizabeth A; Bertram, Paula; Chen, Nanhai; Buller, R Mark L; Atkinson, John P

2010-09-01

Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.

Complement Activation in Relation to Capillary Leakage in Children with Septic Shock and Purpura

PubMed Central

Hazelzet, Jan A.; de Groot, Ronald; van Mierlo, Gerard; Joosten, Koen F. M.; van der Voort, Edwin; Eerenberg, Anke; Suur, Marja H.; Hop, Wim C. J.; Hack, C. Erik

1998-01-01

To assess the relationship between capillary leakage and inflammatory mediators during sepsis, blood samples were taken on hospital admission, as well as 24 and 72 h later, from 52 children (median age, 3.3 years) with severe meningococcal sepsis, of whom 38 survived and 14 died. Parameters related to cytokines (interleukin 6 [IL-6] IL-8, plasma phospholipase A2, and C-reactive protein [CRP]), to neutrophil degranulation (elastase and lactoferrin), to complement activation (C3a, C3b/c, C4b/c, and C3- and C4-CRP complexes), and to complement regulation (functional and inactivated C1 inhibitor and C4BP) were determined. The degree of capillary leakage was derived from the amount of plasma infused and the severity of disease by assessing the pediatric risk of mortality (PRISM) score. Levels of IL-6, IL-8, C3b/c, C3-CRP complexes, and C4BP on admission, adjusted for the duration of skin lesions, were significantly different in survivors and nonsurvivors (C3b/c levels were on average 2.2 times higher in nonsurvivors, and C3-CRP levels were 1.9 times higher in survivors). Mortality was independently related to the levels of C3b/c and C3-CRP complexes. In agreement with this, levels of complement activation products correlated well with the PRISM score or capillary leakage. Thus, these data show that complement activation in patients with severe meningococcal sepsis is associated with a poor outcome and a more severe disease course. Further studies should reveal whether complement activation may be a target for therapeutical intervention in this disease. PMID:9784543

Growth, progression and chromosome instability of Neuroblastoma: a new scenario of tumorigenesis?

PubMed

Tonini, Gian Paolo

2017-01-05

Neuroblastoma is a pediatric cancer with a low survival rate of patients with metastatic stage 4 disease. Tumor aggressiveness and progression have been associated with structural copy number variations (CNVs) that are observed in malignant cells. In contrast, localized Neuroblastomas, which are associated with a low number of structural CNVs but frequent numerical CNVs, are less aggressive, and patients have good outcomes. Finally, whole-genome and whole-exome sequencing of Neuroblastoma tissues have shown few damaging mutations in these tumors. In the present report it is proposed that chromosome instability (CIN) plays a major role in Neuroblastoma tumorigenesis and that CIN is already present in the early phases of tumor development. High CIN can promote several types of chromosomal damage including chromothripsis, gene deletion, amplification and rearrangements, which deregulate gene expression. Indeed, gene rearrangements have been reported as a new scenario in the development of Neuroblastoma, which supports the hypothesis that CIN is an early step preliminary to the late catastrophic events leading to tumor development.

The Group B Streptococcus–Secreted Protein CIP Interacts with C4, Preventing C3b Deposition via the Lectin and Classical Complement Pathways

PubMed Central

Pietrocola, Giampiero; Rindi, Simonetta; Rosini, Roberto; Buccato, Scilla

2016-01-01

The group B Streptococcus (GBS) is a leading cause of neonatal invasive disease. GBS bacteria are surrounded by a thick capsular polysaccharide that is a potent inhibitor of complement deposition via the alternative pathway. Several of its surface molecules can however activate the classical and lectin complement pathways, rendering this species still vulnerable to phagocytic killing. In this study we have identified a novel secreted protein named complement interfering protein (CIP) that downregulates complement activation via the classical and lectin pathways, but not the alternative pathway. The CIP protein showed high affinity toward C4b and inhibited its interaction with C2, presumably preventing the formation of the C4bC2a convertase. Addition of recombinant CIP to GBS cip-negative bacteria resulted in decreased deposition of C3b on their surface and in diminished phagocytic killing in a whole-blood assay. Our data reveal a novel strategy exploited by GBS to counteract innate immunity and could be valuable for the development of anti-infective agents against this important pathogen. PMID:26608922

Analysis of copy number variants by three detection algorithms and their association with body size in horses.

PubMed

Metzger, Julia; Philipp, Ute; Lopes, Maria Susana; da Camara Machado, Artur; Felicetti, Michela; Silvestrelli, Maurizio; Distl, Ottmar

2013-07-18

Copy number variants (CNVs) have been shown to play an important role in genetic diversity of mammals and in the development of many complex phenotypic traits. The aim of this study was to perform a standard comparative evaluation of CNVs in horses using three different CNV detection programs and to identify genomic regions associated with body size in horses. Analysis was performed using the Illumina Equine SNP50 genotyping beadchip for 854 horses. CNVs were detected by three different algorithms, CNVPartition, PennCNV and QuantiSNP. Comparative analysis revealed 50 CNVs that affected 153 different genes mainly involved in sensory perception, signal transduction and cellular components. Genome-wide association analysis for body size showed highly significant deleted regions on ECA1, ECA8 and ECA9. Homologous regions to the detected CNVs on ECA1 and ECA9 have also been shown to be correlated with human height. Comparative analysis of CNV detection algorithms was useful to increase the specificity of CNV detection but had certain limitations dependent on the detection tool. GWAS revealed genome-wide associated CNVs for body size in horses.

Exploration of large, rare copy number variants associated with psychiatric and neurodevelopmental disorders in individuals with anorexia nervosa.

PubMed

Yilmaz, Zeynep; Szatkiewicz, Jin P; Crowley, James J; Ancalade, NaEshia; Brandys, Marek K; van Elburg, Annemarie; de Kovel, Carolien G F; Adan, Roger A H; Hinney, Anke; Hebebrand, Johannes; Gratacos, Monica; Fernandez-Aranda, Fernando; Escaramis, Georgia; Gonzalez, Juan R; Estivill, Xavier; Zeggini, Eleftheria; Sullivan, Patrick F; Bulik, Cynthia M

2017-08-01

Anorexia nervosa (AN) is a serious and heritable psychiatric disorder. To date, studies of copy number variants (CNVs) have been limited and inconclusive because of small sample sizes. We conducted a case-only genome-wide CNV survey in 1983 female AN cases included in the Genetic Consortium for Anorexia Nervosa. Following stringent quality control procedures, we investigated whether pathogenic CNVs in regions previously implicated in psychiatric and neurodevelopmental disorders were present in AN cases. We observed two instances of the well-established pathogenic CNVs in AN cases. In addition, one case had a deletion in the 13q12 region, overlapping with a deletion reported previously in two AN cases. As a secondary aim, we also examined our sample for CNVs over 1 Mbp in size. Out of the 40 instances of such large CNVs that were not implicated previously for AN or neuropsychiatric phenotypes, two of them contained genes with previous neuropsychiatric associations, and only five of them had no associated reports in public CNV databases. Although ours is the largest study of its kind in AN, larger datasets are needed to comprehensively assess the role of CNVs in the etiology of AN.

Global characterization of copy number variants in epilepsy patients from whole genome sequencing

PubMed Central

Meloche, Caroline; Andrade, Danielle M.; Lafreniere, Ron G.; Gravel, Micheline; Spiegelman, Dan; Dionne-Laporte, Alexandre; Boelman, Cyrus; Hamdan, Fadi F.; Michaud, Jacques L.; Rouleau, Guy; Minassian, Berge A.; Bourque, Guillaume; Cossette, Patrick

2018-01-01

Epilepsy will affect nearly 3% of people at some point during their lifetime. Previous copy number variants (CNVs) studies of epilepsy have used array-based technology and were restricted to the detection of large or exonic events. In contrast, whole-genome sequencing (WGS) has the potential to more comprehensively profile CNVs but existing analytic methods suffer from limited accuracy. We show that this is in part due to the non-uniformity of read coverage, even after intra-sample normalization. To improve on this, we developed PopSV, an algorithm that uses multiple samples to control for technical variation and enables the robust detection of CNVs. Using WGS and PopSV, we performed a comprehensive characterization of CNVs in 198 individuals affected with epilepsy and 301 controls. For both large and small variants, we found an enrichment of rare exonic events in epilepsy patients, especially in genes with predicted loss-of-function intolerance. Notably, this genome-wide survey also revealed an enrichment of rare non-coding CNVs near previously known epilepsy genes. This enrichment was strongest for non-coding CNVs located within 100 Kbp of an epilepsy gene and in regions associated with changes in the gene expression, such as expression QTLs or DNase I hypersensitive sites. Finally, we report on 21 potentially damaging events that could be associated with known or new candidate epilepsy genes. Our results suggest that comprehensive sequence-based profiling of CNVs could help explain a larger fraction of epilepsy cases. PMID:29649218

Combined total deficiency of C7 and C4B with systemic lupus erythematosus (SLE).

PubMed Central

Segurado, O G; Arnaiz-Villena, A A; Iglesias-Casarrubios, P; Martinez-Laso, J; Vicario, J L; Fontan, G; Lopez-Trascasa, M

1992-01-01

The first inherited combined total deficiency of C7 and C4B complement components associated with SLE is described in a young female. Functional C7 assays showed a homozygous C7 deficiency in the propositus and her sister, and an heterozygous one in their parents. C4 molecular analyses showed that both the propositus and her mother had two HLA haplotypes carrying only C4A-specific DNA sequences and a normal C4 gene number. Thus, only C4A proteins could be expressed, with resultant normal C4 serum levels. The coexistence of a combined complete C7 and C4B deficiency may therefore abrogate essential functions of the complement cascade presumably related to immune complex handling and solubilization despite an excess of circulating C4A. These findings challenge the putative pathophysiological roles of C4A and C4B and stress the need to perform both functional assays and C4 allotyping in patients with autoimmune pathology and low haemolytic activity without low serum levels of a classical pathway complement component. Images Fig. 1 Fig. 2 PMID:1347491

[Analysis of genomic copy number variations in two unrelated neonates with 8p deletion and duplication associated with congenital heart disease].

PubMed

Mei, Mei; Yang, Lin; Zhan, Guodong; Wang, Huijun; Ma, Duan; Zhou, Wenhao; Huang, Guoying

2014-06-01

To screen for genomic copy number variations (CNVs) in two unrelated neonates with multiple congenital abnormalities using Affymetrix SNP chip and try to find the critical region associated with congenital heart disease. Two neonates were tested for genomic copy number variations by using Cytogenetic SNP chip.Rare CNVs with potential clinical significance were selected of which deletion segments' size was larger than 50 kb and duplication segments' size was larger than 150 kb based on the analysis of ChAs software, without false positive CNVs and segments of normal population. The identified CNVs were compared with those of the cases in DECIPHER and ISCA databases. Eleven rare CNVs with size from 546.6-27 892 kb were identified in the 2 neonates. The deletion region and size of case 1 were 8p23.3-p23.1 (387 912-11 506 771 bp) and 11.1 Mb respectively, the duplication region and size of case 1 were 8p23.1-p11.1 (11 508 387-43 321 279 bp) and 31.8 Mb respectively. The deletion region and size of case 2 were 8p23.3-p23.1 (46 385-7 809 878 bp) and 7.8 Mb respectively, the duplication region and size of case 2 were 8p23.1-p11.21 (12 260 914-40 917 092 bp) and 28.7 Mb respectively. The comparison with Decipher and ISCA databases supported previous viewpoint that 8p23.1 had been associated with congenital heart disease and the region between 7 809 878-11 506 771 bp may play a role in the severe cardiac defects associated with 8p23.1 deletions. Case 1 had serious cardiac abnormalities whose GATA4 was located in the duplication segment and the copy number increased while SOX7 was located in the deletion segment and the copy number decreased. The region between 7 809 878-11 506 771 bp in 8p23.1 is associated with heart defects and copy number variants of SOX7 and GATA4 may result in congenital heart disease.

Evaluation of serum levels of C3 and C4 complement factors in patients with beta thalassemia major in Khuzestan Province, Southwest Iran.

PubMed

Ghafourian, Mehri; Esmaeili, Mehrnosh; Dashti-Gerdabi, Nader; Sadeghi, Alireza; Malekei Naseri, Ali; Kazemi, Akhtar

2017-01-01

Thalassemia syndrome is the most common genetic disorder in the world and infection is the second cause of death in these patients. Measurement of serum C3 and C4 complement factors in serum was done in 60 patients with beta thalassemia major in comparison with 30 healthy subjects as control group. The serum level of C3 and C4 complement factors in 60 patients with beta thalassemia major who were randomly selected from among the patients referred to Shafa Hospital of Ahvaz was evaluated and compared with 30 samples from healthy individuals with no history of recent infectious or autoimmune diseases. It should be noted that single-radial-immunodiffusion assay was used in this study. This study has shown a significant reduction in serum levels of C3 and C4 in patients compared to controls (P value < 0.05). Decreased synthesis or increased consumption of complement factors in patients receiving multiple blood transfusions might lead to continuous contact between the immune system and various antigens, causing nonstop use of complement factors, recurrent infections, changes in parameters of the immune system due to iron overload as well as exposure to infectious factors such as HBV, HCV, HIV, and HTLV through blood transfusion.

CNV-association meta-analysis in 191,161 European adults reveals new loci associated with anthropometric traits.

PubMed

Macé, Aurélien; Tuke, Marcus A; Deelen, Patrick; Kristiansson, Kati; Mattsson, Hannele; Nõukas, Margit; Sapkota, Yadav; Schick, Ursula; Porcu, Eleonora; Rüeger, Sina; McDaid, Aaron F; Porteous, David; Winkler, Thomas W; Salvi, Erika; Shrine, Nick; Liu, Xueping; Ang, Wei Q; Zhang, Weihua; Feitosa, Mary F; Venturini, Cristina; van der Most, Peter J; Rosengren, Anders; Wood, Andrew R; Beaumont, Robin N; Jones, Samuel E; Ruth, Katherine S; Yaghootkar, Hanieh; Tyrrell, Jessica; Havulinna, Aki S; Boers, Harmen; Mägi, Reedik; Kriebel, Jennifer; Müller-Nurasyid, Martina; Perola, Markus; Nieminen, Markku; Lokki, Marja-Liisa; Kähönen, Mika; Viikari, Jorma S; Geller, Frank; Lahti, Jari; Palotie, Aarno; Koponen, Päivikki; Lundqvist, Annamari; Rissanen, Harri; Bottinger, Erwin P; Afaq, Saima; Wojczynski, Mary K; Lenzini, Petra; Nolte, Ilja M; Sparsø, Thomas; Schupf, Nicole; Christensen, Kaare; Perls, Thomas T; Newman, Anne B; Werge, Thomas; Snieder, Harold; Spector, Timothy D; Chambers, John C; Koskinen, Seppo; Melbye, Mads; Raitakari, Olli T; Lehtimäki, Terho; Tobin, Martin D; Wain, Louise V; Sinisalo, Juha; Peters, Annette; Meitinger, Thomas; Martin, Nicholas G; Wray, Naomi R; Montgomery, Grant W; Medland, Sarah E; Swertz, Morris A; Vartiainen, Erkki; Borodulin, Katja; Männistö, Satu; Murray, Anna; Bochud, Murielle; Jacquemont, Sébastien; Rivadeneira, Fernando; Hansen, Thomas F; Oldehinkel, Albertine J; Mangino, Massimo; Province, Michael A; Deloukas, Panos; Kooner, Jaspal S; Freathy, Rachel M; Pennell, Craig; Feenstra, Bjarke; Strachan, David P; Lettre, Guillaume; Hirschhorn, Joel; Cusi, Daniele; Heid, Iris M; Hayward, Caroline; Männik, Katrin; Beckmann, Jacques S; Loos, Ruth J F; Nyholt, Dale R; Metspalu, Andres; Eriksson, Johan G; Weedon, Michael N; Salomaa, Veikko; Franke, Lude; Reymond, Alexandre; Frayling, Timothy M; Kutalik, Zoltán

2017-09-29

There are few examples of robust associations between rare copy number variants (CNVs) and complex continuous human traits. Here we present a large-scale CNV association meta-analysis on anthropometric traits in up to 191,161 adult samples from 26 cohorts. The study reveals five CNV associations at 1q21.1, 3q29, 7q11.23, 11p14.2, and 18q21.32 and confirms two known loci at 16p11.2 and 22q11.21, implicating at least one anthropometric trait. The discovered CNVs are recurrent and rare (0.01-0.2%), with large effects on height (>2.4 cm), weight (>5 kg), and body mass index (BMI) (>3.5 kg/m 2 ). Burden analysis shows a 0.41 cm decrease in height, a 0.003 increase in waist-to-hip ratio and increase in BMI by 0.14 kg/m 2 for each Mb of total deletion burden (P = 2.5 × 10 -10 , 6.0 × 10 -5 , and 2.9 × 10 -3 ). Our study provides evidence that the same genes (e.g., MC4R, FIBIN, and FMO5) harbor both common and rare variants affecting body size and that anthropometric traits share genetic loci with developmental and psychiatric disorders.Individual SNPs have small effects on anthropometric traits, yet the impact of CNVs has remained largely unknown. Here, Kutalik and co-workers perform a large-scale genome-wide meta-analysis of structural variation and find rare CNVs associated with height, weight and BMI with large effect sizes.

Copy number variation at the 7q11.23 segmental duplications is a susceptibility factor for the Williams-Beuren syndrome deletion

PubMed Central

Cuscó, Ivon; Corominas, Roser; Bayés, Mònica; Flores, Raquel; Rivera-Brugués, Núria; Campuzano, Victoria; Pérez-Jurado, Luis A.

2008-01-01

Large copy number variants (CNVs) have been recently found as structural polymorphisms of the human genome of still unknown biological significance. CNVs are significantly enriched in regions with segmental duplications or low-copy repeats (LCRs). Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder caused by a heterozygous deletion of contiguous genes at 7q11.23 mediated by nonallelic homologous recombination (NAHR) between large flanking LCRs and facilitated by a structural variant of the region, a ∼2-Mb paracentric inversion present in 20%–25% of WBS-transmitting progenitors. We now report that eight out of 180 (4.44%) WBS-transmitting progenitors are carriers of a CNV, displaying a chromosome with large deletion of LCRs. The prevalence of this CNV among control individuals and non-transmitting progenitors is much lower (1%, n = 600), thus indicating that it is a predisposing factor for the WBS deletion (odds ratio 4.6-fold, P = 0.002). LCR duplications were found in 2.22% of WBS-transmitting progenitors but also in 1.16% of controls, which implies a non–statistically significant increase in WBS-transmitting progenitors. We have characterized the organization and breakpoints of these CNVs, encompassing ∼100–300 kb of genomic DNA and containing several pseudogenes but no functional genes. Additional structural variants of the region have also been defined, all generated by NAHR between different blocks of segmental duplications. Our data further illustrate the highly dynamic structure of regions rich in segmental duplications, such as the WBS locus, and indicate that large CNVs can act as susceptibility alleles for disease-associated genomic rearrangements in the progeny. PMID:18292220

Characterization of the complement inhibitory function of rhesus rhadinovirus complement control protein (RCP).

PubMed

Okroj, Marcin; Mark, Linda; Stokowska, Anna; Wong, Scott W; Rose, Nicola; Blackbourn, David J; Villoutreix, Bruno O; Spiller, O Brad; Blom, Anna M

2009-01-02

Rhesus rhadinovirus (RRV) is currently the closest known, fully sequenced homolog of human Kaposi sarcoma-associated herpesvirus. Both these viruses encode complement inhibitors as follows: Kaposi sarcoma-associated herpesvirus-complement control protein (KCP) and RRV-complement control protein (RCP). Previously we characterized in detail the functional properties of KCP as a complement inhibitor. Here, we performed comparative analyses for two variants of RCP protein, encoded by RRV strains H26-95 and 17577. Both RCP variants and KCP inhibited human and rhesus complement when tested in hemolytic assays measuring all steps of activation via the classical and the alternative pathway. RCP variants from both RRV strains supported C3b and C4b degradation by factor I and decay acceleration of the classical C3 convertase, similar to KCP. Additionally, the 17577 RCP variant accelerated decay of the alternative C3 convertase, which was not seen for KCP. In contrast to KCP, RCP showed no affinity to heparin and is the first described complement inhibitor in which the binding site for C3b/C4b does not interact with heparin. Molecular modeling shows a structural disruption in the region of RCP that corresponds to the KCP-heparin-binding site. This makes RRV a superior model for future in vivo investigations of complement evasion, as RCP does not play a supportive role in viral attachment as KCP does.

Complement System in Dermatological Diseases – Fire Under the Skin

PubMed Central

Panelius, Jaana; Meri, Seppo

2015-01-01

The complement system plays a key role in several dermatological diseases. Overactivation, deficiency, or abnormality of the control proteins are often related to a skin disease. Autoimmune mechanisms with autoantibodies and a cytotoxic effect of the complement membrane attack complex on epidermal or vascular cells can cause direct tissue damage and inflammation, e.g., in systemic lupus erythematosus (SLE), phospholipid antibody syndrome, and bullous skin diseases like pemphigoid. By evading complement attack, some microbes like Borrelia spirochetes and staphylococci can persist in the skin and cause prolonged symptoms. In this review, we present the most important skin diseases connected to abnormalities in the function of the complement system. Drugs having an effect on the complement system are also briefly described. On one hand, drugs with free hydroxyl on amino groups (e.g., hydralazine, procainamide) could interact with C4A, C4B, or C3 and cause an SLE-like disease. On the other hand, progress in studies on complement has led to novel anti-complement drugs (recombinant C1-inhibitor and anti-C5 antibody, eculizumab) that could alleviate symptoms in diseases associated with excessive complement activation. The main theme of the manuscript is to show how relevant the complement system is as an immune effector system in contributing to tissue injury and inflammation in a broad range of skin disorders. PMID:25688346

Evasion of Complement-Mediated Lysis and Complement C3 Deposition Are Regulated by Francisella tularensis Lipopolysaccharide O Antigen1

PubMed Central

Clay, Corey D.; Soni, Shilpa; Gunn, John S.; Schlesinger, Larry S.

2009-01-01

The bacterium Francisella tularensis (Ft) is a potential weapon of bioterrorism when aerosolized. Macrophage infection is necessary for disease progression and efficient phagocytosis by human macrophages requires serum opsonization by complement. Microbial complement activation leads to surface deposition of a highly regulated protein complex resulting in opsonization or membrane lysis. The nature of complement component C3 deposition, i.e., C3b (opsonization and lysis) or C3bi (opsonization only) fragment deposition, is central to the outcome of activation. In this study, we examine the mechanisms of Ft resistance to complement-mediated lysis, C3 component deposition on the Ft surface, and complement activation. Upon incubation in fresh nonimmune human serum, Schu S4 (Ft subsp. tularensis), Fn (Ft subsp. novicida), and LVS (Ft subsp. holarctica live vaccine strain) were resistant to complement-mediated lysis, but LVSG and LVSR (LVS strains altered in surface carbohydrate structures) were susceptible. C3 deposition, however, occurred on all strains. Complement-susceptible strains had markedly increased C3 fragment deposition, including the persistent presence of C3b compared with C3bi, which indicates that C3b inactivation results in survival of complement-resistant strains. C1q, an essential component of the classical activation pathway, was necessary for lysis of complement-susceptible strains and optimal C3 deposition on all strains. Finally, use of Francisella LPS mutants confirmed O Ag as a major regulator of complement resistance. These data provide evidence that pathogenic Francisella activate complement, but are resistant to complement-mediated lysis in part due to limited C3 deposition, rapid conversion of surface-bound C3b to C3bi, and the presence of LPS O Ag. PMID:18832715

Activated complement components and complement activator molecules on the surface of cell‐derived microparticles in patients with rheumatoid arthritis and healthy individuals

PubMed Central

Biró, Éva; Nieuwland, Rienk; Tak, Paul P; Pronk, Loes M; Schaap, Marianne C L; Sturk, Augueste; Hack, C Erik

2007-01-01

Objectives In vitro, microparticles can activate complement via the classical pathway. If demonstrable ex vivo, this mechanism may contribute to the pathogenesis of rheumatoid arthritis (RA). We therefore investigated the presence of activated complement components and complement activator molecules on the surface of cell‐derived microparticles of RA patients and healthy individuals. Methods Microparticles from synovial fluid (n = 8) and plasma (n = 9) of 10 RA patients and plasma of sex‐ and age‐matched healthy individuals (n = 10) were analysed by flow cytometry for bound complement components (C1q, C4, C3) and complement activator molecules (C‐reactive protein (CRP), serum amyloid P component (SAP), immunoglobulin (Ig) M, IgG). Results Microparticles with bound C1q, C4, and/or C3 were abundant in RA synovial fluid, while in RA and control plasma much lower levels were present. Microparticles with bound C1q correlated with those with bound C3 in synovial fluid (r = 0.961, p = 0.0001), and with those with bound C4 in plasma (RA: r = 0.908, p = 0.0007; control: r = 0.632, p = 0.0498), indicating classical pathway activation. In synovial fluid, microparticles with IgM and IgG correlated with those with C1q (r = 0.728, p = 0.0408; r = 0.952, p = 0.0003, respectively), and in plasma, microparticles with CRP correlated with those with C1q (RA: r = 0.903, p = 0.0021; control: r = 0.683, p = 0.0296), implicating IgG and IgM in the classical pathway activation in RA synovial fluid, and CRP in the low level classical pathway activation in plasma. Conclusions This study demonstrates the presence of bound complement components and activator molecules on microparticles ex vivo, and supports their role in low grade complement activation in plasma and increased complement activation in RA synovial fluid. PMID:17261534

Coagulation cascade and complement system in systemic lupus erythematosus

PubMed Central

Liang, Yan; Xie, Shang-Bo; Wu, Chang-Hao; Hu, Yuan; Zhang, Qin; Li, Si; Fan, Yin-Guang; Leng, Rui-Xue; Pan, Hai-Feng; Xiong, Hua-Bao; Ye, Dong-Qing

2018-01-01

This study was conducted to (1) characterize coagulation cascade and complement system in systemic lupus erythematosus (SLE); (2) evaluate the associations between coagulation cascade, complement system, inflammatory response and SLE disease severity; (3) test the diagnostic value of a combination of D-dimer and C4 for lupus activity. Transcriptomics, proteomics and metabolomics were performed in 24 SLE patients and 24 healthy controls. The levels of ten coagulations, seven complements and three cytokines were measured in 112 SLE patients. Clinical data were collected from 2025 SLE patients. The analysis of multi-omics data revealed the common links for the components of coagulation cascade and complement system. The results of ELISA showed coagulation cascade and complement system had an interaction effect on SLE disease severity, this effect was pronounced among patients with excess inflammation. The analysis of clinical data revealed a combination of D-dimer and C4 provided good diagnostic performance for lupus activity. This study suggested that coagulation cascade and complement system become ‘partners in crime’, contributing to SLE disease severity and identified the diagnostic value of D-dimer combined with C4for lupus activity. PMID:29599912

Structural basis for activation of the complement system by component C4 cleavage

PubMed Central

Kidmose, Rune T.; Laursen, Nick S.; Dobó, József; Kjaer, Troels R.; Sirotkina, Sofia; Yatime, Laure; Sottrup-Jensen, Lars; Thiel, Steffen; Gál, Péter; Andersen, Gregers R.

2012-01-01

An essential aspect of innate immunity is recognition of molecular patterns on the surface of pathogens or altered self through the lectin and classical pathways, two of the three well-established activation pathways of the complement system. This recognition causes activation of the MASP-2 or the C1s serine proteases followed by cleavage of the protein C4. Here we present the crystal structures of the 203-kDa human C4 and the 245-kDa C4⋅MASP-2 substrate⋅enzyme complex. When C4 binds to MASP-2, substantial conformational changes in C4 are induced, and its scissile bond region becomes ordered and inserted into the protease catalytic site in a manner canonical to serine proteases. In MASP-2, an exosite located within the CCP domains recognizes the C4 C345C domain 60 Å from the scissile bond. Mutations in C4 and MASP-2 residues at the C345C–CCP interface inhibit the intermolecular interaction and C4 cleavage. The possible assembly of the huge in vivo enzyme–substrate complex consisting of glycan-bound mannan-binding lectin, MASP-2, and C4 is discussed. Our own and prior functional data suggest that C1s in the classical pathway of complement activated by, e.g., antigen–antibody complexes, also recognizes the C4 C345C domain through a CCP exosite. Our results provide a unified structural framework for understanding the early and essential step of C4 cleavage in the elimination of pathogens and altered self through two major pathways of complement activation. PMID:22949645

Assessing the impact of copy number variants on miRNA genes in autism by Monte Carlo simulation.

PubMed

Marrale, Maurizio; Albanese, Nadia Ninfa; Calì, Francesco; Romano, Valentino

2014-01-01

Autism Spectrum Disorders (ASDs) are childhood neurodevelopmental disorders with complex genetic origins. Previous studies have investigated the role of de novo Copy Number Variants (CNVs) and microRNAs as important but distinct etiological factors in ASD. We developed a novel computational procedure to assess the potential pathogenic role of microRNA genes overlapping de novo CNVs in ASD patients. Here we show that for chromosomes # 1, 2 and 22 the actual number of miRNA loci affected by de novo CNVs in patients was found significantly higher than that estimated by Monte Carlo simulation of random CNV events. Out of 24 miRNA genes over-represented in CNVs from these three chromosomes only hsa-mir-4436b-1 and hsa-mir-4436b-2 have not been detected in CNVs from non-autistic subjects as reported in the Database of Genomic Variants. Altogether the results reported in this study represent a first step towards a full understanding of how a dysregulated expression of the 24 miRNAs genes affect neurodevelopment in autism. We also propose that the procedure used in this study can be effectively applied to CNVs/miRNA genes association data in other genomic disorders beyond autism.

Copy number variations of obesity relevant loci associated with body mass index in young Chinese.

PubMed

Sun, Chen; Cao, Min; Shi, Juan; Li, Lijuan; Miao, Lin; Hong, Jie; Cui, Bin; Ning, Guang

2013-03-10

Obesity is one of the most complex human diseases that are widely concerned and studied. More recently, copy number variations (CNVs) emerge as another important genetic marker to influence various human diseases. To elucidate the relationship between obesity and CNVs, this current study selected obesity-related candidate CNVs and analyzed their association with body mass index (BMI). Results showed that a CNV locus, 8q24.3, was significantly different (P=0.0070) in CNV frequency between the obese and healthy controls in a young eastern Chinese cohort, while no statistical significance was observed in other seven candidate loci including well reported 10q11.22 and 16p11.2 loci. The association of 8q24.3 CNVs with BMI of the subjects only showed marginal significance, while the copy number (CN) of 5p15.33 had a significant correlation with the BMI of the subject. These results suggested that 8q24.3 CN gains was associated with obesity, and 5p15.33 might also contribute to obesity pathogenesis, highlighting the importance of these CNVs for obesity risks, as well as providing new evidence for CNVs in the pathology of common diseases. Copyright © 2012 Elsevier B.V. All rights reserved.

A genome-wide association study of copy number variations with umbilical hernia in swine.

PubMed

Long, Yi; Su, Ying; Ai, Huashui; Zhang, Zhiyan; Yang, Bin; Ruan, Guorong; Xiao, Shijun; Liao, Xinjun; Ren, Jun; Huang, Lusheng; Ding, Nengshui

2016-06-01

Umbilical hernia (UH) is one of the most common congenital defects in pigs, leading to considerable economic loss and serious animal welfare problems. To test whether copy number variations (CNVs) contribute to pig UH, we performed a case-control genome-wide CNV association study on 905 pigs from the Duroc, Landrace and Yorkshire breeds using the Porcine SNP60 BeadChip and penncnv algorithm. We first constructed a genomic map comprising 6193 CNVs that pertain to 737 CNV regions. Then, we identified eight CNVs significantly associated with the risk for UH in the three pig breeds. Six of seven significantly associated CNVs were validated using quantitative real-time PCR. Notably, a rare CNV (CNV14:13030843-13059455) encompassing the NUGGC gene was strongly associated with UH (permutation-corrected P = 0.0015) in Duroc pigs. This CNV occurred exclusively in seven Duroc UH-affected individuals. SNPs surrounding the CNV did not show association signals, indicating that rare CNVs may play an important role in complex pig diseases such as UH. The NUGGC gene has been implicated in human omphalocele and inguinal hernia. Our finding supports that CNVs, including the NUGGC CNV, contribute to the pathogenesis of pig UH. © 2016 Stichting International Foundation for Animal Genetics.

Pneumococcal polysaccharides complexed with C3d bind to human B lymphocytes via complement receptor type 2.

PubMed Central

Griffioen, A W; Rijkers, G T; Janssens-Korpela, P; Zegers, B J

1991-01-01

The immunoregulatory function of the complement system has been the focus of many investigations. In particular, fragments of complement factor C3 have been shown to play a role in B-lymphocyte activation and proliferation, lymphokine production, and the generation of in vitro antibody production. Purified pneumococcal polysaccharides (PS) can induce direct activation of C3 via the alternative pathway. Using sera of C1q-deficient patients and healthy subjects, we demonstrated that C3d, a split product of C3 that is generated after degradation of iC3b, can be bound to PS antigens. The binding of C3d to PS can occur in the absence of specific antibodies. Subsequently, we showed that PS complexed with C3d can be recognized by complement receptor type 2 that is expressed on B cells. Treatment of B cells with a monoclonal antibody recognizing the C3d-binding site of complement receptor type 2 reduces the binding of PS-C3d to the cells. In addition, we showed that PS4 complexed with C3d exerted an increased immunogenicity compared with free PS4. Our results show that the complement system plays a role in the activation of PS-specific B cells, carrying membrane receptors for C3d. Consequently, the complement system plays a regulatory role in the antibody response to T-cell-independent type 2 antigens such as PS. PMID:1826897

Rare De Novo Copy Number Variants in Patients with Congenital Pulmonary Atresia

PubMed Central

Xie, Li; Chen, Jin-Lan; Zhang, Wei-Zhi; Wang, Shou-Zheng; Zhao, Tian-Li; Huang, Can; Wang, Jian; Yang, Jin-Fu; Yang, Yi-Feng; Tan, Zhi-Ping

2014-01-01

Background Ongoing studies using genomic microarrays and next-generation sequencing have demonstrated that the genetic contributions to cardiovascular diseases have been significantly ignored in the past. The aim of this study was to identify rare copy number variants in individuals with congenital pulmonary atresia (PA). Methods and Results Based on the hypothesis that rare structural variants encompassing key genes play an important role in heart development in PA patients, we performed high-resolution genome-wide microarrays for copy number variations (CNVs) in 82 PA patient-parent trios and 189 controls with an Illumina SNP array platform. CNVs were identified in 17/82 patients (20.7%), and eight of these CNVs (9.8%) are considered potentially pathogenic. Five de novo CNVs occurred at two known congenital heart disease (CHD) loci (16p13.1 and 22q11.2). Two de novo CNVs that may affect folate and vitamin B12 metabolism were identified for the first time. A de novo 1-Mb deletion at 17p13.2 may represent a rare genomic disorder that involves mild intellectual disability and associated facial features. Conclusions Rare CNVs contribute to the pathogenesis of PA (9.8%), suggesting that the causes of PA are heterogeneous and pleiotropic. Together with previous data from animal models, our results might help identify a link between CHD and folate-mediated one-carbon metabolism (FOCM). With the accumulation of high-resolution SNP array data, these previously undescribed rare CNVs may help reveal critical gene(s) in CHD and may provide novel insights about CHD pathogenesis. PMID:24826987

Rare Copy Number Variants Are a Common Cause of Short Stature

PubMed Central

Zahnleiter, Diana; Uebe, Steffen; Ekici, Arif B.; Hoyer, Juliane; Wiesener, Antje; Wieczorek, Dagmar; Kunstmann, Erdmute; Reis, André; Doerr, Helmuth-Guenther; Rauch, Anita; Thiel, Christian T.

2013-01-01

Human growth has an estimated heritability of about 80%–90%. Nevertheless, the underlying cause of shortness of stature remains unknown in the majority of individuals. Genome-wide association studies (GWAS) showed that both common single nucleotide polymorphisms and copy number variants (CNVs) contribute to height variation under a polygenic model, although explaining only a small fraction of overall genetic variability in the general population. Under the hypothesis that severe forms of growth retardation might also be caused by major gene effects, we searched for rare CNVs in 200 families, 92 sporadic and 108 familial, with idiopathic short stature compared to 820 control individuals. Although similar in number, patients had overall significantly larger CNVs (p-value<1×10−7). In a gene-based analysis of all non-polymorphic CNVs>50 kb for gene function, tissue expression, and murine knock-out phenotypes, we identified 10 duplications and 10 deletions ranging in size from 109 kb to 14 Mb, of which 7 were de novo (p<0.03) and 13 inherited from the likewise affected parent but absent in controls. Patients with these likely disease causing 20 CNVs were smaller than the remaining group (p<0.01). Eleven (55%) of these CNVs either overlapped with known microaberration syndromes associated with short stature or contained GWAS loci for height. Haploinsufficiency (HI) score and further expression profiling suggested dosage sensitivity of major growth-related genes at these loci. Overall 10% of patients carried a disease-causing CNV indicating that, like in neurodevelopmental disorders, rare CNVs are a frequent cause of severe growth retardation. PMID:23516380

Rare de novo copy number variants in patients with congenital pulmonary atresia.

PubMed

Xie, Li; Chen, Jin-Lan; Zhang, Wei-Zhi; Wang, Shou-Zheng; Zhao, Tian-Li; Huang, Can; Wang, Jian; Yang, Jin-Fu; Yang, Yi-Feng; Tan, Zhi-Ping

2014-01-01

Ongoing studies using genomic microarrays and next-generation sequencing have demonstrated that the genetic contributions to cardiovascular diseases have been significantly ignored in the past. The aim of this study was to identify rare copy number variants in individuals with congenital pulmonary atresia (PA). Based on the hypothesis that rare structural variants encompassing key genes play an important role in heart development in PA patients, we performed high-resolution genome-wide microarrays for copy number variations (CNVs) in 82 PA patient-parent trios and 189 controls with an Illumina SNP array platform. CNVs were identified in 17/82 patients (20.7%), and eight of these CNVs (9.8%) are considered potentially pathogenic. Five de novo CNVs occurred at two known congenital heart disease (CHD) loci (16p13.1 and 22q11.2). Two de novo CNVs that may affect folate and vitamin B12 metabolism were identified for the first time. A de novo 1-Mb deletion at 17p13.2 may represent a rare genomic disorder that involves mild intellectual disability and associated facial features. Rare CNVs contribute to the pathogenesis of PA (9.8%), suggesting that the causes of PA are heterogeneous and pleiotropic. Together with previous data from animal models, our results might help identify a link between CHD and folate-mediated one-carbon metabolism (FOCM). With the accumulation of high-resolution SNP array data, these previously undescribed rare CNVs may help reveal critical gene(s) in CHD and may provide novel insights about CHD pathogenesis.

Explaining the variable penetrance of CNVs: Parental intelligence modulates expression of intellectual impairment caused by the 22q11.2 deletion.

PubMed

Klaassen, Petra; Duijff, Sasja; Swanenburg de Veye, Henriëtte; Beemer, Frits; Sinnema, Gerben; Breetvelt, Elemi; Schappin, Renske; Vorstman, Jacob

2016-09-01

The role of rare genetic variants, in particular copy number variants (CNVs), in the etiology of neurodevelopmental disorders is becoming increasingly clear. While the list of these disorder-related CNVs continues to lengthen, it has also become clear that in nearly all genetic variants the proportion of carriers who express the associated phenotype is far from 100%. To understand this variable penetrance of CNVs it is important to realize that even the largest CNVs represent only a tiny fraction of the entire genome. Therefore, part of the mechanism underlying the variable penetrance of CNVs is likely the modulatory impact of the rest of the genome. In the present study we used the 22q11DS as a model to examine whether the observed penetrance of intellectual impairment-one of the main phenotypes associated with 22q11DS-is modulated by the intellectual level of their parents, for which we used the parents' highest level of education as a proxy. Our results, based on data observed in 171 children with 22q11DS in the age range of 5-15 years, showed a significant association between estimated parental cognitive level and intelligence in offspring (full scale, verbal and performance IQ), with the largest effect size for verbal IQ. These results suggest that possible mechanisms involved in the variable penetrance observed in CNVs include the impact of genetic background and/or environmental influences. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Application of array-comparative genomic hybridization in tetralogy of Fallot

PubMed Central

Liu, Lin; Wang, Hong-Dan; Cui, Cun-Ying; Wu, Dong; Li, Tao; Fan, Tai-Bing; Peng, Bang-Tian; Zhang, Lian-Zhong; Wang, Cheng-Zeng

2016-01-01

Abstract To explore the underlying pathogenesis and provide references for genetic counseling and prenatal gene diagnosis, we analyzed the chromosome karyotypes and genome-wide copy number variations (CNVs) in 86 patients with tetralogy of Fallot (TOF) by G-banding karyotype analysis and array-comparative genomic hybridization (aCGH), respectively. And then quantitative polymerase chain reaction was used to validate these candidate CNVs. Based on their different properties, CNVs were categorized into benign CNVs, suspiciously pathogenic CNVs, and indefinite CNVs. Data analysis was based on public databases such as UCSC, DECIPHER, DGV, ISCA, and OMIM. The karyotype was normal in all the 86 patients with TOF. CNVs were detected in 11 patients by aCGH and quantitative polymerase chain reaction. Patient no. 0001, 0010, and 0029 had 2.52-Mb deletion in the chromosome 22q11.21 region; patient no. 0008 had both 595- and 428-kb duplications, respectively, in 12p12.3p12.2 and 14q23.2q23.3 regions; patient no. 0009 had 1.46-Mb duplication in the 1q21.1q21.2 region; patient no. 0016 had 513-kb duplication in the 1q42.13 region; patient no. 0024 had 292-kb duplication in the 16q11.2 region; patient no. 0026 had 270-kb duplication in the 16q24.1 region; patient no. 0028 had 222-kb deletion in the 7q31.1 region; patient no. 0033 had 1.73-Mb duplication in the 17q12 region; and patient no. 0061 had 5.79-Mb deletion in the 1p36.33p36.31 region. aCGH can accurately detect CNVs in the patients with TOF. This is conducive to genetic counseling and prenatal diagnosis for TOF and provides a new clue and theoretical basis for exploring the pathogenesis of congenital heart disease. PMID:27930557

Application of array-comparative genomic hybridization in tetralogy of Fallot.

PubMed

Liu, Lin; Wang, Hong-Dan; Cui, Cun-Ying; Wu, Dong; Li, Tao; Fan, Tai-Bing; Peng, Bang-Tian; Zhang, Lian-Zhong; Wang, Cheng-Zeng

2016-12-01

To explore the underlying pathogenesis and provide references for genetic counseling and prenatal gene diagnosis, we analyzed the chromosome karyotypes and genome-wide copy number variations (CNVs) in 86 patients with tetralogy of Fallot (TOF) by G-banding karyotype analysis and array-comparative genomic hybridization (aCGH), respectively. And then quantitative polymerase chain reaction was used to validate these candidate CNVs. Based on their different properties, CNVs were categorized into benign CNVs, suspiciously pathogenic CNVs, and indefinite CNVs. Data analysis was based on public databases such as UCSC, DECIPHER, DGV, ISCA, and OMIM.The karyotype was normal in all the 86 patients with TOF. CNVs were detected in 11 patients by aCGH and quantitative polymerase chain reaction. Patient no. 0001, 0010, and 0029 had 2.52-Mb deletion in the chromosome 22q11.21 region; patient no. 0008 had both 595- and 428-kb duplications, respectively, in 12p12.3p12.2 and 14q23.2q23.3 regions; patient no. 0009 had 1.46-Mb duplication in the 1q21.1q21.2 region; patient no. 0016 had 513-kb duplication in the 1q42.13 region; patient no. 0024 had 292-kb duplication in the 16q11.2 region; patient no. 0026 had 270-kb duplication in the 16q24.1 region; patient no. 0028 had 222-kb deletion in the 7q31.1 region; patient no. 0033 had 1.73-Mb duplication in the 17q12 region; and patient no. 0061 had 5.79-Mb deletion in the 1p36.33p36.31 region.aCGH can accurately detect CNVs in the patients with TOF. This is conducive to genetic counseling and prenatal diagnosis for TOF and provides a new clue and theoretical basis for exploring the pathogenesis of congenital heart disease.

The Group B Streptococcus-Secreted Protein CIP Interacts with C4, Preventing C3b Deposition via the Lectin and Classical Complement Pathways.

PubMed

Pietrocola, Giampiero; Rindi, Simonetta; Rosini, Roberto; Buccato, Scilla; Speziale, Pietro; Margarit, Immaculada

2016-01-01

The group B Streptococcus (GBS) is a leading cause of neonatal invasive disease. GBS bacteria are surrounded by a thick capsular polysaccharide that is a potent inhibitor of complement deposition via the alternative pathway. Several of its surface molecules can however activate the classical and lectin complement pathways, rendering this species still vulnerable to phagocytic killing. In this study we have identified a novel secreted protein named complement interfering protein (CIP) that downregulates complement activation via the classical and lectin pathways, but not the alternative pathway. The CIP protein showed high affinity toward C4b and inhibited its interaction with C2, presumably preventing the formation of the C4bC2a convertase. Addition of recombinant CIP to GBS cip-negative bacteria resulted in decreased deposition of C3b on their surface and in diminished phagocytic killing in a whole-blood assay. Our data reveal a novel strategy exploited by GBS to counteract innate immunity and could be valuable for the development of anti-infective agents against this important pathogen. Copyright © 2015 by The American Association of Immunologists, Inc.

Nonrenal and renal activity of systemic lupus erythematosus: a comparison of two anti-C1q and five anti-dsDNA assays and complement C3 and C4.

PubMed

Julkunen, Heikki; Ekblom-Kullberg, Susanne; Miettinen, Aaro

2012-08-01

Associations of different assays for antibodies to C1q (anti-C1q) and to dsDNA (anti-dsDNA) and of complements C3 and C4 with disease activity in patients with systemic lupus erythematosus (SLE) were studied. The clinical manifestations of 223 SLE patients were recorded, and the disease activity was assessed by the SLEDAI score. Anti-C1q were determined by two enzyme-linked immunosorbent assays (ELISA) and anti-dsDNA by a radioimmunoassay (RIA), a Crithidia immunofluorescence (IF) assay and three ELISA assays using human telomere DNA, plasmid DNA circles, or calf thymus DNA as antigens, respectively. Complement C3 and C4 were determined by nephelometry. Control sera were obtained from 98 blood donors. In patients with SLE, the prevalence of anti-C1q was 17-18% and that of anti-dsDNA was 36-69%. Anti-C1q, anti-dsDNA, and complement C3 and C4 correlated well with the overall activity of SLE (r = 0.323-0.351, 0.353-0.566, and -0.372-0.444, respectively; P < 0.001). Sensitivity, specificity, positive predictive value, and negative predictive value for active lupus nephritis among SLE patients were 40-44, 92, 29, and 91-92% for anti-C1q and 48-68, 29-66, 11-16, and 86-91% for anti-dsDNA, respectively. Patients with active nephritis had higher levels of anti-C1q and lower levels of C3 and C4 than patients with inactive nephritis (P = 0.003-0.018). The corresponding associations of anti-dsDNA were somewhat weaker (P = 0.023-0.198). Hematological parameters reflecting disease activity correlated clearly better with anti-dsDNA and complement C3 and C4 than with anti-C1q. Anti-C1q is inferior to anti-dsDNA as a diagnostic test in SLE and in the evaluation of overall clinical activity of the disease. Anti-C1q together with complement C3 and C4 may offer useful additional information to monitor lupus nephritis activity. There are no practical differences between different assays for anti-C1q and anti-dsDNA.

Systemic Lupus Erythematosus and Deficiencies of Early Components of the Complement Classical Pathway

PubMed Central

Macedo, Ana Catarina Lunz; Isaac, Lourdes

2016-01-01

The complement system plays an important role in the innate and acquired immune response against pathogens. It consists of more than 30 proteins found in soluble form or attached to cell membranes. Most complement proteins circulate in inactive forms and can be sequentially activated by the classical, alternative, or lectin pathways. Biological functions, such as opsonization, removal of apoptotic cells, adjuvant function, activation of B lymphocytes, degranulation of mast cells and basophils, and solubilization and clearance of immune complex and cell lysis, are dependent on complement activation. Although the activation of the complement system is important to avoid infections, it also can contribute to the inflammatory response triggered by immune complex deposition in tissues in autoimmune diseases. Paradoxically, the deficiency of early complement proteins from the classical pathway (CP) is strongly associated with development of systemic lupus erythematous (SLE) – mainly C1q deficiency (93%) and C4 deficiency (75%). The aim of this review is to focus on the deficiencies of early components of the CP (C1q, C1r, C1s, C4, and C2) proteins in SLE patients. PMID:26941740

Genome-wide copy number variation study associates metabotropic glutamate receptor gene networks with attention deficit hyperactivity disorder

PubMed Central

Elia, Josephine; Glessner, Joseph T; Wang, Kai; Takahashi, Nagahide; Shtir, Corina J; Hadley, Dexter; Sleiman, Patrick M A; Zhang, Haitao; Kim, Cecilia E; Robison, Reid; Lyon, Gholson J; Flory, James H; Bradfield, Jonathan P; Imielinski, Marcin; Hou, Cuiping; Frackelton, Edward C; Chiavacci, Rosetta M; Sakurai, Takeshi; Rabin, Cara; Middleton, Frank A; Thomas, Kelly A; Garris, Maria; Mentch, Frank; Freitag, Christine M; Steinhausen, Hans-Christoph; Todorov, Alexandre A; Reif, Andreas; Rothenberger, Aribert; Franke, Barbara; Mick, Eric O; Roeyers, Herbert; Buitelaar, Jan; Lesch, Klaus-Peter; Banaschewski, Tobias; Ebstein, Richard P; Mulas, Fernando; Oades, Robert D; Sergeant, Joseph; Sonuga-Barke, Edmund; Renner, Tobias J; Romanos, Marcel; Romanos, Jasmin; Warnke, Andreas; Walitza, Susanne; Meyer, Jobst; Pálmason, Haukur; Seitz, Christiane; Loo, Sandra K; Smalley, Susan L; Biederman, Joseph; Kent, Lindsey; Asherson, Philip; Anney, Richard J L; Gaynor, J William; Shaw, Philip; Devoto, Marcella; White, Peter S; Grant, Struan F A; Buxbaum, Joseph D; Rapoport, Judith L; Williams, Nigel M; Nelson, Stanley F; Faraone, Stephen V; Hakonarson, Hakon

2014-01-01

Attention deficit hyperactivity disorder (ADHD) is a common, heritable neuropsychiatric disorder of unknown etiology. We performed a whole-genome copy number variation (CNV) study on 1,013 cases with ADHD and 4,105 healthy children of European ancestry using 550,000 SNPs. We evaluated statistically significant findings in multiple independent cohorts, with a total of 2,493 cases with ADHD and 9,222 controls of European ancestry, using matched platforms. CNVs affecting metabotropic glutamate receptor genes were enriched across all cohorts (P = 2.1 × 10−9). We saw GRM5 (encoding glutamate receptor, metabotropic 5) deletions in ten cases and one control (P = 1.36 × 10−6). We saw GRM7 deletions in six cases, and we saw GRM8 deletions in eight cases and no controls. GRM1 was duplicated in eight cases. We experimentally validated the observed variants using quantitative RT-PCR. A gene network analysis showed that genes interacting with the genes in the GRM family are enriched for CNVs in ~10% of the cases (P = 4.38 × 10−10) after correction for occurrence in the controls. We identified rare recurrent CNVs affecting glutamatergic neurotransmission genes that were overrepresented in multiple ADHD cohorts. PMID:22138692

The Anticomplementary Activity of ’Fusobacterium polymorphum’ in Normal and C-4 Deficient Sources of Guinea Pig Complement.

DTIC Science & Technology

1977-01-12

A complement consumption assay was used to show that the anticomplementary activity of a cell wall preparation from F. polymorphum in guinea pig complement...tests with C𔃾-deficient guinea pig sera confirmed that F. polymorphum cell walls were capable of generating alternate complement pathway activity in guinea pig sera.

Intravitreal injection of (99)Tc-MDP inhibits the development of laser-induced choroidal neovascularization in rhesus monkeys.

PubMed

Lai, Kunbei; Jin, Chenjin; Tu, Shu; Xiong, Yunfan; Huang, Rui; Ge, Jian

2014-07-01

The aim of this study was to investigate the safety and efficacy of intravitreal injection of (99)Tc-MDP, a decay product of (99m)Tc-MDP, on the development of laser-induced choroidal neovascularization (CNV) in rhesus monkeys. Experimental CNV was induced by argon laser with a small high-energy laser spot. Monkeys were given 50 μL of (99)Tc-MDP at a concentration of 0.005 μg/mL (n = 6) or 0.01 μg/mL (n = 6) by intravitreal injection once a week immediately after laser injury for a period of 56 days. Control animals were treated with the same volume of PBS (n = 6) in the same way. Eyes were monitored by ophthalmic examination, color fundus photography, fluorescence fundus angiography (FFA), optical coherence tomography (OCT) and histology. Incidences of grade 4 CNV lesions as well as the leakage areas of grade 4 CNVs on the late-phase of fluorescein angiograms were measured in a standardized, randomized and masked fashion fortnightly. The maximum widths and heights of grade 4 CNVs were also calculated by histology at the end of the experiment. Toxicity of (99)Tc-MDP on the retina was evaluated by electroretinogram (ERG) and histologic analysis. (99)Tc-MDP reduced the incidences of grade 4 CNVs by 33.33 % and 39.40 % in the 0.005 μg/mL and 0.01 μg/mL groups, respectively, compared with the PBS group on day 28 (P < 0.05; n = 6). The leakage areas of grade 4 CNVs were smaller in the 0.005 μg/mL (0.7136 ± 0.0283 mm(2), p <0.01; n = 6) and 0.01 μg/mL (0.4351 ± 0.0349 mm(2), p < 0.01; n = 6) groups than those in the PBS control group (0.9373 ± 0.0455 mm(2); n = 6) in a dose-dependent manner on day 28. OCT and histology also showed that the sizes of CNVs were smaller in the (99)Tc-MDP treated groups than those in the PBS group. Although intravitreal injection of (99)Tc-MDP led to mild inflammatory reaction in the anterior chamber, histology and ERG findings demonstrated that (99)Tc-MDP did not cause any change in histological structure or function of the retina (p>0.05). Intravitreal injection of (99)Tc-MDP can inhibit the development of laser-induced CNV without toxic effect on retina, suggesting that (99)Tc-MDP has therapeutic potential for CNV related diseases.

Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum

PubMed Central

Castiblanco-Valencia, Mónica M.; Fraga, Tatiana R.; Breda, Leandro C.D.; Vasconcellos, Sílvio A.; Figueira, Cláudio P.; Picardeau, Mathieu; Wunder, Elsio; Ko, Albert I.; Barbosa, Angela S.; Isaac, Lourdes

2017-01-01

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum. PMID:26976804

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

PubMed Central

Valenzuela, Nicole M.; Thomas, Kimberly A.; Mulder, Arend; Parry, Graham C.; Panicker, Sandip; Reed, Elaine F.

2017-01-01

Background Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. Methods Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). Results Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. Conclusions Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR. PMID:28640789

Extensive Copy Number Variations in Admixed Indian Population of African Ancestry: Potential Involvement in Adaptation

PubMed Central

Dash, Debasis; Mukerji, Mitali

2014-01-01

Admixture mapping has been enormously resourceful in identifying genetic variations linked to phenotypes, adaptation, and diseases. In this study through analysis of copy number variable regions (CNVRs), we report extensive restructuring in the genomes of the recently admixed African-Indian population (OG-W-IP) that inhabits a highly saline environment in Western India. The study included subjects from OG-W-IP (OG), five different Indian and three HapMap populations that were genotyped using Affymetrix version 6.0 arrays. Copy number variations (CNVs) detected using Birdsuite were used to define CNVRs. Population structure with respect to CNVRs was delineated using random forest approach. OG genomes have a surprising excess of CNVs in comparison to other studied populations. Individual ancestry proportions computed using STRUCTURE also reveals a unique genetic component in OGs. Population structure analysis with CNV genotypes indicates OG to be distant from both the African and Indian ancestral populations. Interestingly, it shows genetic proximity with respect to CNVs to only one Indian population IE-W-LP4, which also happens to reside in the same geographical region. We also observe a significant enrichment of molecular processes related to ion binding and receptor activity in genes encompassing OG-specific CNVRs. Our results suggest that retention of CNVRs from ancestral natives and de novo acquisition of CNVRs could accelerate the process of adaptation especially in an extreme environment. Additionally, this population would be enormously useful for dissecting genes and delineating the involvement of CNVs in salt adaptation. PMID:25398783

Identification of genomic aberrations in hemangioblastoma by droplet digital PCR and SNP microarray highlights novel candidate genes and pathways for pathogenesis.

PubMed

Mehrian-Shai, Ruty; Yalon, Michal; Moshe, Itai; Barshack, Iris; Nass, Dvorah; Jacob, Jasmine; Dor, Chen; Reichardt, Juergen K V; Constantini, Shlomi; Toren, Amos

2016-01-14

The genetic mechanisms underlying hemangioblastoma development are still largely unknown. We used high-resolution single nucleotide polymorphism microarrays and droplet digital PCR analysis to detect copy number variations (CNVs) in total of 45 hemangioblastoma tumors. We identified 94 CNVs with a median of 18 CNVs per sample. The most frequently gained regions were on chromosomes 1 (p36.32) and 7 (p11.2). These regions contain the EGFR and PRDM16 genes. Recurrent losses were located at chromosome 12 (q24.13), which includes the gene PTPN11. Our findings provide the first high-resolution genome-wide view of chromosomal changes in hemangioblastoma and identify 23 candidate genes: EGFR, PRDM16, PTPN11, HOXD11, HOXD13, FLT3, PTCH, FGFR1, FOXP1, GPC3, HOXC13, HOXC11, MKL1, CHEK2, IRF4, GPHN, IKZF1, RB1, HOXA9, and micro RNA, such as hsa-mir-196a-2 for hemangioblastoma pathogenesis. Furthermore, our data implicate that cell proliferation and angiogenesis promoting pathways may be involved in the molecular pathogenesis of hemangioblastoma.

WholeGenome Sequencing of High-Risk Families to Identify New Mutational Mechanisms of Breast Cancer Predisposition

DTIC Science & Technology

2014-10-01

4 APPENDICES 4 INTRODUCTION: Despite tremendous advances in mutation detection with gene panels...population frequency and overlap with ENCODE regions. 2a. Align reads to the reference sequence (months 4-10) 2b. Identify SNPs, indels, CNVs and

Complement system biomarkers in epilepsy.

PubMed

Kopczynska, Maja; Zelek, Wioleta M; Vespa, Simone; Touchard, Samuel; Wardle, Mark; Loveless, Samantha; Thomas, Rhys H; Hamandi, Khalid; Morgan, B Paul

2018-05-24

To explore whether complement dysregulation occurs in a routinely recruited clinical cohort of epilepsy patients, and whether complement biomarkers have potential to be used as markers of disease severity and seizure control. Plasma samples from 157 epilepsy cases (106 with focal seizures, 46 generalised seizures, 5 unclassified) and 54 controls were analysed. Concentrations of 10 complement analytes (C1q, C3, C4, factor B [FB], terminal complement complex [TCC], iC3b, factor H [FH], Clusterin [Clu], Properdin, C1 Inhibitor [C1Inh] plus C-reactive protein [CRP]) were measured using enzyme linked immunosorbent assay (ELISA). Univariate and multivariate statistical analysis were used to test whether combinations of complement analytes were predictive of epilepsy diagnoses and seizure occurrence. Correlation between number and type of anti-epileptic drugs (AED) and complement analytes was also performed. We found: CONCLUSION: This study adds to evidence implicating complement in pathogenesis of epilepsy and may allow the development of better therapeutics and prognostic markers in the future. Replication in a larger sample set is needed to validate the findings of the study. Copyright © 2018. Published by Elsevier Ltd.

Function of Serum Complement in Drinking Water Arsenic Toxicity

PubMed Central

Islam, Laila N.; Zahid, M. Shamim Hasan; Nabi, A. H. M. Nurun; Hossain, Mahmud

2012-01-01

Serum complement function was evaluated in 125 affected subjects suffering from drinking water arsenic toxicity. Their mean duration of exposure was 7.4 ± 5.3 yrs, and the levels of arsenic in drinking water and urine samples were 216 ± 211 and 223 ± 302 μg/L, respectively. The mean bactericidal activity of complement from the arsenic patients was 92% and that in the unexposed controls was 99% (P < 0.01), but heat-inactivated serum showed slightly elevated activity than in controls. In patients, the mean complement C3 was 1.56 g/L, and C4 was 0.29 g/L compared to 1.68 g/L and 0.25 g/L, respectively, in the controls. The mean IgG in the arsenic patients was 24.3 g/L that was highly significantly elevated (P < 0.001). Arsenic patients showed a significant direct correlation between C3 and bactericidal activity (P = 0.014). Elevated levels of C4 indicated underutilization and possibly impaired activity of the classical complement pathway. We conclude reduced function of serum complement in drinking water arsenic toxicity. PMID:22545044

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice.

PubMed

Bruno, D L; Ganesamoorthy, D; Schoumans, J; Bankier, A; Coman, D; Delatycki, M; Gardner, R J M; Hunter, M; James, P A; Kannu, P; McGillivray, G; Pachter, N; Peters, H; Rieubland, C; Savarirayan, R; Scheffer, I E; Sheffield, L; Tan, T; White, S M; Yeung, A; Bowman, Z; Ngo, C; Choy, K W; Cacheux, V; Wong, L; Amor, D J; Slater, H R

2009-02-01

Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones. Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays. 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as "potentially pathogenic". Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients. Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered "new syndromes" were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype-phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

Copy-number variations are enriched for neurodevelopmental genes in children with developmental coordination disorder.

PubMed

Mosca, Stephen J; Langevin, Lisa Marie; Dewey, Deborah; Innes, A Micheil; Lionel, Anath C; Marshall, Christian C; Scherer, Stephen W; Parboosingh, Jillian S; Bernier, Francois P

2016-12-01

Developmental coordination disorder is a common neurodevelopment disorder that frequently co-occurs with other neurodevelopmental disorders including attention-deficit hyperactivity disorder (ADHD). Copy-number variations (CNVs) have been implicated in a number of neurodevelopmental and psychiatric disorders; however, the proportion of heritability in developmental coordination disorder (DCD) attributed to CNVs has not been explored. This study aims to investigate how CNVs may contribute to the genetic architecture of DCD. CNV analysis was performed on 82 extensively phenotyped Canadian children with DCD, with or without co-occurring ADHD and/or reading disorder, and 2988 healthy European controls using identical genome-wide SNP microarrays and CNV calling algorithms. An increased rate of large and rare genic CNVs (p=0.009) was detected, and there was an enrichment of duplications spanning brain-expressed genes (p=0.039) and genes previously implicated in other neurodevelopmental disorders (p=0.043). Genes and loci of particular interest in this group included: GAP43, RBFOX1, PTPRN2, SHANK3, 16p11.2 and distal 22q11.2. Although no recurrent CNVs were identified, 26% of DCD cases, where sample availability permitted segregation analysis, were found to have a de novo rare CNV. Of the inherited CNVs, 64% were from a parent who also had a neurodevelopmental disorder. These findings suggest that there may be shared susceptibility genes for DCD and other neurodevelopmental disorders and highlight the need for thorough phenotyping when investigating the genetics of neurodevelopmental disorders. Furthermore, these data provide compelling evidence supporting a genetic basis for DCD, and further implicate rare CNVs in the aetiology of neurodevelopmental disorders. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Case history and genome-wide scans for copy number variants in a family with patient having 15q11.1-q11.2 duplication and 22q11.2 deletion, and schizophrenia.

PubMed

Takahashi, Sakae; Suzuki, Takahiro; Nakamura-Tomizuka, Sakura; Osaki, Koichi; Sotome, Yuta; Sagawa, Tomoaki; Uchiyama, Makoto

2015-06-01

Many studies have indicated that chromosomes 15q11 and 22q11 may be associated with the genetic etiologies of schizophrenia. We have followed an adult schizophrenia case with 15q11.1-q11.2 duplication and 22q11.2 deletion. Here we report his clinical history, and copy number variants (CNVs) identified by microarray and real-time PCR in the patient and his parents. This is the first report describing a detailed phenotype of an adult schizophrenic case with both 15q11 and 22q11 CNVs as revealed by novel and trustworthy technologies. Subjects were a 33-year-old male patient with 15q11 and 22q11 CNVs, and his normal parents. He fulfilled the DSM-IV criteria for schizophrenia at age 18 years. He was also diagnosed with 22q11.2 deletion syndrome by fluorescence in situ hybridization (FISH) at age 18 years. To search for CNVs in more detail, whole-genome array-CGH analyses including ∼ 420,000 probes were carried out in the patient and his parents. For validations of the CNVs detected by array-CGH, real-time PCR analyses of these CNVs were performed. The patient had two disease-specific CNVs, 15q11.1-q11.2 duplication (∼ 2.7 Mb) and 22q11.21 deletion (∼ 2.9 Mb). These two regions are important for the development of schizophrenia, and this patient had shown symptoms of schizophrenia. Thus, the two areas may contain causal genes for schizophrenia. © 2015 Wiley Periodicals, Inc.

Family-Based Benchmarking of Copy Number Variation Detection Software.

PubMed

Nutsua, Marcel Elie; Fischer, Annegret; Nebel, Almut; Hofmann, Sylvia; Schreiber, Stefan; Krawczak, Michael; Nothnagel, Michael

2015-01-01

The analysis of structural variants, in particular of copy-number variations (CNVs), has proven valuable in unraveling the genetic basis of human diseases. Hence, a large number of algorithms have been developed for the detection of CNVs in SNP array signal intensity data. Using the European and African HapMap trio data, we undertook a comparative evaluation of six commonly used CNV detection software tools, namely Affymetrix Power Tools (APT), QuantiSNP, PennCNV, GLAD, R-gada and VEGA, and assessed their level of pair-wise prediction concordance. The tool-specific CNV prediction accuracy was assessed in silico by way of intra-familial validation. Software tools differed greatly in terms of the number and length of the CNVs predicted as well as the number of markers included in a CNV. All software tools predicted substantially more deletions than duplications. Intra-familial validation revealed consistently low levels of prediction accuracy as measured by the proportion of validated CNVs (34-60%). Moreover, up to 20% of apparent family-based validations were found to be due to chance alone. Software using Hidden Markov models (HMM) showed a trend to predict fewer CNVs than segmentation-based algorithms albeit with greater validity. PennCNV yielded the highest prediction accuracy (60.9%). Finally, the pairwise concordance of CNV prediction was found to vary widely with the software tools involved. We recommend HMM-based software, in particular PennCNV, rather than segmentation-based algorithms when validity is the primary concern of CNV detection. QuantiSNP may be used as an additional tool to detect sets of CNVs not detectable by the other tools. Our study also reemphasizes the need for laboratory-based validation, such as qPCR, of CNVs predicted in silico.

Population Structure Shapes Copy Number Variation in Malaria Parasites.

PubMed

Cheeseman, Ian H; Miller, Becky; Tan, John C; Tan, Asako; Nair, Shalini; Nkhoma, Standwell C; De Donato, Marcos; Rodulfo, Hectorina; Dondorp, Arjen; Branch, Oralee H; Mesia, Lastenia Ruiz; Newton, Paul; Mayxay, Mayfong; Amambua-Ngwa, Alfred; Conway, David J; Nosten, François; Ferdig, Michael T; Anderson, Tim J C

2016-03-01

If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500 bp to 59 kb, as well as 10,107 flanking, biallelic single-nucleotide polymorphisms. Overall, CNVs were rare, small, and skewed toward low frequency variants, consistent with the deleterious model. Relative to African and South East Asian populations, CNVs were significantly more common in South America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g., DNA helicase and three conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genetic Structures of Copy Number Variants Revealed by Genotyping Single Sperm

PubMed Central

Luo, Minjie; Cui, Xiangfeng; Fredman, David; Brookes, Anthony J.; Azaro, Marco A.; Greenawalt, Danielle M.; Hu, Guohong; Wang, Hui-Yun; Tereshchenko, Irina V.; Lin, Yong; Shentu, Yue; Gao, Richeng; Shen, Li; Li, Honghua

2009-01-01

Background Copy number variants (CNVs) occupy a significant portion of the human genome and may have important roles in meiotic recombination, human genome evolution and gene expression. Many genetic diseases may be underlain by CNVs. However, because of the presence of their multiple copies, variability in copy numbers and the diploidy of the human genome, detailed genetic structure of CNVs cannot be readily studied by available techniques. Methodology/Principal Findings Single sperm samples were used as the primary subjects for the study so that CNV haplotypes in the sperm donors could be studied individually. Forty-eight CNVs characterized in a previous study were analyzed using a microarray-based high-throughput genotyping method after multiplex amplification. Seventeen single nucleotide polymorphisms (SNPs) were also included as controls. Two single-base variants, either allelic or paralogous, could be discriminated for all markers. Microarray data were used to resolve SNP alleles and CNV haplotypes, to quantitatively assess the numbers and compositions of the paralogous segments in each CNV haplotype. Conclusions/Significance This is the first study of the genetic structure of CNVs on a large scale. Resulting information may help understand evolution of the human genome, gain insight into many genetic processes, and discriminate between CNVs and SNPs. The highly sensitive high-throughput experimental system with haploid sperm samples as subjects may be used to facilitate detailed large-scale CNV analysis. PMID:19384415

A feasibility study of colorectal cancer diagnosis via circulating tumor DNA derived CNV detection.

PubMed

Molparia, Bhuvan; Oliveira, Glenn; Wagner, Jennifer L; Spencer, Emily G; Torkamani, Ali

2018-01-01

Circulating tumor DNA (ctDNA) has shown great promise as a biomarker for early detection of cancer. However, due to the low abundance of ctDNA, especially at early stages, it is hard to detect at high accuracies while keeping sequencing costs low. Here we present a pilot stage study to detect large scale somatic copy numbers variations (CNVs), which contribute more molecules to ctDNA signal compared to point mutations, via cell free DNA sequencing. We show that it is possible to detect somatic CNVs in early stage colorectal cancer (CRC) patients and subsequently discriminate them from normal patients. With 25 normal and 24 CRC samples, we achieve 100% specificity (lower bound confidence interval: 86%) and ~79% sensitivity (95% confidence interval: 63% - 95%,), though the performance should be considered with caution given the limited sample size. We report a lack of concordance between the CNVs detected via cfDNA sequencing and CNVs identified in parent tissue samples. However, recent findings suggest that a lack of concordance is expected for CNVs in CRC because of their sub-clonal nature. Finally, the CNVs we detect very likely contribute to cancer progression as they lie in functionally important regions, and have been shown to be associated with CRC specifically. This study paves the path for a larger scale exploration of the potential of CNV detection for both diagnoses and prognoses of cancer.

Assessment of large copy number variants in patients with apparently isolated congenital left-sided cardiac lesions reveals clinically relevant genomic events.

PubMed

Hanchard, Neil A; Umana, Luis A; D'Alessandro, Lisa; Azamian, Mahshid; Poopola, Mojisola; Morris, Shaine A; Fernbach, Susan; Lalani, Seema R; Towbin, Jeffrey A; Zender, Gloria A; Fitzgerald-Butt, Sara; Garg, Vidu; Bowman, Jessica; Zapata, Gladys; Hernandez, Patricia; Arrington, Cammon B; Furthner, Dieter; Prakash, Siddharth K; Bowles, Neil E; McBride, Kim L; Belmont, John W

2017-08-01

Congenital left-sided cardiac lesions (LSLs) are a significant contributor to the mortality and morbidity of congenital heart disease (CHD). Structural copy number variants (CNVs) have been implicated in LSL without extra-cardiac features; however, non-penetrance and variable expressivity have created uncertainty over the use of CNV analyses in such patients. High-density SNP microarray genotyping data were used to infer large, likely-pathogenic, autosomal CNVs in a cohort of 1,139 probands with LSL and their families. CNVs were molecularly confirmed and the medical records of individual carriers reviewed. The gene content of novel CNVs was then compared with public CNV data from CHD patients. Large CNVs (>1 MB) were observed in 33 probands (∼3%). Six of these were de novo and 14 were not observed in the only available parent sample. Associated cardiac phenotypes spanned a broad spectrum without clear predilection. Candidate CNVs were largely non-recurrent, associated with heterozygous loss of copy number, and overlapped known CHD genomic regions. Novel CNV regions were enriched for cardiac development genes, including seven that have not been previously associated with human CHD. CNV analysis can be a clinically useful and molecularly informative tool in LSLs without obvious extra-cardiac defects, and may identify a clinically relevant genomic disorder in a small but important proportion of these individuals. © 2017 Wiley Periodicals, Inc.

A map of copy number variations in Chinese populations.

PubMed

Lou, Haiyi; Li, Shilin; Yang, Yajun; Kang, Longli; Zhang, Xin; Jin, Wenfei; Wu, Bailin; Jin, Li; Xu, Shuhua

2011-01-01

It has been shown that the human genome contains extensive copy number variations (CNVs). Investigating the medical and evolutionary impacts of CNVs requires the knowledge of locations, sizes and frequency distribution of them within and between populations. However, CNV study of Chinese minorities, which harbor the majority of genetic diversity of Chinese populations, has been underrepresented considering the same efforts in other populations. Here we constructed, to our knowledge, a first CNV map in seven Chinese populations representing the major linguistic groups in China with 1,440 CNV regions identified using Affymetrix SNP 6.0 Array. Considerable differences in distributions of CNV regions between populations and substantial population structures were observed. We showed that ∼35% of CNV regions identified in minority ethnic groups are not shared by Han Chinese population, indicating that the contribution of the minorities to genetic architecture of Chinese population could not be ignored. We further identified highly differentiated CNV regions between populations. For example, a common deletion in Dong and Zhuang (44.4% and 50%), which overlaps two keratin-associated protein genes contributing to the structure of hair fibers, was not observed in Han Chinese. Interestingly, the most differentiated CNV deletion between HapMap CEU and YRI containing CCL3L1 gene reported in previous studies was also the highest differentiated regions between Tibetan and other populations. Besides, by jointly analyzing CNVs and SNPs, we found a CNV region containing gene CTDSPL were in almost perfect linkage disequilibrium between flanking SNPs in Tibetan while not in other populations except HapMap CHD. Furthermore, we found the SNP taggability of CNVs in Chinese populations was much lower than that in European populations. Our results suggest the necessity of a full characterization of CNVs in Chinese populations, and the CNV map we constructed serves as a useful resource in further evolutionary and medical studies.

A Map of Copy Number Variations in Chinese Populations

PubMed Central

Yang, Yajun; Kang, Longli; Zhang, Xin; Jin, Wenfei; Wu, Bailin; Jin, Li; Xu, Shuhua

2011-01-01

It has been shown that the human genome contains extensive copy number variations (CNVs). Investigating the medical and evolutionary impacts of CNVs requires the knowledge of locations, sizes and frequency distribution of them within and between populations. However, CNV study of Chinese minorities, which harbor the majority of genetic diversity of Chinese populations, has been underrepresented considering the same efforts in other populations. Here we constructed, to our knowledge, a first CNV map in seven Chinese populations representing the major linguistic groups in China with 1,440 CNV regions identified using Affymetrix SNP 6.0 Array. Considerable differences in distributions of CNV regions between populations and substantial population structures were observed. We showed that ∼35% of CNV regions identified in minority ethnic groups are not shared by Han Chinese population, indicating that the contribution of the minorities to genetic architecture of Chinese population could not be ignored. We further identified highly differentiated CNV regions between populations. For example, a common deletion in Dong and Zhuang (44.4% and 50%), which overlaps two keratin-associated protein genes contributing to the structure of hair fibers, was not observed in Han Chinese. Interestingly, the most differentiated CNV deletion between HapMap CEU and YRI containing CCL3L1 gene reported in previous studies was also the highest differentiated regions between Tibetan and other populations. Besides, by jointly analyzing CNVs and SNPs, we found a CNV region containing gene CTDSPL were in almost perfect linkage disequilibrium between flanking SNPs in Tibetan while not in other populations except HapMap CHD. Furthermore, we found the SNP taggability of CNVs in Chinese populations was much lower than that in European populations. Our results suggest the necessity of a full characterization of CNVs in Chinese populations, and the CNV map we constructed serves as a useful resource in further evolutionary and medical studies. PMID:22087296

Logarithmic phase Escherichia coli K1 efficiently avoids serum killing by promoting C4bp-mediated C3b and C4b degradation

PubMed Central

Wooster, David G; Maruvada, Ravi; Blom, Anna M; Prasadarao, Nemani V

2006-01-01

Meningitis caused by Escherichia coli K1 is a serious illness in neonates with neurological sequelae in up to 50% of survivors. A high degree of bacteremia is required for E. coli K1 to cross the blood–brain barrier, which suggests that the bacterium must evade the host defence mechanisms and survive in the bloodstream. We previously showed that outer membrane protein A (OmpA) of E. coli binds C4b-binding protein (C4bp), an inhibitor of complement activation via the classical pathway. Nevertheless, the exact mechanism by which E. coli K1 survives in serum remains elusive. Here, we demonstrate that log phase (LP) OmpA+E. coli K1 avoids serum bactericidal activity more effectively than postexponential phase bacteria. OmpA–E. coli cannot survive in serum grown to either phase. The increased serum resistance of LP OmpA+E. coli is the result of increased binding of C4bp, with a concomitant decrease in the deposition of C3b and the downstream complement proteins responsible for the formation of the membrane attack complex. C4bp bound to E. coli K1 acts as a cofactor to factor I in the cleavage of both C3b and C4b, which shuts down the ensuing complement cascade. Accordingly, a peptide corresponding to the complement control protein domain 3 of C4bp sequence, was able to compete with C4bp binding to OmpA and cause increased deposition of C3b. Thus, binding of C4bp appears to be responsible for survival of E. coli K1 in human serum. PMID:16556262

Complement component C5a mediates hemorrhage-induced intestinal damage

PubMed Central

Fleming, Sherry D.; Phillips, Lauren M.; Lambris, John D.; Tsokos, George C.

2008-01-01

Background Complement has been implicated in the pathogenesis of intestinal damage and inflammation in multiple animal models. Although the exact mechanism is unknown, inhibition of complement prevents hemodynamic alterations in hemorrhage. Materials/Methods C57Bl/6, complement 5 deficient (C5−/−) and sufficient (C5+/+) mice were subjected to 25% blood loss. In some cases, C57Bl/6 mice were treated with C5a receptor antagonist (C5aRa) post-hemorrhage. Intestinal injury, leukotriene B4, and myeloperoxidase production were assessed for each treatment group of mice. Results Mice subjected to significant blood loss without major trauma develop intestinal inflammation and tissue damage within two hours. We report here that complement 5 (C5) deficient mice are protected from intestinal tissue damage when subjected to hemorrhage (Injury score = 0.36 compared to wildtype hemorrhaged animal injury score = 2.89; p<0.05). We present evidence that C5a represents the effector molecule because C57Bl/6 mice treated with a C5a receptor antagonist displayed limited intestinal injury (Injury score = 0.88), leukotriene B4 (13.16 pg/mg tissue) and myeloperoxidase (115.6 pg/mg tissue) production compared to hemorrhaged C57Bl/6 mice (p<0.05). Conclusion Complement activation is important in the development of hemorrhage-induced tissue injury and C5a generation is critical for tissue inflammation and damage. Thus, therapeutics targeting C5a may be useful therapeutics for hemorrhage-associated injury. PMID:18639891

Screening of copy number variants in the 22q11.2 region of congenital heart disease patients from the São Miguel Island, Azores, revealed the second patient with a triplication.

PubMed

Pires, Renato; Pires, Luís M; Vaz, Sara O; Maciel, Paula; Anjos, Rui; Moniz, Raquel; Branco, Claudia C; Cabral, Rita; Carreira, Isabel M; Mota-Vieira, Luisa

2014-11-07

The rearrangements in the 22q11.2 chromosomal region, responsible for the 22q11.2 deletion and microduplication syndromes, are frequently associated with congenital heart disease (CHD). The present work aimed to identify the genetic basis of CHD in 87 patients from the São Miguel Island, Azores, through the detection of copy number variants (CNVs) in the 22q11.2 region. These structural variants were searched using multiplex ligation-dependent probe amplification (MLPA). In patients with CNVs, we additionally performed fluorescent in situ hybridization (FISH) for the assessment of the exact number of 22q11.2 copies among each chromosome, and array comparative genomic hybridization (array-CGH) for the determination of the exact length of CNVs. We found that four patients (4.6%; A to D) carried CNVs. Patients A and D, both affected with a ventricular septal defect, carried a de novo 2.5 Mb deletion of the 22q11.2 region, which was probably originated by inter-chromosomal (inter-chromatid) non-allelic homologous recombination (NAHR) events in the regions containing low-copy repeats (LCRs). Patient C, with an atrial septal defect, carried a de novo 2.5 Mb duplication of 22q11.2 region, which could have been probably generated during gametogenesis by NAHR or by unequal crossing-over; additionally, this patient presented a benign 288 Kb duplication, which included the TOP3B gene inherited from her healthy mother. Finally, patient B showed a 3 Mb triplication associated with dysmorphic facial features, cognitive deficit and heart defects, a clinical feature not reported in the only case described so far in the literature. The evaluation of patient B's parents revealed a 2.5 Mb duplication in her father, suggesting a paternal inheritance with an extra copy. This report allowed the identification of rare deletion and microduplication syndromes in Azorean CHD patients. Moreover, we report the second patient with a 22q11.2 triplication, and we suggest that patients with triplications of chromosome 22q11.2, although they share some characteristic features with the deletion and microduplication syndromes, present a more severe phenotype probably due to the major dosage of implicated genes.

The lectin pathway in renal disease: old concept and new insights.

PubMed

Gaya da Costa, Mariana; Poppelaars, Felix; Berger, Stefan P; Daha, Mohamed R; Seelen, Marc A

2018-04-26

The complement system is composed of a network of at least 40 proteins, which significantly contributes to health and disease. The lectin pathway (LP) is one of three pathways that can activate the complement system. Next to protection of the host against pathogens, the LP has been shown to play a crucial role in multiple renal diseases as well as during renal replacement therapy. Therefore, several complement-targeted drugs are currently being explored in clinical trials. Among these complement inhibitors, specific LP inhibitors are also being tested in renal abnormalities such as in immunoglobulin A nephropathy and lupus nephritis. Using various in vitro models, Yaseen et al. (Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement component 3 (C3) in absence of C4 and/or C2. FASEB J 2017; 31: 2210-2219) showed that Mannan-associated serine protease2 can directly activate C3 thereby bypassing C2 and C4 in the activation of the LP. These new findings broaden our understanding of the mechanisms of complement activation and could potentially impact our strategies to inhibit the LP in renal diseases. In support of these findings, we present data of human renal biopsies, demonstrating the occurrence of the LP bypass mechanism in vivo. In conclusion, this review provides a detailed overview of the LP and clarifies the recently described bypass mechanism and its relevance. Finally, we speculate on the role of the C4 bypass mechanism in other renal diseases.

Regulation of complement C3 and C4 synthesis in human peritoneal mesothelial cells by peritoneal dialysis fluid

PubMed Central

TANG, S; LEUNG, J C K; CHAN, L Y Y; TSANG, A W L; CHEN, C X R; ZHOU, W; LAI, K N; SACKS, S H

2004-01-01

Although complement is activated in the peritoneal cavity during chronic peritoneal dialysis (PD), little is known about its role in peritoneal defence and injury related to long-term PD. We examined the impact of glucose and commercial peritoneal dialysis solutions on complement expression in HPMCs obtained by primary culture from omental tissues of consented patients undergoing elective abdominal surgery. Constitutive expression of C3 and C4 mRNA in HPMCs was up-regulated upon exposure to 75 mm glucose in a time-dependent manner. C3 and C4 protein was secreted in both apical and basolateral directions. Glucose doses beyond 100 mm markedly down-regulated C3 and C4 expression, and stimulated LDH release dose-dependently. Such cytotoxic effects were attenuated using equivalent doses of mannitol instead of glucose. Treatment with conventional lactate-buffered dialysis solution gave rise to down-regulation of C3 and C4 expression, and heightened LDH release in HPMCs. These effects correlated with the glucose strength of the solution, persisted despite replacement with a bicarbonate-buffered solution, aggravated by glycated albumin, and were partially abrogated by supplementation with 10% fetal bovine serum in the culture system. Our findings suggest that the artificial conditions imposed by PD lead to alterations in local complement synthesis that have implications for the role of the peritoneal mesothelium in both inflammation and defence. PMID:15030518

Evaluation of copy number variation detection for a SNP array platform

PubMed Central

2014-01-01

Background Copy Number Variations (CNVs) are usually inferred from Single Nucleotide Polymorphism (SNP) arrays by use of some software packages based on given algorithms. However, there is no clear understanding of the performance of these software packages; it is therefore difficult to select one or several software packages for CNV detection based on the SNP array platform. We selected four publicly available software packages designed for CNV calling from an Affymetrix SNP array, including Birdsuite, dChip, Genotyping Console (GTC) and PennCNV. The publicly available dataset generated by Array-based Comparative Genomic Hybridization (CGH), with a resolution of 24 million probes per sample, was considered to be the “gold standard”. Compared with the CGH-based dataset, the success rate, average stability rate, sensitivity, consistence and reproducibility of these four software packages were assessed compared with the “gold standard”. Specially, we also compared the efficiency of detecting CNVs simultaneously by two, three and all of the software packages with that by a single software package. Results Simply from the quantity of the detected CNVs, Birdsuite detected the most while GTC detected the least. We found that Birdsuite and dChip had obvious detecting bias. And GTC seemed to be inferior because of the least amount of CNVs it detected. Thereafter we investigated the detection consistency produced by one certain software package and the rest three software suits. We found that the consistency of dChip was the lowest while GTC was the highest. Compared with the CNVs detecting result of CGH, in the matching group, GTC called the most matching CNVs, PennCNV-Affy ranked second. In the non-overlapping group, GTC called the least CNVs. With regards to the reproducibility of CNV calling, larger CNVs were usually replicated better. PennCNV-Affy shows the best consistency while Birdsuite shows the poorest. Conclusion We found that PennCNV outperformed the other three packages in the sensitivity and specificity of CNV calling. Obviously, each calling method had its own limitations and advantages for different data analysis. Therefore, the optimized calling methods might be identified using multiple algorithms to evaluate the concordance and discordance of SNP array-based CNV calling. PMID:24555668

Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum.

PubMed

Castiblanco-Valencia, Mónica M; Fraga, Tatiana R; Breda, Leandro C D; Vasconcellos, Sílvio A; Figueira, Cláudio P; Picardeau, Mathieu; Wunder, Elsio; Ko, Albert I; Barbosa, Angela S; Isaac, Lourdes

2016-05-01

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum. Copyright © 2016 European Federation of Immunological Societies. All rights reserved.

Generation of Anaphylatoxins by Human β-Tryptase from C3, C4, and C51

PubMed Central

Fukuoka, Yoshihiro; Xia, Han-Zhang; Sanchez-Muñoz, Laura B.; Dellinger, Anthony L.; Escribano, Luis; Schwartz, Lawrence B.

2009-01-01

Both mast cells and complement participate in innate and acquired immunity. The current study examines whether β-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of β-tryptase, and the β-tryptase-activating polyanion. The B12 mAb was used to stabilize β-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric β-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by β-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by β-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both β-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by β-tryptase in releasates of activated skin mast cells. Of further biologic interest, β-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest β-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of β-tryptase and complement anaphylatoxins are detected. PMID:18424754

Genomic copy number variations in three Southeast Asian populations.

PubMed

Ku, Chee-Seng; Pawitan, Yudi; Sim, Xueling; Ong, Rick T H; Seielstad, Mark; Lee, Edmund J D; Teo, Yik-Ying; Chia, Kee-Seng; Salim, Agus

2010-07-01

Research on the role of copy number variations (CNVs) in the genetic risk of diseases in Asian populations has been hampered by a relative lack of reference CNV maps for Asian populations outside the East Asians. In this article, we report the population characteristics of CNVs in Chinese, Malay, and Asian Indian populations in Singapore. Using the Illumina Human 1M Beadchip array, we identify 1,174 CNV loci in these populations that corroborated with findings when the same samples were typed on the Affymetrix 6.0 platform. We identify 441 novel loci not previously reported in the Database of Genomic Variations (DGV). We observe a considerable number of loci that span all three populations and were previously unreported, as well as population-specific loci that are quite common in the respective populations. From this we observe the distribution of CNVs in the Asian Indian population to be considerably different from the Chinese and Malay populations. About half of the deletion loci and three-quarters of duplication loci overlap UCSC genes. Tens of loci show population differentiation and overlap with genes previously known to be associated with genetic risk of diseases. One of these loci is the CYP2A6 deletion, previously linked to reduced susceptibility to lung cancer. (c) 2010 Wiley-Liss, Inc.

CNV-TV: a robust method to discover copy number variation from short sequencing reads.

PubMed

Duan, Junbo; Zhang, Ji-Gang; Deng, Hong-Wen; Wang, Yu-Ping

2013-05-02

Copy number variation (CNV) is an important structural variation (SV) in human genome. Various studies have shown that CNVs are associated with complex diseases. Traditional CNV detection methods such as fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH) suffer from low resolution. The next generation sequencing (NGS) technique promises a higher resolution detection of CNVs and several methods were recently proposed for realizing such a promise. However, the performances of these methods are not robust under some conditions, e.g., some of them may fail to detect CNVs of short sizes. There has been a strong demand for reliable detection of CNVs from high resolution NGS data. A novel and robust method to detect CNV from short sequencing reads is proposed in this study. The detection of CNV is modeled as a change-point detection from the read depth (RD) signal derived from the NGS, which is fitted with a total variation (TV) penalized least squares model. The performance (e.g., sensitivity and specificity) of the proposed approach are evaluated by comparison with several recently published methods on both simulated and real data from the 1000 Genomes Project. The experimental results showed that both the true positive rate and false positive rate of the proposed detection method do not change significantly for CNVs with different copy numbers and lengthes, when compared with several existing methods. Therefore, our proposed approach results in a more reliable detection of CNVs than the existing methods.

Erythrocyte-bound C4d in combination with complement and autoantibody status for the monitoring of SLE.

PubMed

Merrill, Joan T; Petri, Michelle A; Buyon, Jill; Ramsey-Goldman, Rosalind; Kalunian, Kenneth; Putterman, Chaim; Conklin, John; Furie, Richard A; Dervieux, Thierry

2018-01-01

We examined the usefulness of erythrocyte-bound C4d (EC4d) to monitor disease activity in SLE. Data and blood samples were collected from three different studies, each of which included longitudinal evaluations using the Physicians Global Assessment (PGA) of disease activity and the Safety of Estrogens in Lupus Erythematosus National Assessment (SELENA) SLE Disease Activity Index (SLEDAI), which was assessed without anti-double-stranded DNA (dsDNA) and low complement C3/C4 (clinical SELENA-SLEDAI). EC4d levels were determined using flow cytometry; other laboratory measures included antibodies to dsDNA, C3 and C4 proteins. Relationships between clinical SELENA-SLEDAI, PGA and the laboratory measures were analysed using linear mixed effect models. The three studies combined enrolled 124 patients with SLE (mean age 42 years, 97% women, 31% Caucasians and 34% African-Americans) followed for an average of 5 consecutive visits (range 2-13 visits). EC4d levels and low C3/C4 status were significantly associated the clinical SELENA-SLEDAI or PGA in each of the three study groups (p<0.05). Multivariate analysis revealed that EC4d levels (estimate=0.94±0.28) and low complement C3/C4 (estimate=1.24±0.43) were both independently and significantly associated with the clinical SELENA-SLEDAI (p<0.01) and PGA. EC4d levels were also associated with the clinical SELENA-SLEDAI (estimate: 1.20±0.29) and PGA (estimate=0.19±0.04) among patients with chronically low or normal C3/C4 (p<0.01). Anti-dsDNA titres were generally associated with disease activity. These data support the association of EC4d with disease activity regardless of complement C3/C4 status and its usefulness in monitoring SLE disease. Additional studies will be required to support these validation data.

Impact of IQ on the diagnostic yield of chromosomal microarray in a community sample of adults with schizophrenia.

PubMed

Lowther, Chelsea; Merico, Daniele; Costain, Gregory; Waserman, Jack; Boyd, Kerry; Noor, Abdul; Speevak, Marsha; Stavropoulos, Dimitri J; Wei, John; Lionel, Anath C; Marshall, Christian R; Scherer, Stephen W; Bassett, Anne S

2017-11-30

Schizophrenia is a severe psychiatric disorder associated with IQ deficits. Rare copy number variations (CNVs) have been established to play an important role in the etiology of schizophrenia. Several of the large rare CNVs associated with schizophrenia have been shown to negatively affect IQ in population-based controls where no major neuropsychiatric disorder is reported. The aim of this study was to examine the diagnostic yield of microarray testing and the functional impact of genome-wide rare CNVs in a community ascertained cohort of adults with schizophrenia and low (< 85) or average (≥ 85) IQ. We recruited 546 adults of European ancestry with schizophrenia from six community psychiatric clinics in Canada. Each individual was assigned to the low or average IQ group based on standardized tests and/or educational attainment. We used rigorous methods to detect genome-wide rare CNVs from high-resolution microarray data. We compared the burden of rare CNVs classified as pathogenic or as a variant of unknown significance (VUS) between each of the IQ groups and the genome-wide burden and functional impact of rare CNVs after excluding individuals with a pathogenic CNV. There were 39/546 (7.1%; 95% confidence interval [CI] = 5.2-9.7%) schizophrenia participants with at least one pathogenic CNV detected, significantly more of whom were from the low IQ group (odds ratio [OR] = 5.01 [2.28-11.03], p = 0.0001). Secondary analyses revealed that individuals with schizophrenia and average IQ had the lowest yield of pathogenic CNVs (n = 9/325; 2.8%), followed by those with borderline intellectual functioning (n = 9/130; 6.9%), non-verbal learning disability (n = 6/29; 20.7%), and co-morbid intellectual disability (n = 15/62; 24.2%). There was no significant difference in the burden of rare CNVs classified as a VUS between any of the IQ subgroups. There was a significantly (p=0.002) increased burden of rare genic duplications in individuals with schizophrenia and low IQ that persisted after excluding individuals with a pathogenic CNV. Using high-resolution microarrays we were able to demonstrate for the first time that the burden of pathogenic CNVs in schizophrenia differs significantly between IQ subgroups. The results of this study have implications for clinical practice and may help inform future rare variant studies of schizophrenia using next-generation sequencing technologies.

Combined pituitary hormone deficiency due to gross deletions in the POU1F1 (PIT-1) and PROP1 genes.

PubMed

Bertko, Eleonore; Klammt, Jürgen; Dusatkova, Petra; Bahceci, Mithat; Gonc, Nazli; Ten Have, Louise; Kandemir, Nurgun; Mansmann, Georg; Obermannova, Barbora; Oostdijk, Wilma; Pfäffle, Heike; Rockstroh-Lippold, Denise; Schlicke, Marina; Tuzcu, Alpaslan Kemal; Pfäffle, Roland

2017-08-01

Pituitary development depends on a complex cascade of interacting transcription factors and signaling molecules. Lesions in this cascade lead to isolated or combined pituitary hormone deficiency (CPHD). The aim of this study was to identify copy number variants (CNVs) in genes known to cause CPHD and to determine their structure. We analyzed 70 CPHD patients from 64 families. Deletions were found in three Turkish families and one family from northern Iraq. In one family we identified a 4.96 kb deletion that comprises the first two exons of POU1F1. In three families a homozygous 15.9 kb deletion including complete PROP1 was discovered. Breakpoints map within highly homologous AluY sequences. Haplotype analysis revealed a shared haplotype of 350 kb among PROP1 deletion carriers. For the first time we were able to assign the boundaries of a previously reported PROP1 deletion. This gross deletion shows strong evidence to originate from a common ancestor in patients with Kurdish descent. No CNVs within LHX3, LHX4, HESX1, GH1 and GHRHR were found. Our data prove multiplex ligation-dependent probe amplification to be a valuable tool for the detection of CNVs as cause of pituitary insufficiencies and should be considered as an analytical method particularly in Kurdish patients.

Chromosomal Microarray Analysis of Consecutive Individuals with Autism Spectrum Disorders Using an Ultra-High Resolution Chromosomal Microarray Optimized for Neurodevelopmental Disorders.

PubMed

Ho, Karen S; Wassman, E Robert; Baxter, Adrianne L; Hensel, Charles H; Martin, Megan M; Prasad, Aparna; Twede, Hope; Vanzo, Rena J; Butler, Merlin G

2016-12-09

Copy number variants (CNVs) detected by chromosomal microarray analysis (CMA) significantly contribute to understanding the etiology of autism spectrum disorder (ASD) and other related conditions. In recognition of the value of CMA testing and its impact on medical management, CMA is in medical guidelines as a first-tier test in the evaluation of children with these disorders. As CMA becomes adopted into routine care for these patients, it becomes increasingly important to report these clinical findings. This study summarizes the results of over 4 years of CMA testing by a CLIA-certified clinical testing laboratory. Using a 2.8 million probe microarray optimized for the detection of CNVs associated with neurodevelopmental disorders, we report an overall CNV detection rate of 28.1% in 10,351 consecutive patients, which rises to nearly 33% in cases without ASD, with only developmental delay/intellectual disability (DD/ID) and/or multiple congenital anomalies (MCA). The overall detection rate for individuals with ASD is also significant at 24.4%. The detection rate and pathogenic yield of CMA vary significantly with the indications for testing, age, and gender, as well as the specialty of the ordering doctor. We note discrete differences in the most common recurrent CNVs found in individuals with or without a diagnosis of ASD.

Functional impact of global rare copy number variation in autism spectrum disorders.

PubMed

Pinto, Dalila; Pagnamenta, Alistair T; Klei, Lambertus; Anney, Richard; Merico, Daniele; Regan, Regina; Conroy, Judith; Magalhaes, Tiago R; Correia, Catarina; Abrahams, Brett S; Almeida, Joana; Bacchelli, Elena; Bader, Gary D; Bailey, Anthony J; Baird, Gillian; Battaglia, Agatino; Berney, Tom; Bolshakova, Nadia; Bölte, Sven; Bolton, Patrick F; Bourgeron, Thomas; Brennan, Sean; Brian, Jessica; Bryson, Susan E; Carson, Andrew R; Casallo, Guillermo; Casey, Jillian; Chung, Brian H Y; Cochrane, Lynne; Corsello, Christina; Crawford, Emily L; Crossett, Andrew; Cytrynbaum, Cheryl; Dawson, Geraldine; de Jonge, Maretha; Delorme, Richard; Drmic, Irene; Duketis, Eftichia; Duque, Frederico; Estes, Annette; Farrar, Penny; Fernandez, Bridget A; Folstein, Susan E; Fombonne, Eric; Freitag, Christine M; Gilbert, John; Gillberg, Christopher; Glessner, Joseph T; Goldberg, Jeremy; Green, Andrew; Green, Jonathan; Guter, Stephen J; Hakonarson, Hakon; Heron, Elizabeth A; Hill, Matthew; Holt, Richard; Howe, Jennifer L; Hughes, Gillian; Hus, Vanessa; Igliozzi, Roberta; Kim, Cecilia; Klauck, Sabine M; Kolevzon, Alexander; Korvatska, Olena; Kustanovich, Vlad; Lajonchere, Clara M; Lamb, Janine A; Laskawiec, Magdalena; Leboyer, Marion; Le Couteur, Ann; Leventhal, Bennett L; Lionel, Anath C; Liu, Xiao-Qing; Lord, Catherine; Lotspeich, Linda; Lund, Sabata C; Maestrini, Elena; Mahoney, William; Mantoulan, Carine; Marshall, Christian R; McConachie, Helen; McDougle, Christopher J; McGrath, Jane; McMahon, William M; Merikangas, Alison; Migita, Ohsuke; Minshew, Nancy J; Mirza, Ghazala K; Munson, Jeff; Nelson, Stanley F; Noakes, Carolyn; Noor, Abdul; Nygren, Gudrun; Oliveira, Guiomar; Papanikolaou, Katerina; Parr, Jeremy R; Parrini, Barbara; Paton, Tara; Pickles, Andrew; Pilorge, Marion; Piven, Joseph; Ponting, Chris P; Posey, David J; Poustka, Annemarie; Poustka, Fritz; Prasad, Aparna; Ragoussis, Jiannis; Renshaw, Katy; Rickaby, Jessica; Roberts, Wendy; Roeder, Kathryn; Roge, Bernadette; Rutter, Michael L; Bierut, Laura J; Rice, John P; Salt, Jeff; Sansom, Katherine; Sato, Daisuke; Segurado, Ricardo; Sequeira, Ana F; Senman, Lili; Shah, Naisha; Sheffield, Val C; Soorya, Latha; Sousa, Inês; Stein, Olaf; Sykes, Nuala; Stoppioni, Vera; Strawbridge, Christina; Tancredi, Raffaella; Tansey, Katherine; Thiruvahindrapduram, Bhooma; Thompson, Ann P; Thomson, Susanne; Tryfon, Ana; Tsiantis, John; Van Engeland, Herman; Vincent, John B; Volkmar, Fred; Wallace, Simon; Wang, Kai; Wang, Zhouzhi; Wassink, Thomas H; Webber, Caleb; Weksberg, Rosanna; Wing, Kirsty; Wittemeyer, Kerstin; Wood, Shawn; Wu, Jing; Yaspan, Brian L; Zurawiecki, Danielle; Zwaigenbaum, Lonnie; Buxbaum, Joseph D; Cantor, Rita M; Cook, Edwin H; Coon, Hilary; Cuccaro, Michael L; Devlin, Bernie; Ennis, Sean; Gallagher, Louise; Geschwind, Daniel H; Gill, Michael; Haines, Jonathan L; Hallmayer, Joachim; Miller, Judith; Monaco, Anthony P; Nurnberger, John I; Paterson, Andrew D; Pericak-Vance, Margaret A; Schellenberg, Gerard D; Szatmari, Peter; Vicente, Astrid M; Vieland, Veronica J; Wijsman, Ellen M; Scherer, Stephen W; Sutcliffe, James S; Betancur, Catalina

2010-07-15

The autism spectrum disorders (ASDs) are a group of conditions characterized by impairments in reciprocal social interaction and communication, and the presence of restricted and repetitive behaviours. Individuals with an ASD vary greatly in cognitive development, which can range from above average to intellectual disability. Although ASDs are known to be highly heritable ( approximately 90%), the underlying genetic determinants are still largely unknown. Here we analysed the genome-wide characteristics of rare (<1% frequency) copy number variation in ASD using dense genotyping arrays. When comparing 996 ASD individuals of European ancestry to 1,287 matched controls, cases were found to carry a higher global burden of rare, genic copy number variants (CNVs) (1.19 fold, P = 0.012), especially so for loci previously implicated in either ASD and/or intellectual disability (1.69 fold, P = 3.4 x 10(-4)). Among the CNVs there were numerous de novo and inherited events, sometimes in combination in a given family, implicating many novel ASD genes such as SHANK2, SYNGAP1, DLGAP2 and the X-linked DDX53-PTCHD1 locus. We also discovered an enrichment of CNVs disrupting functional gene sets involved in cellular proliferation, projection and motility, and GTPase/Ras signalling. Our results reveal many new genetic and functional targets in ASD that may lead to final connected pathways.

Combined pituitary hormone deficiency due to gross deletions in the POU1F1 (PIT-1) and PROP1 genes

PubMed Central

Bertko, Eleonore; Klammt, Jürgen; Dusatkova, Petra; Bahceci, Mithat; Gonc, Nazli; ten Have, Louise; Kandemir, Nurgun; Mansmann, Georg; Obermannova, Barbora; Oostdijk, Wilma; Pfäffle, Heike; Rockstroh-Lippold, Denise; Schlicke, Marina; Tuzcu, Alpaslan Kemal; Pfäffle, Roland

2017-01-01

Pituitary development depends on a complex cascade of interacting transcription factors and signaling molecules. Lesions in this cascade lead to isolated or combined pituitary hormone deficiency (CPHD). The aim of this study was to identify copy number variants (CNVs) in genes known to cause CPHD and to determine their structure. We analyzed 70 CPHD patients from 64 families. Deletions were found in three Turkish families and one family from northern Iraq. In one family we identified a 4.96 kb deletion that comprises the first two exons of POU1F1. In three families a homozygous 15.9 kb deletion including complete PROP1 was discovered. Breakpoints map within highly homologous AluY sequences. Haplotype analysis revealed a shared haplotype of 350 kb among PROP1 deletion carriers. For the first time we were able to assign the boundaries of a previously reported PROP1 deletion. This gross deletion shows strong evidence to originate from a common ancestor in patients with Kurdish descent. No CNVs within LHX3, LHX4, HESX1, GH1 and GHRHR were found. Our data prove multiplex ligation-dependent probe amplification to be a valuable tool for the detection of CNVs as cause of pituitary insufficiencies and should be considered as an analytical method particularly in Kurdish patients. PMID:28356564

Domain structure of human complement C4b extends with increasing NaCl concentration: implications for its regulatory mechanism.

PubMed

Fung, Ka Wai; Wright, David W; Gor, Jayesh; Swann, Marcus J; Perkins, Stephen J

2016-12-01

During the activation of complement C4 to C4b, the exposure of its thioester domain (TED) is crucial for the attachment of C4b to activator surfaces. In the C4b crystal structure, TED forms an Arg 104 -Glu 1032 salt bridge to tether its neighbouring macroglobulin (MG1) domain. Here, we examined the C4b domain structure to test whether this salt bridge affects its conformation. Dual polarisation interferometry of C4b immobilised at a sensor surface showed that the maximum thickness of C4b increased by 0.46 nm with an increase in NaCl concentration from 50 to 175 mM NaCl. Analytical ultracentrifugation showed that the sedimentation coefficient s 20,w of monomeric C4b of 8.41 S in 50 mM NaCl buffer decreased to 7.98 S in 137 mM NaCl buffer, indicating that C4b became more extended. Small angle X-ray scattering reported similar R G values of 4.89-4.90 nm for C4b in 137-250 mM NaCl. Atomistic scattering modelling of the C4b conformation showed that TED and the MG1 domain were separated by 4.7 nm in 137-250 mM NaCl and this is greater than that of 4.0 nm in the C4b crystal structure. Our data reveal that in low NaCl concentrations, both at surfaces and in solution, C4b forms compact TED-MG1 structures. In solution, physiologically relevant NaCl concentrations lead to the separation of the TED and MG1 domain, making C4b less capable of binding to its complement regulators. These conformational changes are similar to those seen previously for complement C3b, confirming the importance of this salt bridge for regulating both C4b and C3b. © 2016 The Author(s).

Multicentre physiological reference intervals for serum concentrations of immunoglobulins A, G and M, complement C3c and C4 measured with Tina-Quant reagents systems.

PubMed

Fuentes-Arderiu, Xavier; Alonso-Gregorio, Eduardo; Alvarez-Funes, Virtudes; Ambrós-Marigómez, Carmen; Coca-Fábregas, Lluís; Cruz-Placer, Marta; Díaz-Fernández, Julián; Pinel-Julián, María Pilar; Gutiérrez-Cecchini, Beatriz; Herrero-Bernal, Pilar; Sempere-Alcocer, Marcos; García-Caballero, Francisca; Del Mar Larrea-Ortiz-Quintana, María; La-Torre-Marcellán, Pedro; Del Señor López-Vélez, María; Mar-Medina, Carmen; Martín-Oncina, Javier; Rodríguez-Hernández, María Victoria; Romero-Sotomayor, María Victoria; Serrano-López, Cándido; Sicilia-Enríquez-de-Salamanca, Adolfo; Velasco-Romero, Ana María; Juvé-Cuxart, Santiago

2007-01-01

Clinical laboratories seeking accreditation for compliance with ISO 15189:2003 need to demonstrate that the physiological reference intervals communicated to all users of the laboratory service are appropriate for the patient population served and for the measurement systems used. In the case of immunological quantities, few articles have been published in peer-reviewed journals. A total of 21 clinical laboratories in different regions of Spain collaborated in identifying reference individuals and determining adult reference intervals for some immunological quantities measured using RD/Hitachi Modular Analytics analysers and Tina-Quant reagent systems. These immunological quantities are the mass concentrations of immunoglobulin A, immunoglobulin G, immunoglobulin M, complement C3c and complement C4 in serum. All the logistic work was carried out in co-operation with the supplier of the reagents and analysers (Roche Diagnostics España, S.L., Sant Cugat del Vallès, Catalonia, Spain). From the set of reference values obtained by each laboratory, multicentre reference limits were estimated non-parametrically. The reference intervals estimated in this study for concentrations of serum components under consideration are: complement C3c, 0.62-1.64 g/L for women and men; complement C4, 0.14-0.72 g/L for women and men; immunoglobulin A, 0.89-4.80 g/L for women and men; immunoglobulin G, 6.5-14.3 g/L for women and men; and immunoglobulin M, 0.48-3.38 g/L for women and 0.41-2.46 g/L for men.

Copy number variants analysis in a cohort of isolated and syndromic developmental delay/intellectual disability reveals novel genomic disorders, position effects and candidate disease genes.

PubMed

Di Gregorio, E; Riberi, E; Belligni, E F; Biamino, E; Spielmann, M; Ala, U; Calcia, A; Bagnasco, I; Carli, D; Gai, G; Giordano, M; Guala, A; Keller, R; Mandrile, G; Arduino, C; Maffè, A; Naretto, V G; Sirchia, F; Sorasio, L; Ungari, S; Zonta, A; Zacchetti, G; Talarico, F; Pappi, P; Cavalieri, S; Giorgio, E; Mancini, C; Ferrero, M; Brussino, A; Savin, E; Gandione, M; Pelle, A; Giachino, D F; De Marchi, M; Restagno, G; Provero, P; Cirillo Silengo, M; Grosso, E; Buxbaum, J D; Pasini, B; De Rubeis, S; Brusco, A; Ferrero, G B

2017-10-01

Array-comparative genomic hybridization (array-CGH) is a widely used technique to detect copy number variants (CNVs) associated with developmental delay/intellectual disability (DD/ID). Identification of genomic disorders in DD/ID. We performed a comprehensive array-CGH investigation of 1,015 consecutive cases with DD/ID and combined literature mining, genetic evidence, evolutionary constraint scores, and functional information in order to assess the pathogenicity of the CNVs. We identified non-benign CNVs in 29% of patients. Amongst the pathogenic variants (11%), detected with a yield consistent with the literature, we found rare genomic disorders and CNVs spanning known disease genes. We further identified and discussed 51 cases with likely pathogenic CNVs spanning novel candidate genes, including genes encoding synaptic components and/or proteins involved in corticogenesis. Additionally, we identified two deletions spanning potential Topological Associated Domain (TAD) boundaries probably affecting the regulatory landscape. We show how phenotypic and genetic analyses of array-CGH data allow unraveling complex cases, identifying rare disease genes, and revealing unexpected position effects. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis

PubMed Central

Ferreira, Viviana P.; Fazito Vale, Vladimir; Pangburn, Michael K.; Abdeladhim, Maha; Ferreira Mendes-Sousa, Antonio; Coutinho-Abreu, Iliano V.; Rasouli, Manoochehr; Brandt, Elizabeth A.; Meneses, Claudio; Lima, Kolyvan Ferreira; Nascimento Araújo, Ricardo; Horácio Pereira, Marcos; Kotsyfakis, Michalis; Oliveira, Fabiano; Kamhawi, Shaden; Ribeiro, Jose M. C.; Gontijo, Nelder F.; Collin, Nicolas; Valenzuela, Jesus G.

2016-01-01

Blood-feeding insects inject potent salivary components including complement inhibitors into their host’s skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases. PMID:26758086

Inactivation of complement by Loxosceles reclusa spider venom.

PubMed

Gebel, H M; Finke, J H; Elgert, K D; Cambell, B J; Barrett, J T

1979-07-01

Zymosan depletion of serum complement in guinea pigs rendered them highly resistant to lesion by Loxosceles reclusa spider venom. Guinea pigs deficient in C4 of the complement system are as sensitive to the venom as normal guinea pigs. The injection of 35 micrograms of whole recluse venom intradermally into guinea pigs lowered their complement level by 35.7%. Brown recluse spider venom in concentrations as slight as 0.02 micrograms protein/ml can totally inactivate one CH50 of guinea pig complement in vitro. Bee, scorpion, and other spider venoms had no influence on the hemolytic titer of complement. Fractionation of recluse spider venom by Sephadex G-200 filtration separated the complement-inactivating property of the venom into three major regions which could be distinguished on the basis of heat stability as well as size. None was neutralized by antivenom. Polyacrylamide gel electrophoresis of venom resolved the complement inactivators into five fractions. Complement inactivated by whole venom or the Sephadex fractions could be restored to hemolytic activity by supplements of fresh serum but not by heat-inactivated serum, pure C3, pure C5, or C3 and C5 in combination.

Antibody-Mediated Rejection in Human Cardiac Allografts: Evaluation of Immunoglobulins and Complement Activation Products C4d and C3d as Markers

PubMed Central

Rodriguez, E. R.; Skojec, Diane V.; Tan, Carmela D.; Zachary, Andrea A.; Kasper, Edward K.; Conte, John V.; Baldwin, William M.

2005-01-01

Antibody-mediated rejection (AMR) in human heart transplantation is an immunopathologic process in which injury to the graft is in part the result of activation of complement and it is poorly responsive to conventional therapy. We evaluated by immunofluorescence (IF), 665 consecutive endomyocardial biopsies from 165 patients for deposits of immunoglobulins and complement. Diffuse IF deposits in a linear capillary pattern greater than 2+ were considered significant. Clinical evidence of graft dysfunction was correlated with complement deposits. IF 2+ or higher was positive for IgG, 66%; IgM, 12%; IgA, 0.6%; C1q, 1.8%; C4d, 9% and C3d, 10%. In 3% of patients, concomitant C4d and C3d correlated with graft dysfunction or heart failure. In these 5 patients AMR occurred 56–163 months after transplantation, and they responded well to therapy for AMR but not to treatment with steroids. Systematic evaluation of endomyocardial biopsies is not improved by the use of antibodies for immunoglobulins or C1q. Concomitant use of C4d and C3d is very useful to diagnose AMR, when correlated with clinical parameters of graft function. AMR in heart transplant patients can occur many months or years after transplant. PMID:16212640

Whole exome sequencing in recurrent early pregnancy loss.

PubMed

Qiao, Ying; Wen, Jiadi; Tang, Flamingo; Martell, Sally; Shomer, Naomi; Leung, Peter C K; Stephenson, Mary D; Rajcan-Separovic, Evica

2016-05-01

Exome sequencing can identify genetic causes of idiopathic recurrent pregnancy loss (RPL). We identified compound heterozygous deleterious mutations affecting DYNC2H1 and ALOX15 in two out of four families with RPL. Both genes have a role in early development. Bioinformatics analysis of all genes with rare and putatively pathogenic mutations in miscarriages and couples showed enrichment in pathways relevant to pregnancy loss, including the complement and coagulation cascades pathways. Next generation sequencing (NGS) is increasingly being used to identify known and novel gene mutations in children with developmental delay and in fetuses with ultrasound-detected anomalies. In contrast, NGS is rarely used to study pregnancy loss. Chromosome microarray analysis detects putatively causative DNA copy number variants (CNVs) in ∼2% of miscarriages and CNVs of unknown significance (predominantly parental in origin) in up to 40% of miscarriages. Therefore, a large number of miscarriages still have an unknown cause. Whole exome sequencing (WES) was performed using Illumina HiSeq 2000 platform on seven euploid miscarriages from four families with RPL. Golden Helix SVS v8.1.5 was used for data assessment and inheritance analysis for deleterious DNA variants predicted to severely disrupt protein-coding genes by introducing a frameshift, loss of the stop codon, gain of the stop codon, changes in splicing or the initial codon. Webgestalt (http://bioinfo.vanderbilt.edu/webgestalt/) was used for pathway and disease association enrichment analysis of a gene pool containing putatively pathogenic variants in miscarriages and couples in comparison to control gene pools. Compound heterozygous mutations in DYNC2H1 and ALOX15 were identified in miscarriages from two families with RPL. DYNC2H1 is involved in cilia biogenesis and has been associated with fetal lethality in humans. ALOX15 is expressed in placenta and its dysregulation has been associated with inflammation, placental, dysfunction, abnormal oxidative stress response and angiogenesis. The pool of putatively pathogenic single nucleotide variants (SNVs) and small insertions and deletions (indels) detected in the miscarriages showed enrichment in 'complement and coagulation cascades pathway', and 'ciliary motility disorders'. We conclude that CNVs, individual SNVs and pool of deleterious gene mutations identified by exome sequencing could contribute to RPL. The size of our sample cohort is small. The functional effect of candidate mutations should be evaluated to determine whether the mutations are causative. This is the first study to assess whether SNVs may contribute to the pathogenesis of miscarriage. Furthermore, our findings suggest that collective effect of mutations in relevant biological pathways could be implicated in RPL. The study was funded by Canadian Institutes of Health Research (grant MOP 106467) and Michael Smith Foundation of Health Research Career Scholar salary award to ERS. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Whole exome sequencing in recurrent early pregnancy loss

PubMed Central

Qiao, Ying; Wen, Jiadi; Tang, Flamingo; Martell, Sally; Shomer, Naomi; Leung, Peter C.K.; Stephenson, Mary D.; Rajcan-Separovic, Evica

2016-01-01

STUDY HYPOTHESIS Exome sequencing can identify genetic causes of idiopathic recurrent pregnancy loss (RPL). STUDY FINDING We identified compound heterozygous deleterious mutations affecting DYNC2H1 and ALOX15 in two out of four families with RPL. Both genes have a role in early development. Bioinformatics analysis of all genes with rare and putatively pathogenic mutations in miscarriages and couples showed enrichment in pathways relevant to pregnancy loss, including the complement and coagulation cascades pathways. WHAT IS KNOWN ALREADY Next generation sequencing (NGS) is increasingly being used to identify known and novel gene mutations in children with developmental delay and in fetuses with ultrasound-detected anomalies. In contrast, NGS is rarely used to study pregnancy loss. Chromosome microarray analysis detects putatively causative DNA copy number variants (CNVs) in ∼2% of miscarriages and CNVs of unknown significance (predominantly parental in origin) in up to 40% of miscarriages. Therefore, a large number of miscarriages still have an unknown cause. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Whole exome sequencing (WES) was performed using Illumina HiSeq 2000 platform on seven euploid miscarriages from four families with RPL. Golden Helix SVS v8.1.5 was used for data assessment and inheritance analysis for deleterious DNA variants predicted to severely disrupt protein-coding genes by introducing a frameshift, loss of the stop codon, gain of the stop codon, changes in splicing or the initial codon. Webgestalt (http://bioinfo.vanderbilt.edu/webgestalt/) was used for pathway and disease association enrichment analysis of a gene pool containing putatively pathogenic variants in miscarriages and couples in comparison to control gene pools. MAIN RESULTS AND THE ROLE OF CHANCE Compound heterozygous mutations in DYNC2H1 and ALOX15 were identified in miscarriages from two families with RPL. DYNC2H1 is involved in cilia biogenesis and has been associated with fetal lethality in humans. ALOX15 is expressed in placenta and its dysregulation has been associated with inflammation, placental, dysfunction, abnormal oxidative stress response and angiogenesis. The pool of putatively pathogenic single nucleotide variants (SNVs) and small insertions and deletions (indels) detected in the miscarriages showed enrichment in ‘complement and coagulation cascades pathway’, and ‘ciliary motility disorders’. We conclude that CNVs, individual SNVs and pool of deleterious gene mutations identified by exome sequencing could contribute to RPL. LIMITATIONS, REASONS FOR CAUTION The size of our sample cohort is small. The functional effect of candidate mutations should be evaluated to determine whether the mutations are causative. WIDER IMPLICATIONS OF THE FINDINGS This is the first study to assess whether SNVs may contribute to the pathogenesis of miscarriage. Furthermore, our findings suggest that collective effect of mutations in relevant biological pathways could be implicated in RPL. STUDY FUNDING AND COMPETING INTEREST(S) The study was funded by Canadian Institutes of Health Research (grant MOP 106467) and Michael Smith Foundation of Health Research Career Scholar salary award to ERS. PMID:26826164

Genome-wide analysis of copy number variants in attention deficit hyperactivity disorder: the role of rare variants and duplications at 15q13.3.

PubMed

Williams, Nigel M; Franke, Barbara; Mick, Eric; Anney, Richard J L; Freitag, Christine M; Gill, Michael; Thapar, Anita; O'Donovan, Michael C; Owen, Michael J; Holmans, Peter; Kent, Lindsey; Middleton, Frank; Zhang-James, Yanli; Liu, Lu; Meyer, Jobst; Nguyen, Thuy Trang; Romanos, Jasmin; Romanos, Marcel; Seitz, Christiane; Renner, Tobias J; Walitza, Susanne; Warnke, Andreas; Palmason, Haukur; Buitelaar, Jan; Rommelse, Nanda; Vasquez, Alejandro Arias; Hawi, Ziarih; Langley, Kate; Sergeant, Joseph; Steinhausen, Hans-Christoph; Roeyers, Herbert; Biederman, Joseph; Zaharieva, Irina; Hakonarson, Hakon; Elia, Josephine; Lionel, Anath C; Crosbie, Jennifer; Marshall, Christian R; Schachar, Russell; Scherer, Stephen W; Todorov, Alexandre; Smalley, Susan L; Loo, Sandra; Nelson, Stanley; Shtir, Corina; Asherson, Philip; Reif, Andreas; Lesch, Klaus-Peter; Faraone, Stephen V

2012-02-01

Attention deficit hyperactivity disorder (ADHD) is a common, highly heritable psychiatric disorder. Because of its multifactorial etiology, however, identifying the genes involved has been difficult. The authors followed up on recent findings suggesting that rare copy number variants (CNVs) may be important for ADHD etiology. The authors performed a genome-wide analysis of large, rare CNVs (<1% population frequency) in children with ADHD (N=896) and comparison subjects (N=2,455) from the IMAGE II Consortium. The authors observed 1,562 individually rare CNVs >100 kb in size, which segregated into 912 independent loci. Overall, the rate of rare CNVs >100 kb was 1.15 times higher in ADHD case subjects relative to comparison subjects, with duplications spanning known genes showing a 1.2-fold enrichment. In accordance with a previous study, rare CNVs >500 kb showed the greatest enrichment (1.28-fold). CNVs identified in ADHD case subjects were significantly enriched for loci implicated in autism and in schizophrenia. Duplications spanning the CHRNA7 gene at chromosome 15q13.3 were associated with ADHD in single-locus analysis. This finding was consistently replicated in an additional 2,242 ADHD case subjects and 8,552 comparison subjects from four independent cohorts from the United Kingdom, the United States, and Canada. Presence of the duplication at 15q13.3 appeared to be associated with comorbid conduct disorder. These findings support the enrichment of large, rare CNVs in ADHD and implicate duplications at 15q13.3 as a novel risk factor for ADHD. With a frequency of 0.6% in the populations investigated and a relatively large effect size (odds ratio=2.22, 95% confidence interval=1.5–3.6), this locus could be an important contributor to ADHD etiology.

Shape-based retrieval of CNV regions in read coverage data.

PubMed

Hong, Sangkyun; Yoon, Jeehee; Hong, Dongwan; Lee, Unjoo; Kim, Baeksop; Park, Sanghyun

2014-01-01

This study proposes a novel copy number variation (CNV) detection method, CNV_shape, based on variations in the shape of the read coverage data which are obtained from millions of short reads aligned to a reference sequence. The proposed method carries out two transforms, mean shift transform and mean slope transform, to extract the shape of a CNV more precisely from real human data, which are vulnerable to experimental and biological noises. The mean shift transform is a procedure for gaining a preliminary estimation of the CNVs by statistically evaluating moving averages of given read coverage data. The mean slope transform extracts candidate CNVs by filtering out non-stationary sub-regions from each of the primary CNVs pre-estimated in the mean shift procedure. Each of the candidate CNVs is merged with neighbours depending on the merging score to be finally identified as a putative CNV, where the merging score is estimated by the ratio of the positions with non-zero values of the mean shift transform to the total length of the region including two neighbouring candidate CNVs and the interval between them. The proposed CNV detection method was validated experimentally with simulated data and real human data. The simulated data with coverage in the range of 1x to 10x were generated for various sampling sizes and p-values. Five individual human genomes were used as real human data. The results show that relatively small CNVs (> 1 kbp) can be detected from low coverage (> 1.7x) data. The results also reveal that, in contrast to conventional methods, performance improvement from 8.18 to 87.90% was achieved in CNV_shape. The outcomes suggest that the proposed method is very effective in reducing noises inherent in real data as well as in detecting CNVs of various sizes and types.

Genome-Wide Analysis of Copy Number Variants in Attention Deficit Hyperactivity Disorder: The Role of Rare Variants and Duplications at 15q13.3

PubMed Central

Franke, Barbara; Mick, Eric; Anney, Richard J.L.; Freitag, Christine M.; Gill, Michael; Thapar, Anita; O'Donovan, Michael C.; Owen, Michael J.; Holmans, Peter; Kent, Lindsey; Middleton, Frank; Zhang-James, Yanli; Liu, Lu; Meyer, Jobst; Nguyen, Thuy Trang; Romanos, Jasmin; Romanos, Marcel; Seitz, Christiane; Renner, Tobias J.; Walitza, Susanne; Warnke, Andreas; Palmason, Haukur; Buitelaar, Jan; Rommelse, Nanda; Vasquez, Alejandro Arias; Hawi, Ziarih; Langley, Kate; Sergeant, Joseph; Steinhausen, Hans-Christoph; Roeyers, Herbert; Biederman, Joseph; Zaharieva, Irina; Hakonarson, Hakon; Elia, Josephine; Lionel, Anath C.; Crosbie, Jennifer; Marshall, Christian R.; Schachar, Russell; Scherer, Stephen W.; Todorov, Alexandre; Smalley, Susan L.; Loo, Sandra; Nelson, Stanley; Shtir, Corina; Asherson, Philip; Reif, Andreas; Lesch, Klaus-Peter

2012-01-01

Objective: Attention deficit hyperactivity disorder (ADHD) is a common, highly heritable psychiatric disorder. Because of its multifactorial etiology, however, identifying the genes involved has been difficult. The authors followed up on recent findings suggesting that rare copy number variants (CNVs) may be important for ADHD etiology. Method: The authors performed a genome-wide analysis of large, rare CNVs (<1% population frequency) in children with ADHD (N=896) and comparison subjects (N=2,455) from the IMAGE II Consortium. Results: The authors observed 1,562 individually rare CNVs >100 kb in size, which segregated into 912 independent loci. Overall, the rate of rare CNVs >100 kb was 1.15 times higher in ADHD case subjects relative to comparison subjects, with duplications spanning known genes showing a 1.2-fold enrichment. In accordance with a previous study, rare CNVs >500 kb showed the greatest enrichment (1.28-fold). CNVs identified in ADHD case subjects were significantly enriched for loci implicated in autism and in schizophrenia. Duplications spanning the CHRNA7 gene at chromosome 15q13.3 were associated with ADHD in single-locus analysis. This finding was consistently replicated in an additional 2,242 ADHD case subjects and 8,552 comparison subjects from four independent cohorts from the United Kingdom, the United States, and Canada. Presence of the duplication at 15q13.3 appeared to be associated with comorbid conduct disorder. Conclusions: These findings support the enrichment of large, rare CNVs in ADHD and implicate duplications at 15q13.3 as a novel risk factor for ADHD. With a frequency of 0.6% in the populations investigated and a relatively large effect size (odds ratio=2.22, 95% confidence interval=1.5–3.6), this locus could be an important contributor to ADHD etiology. PMID:22420048

Early Complementopathy after Multiple Injuries in Humans

PubMed Central

Burk, Anne-Maud; Martin, Myriam; Flierl, Michael A.; Rittirsch, Daniel; Helm, Matthias; Lampl, Lorenz; Bruckner, Uwe; Stahl, Gregory L.; Blom, Anna M.; Perl, Mario; Gebhard, Florian; Huber-Lang, Markus

2012-01-01

After severe tissue injury, innate immunity mounts a robust systemic inflammatory response. However, little is known about the immediate impact of multiple trauma on early complement function in humans. In the present study we hypothesized that multiple trauma results in immediate activation, consumption and dysfunction of the complement cascade and that the resulting severe “complementopathy” may be associated with morbidity and mortality. Therefore a prospective multicenter study with 25 healthy volunteers and 40 polytrauma patients (mean injury severity score [ISS] = 30.3 ± 2.9) was performed. After polytrauma serum was collected as early as possible at the scene, upon admission to the emergency room and 4, 12, 24, 120 and 240 hours post trauma and analysed for the complement profile. Complement hemolytic activity (CH-50) was massively reduced within the first 24 h after injury, recovered only 5 days after trauma and discriminated between lethal and non-lethal 28-day outcome. Serum levels of the complement activation products C3a and C5a were significantly elevated throughout the entire observation period and correlated with the severity of traumatic brain injury and survival. The soluble terminal complement complex SC5b-9 and mannose-binding lectin (MBL) showed a biphasic response after trauma. Key fluid phase inhibitors of complement, such as C4b-binding protein (C4BP) and factor I, were significantly diminished early after trauma. The present data indicate an almost synchronically rapid activation and dysfunction of complement suggesting a trauma-induced “complementopathy” early after injury. These events may participate to the impairment of the innate immune response observed after severe trauma. PMID:22258234

Copy number variation is a fundamental aspect of the placental genome.

PubMed

Hannibal, Roberta L; Chuong, Edward B; Rivera-Mulia, Juan Carlos; Gilbert, David M; Valouev, Anton; Baker, Julie C

2014-05-01

Discovery of lineage-specific somatic copy number variation (CNV) in mammals has led to debate over whether CNVs are mutations that propagate disease or whether they are a normal, and even essential, aspect of cell biology. We show that 1,000 N polyploid trophoblast giant cells (TGCs) of the mouse placenta contain 47 regions, totaling 138 Megabases, where genomic copies are underrepresented (UR). UR domains originate from a subset of late-replicating heterochromatic regions containing gene deserts and genes involved in cell adhesion and neurogenesis. While lineage-specific CNVs have been identified in mammalian cells, classically in the immune system where V(D)J recombination occurs, we demonstrate that CNVs form during gestation in the placenta by an underreplication mechanism, not by recombination nor deletion. Our results reveal that large scale CNVs are a normal feature of the mammalian placental genome, which are regulated systematically during embryogenesis and are propagated by a mechanism of underreplication.

Design, synthesis, and evaluation of A/C/D-ring analogs of the fungal metabolite K-76 as potential complement inhibitors.

PubMed

Kaufman, T S; Srivastava, R P; Sindelar, R D; Scesney, S M; Marsh, H C

1995-04-28

The terpenoid 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydrox y-2',5',5', 8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran] (1a; K-76), a natural product of fungal origin, and its monocarboxylate sodium salt 1c (R = COONa; K-76COONa) inhibit the classical and alternative pathways of complement, and 1c was shown to inhibit the classical pathway at the C5 activation step. In an attempt to elucidate the essential pharmacophore of 1a,c, the natural product was used as a "topographical model" for the design of partial analogs retaining the desired complement inhibiting potency. Therefore, A/C/D-ring analogs have been synthesized, as shown in Scheme 1 using 3-methoxyphenol (3) and limonene chloride (5) as starting materials, which contain functional groups similar to those found on the natural product. The use of (4R)-(+)- and (4S)(-)-limonene chloride (5a,b, respectively) provided two series of compounds differing in the stereochemistry of the C-4 chiral center (limonene moiety numbering). The in vitro assay results of the inhibition of anaphylatoxin production and classical complement-mediated hemolysis revealed that 7-carboxy-2-(R,S)-methyl-2-(1'-methylcyclohexen-(4'R)-yl)-4-met hoxybenzofuran (13a) and 7-carboxy-2-(R,S)-methyl-2-(1'-methylcyclohexen-(4'S)-yl)-4-met hoxybenzofuran (13b) were active in the same range of concentrations as the natural product.

Large-scale deletions of the ABCA1 gene in patients with hypoalphalipoproteinemia.

PubMed

Dron, Jacqueline S; Wang, Jian; Berberich, Amanda J; Iacocca, Michael A; Cao, Henian; Yang, Ping; Knoll, Joan; Tremblay, Karine; Brisson, Diane; Netzer, Christian; Gouni-Berthold, Ioanna; Gaudet, Daniel; Hegele, Robert A

2018-06-04

Copy-number variations (CNVs) have been studied in the context of familial hypercholesterolemia but have not yet been evaluated in patients with extremes of high-density lipoprotein (HDL) cholesterol levels. We evaluated targeted next-generation sequencing data from patients with very low HDL cholesterol (i.e. hypoalphalipoproteinemia) using the VarSeq-CNV caller algorithm to screen for CNVs disrupting the ABCA1, LCAT or APOA1 genes. In four individuals, we found three unique deletions in ABCA1: a heterozygous deletion of exon 4, a heterozygous deletion spanning exons 8 to 31, and a heterozygous deletion of the entire ABCA1 gene. Breakpoints were identified using Sanger sequencing, and the full-gene deletion was also confirmed using exome sequencing and the Affymetrix CytoScanTM HD Array. Before now, large-scale deletions in candidate HDL genes have not been associated with hypoalphalipoproteinemia; our findings indicate that CNVs in ABCA1 may be a previously unappreciated genetic determinant of low HDL cholesterol levels. By coupling bioinformatic analyses with next-generation sequencing data, we can successfully assess the spectrum of genetic determinants of many dyslipidemias, now including hypoalphalipoproteinemia. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Immunization with a Vaccine Combining Herpes Simplex Virus 2 (HSV-2) Glycoprotein C (gC) and gD Subunits Improves the Protection of Dorsal Root Ganglia in Mice and Reduces the Frequency of Recurrent Vaginal Shedding of HSV-2 DNA in Guinea Pigs Compared to Immunization with gD Alone ▿

PubMed Central

Awasthi, Sita; Lubinski, John M.; Shaw, Carolyn E.; Barrett, Shana M.; Cai, Michael; Wang, Fushan; Betts, Michael; Kingsley, Susan; DiStefano, Daniel J.; Balliet, John W.; Flynn, Jessica A.; Casimiro, Danilo R.; Bryan, Janine T.; Friedman, Harvey M.

2011-01-01

Attempts to develop a vaccine to prevent genital herpes simplex virus 2 (HSV-2) disease have been only marginally successful, suggesting that novel strategies are needed. Immunization with HSV-2 glycoprotein C (gC-2) and gD-2 was evaluated in mice and guinea pigs to determine whether adding gC-2 to a gD-2 subunit vaccine would improve protection by producing antibodies that block gC-2 immune evasion from complement. Antibodies produced by gC-2 immunization blocked the interaction between gC-2 and complement C3b, and passive transfer of gC-2 antibody protected complement-intact mice but not C3 knockout mice against HSV-2 challenge, indicating that gC-2 antibody is effective, at least in part, because it prevents HSV-2 evasion from complement. Immunization with gC-2 also produced neutralizing antibodies that were active in the absence of complement; however, the neutralizing titers were higher when complement was present, with the highest titers in animals immunized with both antigens. Animals immunized with the gC-2-plus-gD-2 combination had robust CD4+ T-cell responses to each immunogen. Multiple disease parameters were evaluated in mice and guinea pigs immunized with gC-2 alone, gD-2 alone, or both antigens. In general, gD-2 outperformed gC-2; however, the gC-2-plus-gD-2 combination outperformed gD-2 alone, particularly in protecting dorsal root ganglia in mice and reducing recurrent vaginal shedding of HSV-2 DNA in guinea pigs. Therefore, the gC-2 subunit antigen enhances a gD-2 subunit vaccine by stimulating a CD4+ T-cell response, by producing neutralizing antibodies that are effective in the absence and presence of complement, and by blocking immune evasion domains that inhibit complement activation. PMID:21813597

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA.

PubMed

Thomas, K A; Valenzuela, N M; Gjertson, D; Mulder, A; Fishbein, M C; Parry, G C; Panicker, S; Reed, E F

2015-08-01

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab')2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro. © 2015 The Authors. American Journal of Transplantation Published by Wiley Periodicals, Inc.

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA

PubMed Central

Thomas, K A; Valenzuela, N M; Gjertson, D; Mulder, A; Fishbein, M C; Parry, G C; Panicker, S; Reed, E F

2015-01-01

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab′)2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro. PMID:25904443

Genetic Analyses of a Three Generation Family Segregating Hirschsprung Disease and Iris Heterochromia

PubMed Central

Cheng, Guo; Firmato de Almeida, Manoel; So, Man-Ting; Sham, Pak-Chung; Cherny, Stacey S.; Tam, Paul Kwong-Hang; Garcia-Barceló, Maria-Mercè

2013-01-01

We present the genetic analyses conducted on a three-generation family (14 individuals) with three members affected with isolated-Hirschsprung disease (HSCR) and one with HSCR and heterochromia iridum (syndromic-HSCR), a phenotype reminiscent of Waardenburg-Shah syndrome (WS4). WS4 is characterized by pigmentary abnormalities of the skin, eyes and/or hair, sensorineural deafness and HSCR. None of the members had sensorineural deafness. The family was screened for copy number variations (CNVs) using Illumina-HumanOmni2.5-Beadchip and for coding sequence mutations in WS4 genes (EDN3, EDNRB, or SOX10) and in the main HSCR gene (RET). Confocal microscopy and immunoblotting were used to assess the functional impact of the mutations. A heterozygous A/G transition in EDNRB was identified in 4 affected and 3 unaffected individuals. While in EDNRB isoforms 1 and 2 (cellular receptor) the transition results in the abolishment of translation initiation (M1V), in isoform 3 (only in the cytosol) the replacement occurs at Met91 (M91V) and is predicted benign. Another heterozygous transition (c.-248G/A; -predicted to affect translation efficiency-) in the 5′-untranslated region of EDN3 (EDNRB ligand) was detected in all affected individuals but not in healthy carriers of the EDNRB mutation. Also, a de novo CNVs encompassing DACH1 was identified in the patient with heterochromia iridum and HSCR Since the EDNRB and EDN3 variants only coexist in affected individuals, HSCR could be due to the joint effect of mutations in genes of the same pathway. Iris heterochromia could be due to an independent genetic event and would account for the additional phenotype within the family. PMID:23840513

Hungarian Marfan family with large FBN1 deletion calls attention to copy number variation detection in the current NGS era

PubMed Central

Ágg, Bence; Meienberg, Janine; Kopps, Anna M.; Fattorini, Nathalie; Stengl, Roland; Daradics, Noémi; Pólos, Miklós; Bors, András; Radovits, Tamás; Merkely, Béla; De Backer, Julie; Szabolcs, Zoltán; Mátyás, Gábor

2018-01-01

Copy number variations (CNVs) comprise about 10% of reported disease-causing mutations in Mendelian disorders. Nevertheless, pathogenic CNVs may have been under-detected due to the lack or insufficient use of appropriate detection methods. In this report, on the example of the diagnostic odyssey of a patient with Marfan syndrome (MFS) harboring a hitherto unreported 32-kb FBN1 deletion, we highlight the need for and the feasibility of testing for CNVs (>1 kb) in Mendelian disorders in the current next-generation sequencing (NGS) era. PMID:29850152

Extensive Copy-Number Variation of Young Genes across Stickleback Populations

PubMed Central

Eizaguirre, Christophe; Samonte, Irene E.; Kalbe, Martin; Lenz, Tobias L.; Stoll, Monika; Bornberg-Bauer, Erich; Milinski, Manfred; Reusch, Thorsten B. H.

2014-01-01

Duplicate genes emerge as copy-number variations (CNVs) at the population level, and remain copy-number polymorphic until they are fixed or lost. The successful establishment of such structural polymorphisms in the genome plays an important role in evolution by promoting genetic diversity, complexity and innovation. To characterize the early evolutionary stages of duplicate genes and their potential adaptive benefits, we combine comparative genomics with population genomics analyses to evaluate the distribution and impact of CNVs across natural populations of an eco-genomic model, the three-spined stickleback. With whole genome sequences of 66 individuals from populations inhabiting three distinct habitats, we find that CNVs generally occur at low frequencies and are often only found in one of the 11 populations surveyed. A subset of CNVs, however, displays copy-number differentiation between populations, showing elevated within-population frequencies consistent with local adaptation. By comparing teleost genomes to identify lineage-specific genes and duplications in sticklebacks, we highlight rampant gene content differences among individuals in which over 30% of young duplicate genes are CNVs. These CNV genes are evolving rapidly at the molecular level and are enriched with functional categories associated with environmental interactions, depicting the dynamic early copy-number polymorphic stage of genes during population differentiation. PMID:25474574

Screening for Intellectual Disability Using High-Resolution CMA Technology in a Retrospective Cohort from Central Brazil

PubMed Central

Pereira, Rodrigo Roncato; Pinto, Irene Plaza; Minasi, Lysa Bernardes; de Melo, Aldaires Vieira; da Cruz e Cunha, Damiana Mirian; Cruz, Alex Silva; Ribeiro, Cristiano Luiz; da Silva, Cláudio Carlos; de Melo e Silva, Daniela; da Cruz, Aparecido Divino

2014-01-01

Intellectual disability is a complex, variable, and heterogeneous disorder, representing a disabling condition diagnosed worldwide, and the etiologies are multiple and highly heterogeneous. Microscopic chromosomal abnormalities and well-characterized genetic conditions are the most common causes of intellectual disability. Chromosomal Microarray Analysis analyses have made it possible to identify putatively pathogenic copy number variation that could explain the molecular etiology of intellectual disability. The aim of the current study was to identify possible submicroscopic genomic alterations using a high-density chromosomal microarray in a retrospective cohort of patients with otherwise undiagnosable intellectual disabilities referred by doctors from the public health system in Central Brazil. The CytoScan HD technology was used to detect changes in the genome copy number variation of patients who had intellectual disability and a normal karyotype. The analysis detected 18 CNVs in 60% of patients. Pathogenic CNVs represented about 22%, so it was possible to propose the etiology of intellectual disability for these patients. Likely pathogenic and unknown clinical significance CNVs represented 28% and 50%, respectively. Inherited and de novo CNVs were equally distributed. We report the nature of CNVs in patients from Central Brazil, representing a population not yet screened by microarray technologies. PMID:25061755

Clinical application of chromosomal microarray analysis for the prenatal diagnosis of chromosomal abnormalities and copy number variations in fetuses with congenital heart disease.

PubMed

Xia, Yu; Yang, Yongchao; Huang, Shufang; Wu, Yueheng; Li, Ping; Zhuang, Jian

2018-03-24

This study aimed to determine chromosomal abnormalities and copy number variations (CNVs) in fetuses with congenital heart disease (CHD) by chromosomal microarray analysis (CMA). One hundred and ten cases with CHD detected by prenatal echocardiography were enrolled in the study; 27 cases were simple CHDs, and 83 were complex CHDs. Chromosomal microarray analysis was performed on the Affymetrix CytoScan HD platform. All annotated CNVs were validated by quantitative PCR. Chromosomal microarray analysis identified 6 cases with chromosomal abnormalities, including 2 cases with trisomy 21, 2 cases with trisomy 18, 1 case with trisomy 13, and 1 unusual case of mosaic trisomy 21. Pathogenic CNVs were detected in 15.5% (17/110) of the fetuses with CHDs, including 13 cases with CHD-associated CNVs. We further identified 10 genes as likely novel CHD candidate genes through gene functional enrichment analysis. We also found that pathogenic CMA results impacted the rate of pregnancy termination. This study shows that CMA is particularly effective for identifying chromosomal abnormalities and CNVs in fetuses with CHDs as well as having an effect on obstetrical outcomes. The elucidation of the genetic basis of CHDs will continue to expand our understanding of the etiology of CHDs. © 2018 John Wiley & Sons, Ltd.

Impact of constitutional copy number variants on biological pathway evolution.

PubMed

Poptsova, Maria; Banerjee, Samprit; Gokcumen, Omer; Rubin, Mark A; Demichelis, Francesca

2013-01-23

Inherited Copy Number Variants (CNVs) can modulate the expression levels of individual genes. However, little is known about how CNVs alter biological pathways and how this varies across different populations. To trace potential evolutionary changes of well-described biological pathways, we jointly queried the genomes and the transcriptomes of a collection of individuals with Caucasian, Asian or Yoruban descent combining high-resolution array and sequencing data. We implemented an enrichment analysis of pathways accounting for CNVs and genes sizes and detected significant enrichment not only in signal transduction and extracellular biological processes, but also in metabolism pathways. Upon the estimation of CNV population differentiation (CNVs with different polymorphism frequencies across populations), we evaluated that 22% of the pathways contain at least one gene that is proximal to a CNV (CNV-gene pair) that shows significant population differentiation. The majority of these CNV-gene pairs belong to signal transduction pathways and 6% of the CNV-gene pairs show statistical association between the copy number states and the transcript levels. The analysis suggested possible examples of positive selection within individual populations including NF-kB, MAPK signaling pathways, and Alu/L1 retrotransposition factors. Altogether, our results suggest that constitutional CNVs may modulate subtle pathway changes through specific pathway enzymes, which may become fixed in some populations.

Impact of constitutional copy number variants on biological pathway evolution

PubMed Central

2013-01-01

Background Inherited Copy Number Variants (CNVs) can modulate the expression levels of individual genes. However, little is known about how CNVs alter biological pathways and how this varies across different populations. To trace potential evolutionary changes of well-described biological pathways, we jointly queried the genomes and the transcriptomes of a collection of individuals with Caucasian, Asian or Yoruban descent combining high-resolution array and sequencing data. Results We implemented an enrichment analysis of pathways accounting for CNVs and genes sizes and detected significant enrichment not only in signal transduction and extracellular biological processes, but also in metabolism pathways. Upon the estimation of CNV population differentiation (CNVs with different polymorphism frequencies across populations), we evaluated that 22% of the pathways contain at least one gene that is proximal to a CNV (CNV-gene pair) that shows significant population differentiation. The majority of these CNV-gene pairs belong to signal transduction pathways and 6% of the CNV-gene pairs show statistical association between the copy number states and the transcript levels. Conclusions The analysis suggested possible examples of positive selection within individual populations including NF-kB, MAPK signaling pathways, and Alu/L1 retrotransposition factors. Altogether, our results suggest that constitutional CNVs may modulate subtle pathway changes through specific pathway enzymes, which may become fixed in some populations. PMID:23342974

Significant changes in ITIH4, AHSG, ORM1, and CD46 content in milk fat globule membrane proteins of ketotic dairy cows.

PubMed

Gao, Sansi; Yang, Wei; Yu, Hongjiang; Liu, Runqi; Dong, Zhihao; Zhang, Hongyou; Xia, Cheng; Xu, Chuang

2017-11-01

High concentrations of non-esterified fatty acid (NEFA) and β-hydroxybutyrate (BHBA) in cows' blood caused by ketosis are associated with inflammatory states. We hypothesised that ketosis in postparturient dairy cows would result in altered levels on inflammation-related proteins not only in plasma but also in the milk fat globule membranes (MFGM). Thirty cows were selected from a dairy farm in Heilongjiang, China. Inflammatory milk fat globule membrane proteins were detected using ELISA kits, and a fully automatic biochemical analyser was used to measure the concentrations of BHBA, NEFA, glucose (GLU) and triglyceride (TG) in plasma. MFGM protein from milk of ketotic cows contained significantly different concentrations of acute-phase response proteins (complement C3 (C3), prothrombin (F2), alpha-1-acid glycoprotein (ORM1), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-2-HS-glycoprotein (AHSG), complement C9 (C9), complement regulatory protein variant 4 (CD46)) in comparison with milk from non-ketotic cows. Blood concentrations of C3, complement C9 (C9), tumour necrosis factor α (TNFα), MFGM C3, monocyte differentiation antigen CD14 (CD14) and ORM1 levels were correlated with energy balance. ITIH4 and CD46 increased, and AHSG and ORM1 decreased before the onset of ketosis. These biomarkers offer potential as predictors and monitors of ketosis in at-risk cows.

Assessment of copy number variations in 120 patients with Poland syndrome.

PubMed

Vaccari, Carlotta Maria; Tassano, Elisa; Torre, Michele; Gimelli, Stefania; Divizia, Maria Teresa; Romanini, Maria Victoria; Bossi, Simone; Musante, Ilaria; Valle, Maura; Senes, Filippo; Catena, Nunzio; Bedeschi, Maria Francesca; Baban, Anwar; Calevo, Maria Grazia; Acquaviva, Massimo; Lerone, Margherita; Ravazzolo, Roberto; Puliti, Aldamaria

2016-11-25

Poland Syndrome (PS) is a rare congenital disorder presenting with agenesis/hypoplasia of the pectoralis major muscle variably associated with thoracic and/or upper limb anomalies. Most cases are sporadic, but familial recurrence, with different inheritance patterns, has been observed. The genetic etiology of PS remains unknown. Karyotyping and array-comparative genomic hybridization (CGH) analyses can identify genomic imbalances that can clarify the genetic etiology of congenital and neurodevelopmental disorders. We previously reported a chromosome 11 deletion in twin girls with pectoralis muscle hypoplasia and skeletal anomalies, and a chromosome six deletion in a patient presenting a complex phenotype that included pectoralis muscle hypoplasia. However, the contribution of genomic imbalances to PS remains largely unknown. To investigate the prevalence of chromosomal imbalances in PS, standard cytogenetic and array-CGH analyses were performed in 120 PS patients. Following the application of stringent filter criteria, 14 rare copy number variations (CNVs) were identified in 14 PS patients in different regions outside known common copy number variations: seven genomic duplications and seven genomic deletions, enclosing the two previously reported PS associated chromosomal deletions. These CNVs ranged from 0.04 to 4.71 Mb in size. Bioinformatic analysis of array-CGH data indicated gene enrichment in pathways involved in cell-cell adhesion, DNA binding and apoptosis processes. The analysis also provided a number of candidate genes possibly causing the developmental defects observed in PS patients, among others REV3L, a gene coding for an error-prone DNA polymerase previously associated with Möbius Syndrome with variable phenotypes including pectoralis muscle agenesis. A number of rare CNVs were identified in PS patients, and these involve genes that represent candidates for further evaluation. Rare inherited CNVs may contribute to, or represent risk factors of PS in a multifactorial mode of inheritance.

Chromosomal Microarray With Clinical Diagnostic Utility in Children With Developmental Delay or Intellectual Disability.

PubMed

Lee, Jin Sook; Hwang, Hee; Kim, Soo Yeon; Kim, Ki Joong; Choi, Jin Sun; Woo, Mi Jung; Choi, Young Min; Jun, Jong Kwan; Lim, Byung Chan; Chae, Jong Hee

2018-09-01

Chromosomal microarray (CMA) testing is a first-tier test for patients with developmental delay, autism, or congenital anomalies. It increases diagnostic yield for patients with developmental delay or intellectual disability. In some countries, including Korea, CMA testing is not yet implemented in clinical practice. We assessed the diagnostic utility of CMA testing in a large cohort of patients with developmental delay or intellectual disability in Korea. We conducted a genome-wide microarray analysis of 649 consecutive patients with developmental delay or intellectual disability at the Seoul National University Children's Hospital. Medical records were reviewed retrospectively. Pathogenicity of detected copy number variations (CNVs) was evaluated by referencing previous reports or parental testing using FISH or quantitative PCR. We found 110 patients to have pathogenic CNVs, which included 100 deletions and 31 duplications of 270 kb to 30 Mb. The diagnostic yield was 16.9%, demonstrating the diagnostic utility of CMA testing in clinic. Parental testing was performed in 66 patients, 86.4% of which carried de novo CNVs. In eight patients, pathogenic CNVs were inherited from healthy parents with a balanced translocation, and genetic counseling was provided to these families. We verified five rarely reported deletions on 2p21p16.3, 3p21.31, 10p11.22, 14q24.2, and 21q22.13. This study demonstrated the clinical utility of CMA testing in the genetic diagnosis of patients with developmental delay or intellectual disability. CMA testing should be included as a clinical diagnostic test for all children with developmental delay or intellectual disability. © The Korean Society for Laboratory Medicine.

Extensive copy number variations in admixed Indian population of African ancestry: potential involvement in adaptation.

PubMed

Narang, Ankita; Jha, Pankaj; Kumar, Dhirendra; Kutum, Rintu; Mondal, Anupam Kumar; Dash, Debasis; Mukerji, Mitali

2014-11-13

Admixture mapping has been enormously resourceful in identifying genetic variations linked to phenotypes, adaptation, and diseases. In this study through analysis of copy number variable regions (CNVRs), we report extensive restructuring in the genomes of the recently admixed African-Indian population (OG-W-IP) that inhabits a highly saline environment in Western India. The study included subjects from OG-W-IP (OG), five different Indian and three HapMap populations that were genotyped using Affymetrix version 6.0 arrays. Copy number variations (CNVs) detected using Birdsuite were used to define CNVRs. Population structure with respect to CNVRs was delineated using random forest approach. OG genomes have a surprising excess of CNVs in comparison to other studied populations. Individual ancestry proportions computed using STRUCTURE also reveals a unique genetic component in OGs. Population structure analysis with CNV genotypes indicates OG to be distant from both the African and Indian ancestral populations. Interestingly, it shows genetic proximity with respect to CNVs to only one Indian population IE-W-LP4, which also happens to reside in the same geographical region. We also observe a significant enrichment of molecular processes related to ion binding and receptor activity in genes encompassing OG-specific CNVRs. Our results suggest that retention of CNVRs from ancestral natives and de novo acquisition of CNVRs could accelerate the process of adaptation especially in an extreme environment. Additionally, this population would be enormously useful for dissecting genes and delineating the involvement of CNVs in salt adaptation. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

A Genome Wide Study of Copy Number Variation Associated with Nasopharyngeal Carcinoma in Malaysian Chinese Identifies CNVs at 11q14.3 and 6p21.3 as Candidate Loci.

PubMed

Low, Joyce Siew Yong; Chin, Yoon Ming; Mushiroda, Taisei; Kubo, Michiaki; Govindasamy, Gopala Krishnan; Pua, Kin Choo; Yap, Yoke Yeow; Yap, Lee Fah; Subramaniam, Selva Kumar; Ong, Cheng Ai; Tan, Tee Yong; Khoo, Alan Soo Beng; Ng, Ching Ching

2016-01-01

Nasopharyngeal carcinoma (NPC) is a neoplasm of the epithelial lining of the nasopharynx. Despite various reports linking genomic variants to NPC predisposition, very few reports were done on copy number variations (CNV). CNV is an inherent structural variation that has been found to be involved in cancer predisposition. A discovery cohort of Malaysian Chinese descent (NPC patients, n = 140; Healthy controls, n = 256) were genotyped using Illumina® HumanOmniExpress BeadChip. PennCNV and cnvPartition calling algorithms were applied for CNV calling. Taqman CNV assays and digital PCR were used to validate CNV calls and replicate candidate copy number variant region (CNVR) associations in a follow-up Malaysian Chinese (NPC cases, n = 465; and Healthy controls, n = 677) and Malay cohort (NPC cases, n = 114; Healthy controls, n = 124). Six putative CNVRs overlapping GRM5, MICA/HCP5/HCG26, LILRB3/LILRA6, DPY19L2, RNase3/RNase2 and GOLPH3 genes were jointly identified by PennCNV and cnvPartition. CNVs overlapping GRM5 and MICA/HCP5/HCG26 were subjected to further validation by Taqman CNV assays and digital PCR. Combined analysis in Malaysian Chinese cohort revealed a strong association at CNVR on chromosome 11q14.3 (Pcombined = 1.54x10-5; odds ratio (OR) = 7.27; 95% CI = 2.96-17.88) overlapping GRM5 and a suggestive association at CNVR on chromosome 6p21.3 (Pcombined = 1.29x10-3; OR = 4.21; 95% CI = 1.75-10.11) overlapping MICA/HCP5/HCG26 genes. Our results demonstrated the association of CNVs towards NPC susceptibility, implicating a possible role of CNVs in NPC development.

Micrurus snake venoms activate human complement system and generate anaphylatoxins

PubMed Central

2012-01-01

Background The genus Micrurus, coral snakes (Serpentes, Elapidae), comprises more than 120 species and subspecies distributed from the south United States to the south of South America. Micrurus snake bites can cause death by muscle paralysis and further respiratory arrest within a few hours after envenomation. Clinical observations show mainly neurotoxic symptoms, although other biological activities have also been experimentally observed, including cardiotoxicity, hemolysis, edema and myotoxicity. Results In the present study we have investigated the action of venoms from seven species of snakes from the genus Micrurus on the complement system in in vitro studies. Several of the Micrurus species could consume the classical and/or the lectin pathways, but not the alternative pathway, and C3a, C4a and C5a were generated in sera treated with the venoms as result of this complement activation. Micrurus venoms were also able to directly cleave the α chain of the component C3, but not of the C4, which was inhibited by 1,10 Phenanthroline, suggesting the presence of a C3α chain specific metalloprotease in Micrurus spp venoms. Furthermore, complement activation was in part associated with the cleavage of C1-Inhibitor by protease(s) present in the venoms, which disrupts complement activation control. Conclusion Micrurus venoms can activate the complement system, generating a significant amount of anaphylatoxins, which may assist due to their vasodilatory effects, to enhance the spreading of other venom components during the envenomation process. PMID:22248157

Micrurus snake venoms activate human complement system and generate anaphylatoxins.

PubMed

Tanaka, Gabriela D; Pidde-Queiroz, Giselle; de Fátima D Furtado, Maria; van den Berg, Carmen; Tambourgi, Denise V

2012-01-16

The genus Micrurus, coral snakes (Serpentes, Elapidae), comprises more than 120 species and subspecies distributed from the south United States to the south of South America. Micrurus snake bites can cause death by muscle paralysis and further respiratory arrest within a few hours after envenomation. Clinical observations show mainly neurotoxic symptoms, although other biological activities have also been experimentally observed, including cardiotoxicity, hemolysis, edema and myotoxicity. In the present study we have investigated the action of venoms from seven species of snakes from the genus Micrurus on the complement system in in vitro studies. Several of the Micrurus species could consume the classical and/or the lectin pathways, but not the alternative pathway, and C3a, C4a and C5a were generated in sera treated with the venoms as result of this complement activation. Micrurus venoms were also able to directly cleave the α chain of the component C3, but not of the C4, which was inhibited by 1,10 Phenanthroline, suggesting the presence of a C3α chain specific metalloprotease in Micrurus spp venoms. Furthermore, complement activation was in part associated with the cleavage of C1-Inhibitor by protease(s) present in the venoms, which disrupts complement activation control. Micrurus venoms can activate the complement system, generating a significant amount of anaphylatoxins, which may assist due to their vasodilatory effects, to enhance the spreading of other venom components during the envenomation process.

Plasmin cleaves fibrinogen and the human complement proteins C3b and C5 in the presence of Leptospira interrogans proteins: A new role of LigA and LigB in invasion and complement immune evasion.

PubMed

Castiblanco-Valencia, Mónica Marcela; Fraga, Tatiana Rodrigues; Pagotto, Ana Helena; Serrano, Solange Maria de Toledo; Abreu, Patricia Antonia Estima; Barbosa, Angela Silva; Isaac, Lourdes

2016-05-01

Plasminogen is a single-chain glycoprotein found in human plasma as the inactive precursor of plasmin. When converted to proteolytically active plasmin, plasmin(ogen) regulates both complement and coagulation cascades, thus representing an important target for pathogenic microorganisms. Leptospira interrogans binds plasminogen, which is converted to active plasmin. Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules that interact with extracellular matrix components and complement regulators, including proteins of the FH family and C4BP. In this work, we demonstrate that these multifunctional molecules also bind plasminogen through both N- and C-terminal domains. These interactions are dependent on lysine residues and are affected by ionic strength. Competition assays suggest that plasminogen does not share binding sites with C4BP or FH on Lig proteins at physiological molar ratios. Plasminogen bound to Lig proteins is converted to proteolytic active plasmin in the presence of urokinase-type plasminogen activator (uPA). Lig-bound plasmin is able to cleave the physiological substrates fibrinogen and the complement proteins C3b and C5. Taken together, our data point to a new role of LigA and LigB in leptospiral invasion and complement immune evasion. Plasmin(ogen) acquisition by these versatile proteins may contribute to Leptospira infection, favoring bacterial survival and dissemination inside the host. Copyright © 2016. Published by Elsevier GmbH.

Identification of hot spots in the variola virus complement inhibitor (SPICE) for human complement regulation.

PubMed

Yadav, Viveka Nand; Pyaram, Kalyani; Mullick, Jayati; Sahu, Arvind

2008-04-01

Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is approximately 90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement.

Identification of Hot Spots in the Variola Virus Complement Inhibitor (SPICE) for Human Complement Regulation▿

PubMed Central

Yadav, Viveka Nand; Pyaram, Kalyani; Mullick, Jayati; Sahu, Arvind

2008-01-01

Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is ∼90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement. PMID:18216095

Hypocomplementemia is associated with worse renal survival in ANCA-positive granulomatosis with polyangiitis and microscopic polyangiitis

PubMed Central

Aouba, Achille; Khoy, Kathy; Mariotte, Delphine; Lobbedez, Thierry; Martin Silva, Nicolas

2018-01-01

Recent data suggest the existence of a complement alternative pathway activation in the pathogenesis of antineutrophilic cytoplasmic antibody (ANCA)-associated vasculitis (AAV), a condition that remains poorly understood. This study aims to assess the clinical characteristics and outcomes of granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) patients with regard to their plasma complement levels at diagnosis. A retrospective monocentric study carried out at Caen University Hospital led to the identification of proteinase-3- or myeloperoxidase-ANCA-positive GPA and MPA patients from January 2000 to June 2016 and from September 2011 to June 2016, respectively. All patients with available C3 and C4 levels at diagnosis were included. Patients were categorized in the hypocomplementemia group if their C3 and/or C4 levels at diagnosis were below the lower limit of the normal range. Among the 76 AAV patients (43 GPA, 33 MPA), 4 (5%) had hypocomplementemia, and the 72 remaining patients exhibited normal plasma complement levels. All 4 hypocomplementemia patients had renal involvement. Hypocomplementemia was followed in 1 patient whose post-treatment complement level normalized within 1 month. Among all clinical and ANCA specificity, including relapse-free survival (p = 0.093), only overall and renal survival rates were significantly lower in the hypocomplementemia group (p = 0.0011 and p<0.001, respectively). Hypocomplementemia with low C3 and/or C4 levels at GPA or MPA diagnosis may be responsible for worse survival and renal prognosis. These results argue for larger and prospective studies to better determine the epidemiology of the disease and to assess complement-targeting therapy in these patients. PMID:29621352

Early Components of the Complement Classical Activation Pathway in Human Systemic Autoimmune Diseases

PubMed Central

Lintner, Katherine E.; Wu, Yee Ling; Yang, Yan; Spencer, Charles H.; Hauptmann, Georges; Hebert, Lee A.; Atkinson, John P.; Yu, C. Yung

2016-01-01

The complement system consists of effector proteins, regulators, and receptors that participate in host defense against pathogens. Activation of the complement system, via the classical pathway (CP), has long been recognized in immune complex-mediated tissue injury, most notably systemic lupus erythematosus (SLE). Paradoxically, a complete deficiency of an early component of the CP, as evidenced by homozygous genetic deficiencies reported in human, are strongly associated with the risk of developing SLE or a lupus-like disease. Similarly, isotype deficiency attributable to a gene copy-number (GCN) variation and/or the presence of autoantibodies directed against a CP component or a regulatory protein that result in an acquired deficiency are relatively common in SLE patients. Applying accurate assay methodologies with rigorous data validations, low GCNs of total C4, and heterozygous and homozygous deficiencies of C4A have been shown as medium to large effect size risk factors, while high copy numbers of total C4 or C4A as prevalent protective factors, of European and East-Asian SLE. Here, we summarize the current knowledge related to genetic deficiency and insufficiency, and acquired protein deficiencies for C1q, C1r, C1s, C4A/C4B, and C2 in disease pathogenesis and prognosis of SLE, and, briefly, for other systemic autoimmune diseases. As the complement system is increasingly found to be associated with autoimmune diseases and immune-mediated diseases, it has become an attractive therapeutic target. We highlight the recent developments and offer a balanced perspective concerning future investigations and therapeutic applications with a focus on early components of the CP in human systemic autoimmune diseases. PMID:26913032

Copy Number Variation Is a Fundamental Aspect of the Placental Genome

PubMed Central

Hannibal, Roberta L.; Chuong, Edward B.; Rivera-Mulia, Juan Carlos; Gilbert, David M.; Valouev, Anton; Baker, Julie C.

2014-01-01

Discovery of lineage-specific somatic copy number variation (CNV) in mammals has led to debate over whether CNVs are mutations that propagate disease or whether they are a normal, and even essential, aspect of cell biology. We show that 1,000N polyploid trophoblast giant cells (TGCs) of the mouse placenta contain 47 regions, totaling 138 Megabases, where genomic copies are underrepresented (UR). UR domains originate from a subset of late-replicating heterochromatic regions containing gene deserts and genes involved in cell adhesion and neurogenesis. While lineage-specific CNVs have been identified in mammalian cells, classically in the immune system where V(D)J recombination occurs, we demonstrate that CNVs form during gestation in the placenta by an underreplication mechanism, not by recombination nor deletion. Our results reveal that large scale CNVs are a normal feature of the mammalian placental genome, which are regulated systematically during embryogenesis and are propagated by a mechanism of underreplication. PMID:24785991

The Extracellular Adherence Protein from Staphylococcus aureus Inhibits the Classical and Lectin Pathways of Complement by Blocking Formation of the C3 Pro-Convertase

PubMed Central

Garcia, Brandon L.; Ramyar, Kasra X.; Keightley, Andrew; Ruyken, Maartje; Syriga, Maria; Sfyroera, Georgia; Weber, Alexander B.; Zolkiewski, Michal; Ricklin, Daniel; Lambris, John D.; Rooijakkers, Suzan H.M.; Geisbrecht, Brian V.

2014-01-01

The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. While the majority of staphylococcal complement inhibitors act on the alternative pathway (AP) to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical (CP) and lectin (LP) pathways. We screened a collection of recombinant, secreted staphylococcal proteins to determine if S. aureus produces other molecules that inhibit either the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 pro-convertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits the two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion. PMID:25381436

Reduction in erythrocyte-bound complement activation products and titres of anti-C1q antibodies associate with clinical improvement in systemic lupus erythematosus.

PubMed

Buyon, Jill; Furie, Richard; Putterman, Chaim; Ramsey-Goldman, Rosalind; Kalunian, Kenneth; Barken, Derren; Conklin, John; Dervieux, Thierry

2016-01-01

The relationship between cell-bound complement activation products (CB-CAPs: EC4d, EC3d), anti-C1q, soluble complement C3/C4 and disease activity in systemic lupus erythematosus (SLE) was evaluated. Per protocol, at baseline all SLE subjects enrolled in this longitudinal study presented with active disease and elevated CB-CAPs. At each monthly visit, the non-serological (ns) Safety of Estrogens in Lupus Erythematosus: National Assessment (SELENA-SLEDAI) and the British Isles Lupus Assessment Group (BILAG)-2004 index scores were determined as was a random urinary protein to creatinine ratio (uPCR). Short-form 36 (SF-36) questionnaires were also collected. All soluble markers were determined using immunoassays, while EC4d and EC3d were determined using flow cytometry. Statistical analysis consisted of linear mixed models with random intercept and fixed slopes. A total of 36 SLE subjects (mean age 34 years; 94% female) were enrolled and evaluated monthly for an average 11 visits per subject. Clinical improvements were observed during the study, with significant decreases in ns-SELENA-SLEDAI scores, BILAG-2004 index scores and uPCR, and increases in all domains of SF-36 (p<0.01). The longitudinal decrease in ns-SELENA-SLEDAI and BILAG-2004 index scores was significantly associated with reduced EC4d and EC3d levels, reduced anti-C1q titres and increased serum complement C3/C4 (p<0.05). The changes in uPCR significantly correlated with C3, C4, anti-C1q and EC4d, with EC4d outperforming C3/C4 by a multivariate analysis. The reduced EC4d or EC3d was associated with improvements in at least six out of the eight domains of SF-36 and outperformed C3/C4. Anti-dsDNA titres did not correlate with changes in disease activity. These data indicate that CB-CAPs and anti-C1q are helpful in monitoring patients with SLE.

Binding of Free and Immune Complex-Associated Hepatitis C Virus to Erythrocytes Is Mediated by the Complement System.

PubMed

Salam, Kazi Abdus; Wang, Richard Y; Grandinetti, Teresa; De Giorgi, Valeria; Alter, Harvey J; Allison, Robert D

2018-05-09

Erythrocytes bind circulating immune complexes (IC) and facilitate IC clearance from the circulation. Chronic hepatitis C virus (HCV) infection is associated with IC-related disorders. In this study we investigated the kinetics and mechanism of HCV and HCV-IC binding to and dissociation from erythrocytes. Cell culture-produced HCV was mixed with erythrocytes from healthy blood donors and erythrocyte-associated virus particles were quantified. Purified complement proteins, complement-depleted serum, and complement receptor antibodies were used to investigate complement-mediated HCV-erythrocyte binding. Purified HCV-specific immunoglobulin G from a chronic HCV-infected patient was used to study complement-mediated HCV-IC-erythrocyte binding. Binding of HCV to erythrocytes increased 200 to 1,000 fold after adding complement active human serum in the absence of antibody. Opsonization of free HCV occurred within 10 minutes and peak binding to erythrocytes was observed at 20-30 minutes. Complement protein C1 was required for binding, while C2, C3 and C4 significantly enhanced binding. Complement receptor 1 (CR1, CD35) antibodies blocked the binding of HCV to erythrocytes isolated from chronically infected HCV patients and healthy blood donors. HCV-ICs significantly enhanced complement-mediated binding to erythrocytes compared to unbound HCV. Dissociation of complement-opsonized HCV from erythrocytes depended on the presence of Factor I. HCV released by Factor I bound preferentially to CD19+ B cells compared to other leukocytes. These results demonstrate that complement mediates the binding of free and IC-associated HCV to CR1 on erythrocytes, and provide a mechanistic rationale for investigating the differential phenotypic expression of HCV-IC-related disease. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

Copy number variation profile in the placental and parental genomes of recurrent pregnancy loss families

PubMed Central

Kasak, Laura; Rull, Kristiina; Sõber, Siim; Laan, Maris

2017-01-01

We have previously shown an extensive load of somatic copy number variations (CNVs) in the human placental genome with the highest fraction detected in normal term pregnancies. Hereby, we hypothesized that insufficient promotion of CNVs may impair placental development and lead to recurrent pregnancy loss (RPL). RPL affects ~3% of couples aiming at childbirth and idiopathic RPL represents ~50% of cases. We analysed placental and parental CNV profiles of idiopathic RPL trios (mother-father-placenta) and duos (mother-placenta). Consistent with the hypothesis, the placental genomes of RPL cases exhibited 2-fold less CNVs compared to uncomplicated 1st trimester pregnancies (P = 0.02). This difference mainly arose from lower number of duplications. Overall, 1st trimester control placentas shared only 5.3% of identified CNV regions with RPL cases, whereas the respective fraction with term placentas was 35.1% (P = 1.1 × 10−9). Disruption of the genes NUP98 (embryonic stem cell development) and MTRR (folate metabolism) was detected exclusively in RPL placentas, potentially indicative to novel loci implicated in RPL. Interestingly, genes with higher overall expression were prone to deletions (>3-fold higher median expression compared to genes unaffected by CNVs, P = 6.69 × 10−20). Additionally, large pericentromeric and subtelomeric CNVs in parental genomes emerged as a risk factor for RPL. PMID:28345611

Parallel Evolution of Copy-Number Variation across Continents in Drosophila melanogaster

PubMed Central

Schrider, Daniel R.; Hahn, Matthew W.; Begun, David J.

2016-01-01

Genetic differentiation across populations that is maintained in the presence of gene flow is a hallmark of spatially varying selection. In Drosophila melanogaster, the latitudinal clines across the eastern coasts of Australia and North America appear to be examples of this type of selection, with recent studies showing that a substantial portion of the D. melanogaster genome exhibits allele frequency differentiation with respect to latitude on both continents. As of yet there has been no genome-wide examination of differentiated copy-number variants (CNVs) in these geographic regions, despite their potential importance for phenotypic variation in Drosophila and other taxa. Here, we present an analysis of geographic variation in CNVs in D. melanogaster. We also present the first genomic analysis of geographic variation for copy-number variation in the sister species, D. simulans, in order to investigate patterns of parallel evolution in these close relatives. In D. melanogaster we find hundreds of CNVs, many of which show parallel patterns of geographic variation on both continents, lending support to the idea that they are influenced by spatially varying selection. These findings support the idea that polymorphic CNVs contribute to local adaptation in D. melanogaster. In contrast, we find very few CNVs in D. simulans that are geographically differentiated in parallel on both continents, consistent with earlier work suggesting that clinal patterns are weaker in this species. PMID:26809315

Spores of Mucor ramosissimus, Mucor plumbeus and Mucor circinelloides and their ability to activate human complement system in vitro.

PubMed

Granja, Luiz Fernando Zmetek; Pinto, Lysianne; Almeida, Cátia Amancio; Alviano, Daniela Sales; Da Silva, Maria Helena; Ejzemberg, Regina; Alviano, Celuta Sales

2010-03-01

Complement activation by spores of Mucor ramosissimus, Mucor plumbeus and Mucor circinelloides was studied using absorbed human serum in the presence or absence of chelators (EGTA or EDTA). We found that the spore caused full complement activation when incubated with EGTA-Mg2+ or without chelators, indicating that the alternative pathway is mainly responsible for this response. In order to compare activation profiles from each species, ELISAs for C3 and C4 fragments, mannan binding lectin (MBL), C-reactive protein (CRP) and IgG studies were carried out. All proteins were present on the species tested. Immunofluorescence tests demonstrated the presence of C3 fragments on the surface of all samples, which were confluent throughout fungal surfaces. The same profile of C3, C4, MBL, CRP and IgG deposition, observed in all species, suggests a similar activation behavior for these species.

Copy number variation of individual cattle genomes using next-generation sequencing

USDA-ARS?s Scientific Manuscript database

Copy number variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next-generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one ...

Individualized cattle copy number and segmental duplication maps using next generation sequencing

USDA-ARS?s Scientific Manuscript database

Copy Number Variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one ...

Copy number variation of individual cattle genomes using next-generation sequencing

USDA-ARS?s Scientific Manuscript database

Copy Number Variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often difficult to track. Using a read depth approach based on next generation sequencing, we examined genome-wide copy number differences among five taurine (three Angu...

A high-resolution cattle CNV map by population-scale genome sequencing

USDA-ARS?s Scientific Manuscript database

Copy Number Variations (CNVs) are common genomic structural variations that have been linked to human diseases and phenotypic traits. Prior studies in cattle have produced low-resolution CNV maps. We constructed a draft, high-resolution map of cattle CNVs based on whole genome sequencing data from 7...

Rare DNA copy number variants in cardiovascular malformations with extracardiac abnormalities

USDA-ARS?s Scientific Manuscript database

Clinically significant cardiovascular malformations (CVMs) occur in 5-8 per 1000 live births. Recurrent copy number variations (CNVs) are among the known causes of syndromic CVMs, accounting for an important fraction of cases. We hypothesized that many additional rare CNVs also cause CVMs and can be...

Comparison of CNVs in Buffalo with other species

USDA-ARS?s Scientific Manuscript database

Using a read-depth (RD) and a hybrid read-pair, split-read (RAPTR-SV) CNV detection method, we identified over 1425 unique CNVs in 14 Water Buffalo individual compared to the cattle genome sequence. Total variable sequence of the CNV regions (CNVR) from the RD method approached 59 megabases (~ 2% of...

Effects of freezer storage time on levels of complement biomarkers.

PubMed

Morgan, Angharad R; O'Hagan, Caroline; Touchard, Samuel; Lovestone, Simon; Morgan, B Paul

2017-11-06

There is uncertainty regarding how stable complement analytes are during long-term storage at - 80 °C. As part of our work program we have measured 17 complement biomarkers (C1q, C1 inhibitor, C3, C3a, iC3b, C4, C5, C9, FB, FD, FH, FI, TCC, Bb, sCR1, sCR2, Clusterin) and the benchmark inflammatory marker C-reactive protein (CRP) in a large set of plasma samples (n = 720) that had been collected, processed and subsequently stored at - 80 °C over a period of 6.6-10.6 years, prior to laboratory analysis. The biomarkers were measured using solid-phase enzyme immunoassays with a combination of multiplex assays using the MesoScale Discovery Platform and single-plex enzyme-linked immunosorbent assays (ELISAs). As part of a post hoc analysis of extrinsic factors (co-variables) affecting the analyses we investigated the impact of freezer storage time on the values obtained for each complement analyte. With the exception of five analytes (C4, C9, sCR2, clusterin and CRP), storage time was significantly correlated with measured plasma concentrations. For ten analytes: C3, FI, FB, FD, C5, sCR1, C3a, iC3b, Bb and TCC, storage time was positively correlated with concentration and for three analytes: FH, C1q, and C1 inhibitor, storage time was negatively correlated with concentration. The results suggest that information on storage time should be regarded as an important co-variable and taken into consideration when analysing data to look for associations of complement biomarker levels and disease or other outcomes.

Epididymal C4b-binding protein is processed and degraded during transit through the duct and is not essential for fertility.

PubMed

Nonaka, Mayumi I; Zsigmond, Eva; Kudo, Akihiko; Kawakami, Hayato; Yoshida, Kaoru; Yoshida, Manabu; Kawano, Natsuko; Miyado, Kenji; Nonaka, Masaru; Wetsel, Rick A

2015-04-01

C4b-binding protein (C4BP) is known as one of the circulating complement regulators that prevents excessive activation of the host-defense complement system. We have reported previously that C4BP is expressed abundantly in the rodent epididymis, one of the male reproductive organs connecting the testis and vas deferens, where immature spermatozoa acquire their motility and fertilizing ability during their transit through the duct. Epididymal C4BP (EpC4BP) is synthesized androgen-dependently by the epithelial cells, secreted into the lumen, and bound to the outer membrane of the passing spermatozoa. In this study, we found that EpC4BP is secreted as a large oligomer, similar to the serum C4BP, but is digested during the epididymal transit and is almost lost from both the luminal fluid and the sperm surface in the vas deferens. Such a processing pattern is not known in serum C4BP, suggesting that EpC4BP and serum C4BP might have different functional mechanisms, and that there is a novel function of EpC4BP in reproduction. In addition, the disappearance of EpC4BP from the sperm surface prior to ejaculation suggests that EpC4BP works only in the epididymis and would not work in the female reproductive tract to protect spermatozoa from complement attack. Next, we generated C4BP-deficient (C4BP-/-) mice to examine the possible role of EpC4BP in reproduction. However, the C4BP-/- mice were fertile and no significant differences were observed between the C4BP-/- and wild-type mouse spermatozoa in terms of morphology, motility, and rate of the spontaneous acrosome reaction. These results suggest that EpC4BP is involved in male reproduction, but not essential for sperm maturation. Copyright © 2014 Elsevier GmbH. All rights reserved.

Complement C4 deficiency--a plausible risk factor for non-tuberculous mycobacteria (NTM) infection in apparently immunocompetent patients.

PubMed

Kotilainen, Hannele; Lokki, Marja-Liisa; Paakkanen, Riitta; Seppänen, Mikko; Tukiainen, Pentti; Meri, Seppo; Poussa, Tuija; Eskola, Jussi; Valtonen, Ville; Järvinen, Asko

2014-01-01

Non-tuberculous mycobacteria (NTM) are ubiquitous in the environment and they infect mainly persons with underlying pulmonary diseases but also previously healthy elderly women. Defects in host resistance that lead to pulmonary infections by NTM are relatively unknown. A few genetic defects have been associated with both pulmonary and disseminated mycobacterial infections. Rare disseminated NTM infections have been associated with genetic defects in T-cell mediated immunity and in cytokine signaling in families. We investigated whether there was an association between NTM infections and deficiencies of complement components C4A or C4B that are encoded by major histocompatibility complex (MHC). 50 adult patients with a positive NTM culture with symptoms and findings of a NTM disease were recruited. Patients' clinical history was collected and symptoms and clinical findings were categorized according to 2007 diagnostic criteria of The American Thoracic Society (ATS). To investigate the deficiencies of complement, C4A and C4B gene copy numbers and phenotype frequencies of the C4 allotypes were analyzed. Unselected, healthy, 149 Finnish adults were used as controls. NTM patients had more often C4 deficiencies (C4A or C4B) than controls (36/50 [72%] vs 83/149 [56%], OR = 2.05, 95%CI = 1.019-4.105, p = 0.042). C4 deficiencies for female NTM patients were more common than for controls (29/36 [81%] vs 55/100 [55%], OR = 3.39, 95% CI = 1.358-8.460, p = 0.007). C4 deficiences seemed not to be related to any specific underlying disease or C4 phenotype. C4 deficiency may be a risk factor for NTM infection in especially elderly female patients.

Comprehensive performance comparison of high-resolution array platforms for genome-wide Copy Number Variation (CNV) analysis in humans.

PubMed

Haraksingh, Rajini R; Abyzov, Alexej; Urban, Alexander Eckehart

2017-04-24

High-resolution microarray technology is routinely used in basic research and clinical practice to efficiently detect copy number variants (CNVs) across the entire human genome. A new generation of arrays combining high probe densities with optimized designs will comprise essential tools for genome analysis in the coming years. We systematically compared the genome-wide CNV detection power of all 17 available array designs from the Affymetrix, Agilent, and Illumina platforms by hybridizing the well-characterized genome of 1000 Genomes Project subject NA12878 to all arrays, and performing data analysis using both manufacturer-recommended and platform-independent software. We benchmarked the resulting CNV call sets from each array using a gold standard set of CNVs for this genome derived from 1000 Genomes Project whole genome sequencing data. The arrays tested comprise both SNP and aCGH platforms with varying designs and contain between ~0.5 to ~4.6 million probes. Across the arrays CNV detection varied widely in number of CNV calls (4-489), CNV size range (~40 bp to ~8 Mbp), and percentage of non-validated CNVs (0-86%). We discovered strikingly strong effects of specific array design principles on performance. For example, some SNP array designs with the largest numbers of probes and extensive exonic coverage produced a considerable number of CNV calls that could not be validated, compared to designs with probe numbers that are sometimes an order of magnitude smaller. This effect was only partially ameliorated using different analysis software and optimizing data analysis parameters. High-resolution microarrays will continue to be used as reliable, cost- and time-efficient tools for CNV analysis. However, different applications tolerate different limitations in CNV detection. Our study quantified how these arrays differ in total number and size range of detected CNVs as well as sensitivity, and determined how each array balances these attributes. This analysis will inform appropriate array selection for future CNV studies, and allow better assessment of the CNV-analytical power of both published and ongoing array-based genomics studies. Furthermore, our findings emphasize the importance of concurrent use of multiple analysis algorithms and independent experimental validation in array-based CNV detection studies.

Protection by meningococcal outer membrane protein PorA-specific antibodies and a serogroup B capsular polysaccharide-specific antibody in complement-sufficient and C6-deficient infant rats.

PubMed

Toropainen, Maija; Saarinen, Leena; Vidarsson, Gestur; Käyhty, Helena

2006-05-01

The relative contributions of antibody-induced complement-mediated bacterial lysis and antibody/complement-mediated phagocytosis to host immunity against meningococcal infections are currently unclear. Further, the in vivo effector functions of antibodies may vary depending on their specificity and Fc heavy-chain isotype. In this study, a mouse immunoglobulin G2a (mIgG2a) monoclonal antibody (MN12H2) to meningococcal outer membrane protein PorA (P1.16), its human IgG subclass derivatives (hIgG1 to hIgG4), and an mIgG2a monoclonal antibody (Nmb735) to serogroup B capsular polysaccharide (B-PS) were evaluated for passive protection against meningococcal serogroup B strain 44/76-SL (B:15:P1.7,16) in an infant rat infection model. Complement component C6-deficient (PVG/c-) rats were used to assess the importance of complement-mediated bacterial lysis for protection. The PorA-specific parental mIgG2a and the hIgG1 to hIgG3 derivatives all induced efficient bactericidal activity in vitro in the presence of human or infant rat complement and augmented bacterial clearance in complement-sufficient HsdBrlHan:WIST rats, while the hIgG4 was unable to do so. In C6-deficient PVG/c- rats, lacking complement-mediated bacterial lysis, the augmentation of bacterial clearance by PorA-specific mIgG2a and hIgG1 antibodies was impaired compared to that in the syngeneic complement-sufficient PVG/c+ rat strain. This was in contrast to the case for B-PS-specific mIgG2a, which conferred similar protective activity in both rat strains. These data suggest that while anti-B-PS antibody can provide protection in the infant rats without membrane attack complex formation, the protection afforded by anti-PorA antibody is more dependent on the activation of the whole complement pathway and subsequent bacterial lysis.

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium.

PubMed

Prechl, József; Papp, Krisztián; Hérincs, Zoltán; Péterfy, Hajna; Lóránd, Veronika; Szittner, Zoltán; Estonba, Andone; Rovero, Paolo; Paolini, Ilaria; Del Amo, Jokin; Uribarri, Maria; Alcaro, Maria Claudia; Ruiz-Larrañaga, Otsanda; Migliorini, Paola; Czirják, László

2016-01-01

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.

Natural IgM mediates complement-dependent uptake of Francisella tularensis by human neutrophils via CR1 and CR3 in nonimmune serum

PubMed Central

Schwartz, Justin T.; Barker, Jason H.; Long, Matthew E.; Kaufman, Justin; McCracken, Jenna; Allen, Lee-Ann H.

2012-01-01

A fundamental step in the life cycle of F. tularensis is bacterial entry into host cells. F. tularensis activates complement, and recent data suggest that the classical pathway is required for complement factor C3 deposition on the bacterial surface. Nevertheless, C3 deposition is inefficient and neither the specific serum components necessary for classical pathway activation by F. tularensis in nonimmune human serum, nor the receptors that mediate infection of neutrophils has been defined. Herein human neutrophil uptake of GFP-expressing F. tularensis strains LVS and Schu S4 was quantified with high efficiency by flow cytometry. Using depleted sera and purified complement components we demonstrated first that C1q and C3 were essential for F. tularensis phagocytosis whereas C5 was not. Second, we used purification and immuno-depletion approaches to identify a critical role for natural IgM in this process, and then used a wbtA2 mutant to identify LPS O-antigen and capsule as prominent targets of these antibodies on the bacterial surface. Finally, we demonstrate using receptor-blocking antibodies that CR1 (CD35) and CR3 (CD11b/CD18) acted in concert for phagocytosis of opsonized F. tularensis by human neutrophils, whereas CR3 and CR4 (CD11c/CD18) mediated infection of human monocyte-derived macrophages. Altogether, our data provide fundamental insight into mechanisms of F. tularensis phagocytosis and support a model whereby natural IgM binds to surface capsular and O-antigen polysaccharides of F. tularensis and initiates the classical complement cascade via C1q to promote C3-opsonization of the bacterium and phagocytosis via CR3 and either CR1 or CR4 in a phagocyte-specific manner. PMID:22888138

Pleiotropic Effects of Cell Wall Amidase LytA on Streptococcus pneumoniae Sensitivity to the Host Immune Response

PubMed Central

Ramos-Sevillano, Elisa; Urzainqui, Ana; Campuzano, Susana; Moscoso, Miriam; González-Camacho, Fernando; Domenech, Mirian; Rodríguez de Córdoba, Santiago; Sánchez-Madrid, Francisco; Brown, Jeremy S.; García, Ernesto

2014-01-01

The complement system is a key component of the host immune response for the recognition and clearance of Streptococcus pneumoniae. In this study, we demonstrate that the amidase LytA, the main pneumococcal autolysin, inhibits complement-mediated immunity independently of effects on pneumolysin by a complex process of impaired complement activation, increased binding of complement regulators, and direct degradation of complement C3. The use of human sera depleted of either C1q or factor B confirmed that LytA prevented activation of both the classical and alternative pathways, whereas pneumolysin inhibited only the classical pathway. LytA prevented binding of C1q and the acute-phase protein C-reactive protein to S. pneumoniae, thereby reducing activation of the classical pathway on the bacterial surface. In addition, LytA increased recruitment of the complement downregulators C4BP and factor H to the pneumococcal cell wall and directly cleaved C3b and iC3b to generate degradation products. As a consequence, C3b deposition and phagocytosis increased in the absence of LytA and were markedly enhanced for the lytA ply double mutant, confirming that a combination of LytA and Ply is essential for the establishment of pneumococcal pneumonia and sepsis in a murine model of infection. These data demonstrate that LytA has pleiotropic effects on complement activation, a finding which, in combination with the effects of pneumolysin on complement to assist with pneumococcal complement evasion, confirms a major role of both proteins for the full virulence of the microorganism during septicemia. PMID:25404032

Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies.

PubMed

Nada, R; Kumar, A; Kumar, V G; Gupta, K L; Joshi, K

2016-01-01

Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years.

Role of different pathways of the complement cascade in experimental bullous pemphigoid

PubMed Central

Nelson, Kelly C.; Zhao, Minglang; Schroeder, Pamela R.; Li, Ning; Wetsel, Rick A.; Diaz, Luis A.; Liu, Zhi

2006-01-01

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies directed against the hemidesmosomal proteins BP180 and BP230 and inflammation. Passive transfer of antibodies to the murine BP180 (mBP180) induces a skin disease that closely resembles human BP. In the present study, we defined the roles of the different complement activation pathways in this model system. Mice deficient in the alternative pathway component factor B (Fb) and injected with pathogenic anti-mBP180 IgG developed delayed and less intense subepidermal blisters. Mice deficient in the classical pathway component complement component 4 (C4) and WT mice pretreated with neutralizing antibody against the first component of the classical pathway, C1q, were resistant to experimental BP. These mice exhibited a significantly reduced level of mast cell degranulation and polymorphonuclear neutrophil (PMN) infiltration in the skin. Intradermal administration of compound 48/80, a mast cell degranulating agent, restored BP disease in C4–/– mice. Furthermore, C4–/– mice became susceptible to experimental BP after local injection of PMN chemoattractant IL-8 or local reconstitution with PMNs. These findings provide the first direct evidence to our knowledge that complement activation via the classical and alternative pathways is crucial in subepidermal blister formation in experimental BP. PMID:17024247

Isolation and initial characterisation of complement components C3 and C4 of the nurse shark and the channel catfish.

PubMed

Dodds, A W; Smith, S L; Levine, R P; Willis, A C

1998-01-01

Complement components C3 and C4 have been isolated from the serum of the nurse shark (Ginglymostoma cirratum) and of the channel catfish (Ictalurus punctatus). As in the higher vertebrates, the fish C4 proteins have three-chain structures while the C3 proteins have two-chain structures. All four proteins have intra-chain thioesters located within their highest molecular mass polypeptides. N-terminal sequence analysis of the polypeptides has confirmed the identity of the proteins. In all cases except the catfish C3 alpha-chain, which appears to have a blocked N-terminus, sequence similarities are apparent in comparisons with the chains of C3 and C4 from higher vertebrates. We have confirmed that the activity/protein previously designated C2n is the nurse shark analogue of mammalian C4. This is the first report of structural evidence for C4 in both the bony and cartilaginous fish.

A computational method for detecting copy number variations using scale-space filtering

PubMed Central

2013-01-01

Background As next-generation sequencing technology made rapid and cost-effective sequencing available, the importance of computational approaches in finding and analyzing copy number variations (CNVs) has been amplified. Furthermore, most genome projects need to accurately analyze sequences with fairly low-coverage read data. It is urgently needed to develop a method to detect the exact types and locations of CNVs from low coverage read data. Results Here, we propose a new CNV detection method, CNV_SS, which uses scale-space filtering. The scale-space filtering is evaluated by applying to the read coverage data the Gaussian convolution for various scales according to a given scaling parameter. Next, by differentiating twice and finding zero-crossing points, inflection points of scale-space filtered read coverage data are calculated per scale. Then, the types and the exact locations of CNVs are obtained by analyzing the finger print map, the contours of zero-crossing points for various scales. Conclusions The performance of CNV_SS showed that FNR and FPR stay in the range of 1.27% to 2.43% and 1.14% to 2.44%, respectively, even at a relatively low coverage (0.5x ≤C ≤2x). CNV_SS gave also much more effective results than the conventional methods in the evaluation of FNR, at 3.82% at least and 76.97% at most even when the coverage level of read data is low. CNV_SS source code is freely available from http://dblab.hallym.ac.kr/CNV SS/. PMID:23418726

Structural investigation of C4b-binding protein by molecular modeling: localization of putative binding sites.

PubMed

Villoutreix, B O; Härdig, Y; Wallqvist, A; Covell, D G; García de Frutos, P; Dahlbäck, B

1998-06-01

C4b-binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one beta-chain and seven alpha-chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy-terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its alpha-chains and with protein S through its beta-chain. The principal aim of the present study was to localize regions of C4BP involved in the interaction with C4b, Arp, and heparin. For this purpose, a computer model of the 8 CCP modules of C4BP alpha-chain was constructed, taking into account data from previous electron microscopy (EM) studies. This structure was investigated in the context of known and/or new experimental data. Analysis of the alpha-chain model, together with monoclonal antibody studies and heparin binding experiments, suggests that a patch of positively charged residues, at the interface between the first and second CCP modules, plays an important role in the interaction between C4BP and C4b/Arp/Sir/heparin. Putative binding sites, secondary-structure prediction for the central core, and an overall reevaluation of the size of the C4BP molecule are also presented. An understanding of these intermolecular interactions should contribute to the rational design of potential therapeutic agents aiming at interfering specifically some of these protein-protein interactions.

The Role of Constitutional Copy Number Variants in Breast Cancer

PubMed Central

Walker, Logan C.; Wiggins, George A.R.; Pearson, John F.

2015-01-01

Constitutional copy number variants (CNVs) include inherited and de novo deviations from a diploid state at a defined genomic region. These variants contribute significantly to genetic variation and disease in humans, including breast cancer susceptibility. Identification of genetic risk factors for breast cancer in recent years has been dominated by the use of genome-wide technologies, such as single nucleotide polymorphism (SNP)-arrays, with a significant focus on single nucleotide variants. To date, these large datasets have been underutilised for generating genome-wide CNV profiles despite offering a massive resource for assessing the contribution of these structural variants to breast cancer risk. Technical challenges remain in determining the location and distribution of CNVs across the human genome due to the accuracy of computational prediction algorithms and resolution of the array data. Moreover, better methods are required for interpreting the functional effect of newly discovered CNVs. In this review, we explore current and future application of SNP array technology to assess rare and common CNVs in association with breast cancer risk in humans. PMID:27600231

The human clinical phenotypes of altered CHRNA7 copy number.

PubMed

Gillentine, Madelyn A; Schaaf, Christian P

2015-10-15

Copy number variants (CNVs) have been implicated in multiple neuropsychiatric conditions, including autism spectrum disorder (ASD), schizophrenia, and intellectual disability (ID). Chromosome 15q13 is a hotspot for such CNVs due to the presence of low copy repeat (LCR) elements, which facilitate non-allelic homologous recombination (NAHR). Several of these CNVs have been overrepresented in individuals with neuropsychiatric disorders; yet variable expressivity and incomplete penetrance are commonly seen. Dosage sensitivity of the CHRNA7 gene, which encodes for the α7 nicotinic acetylcholine receptor in the human brain, has been proposed to have a major contribution to the observed cognitive and behavioral phenotypes, as it represents the smallest region of overlap to all the 15q13.3 deletions and duplications. Individuals with zero to four copies of CHRNA7 have been reported in the literature, and represent a range of clinical severity, with deletions causing generally more severe and more highly penetrant phenotypes. Potential mechanisms to account for the variable expressivity within each group of 15q13.3 CNVs will be discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas

PubMed Central

Hou, Yu; Guo, Huahu; Cao, Chen; Li, Xianlong; Hu, Boqiang; Zhu, Ping; Wu, Xinglong; Wen, Lu; Tang, Fuchou; Huang, Yanyi; Peng, Jirun

2016-01-01

Single-cell genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple omics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations (CNVs), DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs generally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells. PMID:26902283

Assessing genome-wide copy number variation in the Han Chinese population.

PubMed

Lu, Jianqi; Lou, Haiyi; Fu, Ruiqing; Lu, Dongsheng; Zhang, Feng; Wu, Zhendong; Zhang, Xi; Li, Changhua; Fang, Baijun; Pu, Fangfang; Wei, Jingning; Wei, Qian; Zhang, Chao; Wang, Xiaoji; Lu, Yan; Yan, Shi; Yang, Yajun; Jin, Li; Xu, Shuhua

2017-10-01

Copy number variation (CNV) is a valuable source of genetic diversity in the human genome and a well-recognised cause of various genetic diseases. However, CNVs have been considerably under-represented in population-based studies, particularly the Han Chinese which is the largest ethnic group in the world. To build a representative CNV map for the Han Chinese population. We conducted a genome-wide CNV study involving 451 male Han Chinese samples from 11 geographical regions encompassing 28 dialect groups, representing a less-biased panel compared with the currently available data. We detected CNVs by using 4.2M NimbleGen comparative genomic hybridisation array and whole-genome deep sequencing of 51 samples to optimise the filtering conditions in CNV discovery. A comprehensive Han Chinese CNV map was built based on a set of high-quality variants (positive predictive value >0.8, with sizes ranging from 369 bp to 4.16 Mb and a median of 5907 bp). The map consists of 4012 CNV regions (CNVRs), and more than half are novel to the 30 East Asian CNV Project and the 1000 Genomes Project Phase 3. We further identified 81 CNVRs specific to regional groups, which was indicative of the subpopulation structure within the Han Chinese population. Our data are complementary to public data sources, and the CNV map may facilitate in the identification of pathogenic CNVs and further biomedical research studies involving the Han Chinese population. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Alternative complement pathway activation during invasive coronary procedures in acute myocardial infarction and stable angina pectoris.

PubMed

Horváth, Zsófia; Csuka, Dorottya; Vargova, Katarina; Kovács, Andrea; Leé, Sarolta; Varga, Lilian; Préda, István; Tóth Zsámboki, Emese; Prohászka, Zoltán; Kiss, Róbert Gábor

2016-12-01

The effect of invasive percutaneous coronary procedures on complement activation has not been elucidated. We enrolled stable angina patients with elective percutaneous coronary intervention (SA-PCI, n=24), diagnostic coronary angiography (CA, n=52) and 23 patients with ST segment elevation myocardial infarction and primary PCI (STEMI-PCI). Complement activation products (C1rC1sC1inh, C3bBbP and SC5b-9) were measured on admission, 6 and 24h after coronary procedures. The alternative pathway product, C3bBbP significantly and reversibly increased 6h after elective PCI (baseline: 7.81AU/ml, 6h: 16.09AU/ml, 24h: 4.27AU/ml, p<0.01, n=23) and diagnostic angiography (baseline: 6.13AU/ml, 6h: 12.08AU/ml, 24h: 5.4AU/ml, p<0.01, n=52). Six hour C3bBbP values correlated with post-procedural CK, creatinine level and the applied contrast material volume (r=0.41, r=0.4, r=0.3, p<0.05, respectively). In STEMI-PCI, baseline C3bBbP level was higher, compared to SA-PCI or CA patients (11.33AU/ml vs. 7.81AU/ml or 6.13AU/ml, p<0.001). Similarly, the terminal complex (SC5b-9) level was already elevated at baseline compared to SA-PCI group (3.49AU/ml vs. 1.87AU/ml, p=0.011). Complement pathway products did not increase further after primary PCI. Elective coronary procedures induced transient alternative complement pathway activation, influenced by the applied contrast volume. In STEMI, the alternative complement pathway is promptly activated during the atherothrombotic event and PCI itself had no further detectable effect. Copyright © 2016 Elsevier B.V. All rights reserved.

Large transcription units unify copy number variants and common fragile sites arising under replication stress.

PubMed

Wilson, Thomas E; Arlt, Martin F; Park, So Hae; Rajendran, Sountharia; Paulsen, Michelle; Ljungman, Mats; Glover, Thomas W

2015-02-01

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus- and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics. © 2015 Wilson et al.; Published by Cold Spring Harbor Laboratory Press.

Copy number variants and genetic polymorphisms in TBX21, GATA3, Rorc, Foxp3 and susceptibility to Behcet's disease and Vogt-Koyanagi-Harada syndrome.

PubMed

Liao, Dan; Hou, Shengping; Zhang, Jun; Fang, Jing; Liu, Yunjia; Bai, Lin; Cao, Qingfeng; Kijlstra, Aize; Yang, Peizeng

2015-04-15

This study aimed to investigate the role of genetic variants including single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) of TBX21, GATA3, Rorc and Foxp3 genes in Behcet's disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese Han population. Genotyping of 25 SNPs was performed by iPLEX system (Sequenom) or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). TaqMan real time PCR was used to assess CNVs. The expression of Rorc and Foxp3 were examined by real-time PCR and cytokine production was measured by ELISA. High Rorc CNV was associated with the susceptibility to BD (P = 8.99 × 10(-8), OR = 3.0), and low Foxp3 CNV predisposed to BD in female patients (P = 1.92 × 10(-5), OR = 3.1). CNVs for the investigated genes were not altered in VKH syndrome. Further functional studies demonstrated that the relative mRNA expression levels of Rorc were increased in individuals with high Rorc copy number, but not for Foxp3. Increased production of IL-1β and IL-6 was found in individuals carrying a high CNV of Rorc. Our study showed that high CNVs of Rorc and low CNVs of Foxp3 confer risk for BD but not for VKH syndrome. The tested 25 SNPs in TBX21, GATA3, Rorc and Foxp3 did not associate with BD and VKH syndrome.

Complex and multi-allelic copy number variation in human disease

PubMed Central

McCarroll, Steven A.

2015-01-01

Hundreds of copy number variants are complex and multi-allelic, in that they have many structural alleles and have rearranged multiple times in the ancestors who contributed chromosomes to current humans. Not only are the relationships of these multi-allelic CNVs (mCNVs) to phenotypes generally unknown, but many mCNVs have not yet been described at the basic levels—alleles, allele frequencies, structural features—that support genetic investigation. To date, most reported disease associations to these variants have been ascertained through candidate gene studies. However, only a few associations have reached the level of acceptance defined by durable replications in many cohorts. This likely stems from longstanding challenges in making precise molecular measurements of the alleles individuals have at these loci. However, approaches for mCNV analysis are improving quickly, and some of the unique characteristics of mCNVs may assist future association studies. Their various structural alleles are likely to have different magnitudes of effect, creating a natural allelic series of growing phenotypic impact and giving investigators a set of natural predictions and testable hypotheses about the extent to which each allele of an mCNV predisposes to a phenotype. Also, mCNVs’ low-to-modest correlation to individual single-nucleotide polymorphisms (SNPs) may make it easier to distinguish between mCNVs and nearby SNPs as the drivers of an association signal, and perhaps, make it possible to preliminarily screen candidate loci, or the entire genome, for the many mCNV–disease relationships that remain to be discovered. PMID:26163405

Detection of clinically relevant copy-number variants by exome sequencing in a large cohort of genetic disorders

PubMed Central

Pfundt, Rolph; del Rosario, Marisol; Vissers, Lisenka E.L.M.; Kwint, Michael P.; Janssen, Irene M.; de Leeuw, Nicole; Yntema, Helger G.; Nelen, Marcel R.; Lugtenberg, Dorien; Kamsteeg, Erik-Jan; Wieskamp, Nienke; Stegmann, Alexander P.A.; Stevens, Servi J.C.; Rodenburg, Richard J.T.; Simons, Annet; Mensenkamp, Arjen R.; Rinne, Tuula; Gilissen, Christian; Scheffer, Hans; Veltman, Joris A.; Hehir-Kwa, Jayne Y.

2017-01-01

Purpose: Copy-number variation is a common source of genomic variation and an important genetic cause of disease. Microarray-based analysis of copy-number variants (CNVs) has become a first-tier diagnostic test for patients with neurodevelopmental disorders, with a diagnostic yield of 10–20%. However, for most other genetic disorders, the role of CNVs is less clear and most diagnostic genetic studies are generally limited to the study of single-nucleotide variants (SNVs) and other small variants. With the introduction of exome and genome sequencing, it is now possible to detect both SNVs and CNVs using an exome- or genome-wide approach with a single test. Methods: We performed exome-based read-depth CNV screening on data from 2,603 patients affected by a range of genetic disorders for which exome sequencing was performed in a diagnostic setting. Results: In total, 123 clinically relevant CNVs ranging in size from 727 bp to 15.3 Mb were detected, which resulted in 51 conclusive diagnoses and an overall increase in diagnostic yield of ~2% (ranging from 0 to –5.8% per disorder). Conclusions: This study shows that CNVs play an important role in a broad range of genetic disorders and that detection via exome-based CNV profiling results in an increase in the diagnostic yield without additional testing, bringing us closer to single-test genomics. Genet Med advance online publication 27 October 2016 PMID:28574513

Large transcription units unify copy number variants and common fragile sites arising under replication stress

PubMed Central

Park, So Hae; Rajendran, Sountharia; Paulsen, Michelle; Ljungman, Mats; Glover, Thomas W.

2015-01-01

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus- and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics. PMID:25373142

iCopyDAV: Integrated platform for copy number variations—Detection, annotation and visualization

PubMed Central

Vogeti, Sriharsha

2018-01-01

Discovery of copy number variations (CNVs), a major category of structural variations, have dramatically changed our understanding of differences between individuals and provide an alternate paradigm for the genetic basis of human diseases. CNVs include both copy gain and copy loss events and their detection genome-wide is now possible using high-throughput, low-cost next generation sequencing (NGS) methods. However, accurate detection of CNVs from NGS data is not straightforward due to non-uniform coverage of reads resulting from various systemic biases. We have developed an integrated platform, iCopyDAV, to handle some of these issues in CNV detection in whole genome NGS data. It has a modular framework comprising five major modules: data pre-treatment, segmentation, variant calling, annotation and visualization. An important feature of iCopyDAV is the functional annotation module that enables the user to identify and prioritize CNVs encompassing various functional elements, genomic features and disease-associations. Parallelization of the segmentation algorithms makes the iCopyDAV platform even accessible on a desktop. Here we show the effect of sequencing coverage, read length, bin size, data pre-treatment and segmentation approaches on accurate detection of the complete spectrum of CNVs. Performance of iCopyDAV is evaluated on both simulated data and real data for different sequencing depths. It is an open-source integrated pipeline available at https://github.com/vogetihrsh/icopydav and as Docker’s image at http://bioinf.iiit.ac.in/icopydav/. PMID:29621297

Rare copy number variants in patients with congenital conotruncal heart defects.

PubMed

Xie, Hongbo M; Werner, Petra; Stambolian, Dwight; Bailey-Wilson, Joan E; Hakonarson, Hakon; White, Peter S; Taylor, Deanne M; Goldmuntz, Elizabeth

2017-03-01

Previous studies using different cardiac phenotypes, technologies and designs suggest a burden of large, rare or de novo copy number variants (CNVs) in subjects with congenital heart defects. We sought to identify disease-related CNVs, candidate genes, and functional pathways in a large number of cases with conotruncal and related defects that carried no known genetic syndrome. Cases and control samples were divided into two cohorts and genotyped to assess each subject's CNV content. Analyses were performed to ascertain differences in overall CNV prevalence and to identify enrichment of specific genes and functional pathways in conotruncal cases relative to healthy controls. Only findings present in both cohorts are presented. From 973 total conotruncal cases, a burden of rare CNVs was detected in both cohorts. Candidate genes from rare CNVs found in both cohorts were identified based on their association with cardiac development or disease, and/or their reported disruption in published studies. Functional and pathway analyses revealed significant enrichment of terms involved in either heart or early embryonic development. Our study tested one of the largest cohorts specifically with cardiac conotruncal and related defects. These results confirm and extend previous findings that CNVs contribute to disease risk for congenital heart defects in general and conotruncal defects in particular. As disease heterogeneity renders identification of single recurrent genes or loci difficult, functional pathway and gene regulation network analyses appear to be more informative. Birth Defects Research 109:271-295, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Rare copy number variants and congenital heart defects in the 22q11.2 deletion syndrome.

PubMed

Mlynarski, Elisabeth E; Xie, Michael; Taylor, Deanne; Sheridan, Molly B; Guo, Tingwei; Racedo, Silvia E; McDonald-McGinn, Donna M; Chow, Eva W C; Vorstman, Jacob; Swillen, Ann; Devriendt, Koen; Breckpot, Jeroen; Digilio, Maria Cristina; Marino, Bruno; Dallapiccola, Bruno; Philip, Nicole; Simon, Tony J; Roberts, Amy E; Piotrowicz, Małgorzata; Bearden, Carrie E; Eliez, Stephan; Gothelf, Doron; Coleman, Karlene; Kates, Wendy R; Devoto, Marcella; Zackai, Elaine; Heine-Suñer, Damian; Goldmuntz, Elizabeth; Bassett, Anne S; Morrow, Bernice E; Emanuel, Beverly S

2016-03-01

The 22q11.2 deletion syndrome (22q11DS; velocardiofacial/DiGeorge syndrome; VCFS/DGS; MIM #192430; 188400) is the most common microdeletion syndrome. The phenotypic presentation of 22q11DS is highly variable; approximately 60-75 % of 22q11DS patients have been reported to have a congenital heart defect (CHD), mostly of the conotruncal type, and/or aortic arch defect. The etiology of the cardiac phenotypic variability is not currently known for the majority of patients. We hypothesized that rare copy number variants (CNVs) outside the 22q11.2 deleted region may modify the risk of being born with a CHD in this sensitized population. Rare CNV analysis was performed using Affymetrix SNP Array 6.0 data from 946 22q11DS subjects with CHDs (n = 607) or with normal cardiac anatomy (n = 339). Although there was no significant difference in the overall burden of rare CNVs, an overabundance of CNVs affecting cardiac-related genes was detected in 22q11DS individuals with CHDs. When the rare CNVs were examined with regard to gene interactions, specific cardiac networks, such as Wnt signaling, appear to be overrepresented in 22q11DS CHD cases but not 22q11DS controls with a normal heart. Collectively, these data suggest that CNVs outside the 22q11.2 region may contain genes that modify risk for CHDs in some 22q11DS patients.

Species Specificity of Vaccinia Virus Complement Control Protein for the Bovine Classical Pathway Is Governed Primarily by Direct Interaction of Its Acidic Residues with Factor I

PubMed Central

Kumar, Jitendra; Yadav, Viveka Nand; Phulera, Swastik; Kamble, Ashish; Gautam, Avneesh Kumar; Panwar, Hemendra Singh

2017-01-01

ABSTRACT Poxviruses display species tropism—variola virus is a human-specific virus, while vaccinia virus causes repeated outbreaks in dairy cattle. Consistent with this, variola virus complement regulator SPICE (smallpox inhibitor of complement enzymes) exhibits selectivity in inhibiting the human alternative complement pathway and vaccinia virus complement regulator VCP (vaccinia virus complement control protein) displays selectivity in inhibiting the bovine alternative complement pathway. In the present study, we examined the species specificity of VCP and SPICE for the classical pathway (CP). We observed that VCP is ∼43-fold superior to SPICE in inhibiting bovine CP. Further, functional assays revealed that increased inhibitory activity of VCP for bovine CP is solely due to its enhanced cofactor activity, with no effect on decay of bovine CP C3-convertase. To probe the structural basis of this specificity, we utilized single- and multi-amino-acid substitution mutants wherein 1 or more of the 11 variant VCP residues were substituted in the SPICE template. Examination of these mutants for their ability to inhibit bovine CP revealed that E108, E120, and E144 are primarily responsible for imparting the specificity and contribute to the enhanced cofactor activity of VCP. Binding and functional assays suggested that these residues interact with bovine factor I but not with bovine C4(H2O) (a moiety conformationally similar to C4b). Mapping of these residues onto the modeled structure of bovine C4b-VCP-bovine factor I supported the mutagenesis data. Taken together, our data help explain why the vaccine strain of vaccinia virus was able to gain a foothold in domesticated animals. IMPORTANCE Vaccinia virus was used for smallpox vaccination. The vaccine-derived virus is now circulating and causing outbreaks in dairy cattle in India and Brazil. However, the reason for this tropism is unknown. It is well recognized that the virus is susceptible to neutralization by the complement classical pathway (CP). Because the virus encodes a soluble complement regulator, VCP, we examined whether this protein displays selectivity in targeting bovine CP. Our data show that it does exhibit selectivity in inhibiting the bovine CP and that this is primarily determined by its amino acids E108, E120, and E144, which interact with bovine serine protease factor I to inactivate bovine C4b—one of the two subunits of CP C3-convertase. Of note, the variola complement regulator SPICE contains positively charged residues at these positions. Thus, these variant residues in VCP help enhance its potency against the bovine CP and thereby the fitness of the virus in cattle. PMID:28724763

Structure of C3b reveals conformational changes that underlie complement activity.

PubMed

Janssen, Bert J C; Christodoulidou, Agni; McCarthy, Andrew; Lambris, John D; Gros, Piet

2006-11-09

Resistance to infection and clearance of cell debris in mammals depend on the activation of the complement system, which is an important component of innate and adaptive immunity. Central to the complement system is the activated form of C3, called C3b, which attaches covalently to target surfaces to amplify complement response, label cells for phagocytosis and stimulate the adaptive immune response. C3b consists of 1,560 amino-acid residues and has 12 domains. It binds various proteins and receptors to effect its functions. However, it is not known how C3 changes its conformation into C3b and thereby exposes its many binding sites. Here we present the crystal structure at 4-A resolution of the activated complement protein C3b and describe the conformational rearrangements of the 12 domains that take place upon proteolytic activation. In the activated form the thioester is fully exposed for covalent attachment to target surfaces and is more than 85 A away from the buried site in native C3 (ref. 5). Marked domain rearrangements in the alpha-chain present an altered molecular surface, exposing hidden and cryptic sites that are consistent with known putative binding sites of factor B and several complement regulators. The structural data indicate that the large conformational changes in the proteolytic activation and regulation of C3 take place mainly in the first conversion step, from C3 to C3b. These insights are important for the development of strategies to treat immune disorders that involve complement-mediated inflammation.

Role of CYP2E1 immunoglobulin G4 subclass antibodies and complement in pathogenesis of idiosyncratic drug-induced hepatitis.

PubMed

Njoku, Dolores B; Mellerson, Jenelle L; Talor, Monica V; Kerr, Douglas R; Faraday, Nauder R; Outschoorn, Ingrid; Rose, Noel R

2006-02-01

Idiosyncratic drug-induced hepatitis (IDDIH) is the third most common cause for acute liver failure in the United States. Previous studies have attempted to identify susceptible patients or early stages of disease with various degrees of success. To determine if total serum immunoglobulin subclasses, CYP2E1-specific subclass autoantibodies, complement components, or immune complexes could distinguish persons with IDDIH from others exposed to drugs, we studied persons exposed to halogenated volatile anesthetics, which have been associated with IDDIH and CYP2E1 autoantibodies. We found that patients with anesthetic-induced IDDIH had significantly elevated levels of CYP2E1-specific immunoglobulin G4 (IgG4) autoantibodies, while anesthetic-exposed healthy persons had significantly elevated levels of CYP2E1-specific IgG1 autoantibodies. Anesthetic IDDIH patients had significantly lower levels of C4a, C3a, and C5a compared to anesthetic-exposed healthy persons. C1q- and C3d-containing immune complexes were significantly elevated in anesthetic-exposed persons. In conclusion, our data suggest that anesthetic-exposed persons develop CYP2E1-specific IgG1 autoantibodies which may form detectable circulating immune complexes subsequently cleared by classical pathway activation of the complement system. Persons susceptible to anesthetic-induced IDDIH develop CYP2E1-specific IgG4 autoantibodies which form small, nonprecipitating immune complexes that escape clearance because of their size or by direct inhibition of complement activation.

CNV analysis in the Lithuanian population.

PubMed

Urnikyte, A; Domarkiene, I; Stoma, S; Ambrozaityte, L; Uktveryte, I; Meskiene, R; Kasiulevičius, V; Burokiene, N; Kučinskas, V

2016-05-04

Although copy number variation (CNV) has received much attention, knowledge about the characteristics of CNVs such as occurrence rate and distribution in the genome between populations and within the same population is still insufficient. In this study, Illumina 770 K HumanOmniExpress-12 v1.0 (and v1.1) arrays were used to examine the diversity and distribution of CNVs in 286 unrelated individuals from the two main ethnolinguistic groups of the Lithuanian population (Aukštaičiai and Žemaičiai) (see Additional file 3). For primary data analysis, the Illumina GenomeStudio™ Genotyping Module v1.9 and two algorithms, cnvPartition 3.2.0 and QuantiSNP 2.0, were used to identify high-confidence CNVs. A total of 478 autosomal CNVs were detected by both algorithms, and those were clustered in 87 copy number variation regions (CNVRs), spanning ~12.5 Mb of the genome (see Table 1). At least 8.6 % of the CNVRs were unique and had not been reported in the Database of Genomic Variants. Most CNVRs (57.5 %) were rare, with a frequency of <1 %, whereas common CNVRs with at least 5 % frequency made up only 1.1 % of all CNVRs identified. About 49 % of non-singleton CNVRs were shared between Aukštaičiai and Žemaičiai, and the remaining CNVRs were specific to each group. Many of the CNVs detected (66 %) overlapped with known UCSC gene regions. The ethnolinguistic groups of the Lithuanian population could not be differentiated based on CNV profiles, which may reflect their geographical proximity and suggest the homogeneity of the Lithuanian population. In addition, putative novel CNVs unique to the Lithuanian population were identified. The results of our study enhance the CNV map of the Lithuanian population.

cn.MOPS: mixture of Poissons for discovering copy number variations in next-generation sequencing data with a low false discovery rate

PubMed Central

Klambauer, Günter; Schwarzbauer, Karin; Mayr, Andreas; Clevert, Djork-Arné; Mitterecker, Andreas; Bodenhofer, Ulrich; Hochreiter, Sepp

2012-01-01

Quantitative analyses of next-generation sequencing (NGS) data, such as the detection of copy number variations (CNVs), remain challenging. Current methods detect CNVs as changes in the depth of coverage along chromosomes. Technological or genomic variations in the depth of coverage thus lead to a high false discovery rate (FDR), even upon correction for GC content. In the context of association studies between CNVs and disease, a high FDR means many false CNVs, thereby decreasing the discovery power of the study after correction for multiple testing. We propose ‘Copy Number estimation by a Mixture Of PoissonS’ (cn.MOPS), a data processing pipeline for CNV detection in NGS data. In contrast to previous approaches, cn.MOPS incorporates modeling of depths of coverage across samples at each genomic position. Therefore, cn.MOPS is not affected by read count variations along chromosomes. Using a Bayesian approach, cn.MOPS decomposes variations in the depth of coverage across samples into integer copy numbers and noise by means of its mixture components and Poisson distributions, respectively. The noise estimate allows for reducing the FDR by filtering out detections having high noise that are likely to be false detections. We compared cn.MOPS with the five most popular methods for CNV detection in NGS data using four benchmark datasets: (i) simulated data, (ii) NGS data from a male HapMap individual with implanted CNVs from the X chromosome, (iii) data from HapMap individuals with known CNVs, (iv) high coverage data from the 1000 Genomes Project. cn.MOPS outperformed its five competitors in terms of precision (1–FDR) and recall for both gains and losses in all benchmark data sets. The software cn.MOPS is publicly available as an R package at http://www.bioinf.jku.at/software/cnmops/ and at Bioconductor. PMID:22302147

Search for Rare Copy-Number Variants in Congenital Heart Defects Identifies Novel Candidate Genes and a Potential Role for FOXC1 in Patients With Coarctation of the Aorta.

PubMed

Sanchez-Castro, Marta; Eldjouzi, Hadja; Charpentier, Eric; Busson, Pierre-François; Hauet, Quentin; Lindenbaum, Pierre; Delasalle-Guyomarch, Béatrice; Baudry, Adrien; Pichon, Olivier; Pascal, Cécile; Lefort, Bruno; Bajolle, Fanny; Pezard, Philippe; Schott, Jean-Jacques; Dina, Christian; Redon, Richard; Gournay, Véronique; Bonnet, Damien; Le Caignec, Cédric

2016-02-01

Congenital heart defects are the most frequent malformations among newborns and a frequent cause of morbidity and mortality. Although genetic variation contributes to congenital heart defects, their precise molecular bases remain unknown in the majority of patients. We analyzed, by high-resolution array comparative genomic hybridization, 316 children with sporadic, nonsyndromic congenital heart defects, including 76 coarctation of the aorta, 159 transposition of the great arteries, and 81 tetralogy of Fallot, as well as their unaffected parents. We identified by array comparative genomic hybridization, and validated by quantitative real-time polymerase chain reaction, 71 rare de novo (n=8) or inherited (n=63) copy-number variants (CNVs; 50 duplications and 21 deletions) in patients. We identified 113 candidate genes for congenital heart defects within these CNVs, including BTRC, CHRNB3, CSRP2BP, ERBB2, ERMARD, GLIS3, PLN, PTPRJ, RLN3, and TCTE3. No de novo CNVs were identified in patients with transposition of the great arteries in contrast to coarctation of the aorta and tetralogy of Fallot (P=0.002; Fisher exact test). A search for transcription factor binding sites showed that 93% of the rare CNVs identified in patients with coarctation of the aorta contained at least 1 gene with FOXC1-binding sites. This significant enrichment (P<0.0001; permutation test) was not observed for the CNVs identified in patients with transposition of the great arteries and tetralogy of Fallot. We hypothesize that these CNVs may alter the expression of genes regulated by FOXC1. Foxc1 belongs to the forkhead transcription factors family, which plays a critical role in cardiovascular development in mice. These data suggest that deregulation of FOXC1 or its downstream genes play a major role in the pathogenesis of coarctation of the aorta in humans. © 2015 American Heart Association, Inc.

cn.MOPS: mixture of Poissons for discovering copy number variations in next-generation sequencing data with a low false discovery rate.

PubMed

Klambauer, Günter; Schwarzbauer, Karin; Mayr, Andreas; Clevert, Djork-Arné; Mitterecker, Andreas; Bodenhofer, Ulrich; Hochreiter, Sepp

2012-05-01

Quantitative analyses of next-generation sequencing (NGS) data, such as the detection of copy number variations (CNVs), remain challenging. Current methods detect CNVs as changes in the depth of coverage along chromosomes. Technological or genomic variations in the depth of coverage thus lead to a high false discovery rate (FDR), even upon correction for GC content. In the context of association studies between CNVs and disease, a high FDR means many false CNVs, thereby decreasing the discovery power of the study after correction for multiple testing. We propose 'Copy Number estimation by a Mixture Of PoissonS' (cn.MOPS), a data processing pipeline for CNV detection in NGS data. In contrast to previous approaches, cn.MOPS incorporates modeling of depths of coverage across samples at each genomic position. Therefore, cn.MOPS is not affected by read count variations along chromosomes. Using a Bayesian approach, cn.MOPS decomposes variations in the depth of coverage across samples into integer copy numbers and noise by means of its mixture components and Poisson distributions, respectively. The noise estimate allows for reducing the FDR by filtering out detections having high noise that are likely to be false detections. We compared cn.MOPS with the five most popular methods for CNV detection in NGS data using four benchmark datasets: (i) simulated data, (ii) NGS data from a male HapMap individual with implanted CNVs from the X chromosome, (iii) data from HapMap individuals with known CNVs, (iv) high coverage data from the 1000 Genomes Project. cn.MOPS outperformed its five competitors in terms of precision (1-FDR) and recall for both gains and losses in all benchmark data sets. The software cn.MOPS is publicly available as an R package at http://www.bioinf.jku.at/software/cnmops/ and at Bioconductor.

Protective immune responses against West Nile virus are primed by distinct complement activation pathways.

PubMed

Mehlhop, Erin; Diamond, Michael S

2006-05-15

West Nile virus (WNV) causes a severe infection of the central nervous system in several vertebrate animals including humans. Prior studies have shown that complement plays a critical role in controlling WNV infection in complement (C) 3(-/-) and complement receptor 1/2(-/-) mice. Here, we dissect the contributions of the individual complement activation pathways to the protection from WNV disease. Genetic deficiencies in C1q, C4, factor B, or factor D all resulted in increased mortality in mice, suggesting that all activation pathways function together to limit WNV spread. In the absence of alternative pathway complement activation, WNV disseminated into the central nervous system at earlier times and was associated with reduced CD8+ T cell responses yet near normal anti-WNV antibody profiles. Animals lacking the classical and lectin pathways had deficits in both B and T cell responses to WNV. Finally, and somewhat surprisingly, C1q was required for productive infection in the spleen but not for development of adaptive immune responses after WNV infection. Our results suggest that individual pathways of complement activation control WNV infection by priming adaptive immune responses through distinct mechanisms.

An initial comparative map of copy number variations in the goat (Capra hircus) genome

PubMed Central

2010-01-01

Background The goat (Capra hircus) represents one of the most important farm animal species. It is reared in all continents with an estimated world population of about 800 million of animals. Despite its importance, studies on the goat genome are still in their infancy compared to those in other farm animal species. Comparative mapping between cattle and goat showed only a few rearrangements in agreement with the similarity of chromosome banding. We carried out a cross species cattle-goat array comparative genome hybridization (aCGH) experiment in order to identify copy number variations (CNVs) in the goat genome analysing animals of different breeds (Saanen, Camosciata delle Alpi, Girgentana, and Murciano-Granadina) using a tiling oligonucleotide array with ~385,000 probes designed on the bovine genome. Results We identified a total of 161 CNVs (an average of 17.9 CNVs per goat), with the largest number in the Saanen breed and the lowest in the Camosciata delle Alpi goat. By aggregating overlapping CNVs identified in different animals we determined CNV regions (CNVRs): on the whole, we identified 127 CNVRs covering about 11.47 Mb of the virtual goat genome referred to the bovine genome (0.435% of the latter genome). These 127 CNVRs included 86 loss and 41 gain and ranged from about 24 kb to about 1.07 Mb with a mean and median equal to 90,292 bp and 49,530 bp, respectively. To evaluate whether the identified goat CNVRs overlap with those reported in the cattle genome, we compared our results with those obtained in four independent cattle experiments. Overlapping between goat and cattle CNVRs was highly significant (P < 0.0001) suggesting that several chromosome regions might contain recurrent interspecies CNVRs. Genes with environmental functions were over-represented in goat CNVRs as reported in other mammals. Conclusions We describe a first map of goat CNVRs. This provides information on a comparative basis with the cattle genome by identifying putative recurrent interspecies CNVs between these two ruminant species. Several goat CNVs affect genes with important biological functions. Further studies are needed to evaluate the functional relevance of these CNVs and their effects on behavior, production, and disease resistance traits in goats. PMID:21083884

Complement Effectors of Inflammation in Cystic Fibrosis Lung Fluid Correlate with Clinical Measures of Disease.

PubMed

Sass, Laura A; Hair, Pamela S; Perkins, Amy M; Shah, Tushar A; Krishna, Neel K; Cunnion, Kenji M

2015-01-01

In cystic fibrosis (CF), lung damage is mediated by a cycle of obstruction, infection, and inflammation. Here we explored complement inflammatory effectors in CF lung fluid. In this study soluble fractions (sols) from sputum samples of 15 CF patients were assayed for complement effectors and analyzed with clinical measurements. The pro-inflammatory peptide C5a was increased 4.8-fold (P = 0.04) in CF sols compared with controls. Incubation of CF sols with P. aeruginosa or S. aureus increased C5a concentration 2.3-fold (P = 0.02). A peptide inhibitor of complement C1 (PIC1) completely blocked the increase in C5a concentration from P. aeruginosa in CF sol in vitro (P = 0.001). C5a concentration in CF sol correlated inversely with body mass index (BMI) percentile in children (r = -0.77, P = 0.04). C3a, which has anti-inflammatory effects, correlated positively with FEV1% predicted (rs = 0.63, P = 0.02). These results suggest that complement effectors may significantly impact inflammation in CF lung fluid.

Glomeruli of Dense Deposit Disease contain components of the alternative and terminal complement pathway

PubMed Central

Sethi, Sanjeev; Gamez, Jeffrey D.; Vrana, Julie A.; Theis, Jason D.; Bergen, H. Robert; Zipfel, Peter F.; Dogan, Ahmet; Smith, Richard J. H.

2009-01-01

Dense Deposit Disease (DDD), or membranoproliferative glomerulonephritis type II, is a rare renal disease characterized by dense deposits in the mesangium and along the glomerular basement membranes that can be seen by electron microscopy. Although these deposits contain complement factor C3, as determined by immunofluorescence microscopy, their precise composition remains unknown. To address this question, we used mass spectrometry to identify the proteins in laser microdissected glomeruli isolated from paraffin-embedded tissue of eight confirmed cases of DDD. Compared to glomeruli from five control patients, we found that all of the glomeruli from patients with DDD contain components of the alternative pathway and terminal complement complex. Factor C9 was uniformly present as well as the two fluid-phase regulators of terminal complement complex clusterin and vitronectin. In contrast, in nine patients with immune complex–mediated membranoproliferative glomerulonephritis, glomerular samples contained mainly immunoglobulins and complement factors C3 and C4. Our study shows that in addition to fluid-phase dysregulation of the alternative pathway, soluble components of the terminal complement complex contribute to glomerular lesions found in DDD. PMID:19177158

Relative importance of complement-mediated bactericidal and opsonic activity for protection against meningococcal disease.

PubMed

Granoff, Dan M

2009-06-24

Killing of Neisseria meningitidis can result from complement-mediated serum bactericidal activity (SBA) or opsonophagocytosis (OPA), or a combination of the two mechanisms. While SBA titers > or =1:4 confer protection, recent evidence suggests that this threshold titer may not be required. For example, the incidence of meningococcal disease declines between ages 1 and 4 years without evidence of acquisition of SBA titers > or =1:4. Meningococcal polysaccharide vaccination also elicited OPA and lowered the risk of disease in patients with late complement component deficiencies whose sera did not support SBA. Sera from healthy adults immunized with an outer membrane vesicle vaccine showed OPA killing of N. meningitidis with C6-depleted complement, and whole blood from complement-sufficient non-immunized adults with SBA titers <1:4 also frequently had killing activity. Collectively the data indicate that SBA titers <1:4 and/or vaccine-induced OPA can confer protection against meningococcal disease.

Relative importance of complement-mediated bactericidal and opsonic activity for protection against meningococcal disease

PubMed Central

Granoff, Dan M.

2009-01-01

Killing of Neisseria meningitidis can result from complement-mediated bactericidal activity (SBA) or opsonophagocytosis (OPA), or a combination of the two mechanisms. While SBA titers ≥1:4 confer protection, recent evidence suggests that this threshold titer may not be required. For example, the incidence of meningococcal disease declines between ages 1 and 4 years without evidence of acquisition of SBA titers ≥1:4. Meningococcal polysaccharide vaccination also elicited OPA and lowered the risk of disease in patients with late complement component deficiencies whose sera did not support SBA. Sera from healthy adults immunized with an outer membrane vesicle vaccine showed OPA killing of N. meningitidis with C6-depleted complement, and whole blood from complement-sufficient non-immunized adults with SBA titers <1:4 also frequently had killing activity. Collectively the data indicate that SBA titers <1:4 and/or vaccine-induced OPA can confer protection against meningococcal disease. PMID:19477054

Cell-derived microparticles and complement activation in preeclampsia versus normal pregnancy.

PubMed

Biró, E; Lok, C A R; Hack, C E; van der Post, J A M; Schaap, M C L; Sturk, A; Nieuwland, R

2007-01-01

Inflammation plays a major role in the vascular dysfunction seen in preeclampsia, and several studies suggest involvement of the complement system. To investigate whether complement activation on the surface of microparticles is increased in plasma of preeclamptic patients versus healthy pregnant controls. Microparticles from plasma of preeclamptic (n=10), healthy pregnant (n=10) and healthy nonpregnant (n=10) women were analyzed by flow cytometry for bound complement components (C1q, C4, C3) and complement activator molecules (C-reactive protein [CRP], serum amyloid P component [SAP], immunoglobulin [Ig]M, IgG). Fluid phase complement activation products and activator molecules were also determined. Levels of microparticles with bound complement components showed no increase in complement activation on the microparticle surface in preeclamptic women, in line with levels of fluid phase complement activation products. In healthy nonpregnant and pregnant women, bound CRP was associated with classical pathway activation on the microparticle surface, and in healthy pregnant women IgM and IgG molecules also contributed. In preeclamptic women, microparticles with bound SAP and those with IgG seemed to contribute to C1q binding without a clear association to further classical pathway activation. Furthermore, significantly increased levels of microparticles with bound CRP were present in preeclamptic compared with healthy pregnant women (median 178x10(6)/L versus 47x10(6)/L, P<0.01), but without concomitant increases in complement activation. We found no evidence of increased complement activation on the microparticle surface in preeclamptic women. Microparticles with bound CRP were significantly increased, but in contrast to healthy pregnant and nonpregnant women, this was not associated with increased classical pathway activation on the surface of the microparticles.

An amphioxus gC1q protein binds human IgG and initiates the classical pathway: Implications for a C1q-mediated complement system in the basal chordate.

PubMed

Gao, Zhan; Li, Mengyang; Ma, Jie; Zhang, Shicui

2014-12-01

The origin of the classical complement pathway remains open during chordate evolution. A C1q-like member, BjC1q, was identified in the basal chordate amphioxus. It is predominantly expressed in the hepatic caecum, hindgut, and notochord, and is significantly upregulated following challenge with bacteria or lipoteichoic acid and LPS. Recombinant BjC1q and its globular head domain specifically interact with lipoteichoic acid and LPS, but BjC1q displays little lectin activity. Moreover, rBjC1q can assemble to form the high molecular weight oligomers necessary for binding to proteases C1r/C1s and for complement activation, and binds human C1r/C1s/mannan-binding lectin-associated serine protease-2 as well as amphioxus serine proteases involved in the cleavage of C4/C2, and C3 activation. Importantly, rBjC1q binds with human IgG as well as an amphioxus Ig domain containing protein, resulting in the activation of the classical complement pathway. This is the first report showing that a C1q-like protein in invertebrates is able to initiate classical pathway, raising the possibility that amphioxus possesses a C1q-mediated complement system. It also suggests a new scenario for the emergence of the classical complement pathway, in contrast to the proposal that the lectin pathway evolved into the classical pathway. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Distribution of Disease-Associated Copy Number Variants across Distinct Disorders of Cognitive Development

ERIC Educational Resources Information Center

Pescosolido, Matthew F.; Gamsiz, Ece D.; Nagpal, Shailender; Morrow, Eric M.

2013-01-01

Objective: The purpose of the present study was to discover the extent to which distinct "DSM" disorders share large, highly recurrent copy number variants (CNVs) as susceptibility factors. We also sought to identify gene mechanisms common to groups of diagnoses and/or specific to a given diagnosis based on associations with CNVs. Method:…

Genome-wide copy number variation in the bovine genome detected using low coverage sequence of popular beef breeds

USDA-ARS?s Scientific Manuscript database

Copy number variations (CNVs) are large insertions, deletions or duplications in the genome that vary between members of a species and are known to affect a wide variety of phenotypic traits. In this study, we identified CNVs in a population of bulls using low coverage next-generation sequence data....

Application of Nexus copy number software for CNV detection and analysis.

PubMed

Darvishi, Katayoon

2010-04-01

Among human structural genomic variation, copy number variants (CNVs) are the most frequently known component, comprised of gains/losses of DNA segments that are generally 1 kb in length or longer. Array-based comparative genomic hybridization (aCGH) has emerged as a powerful tool for detecting genomic copy number variants (CNVs). With the rapid increase in the density of array technology and with the adaptation of new high-throughput technology, a reliable and computationally scalable method for accurate mapping of recurring DNA copy number aberrations has become a main focus in research. Here we introduce Nexus Copy Number software, a platform-independent tool, to analyze the output files of all types of commercial and custom-made comparative genomic hybridization (CGH) and single-nucleotide polymorphism (SNP) arrays, such as those manufactured by Affymetrix, Agilent Technologies, Illumina, and Roche NimbleGen. It also supports data generated by various array image-analysis software tools such as GenePix, ImaGene, and BlueFuse. (c) 2010 by John Wiley & Sons, Inc.

Mutations participating in interallelic complementation in propionic acidemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gravel, R.A.; Akerman, B.R.; Lamhonwah, A.M.

1994-07-01

Deficiency of propionyl-CoA carboxylase (PCC; [alpha][sub 4][beta][sub 4]) results in the rare, autosomal recessive disease propionic acidemia. Cell fusion experiments have revealed two complementation groups, pccA and pccB, corresponding to defects of the PCCA ([alpha]-subunit) and PCCB ([beta]-subunit) genes, respectively. The pccBCC group includes subgroups, pccB and pccC, which are thought to reflect interallelic complementation between certain mutations of the PCCB gene. In this study, the authors have identified the mutations in two pccB, one pccC, and two pccBC cell lines and have deduced those alleles participating in interallelic complementation. One pccB line was a compound hetrozygote of Pro228Leu andmore » Asn536Asp. The latter mutation was also detected in a noncomplementing pccBC line. This leaves Pro228Leu responsible for complementation in the pccB cells. The second pccB line contained an insertional duplication, dupKICK140-143, and a splice mutation IVS+1 G[yields]T, located after Lys466. The authors suggest that the dupKICK mutation is the complementing allele, since the second allele is incompatible with normal splicing. The pccC line studied was homozygous for Arg410Trp, which is necessarily the complementing allele in that line. For a second pccC line, they previously had proposed that [Delta]Ile408 was the complementing allele. They now show that its second allele, [open quotes]Ins[center dot]Del[close quotes], a 14-bp deletion replaced by a 12-bp insertion beginning at codon 407, fails to complement in homozygous form. The authors conclude that the interallelic complementation results from mutations in domains that can interact between [beta]-subunits in the PCC heteromer to restore enzymatic function. On the basis of sequence homology with the Propionibacterium shermanii transcarboxylase 12S subunit, they suggest that the pccC domain, defined by Ile408 and Arg410, may involve the propionyl-CoA binding site. 37 refs., 5 figs., 2 tabs.« less

Phylogenetic aspects of the complement system.

PubMed

Zarkadis, I K; Mastellos, D; Lambris, J D

2001-01-01

During evolution two general systems of immunity have emerged: innate or, natural immunity and adaptive (acquired), or specific immunity. The innate system is phylogenetically older and is found in some form in all multicellular organisms, whereas the adaptive system appeared about 450 million years ago and is found in all vertebrates except jawless fish. The complement system in higher vertebrates plays an important role as an effector of both the innate and the acquired immune response, and also participates in various immunoregulatory processes. In lower vertebrates complement is activated by the alternative and lectin pathways and is primarily involved in the opsonization of foreign material. The Agnatha (the most primitive vertebrate species) possess the alternative and lectin pathways while cartilaginous fish are the first species in which the classical pathway appears following the emergence of immunoglobulins. The rest of the poikilothermic species, ranging from teleosts to reptilians, appear to contain a well-developed complement system resembling that of the homeothermic vertebrates. It seems that most of the complement components have appeared after the duplication of primordial genes encoding C3/C4/C5, fB/C2, C1s/C1r/MASP-1/MASP-2, and C6/C7/C8/C9 molecules, in a process that led to the formation of distinct activation pathways. However, unlike homeotherms, several species of poikilotherms (e.g. trout) have recently been shown to possess multiple forms of complement components (C3, factor B) that are structurally and functionally more diverse than those of higher vertebrates. We hypothesize that this remarkable diversity has allowed these animals to expand their innate capacity for immune recognition and response. Recent studies have also indicated the possible presence of complement receptors in protochordates and lower vertebrates. In conclusion, there is considerable evidence suggesting that the complement system is present in the entire lineage of deuterostomes, and regulatory complement components have been identified in all species beyond the protochordates, indicating that the mechanisms of complement activation and regulation have developed in parallel.

Benign, Pathogenic and Copy Number Variations of Unknown Clinical Significance in Patients with Congenital Malformations and Developmental Delay.

PubMed

Mihaylova, M; Staneva, R; Toncheva, D; Pancheva, M; Hadjidekova, S

2017-06-30

The high frequency (3.0-5.0%) of congenital anomalies (CA) and intellectual disabilities (IDs), make them a serious problem, responsible for a high percentage (33.0%) of neonatal mortality. The genetic cause remains unclear in 40.0% of cases. Recently, molecular karyotyping has become the most powerful method for detection of pathogenic imbalances in patients with multiple CAs and IDs. This method is with high resolution and gives us the opportunity to investigate and identify candidate genes that could explain the genotype-phenotype correlations. This article describes the results from analysis of 81 patients with congenital malformations (CMs), developmental delay (DD) and ID, in which we utilized the CytoChip ISCA oligo microarray, 4 × 44 k, covering the whole genome with a resolution of 70 kb. In the selected group of patients with CAs, 280 copy number variations (CNVs) have been proven, 41 were pathogenic, 118 benign and 121 of unknown clinical significance (average number of variations 3.5). In six patients with established pathogenic variations, our data revealed eight pathogenic aberrations associated with the corresponding phenotype. The interpretation of the other CNVs was made on the basis of their frequency in the investigated group, the size of the variation, content of genes in the region and the type of the CNVs (deletion or duplication).

The Impact of Concomitant Genomic Alterations on Treatment Outcome for Trastuzumab Therapy in HER2-Positive Gastric Cancer

PubMed Central

Lee, Ji Yun; Hong, Mineui; Kim, Seung Tae; Park, Se Hoon; Kang, Won Ki; Kim, Kyoung-Mee; Lee, Jeeyun

2015-01-01

Clinical benefit from trastuzumab and other anti-human epidermal growth factor receptor-2 (HER2) therapies in patients with HER2-positive gastric cancer (GC) remains limited by primary or acquired resistance. We aimed to investigate the impact of concomitant molecular alterations to HER2 amplification on the clinical outcome of trastuzumab-treated patients. Using immunohistochemistry (IHC), copy number variations (CNVs), and Ion Ampliseq Cancer Panel, we analyzed the status of concomitant alterations in 50 HER2-positive advanced GC patients treated with trastuzumab in combination with other chemotherapeutic agents. The percentage of tumor samples with at least one concomitant alteration was 40% as assessed by IHC, 16% by CNVs, and 64% by Ampliseq sequencing. Median progression-free survival (PFS) was 8.0 months (95% confidence interval, 4.8–11.3). Patients were divided into two subgroups according to PFS values with a cutoff point of 8 months; results show that concomitant genomic alterations do not correlate with trastuzumab response. However, CNVs of CCNE1 significantly correlated (p < 0.05) with a shorter survival time. Our findings indicate that additional alterations implemented for prediction of clinical benefit from HER2-targeting agents in GC remained unclear. Further studies will be needed to elucidate the role of each specific biomarker and to optimize therapeutic approaches. PMID:25786580

Complement in Lupus Nephritis: New Perspectives.

PubMed

Bao, Lihua; Cunningham, Patrick N; Quigg, Richard J

2015-09-01

Systemic lupus erythematosus (SLE) is an autoimmune disorder caused by loss of tolerance to self-antigens, the production of autoantibodies and deposition of complement-fixing immune complexes (ICs) in injured tissues. SLE is characterized by a wide range of clinical manifestations and targeted organs, with lupus nephritis being one of the most serious complications. The complement system consists of three pathways and is tightly controlled by a set of regulatory proteins to prevent injudicious complement activation on host tissue. The involvement of the complement system in the pathogenesis of SLE is well accepted; yet, its exact role is still not clear. Complement plays dual roles in the pathogenesis of SLE. On the one hand, the complement system appears to have protective features in that hereditary homozygous deficiencies of classical pathway components, such as C1q and C4, are associated with an increased risk for SLE. On the other hand, IC-mediated activation of complement in affected tissues is clearly evident in both experimental and human SLE along with pathological features that are logical consequences of complement activation. Studies in genetically altered mice have shown that lack of complement inhibitors, such as complement factor H (CFH) or decay-accelerating factor (DAF) accelerates the development of experimental lupus nephritis, while treatment with recombinant protein inhibitors, such as Crry-Ig, CR2-Crry, CR2-DAF and CR2-CFH, ameliorates the disease development. Complement-targeted drugs, including soluble complement receptor 1 (TP10), C1 esterase inhibitor and a monoclonal anti-C5 antibody (eculizumab), have been shown to inhibit complement safely, and are now being investigated in a variety of clinical conditions. SLE is an autoimmune disorder which targets multiple systems. Complement is centrally involved and plays dual roles in the pathogenesis of SLE. Studies from experimental lupus models and clinical trials support the use of complement-targeted therapy in the treatment of SLE.

A Genome Wide Study of Copy Number Variation Associated with Nasopharyngeal Carcinoma in Malaysian Chinese Identifies CNVs at 11q14.3 and 6p21.3 as Candidate Loci

PubMed Central

Low, Joyce Siew Yong; Chin, Yoon Ming; Mushiroda, Taisei; Kubo, Michiaki; Govindasamy, Gopala Krishnan; Pua, Kin Choo; Yap, Yoke Yeow; Yap, Lee Fah; Subramaniam, Selva Kumar; Ong, Cheng Ai; Tan, Tee Yong; Khoo, Alan Soo Beng; Ng, Ching Ching

2016-01-01

Background Nasopharyngeal carcinoma (NPC) is a neoplasm of the epithelial lining of the nasopharynx. Despite various reports linking genomic variants to NPC predisposition, very few reports were done on copy number variations (CNV). CNV is an inherent structural variation that has been found to be involved in cancer predisposition. Methods A discovery cohort of Malaysian Chinese descent (NPC patients, n = 140; Healthy controls, n = 256) were genotyped using Illumina® HumanOmniExpress BeadChip. PennCNV and cnvPartition calling algorithms were applied for CNV calling. Taqman CNV assays and digital PCR were used to validate CNV calls and replicate candidate copy number variant region (CNVR) associations in a follow-up Malaysian Chinese (NPC cases, n = 465; and Healthy controls, n = 677) and Malay cohort (NPC cases, n = 114; Healthy controls, n = 124). Results Six putative CNVRs overlapping GRM5, MICA/HCP5/HCG26, LILRB3/LILRA6, DPY19L2, RNase3/RNase2 and GOLPH3 genes were jointly identified by PennCNV and cnvPartition. CNVs overlapping GRM5 and MICA/HCP5/HCG26 were subjected to further validation by Taqman CNV assays and digital PCR. Combined analysis in Malaysian Chinese cohort revealed a strong association at CNVR on chromosome 11q14.3 (Pcombined = 1.54x10-5; odds ratio (OR) = 7.27; 95% CI = 2.96–17.88) overlapping GRM5 and a suggestive association at CNVR on chromosome 6p21.3 (Pcombined = 1.29x10-3; OR = 4.21; 95% CI = 1.75–10.11) overlapping MICA/HCP5/HCG26 genes. Conclusion Our results demonstrated the association of CNVs towards NPC susceptibility, implicating a possible role of CNVs in NPC development. PMID:26730743

Persistent complement activation on tumor cells in breast cancer.

PubMed Central

Niculescu, F.; Rus, H. G.; Retegan, M.; Vlaicu, R.

1992-01-01

The neoantigens of the C5b-9 complement complex, IgG, C3, C4, S-protein/vitronectin, fibronectin, and macrophages were localized on 17 samples of breast cancer and on 6 samples of benign breast tumors using polyclonal or monoclonal antibodies and the streptavidin-biotin-peroxidase technique. All the tissue samples with carcinoma in each the TNM stages presented C5b-9 deposits on the membranes of tumor cells, thin granules on cell remnants, and diffuse deposits in the necrotic areas. When chemotherapy and radiation therapy preceded surgery, C5b-9 deposits were more intense and extended. The C5b-9 deposits were absent in all the samples with benign lesions. S-protein/vitronectin was present as fibrillar deposits in the connective tissue matrix and as diffuse deposits around the tumor cells, less intense and extended than fibronectin. IgG, C3, and C4 deposits were present only in carcinoma samples. The presence of C5b-9 deposits is indicative of complement activation and its subsequent pathogenetic effects in breast cancer. Images Figure 1 PMID:1374587

Copy number variability in Parkinson's disease: assembling the puzzle through a systems biology approach.

PubMed

La Cognata, Valentina; Morello, Giovanna; D'Agata, Velia; Cavallaro, Sebastiano

2017-01-01

Parkinson's disease (PD), the second most common progressive neurodegenerative disorder of aging, was long believed to be a non-genetic sporadic origin syndrome. The proof that several genetic loci are responsible for rare Mendelian forms has represented a revolutionary breakthrough, enabling to reveal molecular mechanisms underlying this debilitating still incurable condition. While single nucleotide polymorphisms (SNPs) and small indels constitute the most commonly investigated DNA variations accounting for only a limited number of PD cases, larger genomic molecular rearrangements have emerged as significant PD-causing mutations, including submicroscopic Copy Number Variations (CNVs). CNVs constitute a prevalent source of genomic variations and substantially participate in each individual's genomic makeup and phenotypic outcome. However, the majority of genetic studies have focused their attention on single candidate-gene mutations or on common variants reaching a significant statistical level of acceptance. This gene-centric approach is insufficient to uncover the genetic background of polygenic multifactorial disorders like PD, and potentially masks rare individual CNVs that all together might contribute to disease development or progression. In this review, we will discuss literature and bioinformatic data describing the involvement of CNVs on PD pathobiology. We will analyze the most frequent copy number changes in familiar PD genes and provide a "systems biology" overview of rare individual rearrangements that could functionally act on commonly deregulated molecular pathways. Assessing the global genome-wide burden of CNVs in PD patients may reveal new disease-related molecular mechanisms, and open the window to a new possible genetic scenario in the unsolved PD puzzle.

CNVinspector: a web-based tool for the interactive evaluation of copy number variations in single patients and in cohorts.

PubMed

Knierim, Ellen; Schwarz, Jana Marie; Schuelke, Markus; Seelow, Dominik

2013-08-01

Many genetic disorders are caused by copy number variations (CNVs) in the human genome. However, the large number of benign CNV polymorphisms makes it difficult to delineate causative variants for a certain disease phenotype. Hence, we set out to create software that accumulates and visualises locus-specific knowledge and enables clinicians to study their own CNVs in the context of known polymorphisms and disease variants. CNV data from healthy cohorts (Database of Genomic Variants) and from disease-related databases (DECIPHER) were integrated into a joint resource. Data are presented in an interactive web-based application that allows inspection, evaluation and filtering of CNVs in single individuals or in entire cohorts. CNVinspector provides simple interfaces to upload CNV data, compare them with own or published control data and visualise the results in graphical interfaces. Beyond choosing control data from different public studies, platforms and methods, dedicated filter options allow the detection of CNVs that are either enriched in patients or depleted in controls. Alternatively, a search can be restricted to those CNVs that appear in individuals of similar clinical phenotype. For each gene of interest within a CNV, we provide a link to NCBI, ENSEMBL and the GeneDistiller search engine to browse for potential disease-associated genes. With its user-friendly handling, the integration of control data and the filtering options, CNVinspector will facilitate the daily work of clinical geneticists and accelerate the delineation of new syndromes and gene functions. CNVinspector is freely accessible under http://www.cnvinspector.org.

Parallel Evolution of Copy-Number Variation across Continents in Drosophila melanogaster.

PubMed

Schrider, Daniel R; Hahn, Matthew W; Begun, David J

2016-05-01

Genetic differentiation across populations that is maintained in the presence of gene flow is a hallmark of spatially varying selection. In Drosophila melanogaster, the latitudinal clines across the eastern coasts of Australia and North America appear to be examples of this type of selection, with recent studies showing that a substantial portion of the D. melanogaster genome exhibits allele frequency differentiation with respect to latitude on both continents. As of yet there has been no genome-wide examination of differentiated copy-number variants (CNVs) in these geographic regions, despite their potential importance for phenotypic variation in Drosophila and other taxa. Here, we present an analysis of geographic variation in CNVs in D. melanogaster. We also present the first genomic analysis of geographic variation for copy-number variation in the sister species, D. simulans, in order to investigate patterns of parallel evolution in these close relatives. In D. melanogaster we find hundreds of CNVs, many of which show parallel patterns of geographic variation on both continents, lending support to the idea that they are influenced by spatially varying selection. These findings support the idea that polymorphic CNVs contribute to local adaptation in D. melanogaster In contrast, we find very few CNVs in D. simulans that are geographically differentiated in parallel on both continents, consistent with earlier work suggesting that clinal patterns are weaker in this species. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The ICR96 exon CNV validation series: a resource for orthogonal assessment of exon CNV calling in NGS data.

PubMed

Mahamdallie, Shazia; Ruark, Elise; Yost, Shawn; Ramsay, Emma; Uddin, Imran; Wylie, Harriett; Elliott, Anna; Strydom, Ann; Renwick, Anthony; Seal, Sheila; Rahman, Nazneen

2017-01-01

Detection of deletions and duplications of whole exons (exon CNVs) is a key requirement of genetic testing. Accurate detection of this variant type has proved very challenging in targeted next-generation sequencing (NGS) data, particularly if only a single exon is involved. Many different NGS exon CNV calling methods have been developed over the last five years. Such methods are usually evaluated using simulated and/or in-house data due to a lack of publicly-available datasets with orthogonally generated results. This hinders tool comparisons, transparency and reproducibility. To provide a community resource for assessment of exon CNV calling methods in targeted NGS data, we here present the ICR96 exon CNV validation series. The dataset includes high-quality sequencing data from a targeted NGS assay (the TruSight Cancer Panel) together with Multiplex Ligation-dependent Probe Amplification (MLPA) results for 96 independent samples. 66 samples contain at least one validated exon CNV and 30 samples have validated negative results for exon CNVs in 26 genes. The dataset includes 46 exon CNVs in BRCA1 , BRCA2 , TP53 , MLH1 , MSH2 , MSH6 , PMS2 , EPCAM or PTEN , giving excellent representation of the cancer predisposition genes most frequently tested in clinical practice. Moreover, the validated exon CNVs include 25 single exon CNVs, the most difficult type of exon CNV to detect. The FASTQ files for the ICR96 exon CNV validation series can be accessed through the European-Genome phenome Archive (EGA) under the accession number EGAS00001002428.

Copy number variation as a genetic basis for heterotaxy and heterotaxy-spectrum congenital heart defects.

PubMed

Cowan, Jason R; Tariq, Muhammad; Shaw, Chad; Rao, Mitchell; Belmont, John W; Lalani, Seema R; Smolarek, Teresa A; Ware, Stephanie M

2016-12-19

Genomic disorders and rare copy number abnormalities are identified in 15-25% of patients with syndromic conditions, but their prevalence in individuals with isolated birth defects is less clear. A spectrum of congenital heart defects (CHDs) is seen in heterotaxy, a highly heritable and genetically heterogeneous multiple congenital anomaly syndrome resulting from failure to properly establish left-right (L-R) organ asymmetry during early embryonic development. To identify novel genetic causes of heterotaxy, we analysed copy number variants (CNVs) in 225 patients with heterotaxy and heterotaxy-spectrum CHDs using array-based genotyping methods. Clinically relevant CNVs were identified in approximately 20% of patients and encompassed both known and putative heterotaxy genes. Patients were carefully phenotyped, revealing a significant association of abdominal situs inversus with pathogenic or likely pathogenic CNVs, while d-transposition of the great arteries was more frequently associated with common CNVs. Identified cytogenetic abnormalities ranged from large unbalanced translocations to smaller, kilobase-scale CNVs, including a rare, single exon deletion in ZIC3, a gene known to cause X-linked heterotaxy. Morpholino loss-of-function experiments in Xenopus support a role for one of these novel candidates, the platelet isoform of phosphofructokinase-1 (PFKP) in heterotaxy. Collectively, our results confirm a high CNV yield for array-based testing in patients with heterotaxy, and support use of CNV analysis for identification of novel biological processes relevant to human laterality.This article is part of the themed issue 'Provocative questions in left-right asymmetry'. © 2016 The Author(s).

Correction of both spontaneous and DEB-induced chromosome instability in Fanconi anemia FA-C cells by FACC cDNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stavropoulos, D.J.; Tomkins, D.J.; Allingham-Hawkins, D.J.

1994-09-01

Cells from all four Fanconi anemia complementation groups show hypersensitivity to cell-killing by mitomycin C (MMC), diepoxybutane (DEB) and other DNA cross-linking agents, and increased spontaneous and DEB-induced chromosome aberrations (CA). The extent of these phenotypes varies between lymphoblastoid cell lines from different complementation groups. Our data showed that the difference in MMC hypersensitivity and DEB-CA was not always coupled. While 230N (FA-B) had higher DEB-induced CA/cell than 536N (FA-C) (7.42 vs. 4.46 respectively), that latter was much more sensitive to cell-killing by MMC (dose at 10% survival, D{sub 10}: 5.2 vs. 1.2 ng/ml respectively). Strathdes et al. (1992) clonedmore » a cDNA Fanconi anemia complementation group C (FACC) which complemented the hypersensitivity to MMC and DEB cell-killing of FA-C cells (536N) but not cells from the other three complementation groups. The present study was initiated to determine whether chromosome instability in 536N is also complemented by the FACC (FAC3) cDNA. The pREP4-FAC3 vector was transfected into 536N and transfectants selected with hygromycin B. The DEB D{sub 10} of 536N (1.0 {mu}M) was corrected to the control level (16.2 {mu}M for 3TO) by FACC (15.1 {mu}M for 536N-FACC), as previously demonstrated. Chromosome instability (cab, cse, ctb, cte) was determined without and with 0.1 {mu}g/ml DEB treatment. Spontaneous CA of 536N (0.30 aberrations/cell) was corrected to the control level (0.04 for 3TO) by FACC (0.06 for 536N-FACC). Similarly, the DEB-induced CA was corrected (2.74 for 536N vs. 0.06 and 0.02 for 3TO and 536N-FACC respectively). Thus, at least for FA complementation group C, hypersensitivity to cell-killing and chromosome instability are not dissociated and are most likely caused by the same gene defect.« less

Copy number analysis of NIPBL in a cohort of 510 patients reveals rare copy number variants and a mosaic deletion.

PubMed

Cheng, Yu-Wei; Tan, Christopher A; Minor, Agata; Arndt, Kelly; Wysinger, Latrice; Grange, Dorothy K; Kozel, Beth A; Robin, Nathaniel H; Waggoner, Darrel; Fitzpatrick, Carrie; Das, Soma; Del Gaudio, Daniela

2014-03-01

Cornelia de Lange syndrome (CdLS) is a genetically heterogeneous disorder characterized by growth retardation, intellectual disability, upper limb abnormalities, hirsutism, and characteristic facial features. In this study we explored the occurrence of intragenic NIPBL copy number variations (CNVs) in a cohort of 510 NIPBL sequence-negative patients with suspected CdLS. Copy number analysis was performed by custom exon-targeted oligonucleotide array-comparative genomic hybridization and/or MLPA. Whole-genome SNP array was used to further characterize rearrangements extending beyond the NIPBL gene. We identified NIPBL CNVs in 13 patients (2.5%) including one intragenic duplication and a deletion in mosaic state. Breakpoint sequences in two patients provided further evidence of a microhomology-mediated replicative mechanism as a potential predominant contributor to CNVs in NIPBL. Patients for whom clinical information was available share classical CdLS features including craniofacial and limb defects. Our experience in studying the frequency of NIBPL CNVs in the largest series of patients to date widens the mutational spectrum of NIPBL and emphasizes the clinical utility of performing NIPBL deletion/duplication analysis in patients with CdLS.

Host Defense against Opportunist Microorganisms Following Trauma.

DTIC Science & Technology

1979-06-01

patients were total hemolytic complement (CH5 0 ), C3 conversion by inulin and cobra venom factor (CoVF), and itmunochemical concentrations of Clq, C4, C2...were normal or elevated for the entire study period. C3 conversion by inulin and CoVF and the concentration of properdin were reduced in the sera of the...measured in all patients were total hemolytic complement (CH5 0), C3 conversion by inulin and cobra venom factor (CoVF), and inunochemical

Role of CYP2E1 Immunoglobulin G4 Subclass Antibodies and Complement in Pathogenesis of Idiosyncratic Drug-Induced Hepatitis

PubMed Central

Njoku, Dolores B.; Mellerson, Jenelle L.; Talor, Monica V.; Kerr, Douglas R.; Faraday, Nauder R.; Outschoorn, Ingrid; Rose, Noel R.

2006-01-01

Idiosyncratic drug-induced hepatitis (IDDIH) is the third most common cause for acute liver failure in the United States. Previous studies have attempted to identify susceptible patients or early stages of disease with various degrees of success. To determine if total serum immunoglobulin subclasses, CYP2E1-specific subclass autoantibodies, complement components, or immune complexes could distinguish persons with IDDIH from others exposed to drugs, we studied persons exposed to halogenated volatile anesthetics, which have been associated with IDDIH and CYP2E1 autoantibodies. We found that patients with anesthetic-induced IDDIH had significantly elevated levels of CYP2E1-specific immunoglobulin G4 (IgG4) autoantibodies, while anesthetic-exposed healthy persons had significantly elevated levels of CYP2E1-specific IgG1 autoantibodies. Anesthetic IDDIH patients had significantly lower levels of C4a, C3a, and C5a compared to anesthetic-exposed healthy persons. C1q- and C3d-containing immune complexes were significantly elevated in anesthetic-exposed persons. In conclusion, our data suggest that anesthetic-exposed persons develop CYP2E1-specific IgG1 autoantibodies which may form detectable circulating immune complexes subsequently cleared by classical pathway activation of the complement system. Persons susceptible to anesthetic-induced IDDIH develop CYP2E1-specific IgG4 autoantibodies which form small, nonprecipitating immune complexes that escape clearance because of their size or by direct inhibition of complement activation. PMID:16467335

Identification of a central role for complement in osteoarthritis

PubMed Central

Wang, Qian; Rozelle, Andrew L.; Lepus, Christin M.; Scanzello, Carla R.; Song, Jason J.; Larsen, D. Meegan; Crish, James F.; Bebek, Gurkan; Ritter, Susan Y.; Lindstrom, Tamsin M.; Hwang, Inyong; Wong, Heidi H.; Punzi, Leonardo; Encarnacion, Angelo; Shamloo, Mehrdad; Goodman, Stuart B.; Wyss-Coray, Tony; Goldring, Steven R.; Banda, Nirmal K.; Thurman, Joshua M.; Gobezie, Reuben; Crow, Mary K.; Holers, V. Michael; Lee, David M.; Robinson, William H.

2011-01-01

Osteoarthritis, characterized by the breakdown of articular cartilage in synovial joints, has long been viewed as the result of “wear and tear”1. Although low-grade inflammation is detected in osteoarthritis, its role is unclear2–4. Here we identify a central role for the inflammatory complement system in the pathogenesis of osteoarthritis. Through proteomic and transcriptomic analyses of synovial fluids and membranes from individuals with osteoarthritis, we find that expression and activation of complement is abnormally high in human osteoarthritic joints. Using mice genetically deficient in C5, C6, or CD59a, we show that complement, and specifically the membrane attack complex (MAC)-mediated arm of complement, is critical to the development of arthritis in three different mouse models of osteoarthritis. Pharmacological modulation of complement in wild-type mice confirmed the results obtained with genetically deficient mice. Expression of inflammatory and degradative molecules was lower in chondrocytes from destabilized joints of C5-deficient mice than C5-sufficient mice, and MAC induced production of these molecules in cultured chondrocytes. Furthermore, MAC co-localized with matrix metalloprotease (MMP)-13 and with activated extracellular signal-regulated kinase (ERK) around chondrocytes in human osteoarthritic cartilage. Our findings indicate that dysregulation of complement in synovial joints plays a critical role in the pathogenesis of osteoarthritis. PMID:22057346

Immunological properties of glycolipids from membranes of Acholeplasma laidlawii.

PubMed Central

Ryan, M D; Noker, P; Matz, L L

1975-01-01

Glycolipids, the predominant class of lipids in the membranes of Acholeplasma laidlawii, are the haptenic determinants that react with anti-A. Laidlawii serum to fix complement. The predominant complement-fixing activity of the membrane glycolipids was associated with the monoglucoysyl diglyceride, diglucosyl diglyceride, glycerlphosphoryl diglucosyl diglyceride (GPDD), and an unknown lipid B, which did not react with ninhydrin but release glucose and glycerol and traces of phosphorus upon hydrolysis. The glycolipids monoglucosyl diglyceride and diglucosyl diglyceride or GPDD and unknown lipid B were paired as a result of their cross-reactions with selective antisera prepared with the aid of reconstituted membrane complexes containing membrane lipids. Reconstituted membrane complexes assembled from [14C]monoglucosyl diglyceride and delipidated membrane proteins gave optimal complement fixation titers before saturation of the complexes with the ]14C]monoglucosyl diglyceride. The phosphoglycolipid of the membrane, GPDD, was anticomplementary as a pure lipid, a cholesterol liposome, and a reconstituted membrane complex. This anticomplementary activity, which was caused by 3 mug of pure GPDD, affected both human and guinea pig complement. Although human C1, C4, C3, and C5 were not inhibited by GPDD, C2 was inhibited 10-fold by reconstituted membrane complexes containing 150 mug of GPDD. A role for this phosphoglycolipid is discussed in the hypothetical mechanism of inhibition of C2 attachment to SAC1, 4 sites. PMID:1193716

Genome wide analysis reveals single nucleotide polymorphisms associated with fatness and putative novel copy number variants in three pig breeds

PubMed Central

2013-01-01

Background Obesity, excess fat tissue in the body, can underlie a variety of medical complaints including heart disease, stroke and cancer. The pig is an excellent model organism for the study of various human disorders, including obesity, as well as being the foremost agricultural species. In order to identify genetic variants associated with fatness, we used a selective genomic approach sampling DNA from animals at the extreme ends of the fat and lean spectrum using estimated breeding values derived from a total population size of over 70,000 animals. DNA from 3 breeds (Sire Line Large White, Duroc and a white Pietrain composite line (Titan)) was used to interrogate the Illumina Porcine SNP60 Genotyping Beadchip in order to identify significant associations in terms of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs). Results By sampling animals at each end of the fat/lean EBV (estimate breeding value) spectrum the whole population could be assessed using less than 300 animals, without losing statistical power. Indeed, several significant SNPs (at the 5% genome wide significance level) were discovered, 4 of these linked to genes with ontologies that had previously been correlated with fatness (NTS, FABP6, SST and NR3C2). Quantitative analysis of the data identified putative CNV regions containing genes whose ontology suggested fatness related functions (MCHR1, PPARα, SLC5A1 and SLC5A4). Conclusions Selective genotyping of EBVs at either end of the phenotypic spectrum proved to be a cost effective means of identifying SNPs and CNVs associated with fatness and with estimated major effects in a large population of animals. PMID:24225222

Anti-complementary constituents of Houttuynia cordata and their targets in complement activation cascade.

PubMed

Jiang, Yun; Lu, Yan; Zhang, Yun-Yi; Chen, Dao-Feng

2014-01-01

Activity-guided fractionation for complement inhibitors led to the isolation of 23 known compounds from Houttuynia cordata Thunb. Seven flavonoids, two alkaloids, one coumarin and two phenols showed anti-complementary activity. Preliminary inhibitory mechanism of four flavonoids, including quercitrin, afzelin, isoquercitrin and quercetin in the complement activation cascade were examined for the first time. The results indicated that the target components of flavonols are different from those of flavonosides, and the glycoside moieties may be necessary to block C3 and C4 components.

Targeting the Human Complement Membrane Attack Complex to Selectively Kill Prostate Cancer Cells

DTIC Science & Technology

2013-10-01

prostate cancer cells in vitro . Evaluate CD59 expression in human prostate cancer microarrays. Aim 4: Evaluate toxicity and efficacy of the lead PAC5...fragment in vitro . Since PSA is the major chymotrypsin-like serine protease in the seminal plasma and prostatic fluid, we hypothesized that PSA was...that the evolution -related complement protein C5, but not C4, is a substrate of PSA as well. *Department of Pharmacology and Molecular Sciences, The

[Variations of peripheral blood autoantibody, immunoglobuliln, and complement levels in patients with non-lactational mastitis and their clinical significances].

PubMed

Xu, Rui; Guo, Qian-Qian; Yang, Le-Ping; Lai, Mi-Lin; Tong, Lin

2016-08-20

To detect the variations in peripheral blood levels of autoantibodies, immunoglobulilns and complements in patients with non-lactational mastitis and investigate whether non-lactational mastitis is an autoimmune disease with immune dysfunction. Seven-eight patients with non-lactational mastitis treated in our hospital between September 2013 and May 2015 and 88 healthy women (control) were examined for peripheral blood levels of antinuclear antibody (ANA), anti-histone antibody (AHA), immunoglobulins (IgA, IgM, and IgG) and complements (C3, C4, and total complements). s Of the 78 patients with non-lactational mastitis, 50 (64.10%) were positive of ANA showing mainly the granular and cytoplasmic granular fluorescence patterns, and the positivity rate was significantly higher than that in the control group (P<0.000). Twenty-eight (36.00%) of the patients were positive of AHA, a rate significantly higher than that in the control group (P<0.000). The levels of IgA, IgM, C4, and total complements levels were all significantly elevated in the patients compared with those in the control group (P<0.05). Patients with non-lactational mastitis have abnormal changes in peripheral blood levels of immunoglobulins and complements with high positivity rates for ANA and AHA, indicating that non-lactational mastitis is an autoimmune disease with immune dysfunction.

Effects of partial replacement of fish meal by yeast hydrolysate on complement system and stress resistance in juvenile Jian carp (Cyprinus carpio var. Jian).

PubMed

Yuan, Xiang-Yang; Liu, Wen-Bin; Liang, Chao; Sun, Cun-Xin; Xue, Yun-Fei; Wan, Zu-De; Jiang, Guang-Zhen

2017-08-01

A 10-week feeding trial was carried out to investigate the effects of dietary fish meal replacement by yeast hydrolysate (YH) on growth performance, complement system and stress resistance of juvenile Jian carp (Cyprinus carpio var. Jian) (initial average weight 19.44 ± 0.06 g). In the study, there were five groups: one control group was fed with a basal diet (YH0), and four treatment groups were fed with dietary fish meal replaced by 1% YH (YH1), 3% (YH3), 5% (YH5) and 7% (YH7), respectively. Each group had four replicates. At the end of feeding trial, twelve fish from each group (three fish per replicate) were randomly selected for assessing the growth and immunity. Meanwhile, 20 fish per replicate were injected by Aeromonas hydrophila. The results showed that (1) Replacement levels of YH significantly affected the growth of the fish with the highest values of weight gain (WG) occurred in fish fed YH3 diet. However, no significant difference in feed conversion ratios (FCR) was observed among all groups. (2) Pre-stressed plasma lysozyme activity, total protein and albumin contents and complement component 3 (C3) and complement component 4 (C4) levels of fish fed YH3 diet were significantly higher than those of fish fed YH0 diet. However, post-stressed immune parameters of fish in all groups were significantly lower. (3) There was a trend that the expression levels of the complement-related genes (c1r/s-A, c4-1, c3-H1, c5-1, fb/c2-A, mbl-2 and masp) initially increased and then decreased except mbl-2 and masp, with the maximum values observed in fish fed YH3 diet. Before stress, the expression levels of the inflammation-related genes (alp, il-1β and tnf-α) in the hepatopancreas and spleen of fish fed YH1 diet and YH7 diet were significant higher than that of fish fed YH0 diet. After stress, no significant difference in the expression levels of those genes was observed among all groups. These results indicated that FM replacement by YH could improve growth performance, enhance innate immunity, and activate complement via the alternative complement pathway (ACP) and the classical complement pathway (CCP). Copyright © 2017 Elsevier Ltd. All rights reserved.

Complement activation promotes muscle inflammation during modified muscle use

NASA Technical Reports Server (NTRS)

Frenette, J.; Cai, B.; Tidball, J. G.

2000-01-01

Modified muscle use can result in muscle inflammation that is triggered by unidentified events. In the present investigation, we tested whether the activation of the complement system is a component of muscle inflammation that results from changes in muscle loading. Modified rat hindlimb muscle loading was achieved by removing weight-bearing from the hindlimbs for 10 days followed by reloading through normal ambulation. Experimental animals were injected with the recombinant, soluble complement receptor sCR1 to inhibit complement activation. Assays for complement C4 or factor B in sera showed that sCR1 produced large reductions in the capacity for activation of the complement system through both the classical and alternative pathways. Analysis of complement C4 concentration in serum in untreated animals showed that the classical pathway was activated during the first 2 hours of reloading. Analysis of factor B concentration in untreated animals showed activation of the alternative pathway at 6 hours of reloading. Administration of sCR1 significantly attenuated the invasion of neutrophils (-49%) and ED1(+) macrophages (-52%) that occurred in nontreated animals after 6 hours of reloading. The presence of sCR1 also reduced significantly the degree of edema by 22% as compared to untreated animals. Together, these data show that increased muscle loading activated the complement system which then briefly contributes to the early recruitment of inflammatory cells during modified muscle loading.

Translational Mini-Review Series on Complement Factor H: Structural and functional correlations for factor H

PubMed Central

Schmidt, C Q; Herbert, A P; Hocking, H G; Uhrín, D; Barlow, P N

2008-01-01

The 155-kDa glycoprotein, complement factor H (CFH), is a regulator of complement activation that is abundant in human plasma. Three-dimensional structures of over half the 20 complement control protein (CCP) modules in CFH have been solved in the context of single-, double- and triple-module segments. Proven binding sites for C3b occupy the N and C termini of this elongated molecule and may be brought together by a bend in CFH mediated by its central CCP modules. The C-terminal CCP 20 is key to the ability of the molecule to adhere to polyanionic markers on self-surfaces where CFH acts to regulate amplification of the alternative pathway of complement. The surface patch on CCP 20 that binds to model glycosaminoglycans has been mapped using nuclear magnetic resonance (NMR), as has a second glycosaminoglycan-binding patch on CCP 7. These patches include many of the residue positions at which sequence variations have been linked to three complement-mediated disorders: dense deposit disease, age-related macular degeneration and atypical haemolytic uraemic syndrome. In one plausible model, CCP 20 anchors CFH to self-surfaces via a C3b/polyanion composite binding site, CCP 7 acts as a ‘proof-reader’ to help discriminate self- from non-self patterns of sulphation, and CCPs 1–4 disrupt C3/C5 convertase formation and stability. PMID:18081691

The profile of adsorbed plasma and serum proteins on methacrylic acid copolymer beads: Effect on complement activation.

PubMed

Wells, Laura A; Guo, Hongbo; Emili, Andrew; Sefton, Michael V

2017-02-01

Polymer beads made of 45% methacrylic acid co methyl methacrylate (MAA beads) promote vascular regenerative responses in contrast to control materials without methacrylic acid (here polymethyl methacrylate beads, PMMA). In vitro and in vivo studies suggest that MAA copolymers induce differences in macrophage phenotype and polarization and inflammatory responses, presumably due to protein adsorption differences between the beads. To explore differences in protein adsorption in an unbiased manner, we used high resolution shotgun mass spectrometry to identify and compare proteins that adsorb from human plasma or serum onto MAA and PMMA beads. From plasma, MAA beads adsorbed many complement proteins, such as C1q, C4-related proteins and the complement inhibitor factor H, while PMMA adsorbed proteins, such as albumin, C3 and apolipoproteins. Because of the differences in complement protein adsorption, follow-up studies focused on using ELISA to assess complement activation. When incubated in serum, MAA beads generated significantly lower levels of soluble C5b9 and C3a/C3a desarg in comparison to PMMA beads, indicating a decrease in complement activation with MAA beads. The differences in adsorbed protein on the two materials likely alter subsequent cell-material interactions that ultimately result in different host responses and local vascularization. Copyright © 2016 Elsevier Ltd. All rights reserved.

Several Human Liver Cell Expressed Apolipoproteins Complement HCV Virus Production with Varying Efficacy Conferring Differential Specific Infectivity to Released Viruses.

PubMed

Hueging, Kathrin; Weller, Romy; Doepke, Mandy; Vieyres, Gabrielle; Todt, Daniel; Wölk, Benno; Vondran, Florian W R; Geffers, Robert; Lauber, Chris; Kaderali, Lars; Penin, François; Pietschmann, Thomas

2015-01-01

Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to distinct characteristics of these apolipoproteins that influence HCV assembly and cell entry. This will guide future research to precisely pinpoint how apolipoproteins function during virus assembly and cell entry.

Several Human Liver Cell Expressed Apolipoproteins Complement HCV Virus Production with Varying Efficacy Conferring Differential Specific Infectivity to Released Viruses

PubMed Central

Doepke, Mandy; Vieyres, Gabrielle; Todt, Daniel; Wölk, Benno; Vondran, Florian W. R.; Geffers, Robert; Lauber, Chris; Kaderali, Lars; Penin, François; Pietschmann, Thomas

2015-01-01

Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to distinct characteristics of these apolipoproteins that influence HCV assembly and cell entry. This will guide future research to precisely pinpoint how apolipoproteins function during virus assembly and cell entry. PMID:26226615

Low copy numbers of complement C4 and homozygous deficiency of C4A may predispose to severe disease and earlier disease onset in patients with systemic lupus erythematosus.

PubMed

Jüptner, M; Flachsbart, F; Caliebe, A; Lieb, W; Schreiber, S; Zeuner, R; Franke, A; Schröder, J O

2018-04-01

Objectives Low copy numbers and deletion of complement C4 genes are potent risk factors for systemic lupus erythematosus (SLE). However, it is not known whether this genetic association affects the clinical outcome. We investigated C4 copy number variation and its relationship to clinical and serological features in a Northern European lupus cohort. Methods We genotyped the C4 gene locus using polymerase chain reaction (PCR)-based TaqMan assays in 169 patients with SLE classified according to the 1997 revised American College of Rheumatology (ACR) criteria and in 520 matched controls. In the patient group the mean C4 serum protein concentrations nephelometrically measured during a 12-month period prior to genetic analysis were compared to C4 gene copy numbers. Severity of disease was classified according to the intensity of the immunosuppressive regimens applied and compared to C4 gene copy numbers, too. In addition, we performed a TaqMan based analysis of three lupus-associated single-nucleotide polymorphisms (SNPs) located inside the major histocompatibility complex (MHC) to investigate the independence of complement C4 in association with SLE. Results Homozygous deficiency of the C4A isotype was identified as the strongest risk factor for SLE (odds ratio (OR) = 5.329; p = 7.7 × 10 -3 ) in the case-control comparison. Moreover, two copies of total C4 were associated with SLE (OR = 3.699; p = 6.8 × 10 -3 ). C4 serum levels were strongly related to C4 gene copy numbers in patients, the mean concentration ranging from 0.110 g/l (two copies) to 0.256 g/l (five to six copies; p = 4.9 × 10 -6 ). Two copies of total C4 and homozygous deletion of C4A were associated with a disease course requiring cyclophosphamide therapy (OR = 4.044; p = 0.040 and OR = 5.798; p = 0.034, respectively). Homozygous deletion of C4A was associated with earlier onset of SLE (median 24 vs. 34 years; p = 0.019) but not significant after correction for multiple testing. SNP analysis revealed a significant association of HLA-DRB1*0301 with SLE (OR = 2.231; p = 1.33 × 10 -5 ). Conclusions Our findings confirm the important role of complement C4 genes in the development of SLE. Beyond the impact on the susceptibility for lupus, C4 copy numbers may be related to earlier onset and a more severe course of the disease. The association of homozygous deletion of C4A and SLE is accompanied by the presence of HLA-DRB1*0301 without a proven pathophysiological mechanism.

Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy.

PubMed

Zhao, Limei; Shao, Shuai; Chen, Yi; Sun, Ximeng; Sun, Ran; Huang, Jingjing; Zhan, Bin; Zhu, Xinping

2017-01-01

As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin ( Ts -CRT), a Ca 2+ -binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts -CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts -CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts -CRT (r Ts -CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte-macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts -CRT on the surface of newborn larvae (NBL) of T. spiralis with anti- Ts -CRT antibody increased the C1q-mediated adherence of monocyte-macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis -expressed Ts -CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages.

Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy

PubMed Central

Zhao, Limei; Shao, Shuai; Chen, Yi; Sun, Ximeng; Sun, Ran; Huang, Jingjing; Zhan, Bin; Zhu, Xinping

2017-01-01

As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin (Ts-CRT), a Ca2+-binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts-CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts-CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts-CRT (rTs-CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte–macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts-CRT on the surface of newborn larvae (NBL) of T. spiralis with anti-Ts-CRT antibody increased the C1q-mediated adherence of monocyte–macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis-expressed Ts-CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages. PMID:28620388

The functional genetic link of NLGN4X knockdown and neurodevelopment in neural stem cells

PubMed Central

Shi, Lingling; Chang, Xiao; Zhang, Peilin; Coba, Marcelo P.; Lu, Wange; Wang, Kai

2013-01-01

Genetic mutations in NLGN4X (neuroligin 4), including point mutations and copy number variants (CNVs), have been associated with susceptibility to autism spectrum disorders (ASDs). However, it is unclear how mutations in NLGN4X result in neurodevelopmental defects. Here, we used neural stem cells (NSCs) as in vitro models to explore the impacts of NLGN4X knockdown on neurodevelopment. Using two shRNAmir-based vectors targeting NLGN4X and one control shRNAmir vector, we modulated NLGN4X expression and differentiated these NSCs into mature neurons. We monitored the neurodevelopmental process at Weeks 0, 0.5, 1, 2, 4 and 6, based on morphological analysis and whole-genome gene expression profiling. At the cellular level, in NSCs with NLGN4X knockdown, we observed increasingly delayed neuronal development and compromised neurite formation, starting from Week 2 through Week 6 post differentiation. At the molecular level, we identified multiple pathways, such as neurogenesis, neuron differentiation and muscle development, which are increasingly disturbed in cells with NLGN4X knockdown. Notably, several postsynaptic genes, including DLG4, NLGN1 and NLGN3, also have decreased expression. Based on in vitro models, NLGN4X knockdown directly impacts neurodevelopmental process during the formation of neurons and their connections. Our functional genomics study highlights the utility of NSCs models in understanding the functional roles of CNVs in affecting neurodevelopment and conferring susceptibility to neurodevelopmental diseases. PMID:23710042

The functional genetic link of NLGN4X knockdown and neurodevelopment in neural stem cells.

PubMed

Shi, Lingling; Chang, Xiao; Zhang, Peilin; Coba, Marcelo P; Lu, Wange; Wang, Kai

2013-09-15

Genetic mutations in NLGN4X (neuroligin 4), including point mutations and copy number variants (CNVs), have been associated with susceptibility to autism spectrum disorders (ASDs). However, it is unclear how mutations in NLGN4X result in neurodevelopmental defects. Here, we used neural stem cells (NSCs) as in vitro models to explore the impacts of NLGN4X knockdown on neurodevelopment. Using two shRNAmir-based vectors targeting NLGN4X and one control shRNAmir vector, we modulated NLGN4X expression and differentiated these NSCs into mature neurons. We monitored the neurodevelopmental process at Weeks 0, 0.5, 1, 2, 4 and 6, based on morphological analysis and whole-genome gene expression profiling. At the cellular level, in NSCs with NLGN4X knockdown, we observed increasingly delayed neuronal development and compromised neurite formation, starting from Week 2 through Week 6 post differentiation. At the molecular level, we identified multiple pathways, such as neurogenesis, neuron differentiation and muscle development, which are increasingly disturbed in cells with NLGN4X knockdown. Notably, several postsynaptic genes, including DLG4, NLGN1 and NLGN3, also have decreased expression. Based on in vitro models, NLGN4X knockdown directly impacts neurodevelopmental process during the formation of neurons and their connections. Our functional genomics study highlights the utility of NSCs models in understanding the functional roles of CNVs in affecting neurodevelopment and conferring susceptibility to neurodevelopmental diseases.

Copy number variation analysis implicates the cell polarity gene glypican 5 as a human spina bifida candidate gene

PubMed Central

Bassuk, Alexander G.; Muthuswamy, Lakshmi B.; Boland, Riley; Smith, Tiffany L.; Hulstrand, Alissa M.; Northrup, Hope; Hakeman, Matthew; Dierdorff, Jason M.; Yung, Christina K.; Long, Abby; Brouillette, Rachel B.; Au, Kit Sing; Gurnett, Christina; Houston, Douglas W.; Cornell, Robert A.; Manak, J. Robert

2013-01-01

Neural tube defects (NTDs) are common birth defects of complex etiology. Family and population-based studies have confirmed a genetic component to NTDs. However, despite more than three decades of research, the genes involved in human NTDs remain largely unknown. We tested the hypothesis that rare copy number variants (CNVs), especially de novo germline CNVs, are a significant risk factor for NTDs. We used array-based comparative genomic hybridization (aCGH) to identify rare CNVs in 128 Caucasian and 61 Hispanic patients with non-syndromic lumbar-sacral myelomeningocele. We also performed aCGH analysis on the parents of affected individuals with rare CNVs where parental DNA was available (42 sets). Among the eight de novo CNVs that we identified, three generated copy number changes of entire genes. One large heterozygous deletion removed 27 genes, including PAX3, a known spina bifida-associated gene. A second CNV altered genes (PGPD8, ZC3H6) for which little is known regarding function or expression. A third heterozygous deletion removed GPC5 and part of GPC6, genes encoding glypicans. Glypicans are proteoglycans that modulate the activity of morphogens such as Sonic Hedgehog (SHH) and bone morphogenetic proteins (BMPs), both of which have been implicated in NTDs. Additionally, glypicans function in the planar cell polarity (PCP) pathway, and several PCP genes have been associated with NTDs. Here, we show that GPC5 orthologs are expressed in the neural tube, and that inhibiting their expression in frog and fish embryos results in NTDs. These results implicate GPC5 as a gene required for normal neural tube development. PMID:23223018

A Discovery Resource of Rare Copy Number Variations in Individuals with Autism Spectrum Disorder

PubMed Central

Prasad, Aparna; Merico, Daniele; Thiruvahindrapuram, Bhooma; Wei, John; Lionel, Anath C.; Sato, Daisuke; Rickaby, Jessica; Lu, Chao; Szatmari, Peter; Roberts, Wendy; Fernandez, Bridget A.; Marshall, Christian R.; Hatchwell, Eli; Eis, Peggy S.; Scherer, Stephen W.

2012-01-01

The identification of rare inherited and de novo copy number variations (CNVs) in human subjects has proven a productive approach to highlight risk genes for autism spectrum disorder (ASD). A variety of microarrays are available to detect CNVs, including single-nucleotide polymorphism (SNP) arrays and comparative genomic hybridization (CGH) arrays. Here, we examine a cohort of 696 unrelated ASD cases using a high-resolution one-million feature CGH microarray, the majority of which were previously genotyped with SNP arrays. Our objective was to discover new CNVs in ASD cases that were not detected by SNP microarray analysis and to delineate novel ASD risk loci via combined analysis of CGH and SNP array data sets on the ASD cohort and CGH data on an additional 1000 control samples. Of the 615 ASD cases analyzed on both SNP and CGH arrays, we found that 13,572 of 21,346 (64%) of the CNVs were exclusively detected by the CGH array. Several of the CGH-specific CNVs are rare in population frequency and impact previously reported ASD genes (e.g., NRXN1, GRM8, DPYD), as well as novel ASD candidate genes (e.g., CIB2, DAPP1, SAE1), and all were inherited except for a de novo CNV in the GPHN gene. A functional enrichment test of gene-sets in ASD cases over controls revealed nucleotide metabolism as a potential novel pathway involved in ASD, which includes several candidate genes for follow-up (e.g., DPYD, UPB1, UPP1, TYMP). Finally, this extensively phenotyped and genotyped ASD clinical cohort serves as an invaluable resource for the next step of genome sequencing for complete genetic variation detection. PMID:23275889

Solid-phase classical complement activation by C-reactive protein (CRP) is inhibited by fluid-phase CRP-C1q interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sjoewall, Christopher; Wetteroe, Jonas; Bengtsson, Torbjoern

2007-01-05

C-reactive protein (CRP) interacts with phosphorylcholine (PC), Fc{gamma} receptors, complement factor C1q and cell nuclear constituents, yet its biological roles are insufficiently understood. The aim was to characterize CRP-induced complement activation by ellipsometry. PC conjugated with keyhole limpet hemocyanin (PC-KLH) was immobilized to cross-linked fibrinogen. A low-CRP serum with different amounts of added CRP was exposed to the PC-surfaces. The total serum protein deposition was quantified and deposition of IgG, C1q, C3c, C4, factor H, and CRP detected with polyclonal antibodies. The binding of serum CRP to PC-KLH dose-dependently triggered activation of the classical pathway. Unexpectedly, the activation was efficientlymore » down-regulated at CRP levels >150 mg/L. Using radial immunodiffusion, CRP-C1q interaction was observed in serum samples with high CRP concentrations. We propose that the underlying mechanism depends on fluid-phase interaction between C1q and CRP. This might constitute another level of complement regulation, which has implications for systemic lupus erythematosus where CRP is often low despite flare-ups.« less

The Lectin Pathway of Complement and Rheumatic Heart Disease

PubMed Central

Beltrame, Marcia Holsbach; Catarino, Sandra Jeremias; Goeldner, Isabela; Boldt, Angelica Beate Winter; de Messias-Reason, Iara José

2014-01-01

The innate immune system is the first line of host defense against infection and is comprised of humoral and cellular mechanisms that recognize potential pathogens within minutes or hours of entry. The effector components of innate immunity include epithelial barriers, phagocytes, and natural killer cells, as well as cytokines and the complement system. Complement plays an important role in the immediate response against microorganisms, including Streptococcus sp. The lectin pathway is one of three pathways by which the complement system can be activated. This pathway is initiated by the binding of mannose-binding lectin (MBL), collectin 11 (CL-K1), and ficolins (Ficolin-1, Ficolin-2, and Ficolin-3) to microbial surface oligosaccharides and acetylated residues, respectively. Upon binding to target molecules, MBL, CL-K1, and ficolins form complexes with MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2), which cleave C4 and C2 forming the C3 convertase (C4b2a). Subsequent activation of complement cascade leads to opsonization, phagocytosis, and lysis of target microorganisms through the formation of the membrane-attack complex. In addition, activation of complement may induce several inflammatory effects, such as expression of adhesion molecules, chemotaxis and activation of leukocytes, release of reactive oxygen species, and secretion of cytokines and chemokines. In this chapter, we review the general aspects of the structure, function, and genetic polymorphism of lectin-pathway components and discuss most recent understanding on the role of the lectin pathway in the predisposition and clinical progression of Rheumatic Fever. PMID:25654073

Analysis of Multiallelic CNVs by Emulsion Haplotype Fusion PCR.

PubMed

Tyson, Jess; Armour, John A L

2017-01-01

Emulsion-fusion PCR recovers long-range sequence information by combining products in cis from individual genomic DNA molecules. Emulsion droplets act as very numerous small reaction chambers in which different PCR products from a single genomic DNA molecule are condensed into short joint products, to unite sequences in cis from widely separated genomic sites. These products can therefore provide information about the arrangement of sequences and variants at a larger scale than established long-read sequencing methods. The method has been useful in defining the phase of variants in haplotypes, the typing of inversions, and determining the configuration of sequence variants in multiallelic CNVs. In this description we outline the rationale for the application of emulsion-fusion PCR methods to the analysis of multiallelic CNVs, and give practical details for our own implementation of the method in that context.

Array CGH analysis of a cohort of Russian patients with intellectual disability.

PubMed

Kashevarova, Anna A; Nazarenko, Lyudmila P; Skryabin, Nikolay A; Salyukova, Olga A; Chechetkina, Nataliya N; Tolmacheva, Ekaterina N; Sazhenova, Elena A; Magini, Pamela; Graziano, Claudio; Romeo, Giovanni; Kučinskas, Vaidutis; Lebedev, Igor N

2014-02-15

The use of array comparative genomic hybridization (array CGH) as a diagnostic tool in molecular genetics has facilitated the identification of many new microdeletion/microduplication syndromes (MMSs). Furthermore, this method has allowed for the identification of copy number variations (CNVs) whose pathogenic role has yet to be uncovered. Here, we report on our application of array CGH for the identification of pathogenic CNVs in 79 Russian children with intellectual disability (ID). Twenty-six pathogenic or likely pathogenic changes in copy number were detected in 22 patients (28%): 8 CNVs corresponded to known MMSs, and 17 were not associated with previously described syndromes. In this report, we describe our findings and comment on genes potentially associated with ID that are located within the CNV regions. Copyright © 2013 Elsevier B.V. All rights reserved.

Steroids and triterpenes from the fruit bodies of Ganoderma lucidum and their anti-complement activity.

PubMed

Seo, Hyo Won; Hung, Tran Manh; Na, MinKyun; Jung, Hyun Ju; Kim, Jin Cheol; Choi, Jae Sue; Kim, Jung Hee; Lee, Hyeong-Kyu; Lee, IkSoo; Bae, KiHwan; Hattori, Masao; Min, Byung Sun

2009-11-01

To determine the anti-complement activity of natural triterpenes, chromatographic separation of the EtOAc-soluble fraction from the fruiting body of Ganoderma lucidum led to the isolation of three steroids and five triterpenoids. They were identified as ergosterol peroxide (1), ergosterol (2), genoderic acid Sz (3), stella sterol (4), ganoderic aic C1 (5), ganoderic acid A (6), methyl ganoderate A (7), and lucidenic acid A (8) based on spectroscopic evidence and physicochemical properties. These compounds were examined for their anti-complement activity against the classical pathway of the complement system. Compounds 2 and 3 showed potent anti-complement activity with IC50 values of 52.0 and 44.6 microM, respectively. Compound 1 exhibited significant inhibitory activity with an IC50 value of 126.8 microM, whereas compounds 4-8 were inactive. Our findings suggested that in addition to the ketone group at C-3, the delta7(8), delta9(11)-lanostadiene type triterpene also plays an important role in inhibiting the hemolytic activity of human serum against erythrocytes.

The Lectin Complement Pathway Is Involved in Protection Against Enteroaggregative Escherichia coli Infection.

PubMed

Adler Sørensen, Camilla; Rosbjerg, Anne; Hebbelstrup Jensen, Betina; Krogfelt, Karen Angeliki; Garred, Peter

2018-01-01

Enteroaggregative Escherichia coli (EAEC) causes acute and persistent diarrhea worldwide. Still, the involvement of host factors in EAEC infections is unresolved. Binding of recognition molecules from the lectin pathway of complement to EAEC strains have been observed, but the importance is not known. Our aim was to uncover the involvement of these molecules in innate complement dependent immune protection toward EAEC. Binding of mannose-binding lectin, ficolin-1, -2, and -3 to four prototypic EAEC strains, and ficolin-2 binding to 56 clinical EAEC isolates were screened by a consumption-based ELISA method. Flow cytometry was used to determine deposition of C4b, C3b, and the bactericidal C5b-9 membrane attack complex (MAC) on the bacteria in combination with different complement inhibitors. In addition, the direct serum bactericidal effect was assessed. Screening of the prototypic EAEC strains revealed that ficolin-2 was the major binder among the lectin pathway recognition molecules. However, among the clinical EAEC isolates only a restricted number ( n  = 5) of the isolates bound ficolin-2. Using the ficolin-2 binding isolate C322-17 as a model, we found that incubation with normal human serum led to deposition of C4b, C3b, and to MAC formation. No inhibition of complement deposition was observed when a C1q inhibitor was added, while partial inhibition was observed when ficolin-2 or factor D inhibitors were used separately. Combining the inhibitors against ficolin-2 and factor D led to virtually complete inhibition of complement deposition and protection against direct bacterial killing. These results demonstrate that ficolin-2 may play an important role in innate immune protection against EAEC when an appropriate ligand is exposed, but many EAEC strains evade lectin pathway recognition and may, therefore, circumvent this strategy of innate host immune protection.

T cell subsets and immunoglobulin G levels are associated with the infection status of systemic lupus erythematosus patients

PubMed Central

Wu, Lifen; Wang, Xinru; Chen, Fenghua; Lv, Xing; Sun, Wenwen; Guo, Ying; Hou, Hou; Ji, Haiyan; Wei, Wei; Gong, Lu

2017-01-01

Systemic lupus erythematosus (SLE) is a chronic, autoimmune disorder that affects nearly all organs and tissues. As knowledge about the mechanism of SLE has increased, some immunosuppressive agents have become routinely used in clinical care, and infections have become one of the direct causes of mortality in SLE patients. To identify the risk factors indicative of infection in SLE patients, a case control study of our hospital's medical records between 2011 and 2013 was performed. We reviewed the records of 117 SLE patients with infection and 61 SLE patients without infection. Changes in the levels of T cell subsets, immunoglobulin G (IgG), complement C3, complement C4, globulin, and anti-double-stranded DNA (anti-ds-DNA) were detected. CD4+ and CD4+/CD8+ T cell levels were significantly lower and CD8+ T cell levels were significantly greater in SLE patients with infection than in SLE patients without infection. Additionally, the concentrations of IgG in SLE patients with infection were significantly lower than those in SLE patients without infection. However, complement C3, complement C4, globulin, and anti-ds-DNA levels were not significantly different in SLE patients with and without infection. Therefore, clinical testing for T cell subsets and IgG is potentially useful for identifying the presence of infection in SLE patients and for distinguishing a lupus flare from an acute infection. PMID:29267496

Genome-wide analyses of LINE–LINE-mediated nonallelic homologous recombination

PubMed Central

Startek, Michał; Szafranski, Przemyslaw; Gambin, Tomasz; Campbell, Ian M.; Hixson, Patricia; Shaw, Chad A.; Stankiewicz, Paweł; Gambin, Anna

2015-01-01

Nonallelic homologous recombination (NAHR), occurring between low-copy repeats (LCRs) >10 kb in size and sharing >97% DNA sequence identity, is responsible for the majority of recurrent genomic rearrangements in the human genome. Recent studies have shown that transposable elements (TEs) can also mediate recurrent deletions and translocations, indicating the features of substrates that mediate NAHR may be significantly less stringent than previously believed. Using >4 kb length and >95% sequence identity criteria, we analyzed of the genome-wide distribution of long interspersed element (LINE) retrotransposon and their potential to mediate NAHR. We identified 17 005 directly oriented LINE pairs located <10 Mbp from each other as potential NAHR substrates, placing 82.8% of the human genome at risk of LINE–LINE-mediated instability. Cross-referencing these regions with CNVs in the Baylor College of Medicine clinical chromosomal microarray database of 36 285 patients, we identified 516 CNVs potentially mediated by LINEs. Using long-range PCR of five different genomic regions in a total of 44 patients, we confirmed that the CNV breakpoints in each patient map within the LINE elements. To additionally assess the scale of LINE–LINE/NAHR phenomenon in the human genome, we tested DNA samples from six healthy individuals on a custom aCGH microarray targeting LINE elements predicted to mediate CNVs and identified 25 LINE–LINE rearrangements. Our data indicate that LINE–LINE-mediated NAHR is widespread and under-recognized, and is an important mechanism of structural rearrangement contributing to human genomic variability. PMID:25613453

Use of next-generation sequencing to detect LDLR gene copy number variation in familial hypercholesterolemia[S

PubMed Central

Iacocca, Michael A.; Wang, Jian; Dron, Jacqueline S.; Robinson, John F.; McIntyre, Adam D.; Cao, Henian

2017-01-01

Familial hypercholesterolemia (FH) is a heritable condition of severely elevated LDL cholesterol, caused predominantly by autosomal codominant mutations in the LDL receptor gene (LDLR). In providing a molecular diagnosis for FH, the current procedure often includes targeted next-generation sequencing (NGS) panels for the detection of small-scale DNA variants, followed by multiplex ligation-dependent probe amplification (MLPA) in LDLR for the detection of whole-exon copy number variants (CNVs). The latter is essential because ∼10% of FH cases are attributed to CNVs in LDLR; accounting for them decreases false negative findings. Here, we determined the potential of replacing MLPA with bioinformatic analysis applied to NGS data, which uses depth-of-coverage analysis as its principal method to identify whole-exon CNV events. In analysis of 388 FH patient samples, there was 100% concordance in LDLR CNV detection between these two methods: 38 reported CNVs identified by MLPA were also successfully detected by our NGS method, while 350 samples negative for CNVs by MLPA were also negative by NGS. This result suggests that MLPA can be removed from the routine diagnostic screening for FH, significantly reducing associated costs, resources, and analysis time, while promoting more widespread assessment of this important class of mutations across diagnostic laboratories. PMID:28874442

Phenotypic Analysis of Korean Patients with Abnormal Chromosomal Microarray in Patients with Unexplained Developmental Delay/Intellectual Disability.

PubMed

Kim, Hyo Jeong; Park, Chang Il; Lim, Jae Woo; Lee, Gyung Min; Cho, Eunhae; Kim, Hyon J

2018-05-01

The present study aimed to investigate chromosomal microarray (CMA) and clinical data in patients with unexplained developmental delay/intellectual disability (DD/ID) accompanying dysmorphism, congenital anomalies, or epilepsy. We also aimed to evaluate phenotypic clues in patients with pathogenic copy number variants (CNVs). We collected clinical and CMA data from patients at Konyang University Hospital between September 2013 and October 2014. We included patients who had taken the CMA test to evaluate the etiology of unexplained DD/ID. All of the 50 patients identified had DD/ID. Thirty-nine patients had dysmorphism, 19 patients suffered from epilepsy, and 12 patients had congenital anomalies. Twenty-nine of the 50 patients (58%) showed abnormal results. Eighteen (36%) were considered to have pathogenic CNVs. Dysmorphism (p=0.028) was significantly higher in patients with pathogenic CNVs than in those with normal CMA. Two or more clinical features were presented by 61.9% (13/21) of the patients with normal CMA and by 83.3% (15/18) of the patients with pathogenic CMA. Dysmorphism can be a phenotypic clue to pathogenic CNVs. Furthermore, pathogenic CNV might be more frequently found if patients have two or more clinical features in addition to DD/ID. © Copyright: Yonsei University College of Medicine 2018.

CNV-CH: A Convex Hull Based Segmentation Approach to Detect Copy Number Variations (CNV) Using Next-Generation Sequencing Data

PubMed Central

De, Rajat K.

2015-01-01

Copy number variation (CNV) is a form of structural alteration in the mammalian DNA sequence, which are associated with many complex neurological diseases as well as cancer. The development of next generation sequencing (NGS) technology provides us a new dimension towards detection of genomic locations with copy number variations. Here we develop an algorithm for detecting CNVs, which is based on depth of coverage data generated by NGS technology. In this work, we have used a novel way to represent the read count data as a two dimensional geometrical point. A key aspect of detecting the regions with CNVs, is to devise a proper segmentation algorithm that will distinguish the genomic locations having a significant difference in read count data. We have designed a new segmentation approach in this context, using convex hull algorithm on the geometrical representation of read count data. To our knowledge, most algorithms have used a single distribution model of read count data, but here in our approach, we have considered the read count data to follow two different distribution models independently, which adds to the robustness of detection of CNVs. In addition, our algorithm calls CNVs based on the multiple sample analysis approach resulting in a low false discovery rate with high precision. PMID:26291322

MSeq-CNV: accurate detection of Copy Number Variation from Sequencing of Multiple samples.

PubMed

Malekpour, Seyed Amir; Pezeshk, Hamid; Sadeghi, Mehdi

2018-03-05

Currently a few tools are capable of detecting genome-wide Copy Number Variations (CNVs) based on sequencing of multiple samples. Although aberrations in mate pair insertion sizes provide additional hints for the CNV detection based on multiple samples, the majority of the current tools rely only on the depth of coverage. Here, we propose a new algorithm (MSeq-CNV) which allows detecting common CNVs across multiple samples. MSeq-CNV applies a mixture density for modeling aberrations in depth of coverage and abnormalities in the mate pair insertion sizes. Each component in this mixture density applies a Binomial distribution for modeling the number of mate pairs with aberration in the insertion size and also a Poisson distribution for emitting the read counts, in each genomic position. MSeq-CNV is applied on simulated data and also on real data of six HapMap individuals with high-coverage sequencing, in 1000 Genomes Project. These individuals include a CEU trio of European ancestry and a YRI trio of Nigerian ethnicity. Ancestry of these individuals is studied by clustering the identified CNVs. MSeq-CNV is also applied for detecting CNVs in two samples with low-coverage sequencing in 1000 Genomes Project and six samples form the Simons Genome Diversity Project.

CNV-CH: A Convex Hull Based Segmentation Approach to Detect Copy Number Variations (CNV) Using Next-Generation Sequencing Data.

PubMed

Sinha, Rituparna; Samaddar, Sandip; De, Rajat K

2015-01-01

Copy number variation (CNV) is a form of structural alteration in the mammalian DNA sequence, which are associated with many complex neurological diseases as well as cancer. The development of next generation sequencing (NGS) technology provides us a new dimension towards detection of genomic locations with copy number variations. Here we develop an algorithm for detecting CNVs, which is based on depth of coverage data generated by NGS technology. In this work, we have used a novel way to represent the read count data as a two dimensional geometrical point. A key aspect of detecting the regions with CNVs, is to devise a proper segmentation algorithm that will distinguish the genomic locations having a significant difference in read count data. We have designed a new segmentation approach in this context, using convex hull algorithm on the geometrical representation of read count data. To our knowledge, most algorithms have used a single distribution model of read count data, but here in our approach, we have considered the read count data to follow two different distribution models independently, which adds to the robustness of detection of CNVs. In addition, our algorithm calls CNVs based on the multiple sample analysis approach resulting in a low false discovery rate with high precision.

Novel Population Specific Autosomal Copy Number Variation and Its Functional Analysis amongst Negritos from Peninsular Malaysia

PubMed Central

Mokhtar, Siti Shuhada; Marshall, Christian R.; Phipps, Maude E.; Thiruvahindrapuram, Bhooma; Lionel, Anath C.; Scherer, Stephen W.; Peng, Hoh Boon

2014-01-01

Copy number variation (CNV) has been recognized as a major contributor to human genome diversity. It plays an important role in determining phenotypes and has been associated with a number of common and complex diseases. However CNV data from diverse populations is still limited. Here we report the first investigation of CNV in the indigenous populations from Peninsular Malaysia. We genotyped 34 Negrito genomes from Peninsular Malaysia using the Affymetrix SNP 6.0 microarray and identified 48 putative novel CNVs, consisting of 24 gains and 24 losses, of which 5 were identified in at least 2 unrelated samples. These CNVs appear unique to the Negrito population and were absent in the DGV, HapMap3 and Singapore Genome Variation Project (SGVP) datasets. Analysis of gene ontology revealed that genes within these CNVs were enriched in the immune system (GO:0002376), response to stimulus mechanisms (GO:0050896), the metabolic pathways (GO:0001852), as well as regulation of transcription (GO:0006355). Copy number gains in CNV regions (CNVRs) enriched with genes were significantly higher than the losses (P value <0.001). In view of the small population size, relative isolation and semi-nomadic lifestyles of this community, we speculate that these CNVs may be attributed to recent local adaptation of Negritos from Peninsular Malaysia. PMID:24956385

Novel population specific autosomal copy number variation and its functional analysis amongst Negritos from Peninsular Malaysia.

PubMed

Mokhtar, Siti Shuhada; Marshall, Christian R; Phipps, Maude E; Thiruvahindrapuram, Bhooma; Lionel, Anath C; Scherer, Stephen W; Peng, Hoh Boon

2014-01-01

Copy number variation (CNV) has been recognized as a major contributor to human genome diversity. It plays an important role in determining phenotypes and has been associated with a number of common and complex diseases. However CNV data from diverse populations is still limited. Here we report the first investigation of CNV in the indigenous populations from Peninsular Malaysia. We genotyped 34 Negrito genomes from Peninsular Malaysia using the Affymetrix SNP 6.0 microarray and identified 48 putative novel CNVs, consisting of 24 gains and 24 losses, of which 5 were identified in at least 2 unrelated samples. These CNVs appear unique to the Negrito population and were absent in the DGV, HapMap3 and Singapore Genome Variation Project (SGVP) datasets. Analysis of gene ontology revealed that genes within these CNVs were enriched in the immune system (GO:0002376), response to stimulus mechanisms (GO:0050896), the metabolic pathways (GO:0001852), as well as regulation of transcription (GO:0006355). Copy number gains in CNV regions (CNVRs) enriched with genes were significantly higher than the losses (P value <0.001). In view of the small population size, relative isolation and semi-nomadic lifestyles of this community, we speculate that these CNVs may be attributed to recent local adaptation of Negritos from Peninsular Malaysia.

SIGN-R1 and complement factors are involved in the systemic clearance of radiation-induced apoptotic cells in whole-body irradiated mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Jin-Yeon; Loh, SoHee; Cho, Eun-hee

Although SIGN-R1-mediated complement activation pathway has been shown to enhance the systemic clearance of apoptotic cells, the role of SIGN-R1 in the clearance of radiation-induced apoptotic cells has not been characterized and was investigated in this study. Our data indicated that whole-body γ-irradiation of mice increased caspase-3{sup +} apoptotic lymphocyte numbers in secondary lymphoid organs. Following γ-irradiation, SIGN-R1 and complements (C4 and C3) were simultaneously increased only in the mice spleen tissue among the assessed tissues. In particular, C3 was exclusively activated in the spleen. The delayed clearance of apoptotic cells was markedly prevalent in the spleen and liver ofmore » SIGN-R1 KO mice, followed by a significant increase of CD11b{sup +} cells. These results indicate that SIGN-R1 and complement factors play an important role in the systemic clearance of radiation-induced apoptotic innate immune cells to maintain tissue homeostasis after γ-irradiation. - Highlights: • Splenic SIGN-R1{sup +} macrophages are activated after γ-irradiation. • C3 and C4 levels increased and C3 was activated in the spleen after γ-irradiation. • SIGN-R1 mediated the systemic clearance of radiation-induced apoptotic cells in spleen and liver.« less

Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development

PubMed Central

Jäckel, Sven; Saffarzadeh, Mona; Langer, Florian

2017-01-01

Expanding evidence indicates multiple interactions between the hemostatic system and innate immunity, and the coagulation and complement cascades. Here we show in a tissue factor (TF)–dependent model of flow restriction-induced venous thrombosis that complement factors make distinct contributions to platelet activation and fibrin deposition. Complement factor 3 (C3) deficiency causes prolonged bleeding, reduced thrombus incidence, thrombus size, fibrin and platelet deposition in the ligated inferior vena cava, and diminished platelet activation in vitro. Initial fibrin deposition at the vessel wall over 6 hours in this model was dependent on protein disulfide isomerase (PDI) and TF expression by myeloid cells, but did not require neutrophil extracellular trap formation involving peptidyl arginine deiminase 4. In contrast to C3−/− mice, C5-deficient mice had no apparent defect in platelet activation in vitro, and vessel wall platelet deposition and initial hemostasis in vivo. However, fibrin formation, the exposure of negatively charged phosphatidylserine (PS) on adherent leukocytes, and clot burden after 48 hours were significantly reduced in C5−/− mice compared with wild-type controls. These results delineate that C3 plays specific roles in platelet activation independent of formation of the terminal complement complex and provide in vivo evidence for contributions of complement-dependent membrane perturbations to prothrombotic TF activation on myeloid cells. PMID:28223279

Effects of CTLA4-Fc on glomerular injury in humorally-mediated glomerulonephritis in BALB/c mice

PubMed Central

Kitching, A R; Huang, X R; Ruth, A-J; Tipping, P G; Holdsworth, S R

2002-01-01

The effect of cytotoxic T-lymphocyte-associated molecule 4-immunoglobulin fusion protein (CTLA4-Fc) on humorally-mediated glomerulonephritis was studied in accelerated anti-glomerular basement membrane (anti-GBM) glomerulonephritis induced in BALB/c mice. This strain of mice develops antibody and complement dependent glomerulonephritis under this protocol. Sensitized BALB/c mice developed high levels of circulating autologous antibody titres, intense glomerular deposition of mouse immunoglobulin and complement, significant proteinuria, renal impairment, significant glomerular necrosis and a minor component of crescent formation 10 days after challenge with a nephritogenic antigen (sheep anti-GBM globulin). Early treatment during the primary immune response, or continuous treatment throughout the disease with CTLA4-Fc, significantly suppressed mouse anti-sheep globulin antibody titres in serum, and immunoglobulin and complement deposition in glomeruli. The degree of glomerular necrosis was improved and proteinuria was reduced, particularly in the earlier stages of disease. Late treatment by CTLA4-Fc starting one day after challenge with sheep anti-mouse GBM did not affect antibody production and did not attenuate glomerulonephritis. The low level of crescent formation found in BALB/c mice developing glomerulonephritis was not prevented by the administration of CTLA4-Fc. These results demonstrate that CTLA4-Fc is of benefit in this model of glomerulonephritis by its capacity to attenuate antibody production, without affecting the minor degree of cell-mediated glomerular injury. PMID:12067297

Effects of CTLA4-Fc on glomerular injury in humorally-mediated glomerulonephritis in BALB/c mice.

PubMed

Kitching, A R; Huang, X R; Ruth, A-J; Tipping, P G; Holdsworth, S R

2002-06-01

The effect of cytotoxic T-lymphocyte-associated molecule 4-immunoglobulin fusion protein (CTLA4-Fc) on humorally-mediated glomerulonephritis was studied in accelerated anti-glomerular basement membrane (anti-GBM) glomerulonephritis induced in BALB/c mice. This strain of mice develops antibody and complement dependent glomerulonephritis under this protocol. Sensitized BALB/c mice developed high levels of circulating autologous antibody titres, intense glomerular deposition of mouse immunoglobulin and complement, significant proteinuria, renal impairment, significant glomerular necrosis and a minor component of crescent formation 10 days after challenge with a nephritogenic antigen (sheep anti-GBM globulin). Early treatment during the primary immune response, or continuous treatment throughout the disease with CTLA4-Fc, significantly suppressed mouse anti-sheep globulin antibody titres in serum, and immunoglobulin and complement deposition in glomeruli. The degree of glomerular necrosis was improved and proteinuria was reduced, particularly in the earlier stages of disease. Late treatment by CTLA4-Fc starting one day after challenge with sheep anti-mouse GBM did not affect antibody production and did not attenuate glomerulonephritis. The low level of crescent formation found in BALB/c mice developing glomerulonephritis was not prevented by the administration of CTLA4-Fc. These results demonstrate that CTLA4-Fc is of benefit in this model of glomerulonephritis by its capacity to attenuate antibody production, without affecting the minor degree of cell-mediated glomerular injury.

Shark complement: an assessment.

PubMed

Smith, S L

1998-12-01

The classical (CCP) and alternative (ACP) pathways of complement activation have been established for the nurse shark (Ginglymostoma cirratum). The isolation of a cDNA clone encoding a mannan-binding protein-associated serine protease (MASP)-1-like protein from the Japanese dogfish (Triakis scyllia) suggests the presence of a lectin pathway. The CCP consists of six functionally distinct components: C1n, C2n, C3n, C4n, C8n and C9n, and is activated by immune complexes in the presence of Ca++ and Mg++ ions. The ACP is antibody independent, requiring Mg++ ions and a heat-labile 90 kDa factor B-like protein for activity. Proteins considered homologues of C1q, C3 and C4 (C2n) of the mammalian complement system have been isolated from nurse shark serum. Shark C1q is composed of at least two chain types each showing 50% identity to human C1q chains A and B. Partial sequence of the globular domain of one of the chains shows it to be C1q-like rather than like mannan-binding protein. N-terminal amino acid sequences of the alpha and beta chain of shark C3 and C4 molecules show significant identity with corresponding human C3 and C4 chains. A sequence representing shark C4 gamma chain, shows little similarity to human C4 gamma chain. The terminal shark components C8n and C9n are functional analogues of mammalian C8 and C9. Anaphylatoxin activity has been demonstrated in activated shark serum, and porcine C5a desArg induces shark leucocyte chemotaxis. The deduced amino acid sequence of a partial C3 cDNA clone from the nurse shark shows 50%, 30% and 24% homology with the corresponding region of mammalian C3, C4 and alpha 2-macroglobulin. Deduced amino acid sequence data from partial Bf/C2 cDNA clones, two from the nurse shark and one from the Japanese dogfish, suggest that at least one species of elasmobranch has two distinct Bf/C2 genes.

Mutation screening of Chinese Treacher Collins syndrome patients identified novel TCOF1 mutations.

PubMed

Chen, Ying; Guo, Luo; Li, Chen-Long; Shan, Jing; Xu, Hai-Song; Li, Jie-Ying; Sun, Shan; Hao, Shao-Juan; Jin, Lei; Chai, Gang; Zhang, Tian-Yu

2018-04-01

Treacher Collins syndrome (TCS) (OMIM 154500) is a rare congenital craniofacial disorder with an autosomal dominant manner of inheritance in most cases. To date, three pathogenic genes (TCOF1, POLR1D and POLR1C) have been identified. In this study, we conducted mutational analysis on Chinese TCS patients to reveal a mutational spectrum of known causative genes and show phenotype-genotype data to provide more information for gene counselling and future studies on the pathogenesis of TCS. Twenty-two TCS patients were recruited from two tertiary referral centres, and Sanger sequencing for the coding exons and exon-intron boundaries of TCOF1, POLR1D and POLR1C was performed. For patients without small variants, further copy number variations (CNVs) analysis was conducted using high-density SNP array platforms. The Sanger sequencing overall mutation detection rate was as high as 86.3% (19/22) for our cohort. Fifteen TCOF1 pathogenic variants, including ten novel mutations, were identified in nineteen patients. No causative mutations in POLR1D and POLR1C genes and no CNVs mutations were detected. A suspected autosomal dominant inheritance case that implies germinal mosaicism was described. Our study confirmed that TCOF1 was the main disease-causing gene for the Chinese TCS population and revealed its mutation spectrum. We also addressed the need for more studies of mosaicism in TCS cases, which could explain the mechanism of autosomal dominant inheritance in TCS cases and benefit the prevention of TCS.

Cold activation of complement for monitoring the response to interferon in patients with chronic hepatitis C.

PubMed

Akahane, Y; Miyazaki, Y; Naitoh, S; Takeda, K; Tsuda, F; Okamoto, H; Itoh, K; Miyakawa, Y; Mayumi, M

1996-02-01

Because of its specific association with hepatitis C virus (HCV) infection, the cold activation of complement is an easy and inexpensive indicator of HCV viremia. It was evaluated for eligibility as a marker of response to interferon in patients with hepatitis C. The cold activation of complement was determined by the loss or decrease of hemolytic activity with the microtitration method in sera that had been stored at 4 degrees C overnight. We observed the loss of hemolytic activity by the cold activation of complement in 236 (72%) and a decrease in 56 (17%) of 327 sera from patients with HCV-associated chronic liver disease, which was much more (p < 0.001) that in 1 (1%) and 13 (14%), respectively, of 49 sera from patients with chronic liver disease associated with hepatitis B virus infection. Interferon-alpha (total dose 516 x 10(6) units) or interferon-alpha 2b (774 x 10(6) units) was given to 67 patients with chronic hepatitis C, of whom 56 had the cold activation of complement. The response to interferon was evaluated by the clearance of serum HCV RNA at 6 months after the completion of therapy. The cold activation of complement disappeared in 18 patients, of whom 15 (86%) responded. It persisted or fluctuated in the remaining 38 patients, only six (16%) of whom responded to interferon (p < 0.001). The cold activation of complement once disappeared at the completion of interferon and then reappeared in patients who relapsed after completing interferon therapy. These results indicate that the cold activation of complement may be associated with the presence of HCV in blood and a lower rate of durable response after completion of interferon therapy.

Array-CGH analysis in Rwandan patients presenting development delay/intellectual disability with multiple congenital anomalies.

PubMed

Uwineza, Annette; Caberg, Jean-Hubert; Hitayezu, Janvier; Hellin, Anne Cecile; Jamar, Mauricette; Dideberg, Vinciane; Rusingiza, Emmanuel K; Bours, Vincent; Mutesa, Leon

2014-07-12

Array-CGH is considered as the first-tier investigation used to identify copy number variations. Right now, there is no available data about the genetic etiology of patients with development delay/intellectual disability and congenital malformation in East Africa. Array comparative genomic hybridization was performed in 50 Rwandan patients with development delay/intellectual disability and multiple congenital abnormalities, using the Agilent's 180 K microarray platform. Fourteen patients (28%) had a global development delay whereas 36 (72%) patients presented intellectual disability. All patients presented multiple congenital abnormalities. Clinically significant copy number variations were found in 13 patients (26%). Size of CNVs ranged from 0,9 Mb to 34 Mb. Six patients had CNVs associated with known syndromes, whereas 7 patients presented rare genomic imbalances. This study showed that CNVs are present in African population and show the importance to implement genetic testing in East-African countries.

[Copy number variation: markers and predictors for type 2 diabetes].

PubMed

Ramírez-Valverde, Alan Gilberto; Antúnez-Ortiz, Diana Lizzete; Méndez-Beleche, Alberto; Flores-Alfaro, Eugenia; Ascencio-Montiel, Iván Jesús; Cruz, Miguel

2015-01-01

Type 2 diabetes (T2D) is a disease characterized by a deficiency in production or action of insulin. It is the result mainly of the interaction of the environment, lifestyle, as well as genetic factors. It is considered as one of the major health issues in the world because it affects severely the psychological well-being and overall life quality. Recently it has been shown that DNA copy number variations (CNVs) are associated with several diseases, including obesity and T2D. The CNVs are present from 9 to 18 % of the genome and can modify the expression levels of mRNA and proteins encoded by genes located near their localization. Less is known about their contribution to the pathogenesis of metabolic diseases, which is necessary to characterize so that these variations can be potentially used as biomarkers of genetic risk CNVs of T2D.

Anaconda: AN automated pipeline for somatic COpy Number variation Detection and Annotation from tumor exome sequencing data.

PubMed

Gao, Jianing; Wan, Changlin; Zhang, Huan; Li, Ao; Zang, Qiguang; Ban, Rongjun; Ali, Asim; Yu, Zhenghua; Shi, Qinghua; Jiang, Xiaohua; Zhang, Yuanwei

2017-10-03

Copy number variations (CNVs) are the main genetic structural variations in cancer genome. Detecting CNVs in genetic exome region is efficient and cost-effective in identifying cancer associated genes. Many tools had been developed accordingly and yet these tools lack of reliability because of high false negative rate, which is intrinsically caused by genome exonic bias. To provide an alternative option, here, we report Anaconda, a comprehensive pipeline that allows flexible integration of multiple CNV-calling methods and systematic annotation of CNVs in analyzing WES data. Just by one command, Anaconda can generate CNV detection result by up to four CNV detecting tools. Associated with comprehensive annotation analysis of genes involved in shared CNV regions, Anaconda is able to deliver a more reliable and useful report in assistance with CNV-associate cancer researches. Anaconda package and manual can be freely accessed at http://mcg.ustc.edu.cn/bsc/ANACONDA/ .

Vitamin D. Treatment of Prostate Cancer: The Inhibitory Role of IGFBP-3

DTIC Science & Technology

2005-01-01

cassette, sub-family C (CFTR/MRP), member 6 AA424804 Flag 1.62 2.36 Xeroderma pigmentosum , complementation group C (XPC) AA287323 1.44 1.83 2.25...expressed gene I (POVI) T72067 2.37 7.30 Xeroderma pigmentosum , complementation group C (XPC) AA287323 2.25 11.2 Claudin 4 AA506754 2.21 7.30 UDP...Diego: Academic Press; 2001. in DNA damage response ( xeroderma pigmentosum pp 257-303. compledamentatgerouponse C e r [e 3. Miller GJ. Vitamin D and

Serum complement C3 strongly correlates with whole-body insulin sensitivity in rheumatoid arthritis.

PubMed

Ursini, Francesco; D'Angelo, Salvatore; Russo, Emilio; Arturi, Franco; D'Antona, Lucia; Bruno, Caterina; Naty, Saverio; De Sarro, Giovambattista; Olivieri, Ignazio; Grembiale, Rosa Daniela

2017-01-01

Rheumatoid arthritis (RA) is characterised by an excess of cardiovascular diseases (CVD) risk, attributable to a synergy between under-diagnosed traditional risk factors (i.e. insulin resistance) and inflammatory disease activity. The aim of the present study was to evaluate the correlation between inflammatory measures and insulin sensitivity in RA patients. Forty non-diabetic RA patients (19 males) were recruited. All patients underwent anthropometric measurements, laboratory evaluation and oral glucose tolerance test (OGTT). Insulin sensitivity index (ISI) was calculated with the equation proposed by Matsuda et al., from dynamic values of glucose and insulin obtained during OGTT. In the univariate analysis, lnISI correlated inversely with age, BMI, waist circumference, sBP, ESR, lnCRP and complement C3, but not with disease duration, dBP or complement C4. In non-obese patients (BMI <30 kg/m2, n=28), only age, BMI, lnCRP and C3 maintained their correlation with lnISI. In a stepwise multiple regression using lnISI as the dependent variable and BMI, age, lnCRP and complement C3 as predictors, only BMI and C3 entered the equation and accounted for 38.2% of the variance in lnISI. In non-obese patients, only C3 entered the regression equation, accounting for 32.2% of the variance in lnISI. Using a ROC curve, we identified the best cut-off for complement C3 of 1.22 g/L that yielded a sensitivity of 67% and a specificity of 79% for classification of insulin resistant patients. In RA patients, complement C3 correlates strongly with insulin sensitivity, in both obese and non-obese individuals.

Xp11.2 microduplications including IQSEC2, TSPYL2 and KDM5C genes in patients with neurodevelopmental disorders

PubMed Central

Moey, Ching; Hinze, Susan J; Brueton, Louise; Morton, Jenny; McMullan, Dominic J; Kamien, Benjamin; Barnett, Christopher P; Brunetti-Pierri, Nicola; Nicholl, Jillian; Gecz, Jozef; Shoubridge, Cheryl

2016-01-01

Copy number variations are a common cause of intellectual disability (ID). Determining the contribution of copy number variants (CNVs), particularly gains, to disease remains challenging. Here, we report four males with ID with sub-microscopic duplications at Xp11.2 and review the few cases with overlapping duplications reported to date. We established the extent of the duplicated regions in each case encompassing a minimum of three known disease genes TSPYL2, KDM5C and IQSEC2 with one case also duplicating the known disease gene HUWE1. Patients with a duplication encompassing TSPYL2, KDM5C and IQSEC2 without gains of nearby SMC1A and HUWE1 genes have not been reported thus far. All cases presented with ID and significant deficits of speech development. Some patients also manifested behavioral disturbances such as hyperactivity and attention-deficit/hyperactivity disorder. Lymphoblastic cell lines from patients show markedly elevated levels of TSPYL2, KDM5C and SMC1A, transcripts consistent with the extent of their CNVs. The duplicated region in our patients contains several genes known to escape X-inactivation, including KDM5C, IQSEC2 and SMC1A. In silico analysis of expression data in selected gene expression omnibus series indicates that dosage of these genes, especially IQSEC2, is similar in males and females despite the fact they escape from X-inactivation in females. Taken together, the data suggest that gains in Xp11.22 including IQSEC2 cause ID and are associated with hyperactivity and attention-deficit/hyperactivity disorder, and are likely to be dosage-sensitive in males. PMID:26059843

Xp11.2 microduplications including IQSEC2, TSPYL2 and KDM5C genes in patients with neurodevelopmental disorders.

PubMed

Moey, Ching; Hinze, Susan J; Brueton, Louise; Morton, Jenny; McMullan, Dominic J; Kamien, Benjamin; Barnett, Christopher P; Brunetti-Pierri, Nicola; Nicholl, Jillian; Gecz, Jozef; Shoubridge, Cheryl

2016-03-01

Copy number variations are a common cause of intellectual disability (ID). Determining the contribution of copy number variants (CNVs), particularly gains, to disease remains challenging. Here, we report four males with ID with sub-microscopic duplications at Xp11.2 and review the few cases with overlapping duplications reported to date. We established the extent of the duplicated regions in each case encompassing a minimum of three known disease genes TSPYL2, KDM5C and IQSEC2 with one case also duplicating the known disease gene HUWE1. Patients with a duplication encompassing TSPYL2, KDM5C and IQSEC2 without gains of nearby SMC1A and HUWE1 genes have not been reported thus far. All cases presented with ID and significant deficits of speech development. Some patients also manifested behavioral disturbances such as hyperactivity and attention-deficit/hyperactivity disorder. Lymphoblastic cell lines from patients show markedly elevated levels of TSPYL2, KDM5C and SMC1A, transcripts consistent with the extent of their CNVs. The duplicated region in our patients contains several genes known to escape X-inactivation, including KDM5C, IQSEC2 and SMC1A. In silico analysis of expression data in selected gene expression omnibus series indicates that dosage of these genes, especially IQSEC2, is similar in males and females despite the fact they escape from X-inactivation in females. Taken together, the data suggest that gains in Xp11.22 including IQSEC2 cause ID and are associated with hyperactivity and attention-deficit/hyperactivity disorder, and are likely to be dosage-sensitive in males.

Pathogenetic mechanisms in dengue haemorrhagic fever: Report of an international collaborative study*

PubMed Central

1973-01-01

In a study of 55 persons with dengue haemorrhagic fever—36 of whom showed the dengue shock syndrome—clinical, haematological, virological, and serological changes were correlated with serial measurements of complement components and immunopathological studies. Viruses dengue-1 or dengue-2 were isolated from the sera of 9 patients. Serological responses indicative of secondary dengue virus infections were observed in 53 patients; 2 (infants) had primary infections. During the acute phase of the disease, dengue antibody titres rose logarithmically. Marked depression of complement components, especially C3, was observed. Activation of both the classical and alternative complement pathways was demonstrated, with depression of both C4 and C3 proactivator levels in most instances, although in some cases it appeared that one mechanism was involved to a greater extent than the other. The level of depression of C3 was correlated with the severity of the disease. Relatively stable transferrin levels indicated that depletion of complement proteins was not primarily due to extravasation. Fibrinogen levels were depressed and fibrinogen split products were found in the plasma. The accumulated data provide further evidence of the central role that activated complement components play in the pathogenesis of dengue haemorrhagic fever. PMID:4575523

Association between Leptin and Complement in Hepatitis C Patients with Viral Clearance: Homeostasis of Metabolism and Immunity.

PubMed

Chang, Ming-Ling; Kuo, Chia-Jung; Huang, Hsin-Chih; Chu, Yin-Yi; Chiu, Cheng-Tang

2016-01-01

The association between leptin and complement in hepatitis C virus (HCV) infection remains unknown. A prospective study was conducted including 474 (250 genotype 1, 224 genotype 2) consecutive chronic hepatitis C (CHC) patients who had completed an anti-HCV therapy course and undergone pre-therapy and 24-week post-therapy assessments of interferon λ3-rs12979860 and HCV RNA/genotypes, anthropometric measurements, metabolic and liver profiles, and complement component 3 (C3), C4, and leptin levels. Of the 474 patients, 395 had a sustained virological response (SVR). Pre-therapy leptin levels did not differ between patients with and without an SVR. Univariate and multivariate analyses showed that sex (pre- and post-therapy, p<0.001), body mass index (BMI) (pre- and post-therapy, p<0.001), and C3 levels (pre-therapy, p = 0.027; post-therapy, p = 0.02) were independently associated with leptin levels with or without HCV infection. Pre-therapy BMI, total cholesterol (TC), C4 levels, and the rs12979860 genotype were independently associated with pre-therapy C3 levels in all patients. Post-therapy BMI, alanine aminotransferase, TC, C4 levels, white blood cell counts, and hepatic steatosis were independently associated with the post-therapy C3 levels of SVR patients. Compared with pre-therapy levels, SVR patients showed higher 24-week post-therapy C4 (20.32+/-7.30 vs. 21.55+/-7.07 mg/dL, p<0.001) and TC (171.68+/-32.67 vs. 186.97+/-36.09 mg/dL, p<0.001) levels; however, leptin and C3 levels remained unchanged after therapy in patients with and without an SVR. Leptin and C3 may maintain immune and metabolic homeostasis through association with C4 and TC. Positive alterations in C4 and TC levels reflect viral clearance after therapy in CHC patients.

Transcriptome Analysis of the Innate Immunity-Related Complement System in Spleen Tissue of Ctenopharyngodon idella Infected with Aeromonas hydrophila

PubMed Central

Dang, Yunfei; Xu, Xiaoyan; Shen, Yubang; Hu, Moyan; Zhang, Meng; Li, Lisen; Lv, Liqun; Li, Jiale

2016-01-01

The grass carp (Ctenopharyngodon idella) is an important commercial farmed herbivorous fish species in China, but is susceptible to Aeromonas hydrophila infections. In the present study, we performed de novo RNA-Seq sequencing of spleen tissue from specimens of a disease-resistant family, which were given intra-peritoneal injections containing PBS with or without a dose of A. hydrophila. The fish were sampled from the control group at 0 h, and from the experimental group at 4, 8, 12, 24, 48 and 72 h. 122.18 million clean reads were obtained from the normalized cDNA libraries; these were assembled into 425,260 contigs and then 191,795 transcripts. Of those, 52,668 transcripts were annotated with the NCBI Nr database, and 41,347 of the annotated transcripts were assigned into 90 functional groups. 20,569 unigenes were classified into six main categories, including 38 secondary KEGG pathways. 2,992 unigenes were used in the analysis of differentially expressed genes (DEGs). 89 of the putative DEGs were related to the immune system and 41 of them were involved in the complement and coagulation cascades pathway. This study provides insights into the complement and complement-related pathways involved in innate immunity, through expression profile analysis of the genomic resources in C. idella. We conclude that complement and complement-related genes play important roles during defense against A. hydrophila infection. The immune response is activated at 4 h after the bacterial injections, indicating that the complement pathways are activated at the early stage of bacterial infection. The study has improved our understanding of the immune response mechanisms in C. idella to bacterial pathogens. PMID:27383749

Immunization with Recombinantly Expressed LRP4 Induces Experimental Autoimmune Myasthenia Gravis in C57BL/6 Mice.

PubMed

Ulusoy, Canan; Çavuş, Filiz; Yılmaz, Vuslat; Tüzün, Erdem

2017-07-01

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ), characterized with muscle weakness. While MG develops due to acetylcholine receptor (AChR) antibodies in most patients, antibodies to muscle-specific receptor tyrosine kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4) may also be identified. Experimental autoimmune myasthenia gravis (EAMG) has been previously induced by both LRP4 immunization and passive transfer of LRP4 antibodies. Our aim was to confirm previous results and to test the pathogenic effects of LRP4 immunization in a commonly used mouse strain C57BL/6 (B6) using a recombinantly expressed human LRP4 protein. B6 mice were immunized with human LRP4 in CFA, Torpedo Californica AChR in CFA or only CFA. Clinical and pathogenic aspects of EAMG were compared among groups. LRP4- and AChR-immunized mice showed comparable EAMG clinical severity. LRP4-immunized mice displayed serum antibodies to LRP4 and NMJ IgG and complement factor C3 deposits. IgG2 was the dominant anti-LRP4 isotype. Cultured lymph node cells of LRP4- and AChR-immunized mice gave identical pro-inflammatory cytokine (IL-6, IFN-γ and IL-17) responses to LRP4 and AChR stimulation, respectively. Our results confirm the EAMG-inducing action of LRP4 immunization and identify B6 as a LRP4-EAMG-susceptible mouse strain. Demonstration of complement fixing anti-LRP4 antibodies in sera and complement/IgG deposits at the NMJ of LRP4-immunized mice indicates complement activation as a putative pathogenic mechanism. We have thus developed a practical LRP4-induced EAMG model using a non-conformational protein and a widely available mouse strain for future investigation of LRP4-related MG.

A previously unrecognized role of C3a in proteinuric progressive nephropathy

PubMed Central

Morigi, Marina; Locatelli, Monica; Rota, Cinzia; Buelli, Simona; Corna, Daniela; Rizzo, Paola; Abbate, Mauro; Conti, Debora; Perico, Luca; Longaretti, Lorena; Benigni, Ariela; Zoja, Carlamaria; Remuzzi, Giuseppe

2016-01-01

Podocyte loss is the initial event in the development of glomerulosclerosis, the structural hallmark of progressive proteinuric nephropathies. Understanding mechanisms underlying glomerular injury is the key challenge for identifying novel therapeutic targets. In mice with protein-overload induced by bovine serum albumin (BSA), we evaluated whether the alternative pathway (AP) of complement mediated podocyte depletion and podocyte-dependent parietal epithelial cell (PEC) activation causing glomerulosclerosis. Factor H (Cfh−/−) or factor B-deficient mice were studied in comparison with wild-type (WT) littermates. WT+BSA mice showed podocyte depletion accompanied by glomerular complement C3 and C3a deposits, PEC migration to capillary tuft, proliferation, and glomerulosclerosis. These changes were more prominent in Cfh−/− +BSA mice. The pathogenic role of AP was documented by data that factor B deficiency preserved glomerular integrity. In protein-overload mice, PEC dysregulation was associated with upregulation of CXCR4 and GDNF/c-Ret axis. In vitro studies provided additional evidence of a direct action of C3a on proliferation and CXCR4-related migration of PECs. These effects were enhanced by podocyte-derived GDNF. In patients with proteinuric nephropathy, glomerular C3/C3a paralleled PEC activation, CXCR4 and GDNF upregulation. These results indicate that mechanistically uncontrolled AP complement activation is not dispensable for podocyte-dependent PEC activation resulting in glomerulosclerosis. PMID:27345360

Use of next-generation sequencing to detect LDLR gene copy number variation in familial hypercholesterolemia.

PubMed

Iacocca, Michael A; Wang, Jian; Dron, Jacqueline S; Robinson, John F; McIntyre, Adam D; Cao, Henian; Hegele, Robert A

2017-11-01

Familial hypercholesterolemia (FH) is a heritable condition of severely elevated LDL cholesterol, caused predominantly by autosomal codominant mutations in the LDL receptor gene ( LDLR ). In providing a molecular diagnosis for FH, the current procedure often includes targeted next-generation sequencing (NGS) panels for the detection of small-scale DNA variants, followed by multiplex ligation-dependent probe amplification (MLPA) in LDLR for the detection of whole-exon copy number variants (CNVs). The latter is essential because ∼10% of FH cases are attributed to CNVs in LDLR ; accounting for them decreases false negative findings. Here, we determined the potential of replacing MLPA with bioinformatic analysis applied to NGS data, which uses depth-of-coverage analysis as its principal method to identify whole-exon CNV events. In analysis of 388 FH patient samples, there was 100% concordance in LDLR CNV detection between these two methods: 38 reported CNVs identified by MLPA were also successfully detected by our NGS method, while 350 samples negative for CNVs by MLPA were also negative by NGS. This result suggests that MLPA can be removed from the routine diagnostic screening for FH, significantly reducing associated costs, resources, and analysis time, while promoting more widespread assessment of this important class of mutations across diagnostic laboratories. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

panelcn.MOPS: Copy-number detection in targeted NGS panel data for clinical diagnostics.

PubMed

Povysil, Gundula; Tzika, Antigoni; Vogt, Julia; Haunschmid, Verena; Messiaen, Ludwine; Zschocke, Johannes; Klambauer, Günter; Hochreiter, Sepp; Wimmer, Katharina

2017-07-01

Targeted next-generation-sequencing (NGS) panels have largely replaced Sanger sequencing in clinical diagnostics. They allow for the detection of copy-number variations (CNVs) in addition to single-nucleotide variants and small insertions/deletions. However, existing computational CNV detection methods have shortcomings regarding accuracy, quality control (QC), incidental findings, and user-friendliness. We developed panelcn.MOPS, a novel pipeline for detecting CNVs in targeted NGS panel data. Using data from 180 samples, we compared panelcn.MOPS with five state-of-the-art methods. With panelcn.MOPS leading the field, most methods achieved comparably high accuracy. panelcn.MOPS reliably detected CNVs ranging in size from part of a region of interest (ROI), to whole genes, which may comprise all ROIs investigated in a given sample. The latter is enabled by analyzing reads from all ROIs of the panel, but presenting results exclusively for user-selected genes, thus avoiding incidental findings. Additionally, panelcn.MOPS offers QC criteria not only for samples, but also for individual ROIs within a sample, which increases the confidence in called CNVs. panelcn.MOPS is freely available both as R package and standalone software with graphical user interface that is easy to use for clinical geneticists without any programming experience. panelcn.MOPS combines high sensitivity and specificity with user-friendliness rendering it highly suitable for routine clinical diagnostics. © 2017 The Authors. Human Mutation published by Wiley Periodicals, Inc.

panelcn.MOPS: Copy‐number detection in targeted NGS panel data for clinical diagnostics

PubMed Central

Povysil, Gundula; Tzika, Antigoni; Vogt, Julia; Haunschmid, Verena; Messiaen, Ludwine; Zschocke, Johannes; Klambauer, Günter; Wimmer, Katharina

2017-01-01

Abstract Targeted next‐generation‐sequencing (NGS) panels have largely replaced Sanger sequencing in clinical diagnostics. They allow for the detection of copy‐number variations (CNVs) in addition to single‐nucleotide variants and small insertions/deletions. However, existing computational CNV detection methods have shortcomings regarding accuracy, quality control (QC), incidental findings, and user‐friendliness. We developed panelcn.MOPS, a novel pipeline for detecting CNVs in targeted NGS panel data. Using data from 180 samples, we compared panelcn.MOPS with five state‐of‐the‐art methods. With panelcn.MOPS leading the field, most methods achieved comparably high accuracy. panelcn.MOPS reliably detected CNVs ranging in size from part of a region of interest (ROI), to whole genes, which may comprise all ROIs investigated in a given sample. The latter is enabled by analyzing reads from all ROIs of the panel, but presenting results exclusively for user‐selected genes, thus avoiding incidental findings. Additionally, panelcn.MOPS offers QC criteria not only for samples, but also for individual ROIs within a sample, which increases the confidence in called CNVs. panelcn.MOPS is freely available both as R package and standalone software with graphical user interface that is easy to use for clinical geneticists without any programming experience. panelcn.MOPS combines high sensitivity and specificity with user‐friendliness rendering it highly suitable for routine clinical diagnostics. PMID:28449315

Genetic Analyses in Small-for-Gestational-Age Newborns.

PubMed

Stalman, Susanne E; Solanky, Nita; Ishida, Miho; Alemán-Charlet, Cristina; Abu-Amero, Sayeda; Alders, Marielle; Alvizi, Lucas; Baird, William; Demetriou, Charalambos; Henneman, Peter; James, Chela; Knegt, Lia C; Leon, Lydia J; Mannens, Marcel M A M; Mul, Adi N; Nibbering, Nicole A; Peskett, Emma; Rezwan, Faisal I; Ris-Stalpers, Carrie; van der Post, Joris A M; Kamp, Gerdine A; Plötz, Frans B; Wit, Jan M; Stanier, Philip; Moore, Gudrun E; Hennekam, Raoul C

2018-03-01

Small for gestational age (SGA) can be the result of fetal growth restriction, which is associated with perinatal morbidity and mortality. Mechanisms that control prenatal growth are poorly understood. The aim of the current study was to gain more insight into prenatal growth failure and determine an effective diagnostic approach in SGA newborns. We hypothesized that one or more copy number variations (CNVs) and disturbed methylation and sequence variants may be present in genes associated with fetal growth. A prospective cohort study of subjects with a low birth weight for gestational age. The study was conducted at an academic pediatric research institute. A total of 21 SGA newborns with a mean birth weight below the first centile and a control cohort of 24 appropriate-for-gestational-age newborns were studied. Array comparative genomic hybridization, genome-wide methylation studies, and exome sequencing were performed. The numbers of CNVs, methylation disturbances, and sequence variants. The genetic analyses demonstrated three CNVs, one systematically disturbed methylation pattern, and one sequence variant explaining SGA. Additional methylation disturbances and sequence variants were present in 20 patients. In 19 patients, multiple abnormalities were found. Our results confirm the influence of a large number of mechanisms explaining dysregulation of fetal growth. We concluded that CNVs, methylation disturbances, and sequence variants all contribute to prenatal growth failure. These genetic workups can be an effective diagnostic approach in SGA newborns.

Rare pseudoautosomal copy-number variations involving SHOX and/or its flanking regions in individuals with and without short stature.

PubMed

Fukami, Maki; Naiki, Yasuhiro; Muroya, Koji; Hamajima, Takashi; Soneda, Shun; Horikawa, Reiko; Jinno, Tomoko; Katsumi, Momori; Nakamura, Akie; Asakura, Yumi; Adachi, Masanori; Ogata, Tsutomu; Kanzaki, Susumu

2015-09-01

Pseudoautosomal region 1 (PAR1) contains SHOX, in addition to seven highly conserved non-coding DNA elements (CNEs) with cis-regulatory activity. Microdeletions involving SHOX exons 1-6a and/or the CNEs result in idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). Here, we report six rare copy-number variations (CNVs) in PAR1 identified through copy-number analyzes of 245 ISS/LWD patients and 15 unaffected individuals. The six CNVs consisted of three microduplications encompassing SHOX and some of the CNEs, two microduplications in the SHOX 3'-region affecting one or four of the downstream CNEs, and a microdeletion involving SHOX exon 6b and its neighboring CNE. The amplified DNA fragments of two SHOX-containing duplications were detected at chromosomal regions adjacent to the original positions. The breakpoints of a SHOX-containing duplication resided within Alu repeats. A microduplication encompassing four downstream CNEs was identified in an unaffected father-daughter pair, whereas the other five CNVs were detected in ISS patients. These results suggest that microduplications involving SHOX cause ISS by disrupting the cis-regulatory machinery of this gene and that at least some of microduplications in PAR1 arise from Alu-mediated non-allelic homologous recombination. The pathogenicity of other rare PAR1-linked CNVs, such as CNE-containing microduplications and exon 6b-flanking microdeletions, merits further investigation.

Feature co-localization landscape of the human genome

PubMed Central

Ng, Siu-Kin; Hu, Taobo; Long, Xi; Chan, Cheuk-Hin; Tsang, Shui-Ying; Xue, Hong

2016-01-01

Although feature co-localizations could serve as useful guide-posts to genome architecture, a comprehensive and quantitative feature co-localization map of the human genome has been lacking. Herein we show that, in contrast to the conventional bipartite division of genomic sequences into genic and inter-genic regions, pairwise co-localizations of forty-two genomic features in the twenty-two autosomes based on 50-kb to 2,000-kb sequence windows indicate a tripartite zonal architecture comprising Genic zones enriched with gene-related features and Alu-elements; Proximal zones enriched with MIR- and L2-elements, transcription-factor-binding-sites (TFBSs), and conserved-indels (CIDs); and Distal zones enriched with L1-elements. Co-localizations between single-nucleotide-polymorphisms (SNPs) and copy-number-variations (CNVs) reveal a fraction of sequence windows displaying steeply enhanced levels of SNPs, CNVs and recombination rates that point to active adaptive evolution in such pathways as immune response, sensory perceptions, and cognition. The strongest positive co-localization observed between TFBSs and CIDs suggests a regulatory role of CIDs in cooperation with TFBSs. The positive co-localizations of cancer somatic CNVs (CNVT) with all Proximal zone and most Genic zone features, in contrast to the distinctly more restricted co-localizations exhibited by germline CNVs (CNVG), reveal disparate distributions of CNVTs and CNVGs indicative of dissimilarity in their underlying mechanisms. PMID:26854351

A large-scale survey of genetic copy number variations among Han Chinese residing in Taiwan

PubMed Central

Lin, Chien-Hsing; Li, Ling-Hui; Ho, Sheng-Feng; Chuang, Tzu-Po; Wu, Jer-Yuarn; Chen, Yuan-Tsong; Fann, Cathy SJ

2008-01-01

Background Copy number variations (CNVs) have recently been recognized as important structural variations in the human genome. CNVs can affect gene expression and thus may contribute to phenotypic differences. The copy number inferring tool (CNIT) is an effective hidden Markov model-based algorithm for estimating allele-specific copy number and predicting chromosomal alterations from single nucleotide polymorphism microarrays. The CNIT algorithm, which was constructed using data from 270 HapMap multi-ethnic individuals, was applied to identify CNVs from 300 unrelated Han Chinese individuals in Taiwan. Results Using stringent selection criteria, 230 regions with variable copy numbers were identified in the Han Chinese population; 133 (57.83%) had been reported previously, 64 displayed greater than 1% CNV allele frequency. The average size of the CNV regions was 322 kb (ranging from 1.48 kb to 5.68 Mb) and covered a total of 2.47% of the human genome. A total of 196 of the CNV regions were simple deletions and 27 were simple amplifications. There were 449 genes and 5 microRNAs within these CNV regions; some of these genes are known to be associated with diseases. Conclusion The identified CNVs are characteristic of the Han Chinese population and should be considered when genetic studies are conducted. The CNV distribution in the human genome is still poorly characterized, and there is much diversity among different ethnic populations. PMID:19108714

Age-related macular degeneration and modification of systemic complement factor H production through liver transplantation.

PubMed

Khandhadia, Samir; Hakobyan, Svetlana; Heng, Ling Z; Gibson, Jane; Adams, David H; Alexander, Graeme J; Gibson, Jonathan M; Martin, Keith R; Menon, Geeta; Nash, Kathryn; Sivaprasad, Sobha; Ennis, Sarah; Cree, Angela J; Morgan, B Paul; Lotery, Andrew J

2013-08-01

To investigate whether modification of liver complement factor H (CFH) production, by alteration of liver CFH Y402H genotype through liver transplantation (LT), influences the development of age-related macular degeneration (AMD). Multicenter, cross-sectional study. We recruited 223 Western European patients ≥ 55 years old who had undergone LT ≥ 5 years previously. We determined AMD status using a standard grading system. Recipient CFH Y402H genotype was obtained from DNA extracted from recipient blood samples. Donor CFH Y402H genotype was inferred from recipient plasma CFH Y402H protein allotype, measured using enzyme-linked immunosorbent assays. This approach was verified by genotyping donor tissue from a subgroup of patients. Systemic complement activity was ascertained by measuring levels of plasma complement proteins using an enzyme-linked immunosorbent assay, including substrates (C3, C4), activation products (C3a, C4a, and terminal complement complex), and regulators (total CFH, C1 inhibitor). We evaluated AMD status and recipient and donor CFH Y402H genotype. In LT patients, AMD was associated with recipient CFH Y402H genotype (P = 0.036; odds ratio [OR], 1.6; 95% confidence interval [CI], 1.0-2.4) but not with donor CFH Y402H genotype (P = 0.626), after controlling for age, sex, smoking status, and body mass index. Recipient plasma CFH Y402H protein allotype predicted donor CFH Y402H genotype with 100% accuracy (n = 49). Plasma complement protein or activation product levels were similar in LT patients with and without AMD. Compared with previously reported prevalence figures (Rotterdam Study), LT patients demonstrated a high prevalence of both AMD (64.6% vs 37.1%; OR, 3.09; P<0.001) and the CFH Y402H sequence variation (41.9% vs 36.2%; OR, 1.27; P = 0.014). Presence of AMD is not associated with modification of hepatic CFH production. In addition, AMD is not associated with systemic complement activity in LT patients. These findings suggest that local intraocular complement activity is of greater importance in AMD pathogenesis. The high AMD prevalence observed in LT patients may be associated with the increased frequency of the CFH Y402H sequence variation. The authors have no proprietary or commercial interest in any materials discussed in this article. Copyright © 2013 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

Unique structure of iC3b resolved at a resolution of 24 Å by 3D-electron microscopy.

PubMed

Alcorlo, Martin; Martínez-Barricarte, Ruben; Fernández, Francisco J; Rodríguez-Gallego, César; Round, Adam; Vega, M Cristina; Harris, Claire L; de Cordoba, Santiago Rodríguez; Llorca, Oscar

2011-08-09

Activation of C3, deposition of C3b on the target surface, and subsequent amplification by formation of a C3-cleaving enzyme (C3-convertase; C3bBb) triggers the effector functions of complement that result in inflammation and cell lysis. Concurrently, surface-bound C3b is proteolyzed to iC3b by factor I and appropriate cofactors. iC3b then interacts with the complement receptors (CR) of the Ig superfamily, CR2 (CD21), CR3 (CD11b/CD18), and CR4 (CD11c/CD18) on leukocytes, down-modulating inflammation, enhancing B cell-mediated immunity, and targeting pathogens for clearance by phagocytosis. Using EM and small-angle X-ray scattering, we now present a medium-resolution structure of iC3b (24 Å). iC3b displays a unique conformation with structural features distinct from any other C3 fragment. The macroglobulin ring in iC3b is similar to that in C3b, whereas the TED (thioester-containing domain) domain and the remnants of the CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain have moved to locations more similar to where they were in native C3. A consequence of this large conformational change is the disruption of the factor B binding site, which renders iC3b unable to assemble a C3-convertase. This structural model also justifies the decreased interaction between iC3b and complement regulators and the recognition of iC3b by the CR of the Ig superfamily, CR2, CR3, and CR4. These data further illustrate the extraordinary conformational versatility of C3 to accommodate a great diversity of functional activities.

SCRIB and PUF60 Are Primary Drivers of the Multisystemic Phenotypes of the 8q24.3 Copy-Number Variant

PubMed Central

Dauber, Andrew; Golzio, Christelle; Guenot, Cécile; Jodelka, Francine M.; Kibaek, Maria; Kjaergaard, Susanne; Leheup, Bruno; Martinet, Danielle; Nowaczyk, Malgorzata J.M.; Rosenfeld, Jill A.; Zeesman, Susan; Zunich, Janice; Beckmann, Jacques S.; Hirschhorn, Joel N.; Hastings, Michelle L.; Jacquemont, Sebastien; Katsanis, Nicholas

2013-01-01

Copy-number variants (CNVs) represent a significant interpretative challenge, given that each CNV typically affects the dosage of multiple genes. Here we report on five individuals with coloboma, microcephaly, developmental delay, short stature, and craniofacial, cardiac, and renal defects who harbor overlapping microdeletions on 8q24.3. Fine mapping localized a commonly deleted 78 kb region that contains three genes: SCRIB, NRBP2, and PUF60. In vivo dissection of the CNV showed discrete contributions of the planar cell polarity effector SCRIB and the splicing factor PUF60 to the syndromic phenotype, and the combinatorial suppression of both genes exacerbated some, but not all, phenotypic components. Consistent with these findings, we identified an individual with microcephaly, short stature, intellectual disability, and heart defects with a de novo c.505C>T variant leading to a p.His169Tyr change in PUF60. Functional testing of this allele in vivo and in vitro showed that the mutation perturbs the relative dosage of two PUF60 isoforms and, subsequently, the splicing efficiency of downstream PUF60 targets. These data inform the functions of two genes not associated previously with human genetic disease and demonstrate how CNVs can exhibit complex genetic architecture, with the phenotype being the amalgam of both discrete dosage dysfunction of single transcripts and also of binary genetic interactions. PMID:24140112

Genome-wide DNA polymorphism in the indica rice varieties RGD-7S and Taifeng B as revealed by whole genome re-sequencing.

PubMed

Fu, Chong-Yun; Liu, Wu-Ge; Liu, Di-Lin; Li, Ji-Hua; Zhu, Man-Shan; Liao, Yi-Long; Liu, Zhen-Rong; Zeng, Xue-Qin; Wang, Feng

2016-03-01

Next-generation sequencing technologies provide opportunities to further understand genetic variation, even within closely related cultivars. We performed whole genome resequencing of two elite indica rice varieties, RGD-7S and Taifeng B, whose F1 progeny showed hybrid weakness and hybrid vigor when grown in the early- and late-cropping seasons, respectively. Approximately 150 million 100-bp pair-end reads were generated, which covered ∼86% of the rice (Oryza sativa L. japonica 'Nipponbare') reference genome. A total of 2,758,740 polymorphic sites including 2,408,845 SNPs and 349,895 InDels were detected in RGD-7S and Taifeng B, respectively. Applying stringent parameters, we identified 961,791 SNPs and 46,640 InDels between RGD-7S and Taifeng B (RGD-7S/Taifeng B). The density of DNA polymorphisms was 256.8 SNPs and 12.5 InDels per 100 kb for RGD-7S/Taifeng B. Copy number variations (CNVs) were also investigated. In RGD-7S, 1989 of 2727 CNVs were overlapped in 218 genes, and 1231 of 2010 CNVs were annotated in 175 genes in Taifeng B. In addition, we verified a subset of InDels in the interval of hybrid weakness genes, Hw3 and Hw4, and obtained some polymorphic InDel markers, which will provide a sound foundation for cloning hybrid weakness genes. Analysis of genomic variations will also contribute to understanding the genetic basis of hybrid weakness and heterosis.

Analysis of copy number variations in Holstein cows identify potential mechanisms contributing to differences in residual feed intake.

PubMed

Hou, Yali; Bickhart, Derek M; Chung, Hoyoung; Hutchison, Jana L; Norman, H Duane; Connor, Erin E; Liu, George E

2012-11-01

Genomic structural variation is an important and abundant source of genetic and phenotypic variation. In this study, we performed an initial analysis of copy number variations (CNVs) using BovineHD SNP genotyping data from 147 Holstein cows identified as having high or low feed efficiency as estimated by residual feed intake (RFI). We detected 443 candidate CNV regions (CNVRs) that represent 18.4 Mb (0.6 %) of the genome. To investigate the functional impacts of CNVs, we created two groups of 30 individual animals with extremely low or high estimated breeding values (EBVs) for RFI, and referred to these groups as low intake (LI; more efficient) or high intake (HI; less efficient), respectively. We identified 240 (~9.0 Mb) and 274 (~10.2 Mb) CNVRs from LI and HI groups, respectively. Approximately 30-40 % of the CNVRs were specific to the LI group or HI group of animals. The 240 LI CNVRs overlapped with 137 Ensembl genes. Network analyses indicated that the LI-specific genes were predominantly enriched for those functioning in the inflammatory response and immunity. By contrast, the 274 HI CNVRs contained 177 Ensembl genes. Network analyses indicated that the HI-specific genes were particularly involved in the cell cycle, and organ and bone development. These results relate CNVs to two key variables, namely immune response and organ and bone development. The data indicate that greater feed efficiency relates more closely to immune response, whereas cattle with reduced feed efficiency may have a greater capacity for organ and bone development.

Distribution, functional impact, and origin mechanisms of copy number variation in the barley genome

PubMed Central

2013-01-01

Background There is growing evidence for the prevalence of copy number variation (CNV) and its role in phenotypic variation in many eukaryotic species. Here we use array comparative genomic hybridization to explore the extent of this type of structural variation in domesticated barley cultivars and wild barleys. Results A collection of 14 barley genotypes including eight cultivars and six wild barleys were used for comparative genomic hybridization. CNV affects 14.9% of all the sequences that were assessed. Higher levels of CNV diversity are present in the wild accessions relative to cultivated barley. CNVs are enriched near the ends of all chromosomes except 4H, which exhibits the lowest frequency of CNVs. CNV affects 9.5% of the coding sequences represented on the array and the genes affected by CNV are enriched for sequences annotated as disease-resistance proteins and protein kinases. Sequence-based comparisons of CNV between cultivars Barke and Morex provided evidence that DNA repair mechanisms of double-strand breaks via single-stranded annealing and synthesis-dependent strand annealing play an important role in the origin of CNV in barley. Conclusions We present the first catalog of CNVs in a diploid Triticeae species, which opens the door for future genome diversity research in a tribe that comprises the economically important cereal species wheat, barley, and rye. Our findings constitute a valuable resource for the identification of CNV affecting genes of agronomic importance. We also identify potential mechanisms that can generate variation in copy number in plant genomes. PMID:23758725

Rare Copy Number Variation in Treatment-Resistant Major Depressive Disorder

PubMed Central

O’Dushlaine, Colm; Ripke, Stephan; Ruderfer, Douglas M.; Hamilton, Steven P.; Fava, Maurizio; Iosifescu, Dan V.; Kohane, Isaac S.; Churchill, Susanne E.; Castro, Victor M.; Clements, Caitlin C.; Blumenthal, Sarah R.; Murphy, Shawn N.; Smoller, Jordan W.; Perlis, Roy H.

2014-01-01

Background While antidepressant treatment response appears to be partially heritable, no consistent genetic associations have been identified. Large, rare copy number variants (CNVs) play a role in other neuropsychiatric diseases, so we assessed their association with treatment-resistant depression (TRD). Methods We analyzed data from two genome-wide association studies comprising 1263 Caucasian patients with major depressive disorder. One was drawn from a large health system by applying natural language processing to electronic health records (i2b2 cohort). The second consisted of a multicenter study of sequential antidepressant treatments, Sequenced Treatment Alternatives to Relieve Depression. The Birdsuite package was used to identify rare deletions and duplications. Individuals without symptomatic remission, despite two antidepressant treatment trials, were contrasted with those who remitted with a first treatment trial. Results CNV data were derived for 778 subjects in the i2b2 cohort, including 300 subjects (37%) with TRD, and 485 subjects in Sequenced Treatment Alternatives to Relieve Depression cohort, including 152 (31%) with TRD. CNV burden analyses identified modest enrichment of duplications in cases (empirical p = .04 for duplications of 100–200 kilobase) and a particular deletion region spanning gene PABPC4L (empirical p = .02, 6 cases: 0 controls). Pathway analysis suggested enrichment of CNVs intersecting genes regulating actin cytoskeleton. However, none of these associations survived genome-wide correction. Conclusions Contribution of rare CNVs to TRD appears to be modest, individually or in aggregate. The electronic health record-based methodology demonstrated here should facilitate collection of larger TRD cohorts necessary to further characterize these effects. PMID:24529801

In vitro inactivation of complement by a serum factor present in Junin-virus infected guinea-pigs.

PubMed Central

Rimoldi, M T; de Bracco, M M

1980-01-01

A serum factor(s) of guinea-pigs infected with Junin virus, the etiological agent of Argentine haemorrhagic fever, is endowed with a potent anticomplementary activity. It is resistant to heat (56 degrees, 30 min) and elutes from a Sephadex G-200 column between albumin and haemoglobin. It is ineffective in the presence of EDTA or EGTA and does not sediment at 82,000 g. It has no direct effect on C4 unless functional Cl is present. However, it induces Cl activation that consumes C4 haemolytic activity in normal human and guinea-pig sera. The evidence presented in this report demonstrates that the complement activation observed in experimental Argentine haemorrhagic fever is at least in part due to a direct effect of this serum factor on the classical complement pathway. PMID:6247264

SLC2A3 single-nucleotide polymorphism and duplication influence cognitive processing and population-specific risk for attention-deficit/hyperactivity disorder.

PubMed

Merker, Sören; Reif, Andreas; Ziegler, Georg C; Weber, Heike; Mayer, Ute; Ehlis, Ann-Christine; Conzelmann, Annette; Johansson, Stefan; Müller-Reible, Clemens; Nanda, Indrajit; Haaf, Thomas; Ullmann, Reinhard; Romanos, Marcel; Fallgatter, Andreas J; Pauli, Paul; Strekalova, Tatyana; Jansch, Charline; Vasquez, Alejandro Arias; Haavik, Jan; Ribasés, Marta; Ramos-Quiroga, Josep Antoni; Buitelaar, Jan K; Franke, Barbara; Lesch, Klaus-Peter

2017-07-01

Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neurodevelopmental disorder with profound cognitive, behavioral, and psychosocial impairments with persistence across the life cycle. Our initial genome-wide screening approach for copy number variants (CNVs) in ADHD implicated a duplication of SLC2A3, encoding glucose transporter-3 (GLUT3). GLUT3 plays a critical role in cerebral glucose metabolism, providing energy for the activity of neurons, which, in turn, moderates the excitatory-inhibitory balance impacting both brain development and activity-dependent neural plasticity. We therefore aimed to provide additional genetic and functional evidence for GLUT3 dysfunction in ADHD. Case-control association analyses of SLC2A3 single-nucleotide polymorphisms (SNPs) and CNVs were conducted in several European cohorts of patients with childhood and adult ADHD (SNP, n = 1,886 vs. 1,988; CNV, n = 1,692 vs. 1,721). These studies were complemented by SLC2A3 expression analyses in peripheral cells, functional EEG recordings during neurocognitive tasks, and ratings of food energy content. Meta-analysis of all cohorts detected an association of SNP rs12842 with ADHD. While CNV analysis detected a population-specific enrichment of SLC2A3 duplications only in German ADHD patients, the CNV + rs12842 haplotype influenced ADHD risk in both the German and Spanish cohorts. Duplication carriers displayed elevated SLC2A3 mRNA expression in peripheral blood cells and altered event-related potentials reflecting deficits in working memory and cognitive response control, both endophenotypic traits of ADHD, and an underestimation of energy units of high-caloric food. Taken together, our results indicate that both common and rare SLC2A3 variation impacting regulation of neuronal glucose utilization and energy homeostasis may result in neurocognitive deficits known to contribute to ADHD risk. © 2017 Association for Child and Adolescent Mental Health.

Accurately Assessing the Risk of Schizophrenia Conferred by Rare Copy-Number Variation Affecting Genes with Brain Function

PubMed Central

Raychaudhuri, Soumya; Korn, Joshua M.; McCarroll, Steven A.; Altshuler, David; Sklar, Pamela; Purcell, Shaun; Daly, Mark J.

2010-01-01

Investigators have linked rare copy number variation (CNVs) to neuropsychiatric diseases, such as schizophrenia. One hypothesis is that CNV events cause disease by affecting genes with specific brain functions. Under these circumstances, we expect that CNV events in cases should impact brain-function genes more frequently than those events in controls. Previous publications have applied “pathway” analyses to genes within neuropsychiatric case CNVs to show enrichment for brain-functions. While such analyses have been suggestive, they often have not rigorously compared the rates of CNVs impacting genes with brain function in cases to controls, and therefore do not address important confounders such as the large size of brain genes and overall differences in rates and sizes of CNVs. To demonstrate the potential impact of confounders, we genotyped rare CNV events in 2,415 unaffected controls with Affymetrix 6.0; we then applied standard pathway analyses using four sets of brain-function genes and observed an apparently highly significant enrichment for each set. The enrichment is simply driven by the large size of brain-function genes. Instead, we propose a case-control statistical test, cnv-enrichment-test, to compare the rate of CNVs impacting specific gene sets in cases versus controls. With simulations, we demonstrate that cnv-enrichment-test is robust to case-control differences in CNV size, CNV rate, and systematic differences in gene size. Finally, we apply cnv-enrichment-test to rare CNV events published by the International Schizophrenia Consortium (ISC). This approach reveals nominal evidence of case-association in neuronal-activity and the learning gene sets, but not the other two examined gene sets. The neuronal-activity genes have been associated in a separate set of schizophrenia cases and controls; however, testing in independent samples is necessary to definitively confirm this association. Our method is implemented in the PLINK software package. PMID:20838587

Characteristic face: a key indicator for direct diagnosis of 22q11.2 deletions in Chinese velocardiofacial syndrome patients.

PubMed

Wu, Dandan; Chen, Yang; Xu, Chen; Wang, Ke; Wang, Huijun; Zheng, Fengyun; Ma, Duan; Wang, Guomin

2013-01-01

Velocardiofacial syndrome (VCFS) is a disease in human with an expansive phenotypic spectrum and diverse genetic mechanisms mainly associated with copy number variations (CNVs) on 22q11.2 or other chromosomes. However, the correlations between CNVs and phenotypes remain ambiguous. This study aims to analyze the types and sizes of CNVs in VCFS patients, to define whether correlations exist between CNVs and clinical manifestations in Chinese VCFS patients. In total, 55 clinically suspected Chinese VCFS patients and 100 normal controls were detected by multiplex ligation-dependent probe amplification (MLPA). The data from MLPA and all the detailed clinical features of the objects were documented and analyzed. A total of 44 patients (80.0%) were diagnosed with CNVs on 22q11.2. Among them, 43 (78.2%) presented with 22q11.2 heterozygous deletions, of whom 40 (93.0%) had typical 3-Mb deletion, and 3 (7.0%) exhibited proximal 1.5-Mb deletion; no patient was found with atypical deletion on 22q11.2. One patient (1.8%) presented with a 3-Mb duplication mapping to the typical 3-Mb region on 22q11.2, while none of the chromosomal abnormalities in the MLPA kit were found in the other 11 patients and 100 normal controls. All the 43 patients with 22q11.2 deletions displayed characteristic face and palatal anomalies; 37 of them (86.0%) had cognitive or behavioral disorders, and 23 (53.5%) suffered from immune deficiencies; 10 patients (23.3%) manifested congenital heart diseases. Interestingly, all patients with the characteristic face had 22q11.2 heterozygous deletions, but no difference in phenotypic spectrum was observed between 3-Mb and 1.5-Mb deletions. Our data suggest that the characteristic face can be used as a key indicator for direct diagnosis of 22q11.2 deletions in Chinese VCFS patients.

The Role of Copy Number Variation in Susceptibility to Amyotrophic Lateral Sclerosis: Genome-Wide Association Study and Comparison with Published Loci

PubMed Central

Wain, Louise V.; Pedroso, Inti; Landers, John E.; Breen, Gerome; Shaw, Christopher E.; Leigh, P. Nigel; Brown, Robert H.

2009-01-01

Background The genetic contribution to sporadic amyotrophic lateral sclerosis (ALS) has not been fully elucidated. There are increasing efforts to characterise the role of copy number variants (CNVs) in human diseases; two previous studies concluded that CNVs may influence risk of sporadic ALS, with multiple rare CNVs more important than common CNVs. A little-explored issue surrounding genome-wide CNV association studies is that of post-calling filtering and merging of raw CNV calls. We undertook simulations to define filter thresholds and considered optimal ways of merging overlapping CNV calls for association testing, taking into consideration possibly overlapping or nested, but distinct, CNVs and boundary estimation uncertainty. Methodology and Principal Findings In this study we screened Illumina 300K SNP genotyping data from 730 ALS cases and 789 controls for copy number variation. Following quality control filters using thresholds defined by simulation, a total of 11321 CNV calls were made across 575 cases and 621 controls. Using region-based and gene-based association analyses, we identified several loci showing nominally significant association. However, the choice of criteria for combining calls for association testing has an impact on the ranking of the results by their significance. Several loci which were previously reported as being associated with ALS were identified here. However, of another 15 genes previously reported as exhibiting ALS-specific copy number variation, only four exhibited copy number variation in this study. Potentially interesting novel loci, including EEF1D, a translation elongation factor involved in the delivery of aminoacyl tRNAs to the ribosome (a process which has previously been implicated in genetic studies of spinal muscular atrophy) were identified but must be treated with caution due to concerns surrounding genomic location and platform suitability. Conclusions and Significance Interpretation of CNV association findings must take into account the effects of filtering and combining CNV calls when based on early genome-wide genotyping platforms and modest study sizes. PMID:19997636

CNV discovery for milk composition traits in dairy cattle using whole genome resequencing.

PubMed

Gao, Yahui; Jiang, Jianping; Yang, Shaohua; Hou, Yali; Liu, George E; Zhang, Shengli; Zhang, Qin; Sun, Dongxiao

2017-03-29

Copy number variations (CNVs) are important and widely distributed in the genome. CNV detection opens a new avenue for exploring genes associated with complex traits in humans, animals and plants. Herein, we present a genome-wide assessment of CNVs that are potentially associated with milk composition traits in dairy cattle. In this study, CNVs were detected based on whole genome re-sequencing data of eight Holstein bulls from four half- and/or full-sib families, with extremely high and low estimated breeding values (EBVs) of milk protein percentage and fat percentage. The range of coverage depth per individual was 8.2-11.9×. Using CNVnator, we identified a total of 14,821 CNVs, including 5025 duplications and 9796 deletions. Among them, 487 differential CNV regions (CNVRs) comprising ~8.23 Mb of the cattle genome were observed between the high and low groups. Annotation of these differential CNVRs were performed based on the cattle genome reference assembly (UMD3.1) and totally 235 functional genes were found within the CNVRs. By Gene Ontology and KEGG pathway analyses, we found that genes were significantly enriched for specific biological functions related to protein and lipid metabolism, insulin/IGF pathway-protein kinase B signaling cascade, prolactin signaling pathway and AMPK signaling pathways. These genes included INS, IGF2, FOXO3, TH, SCD5, GALNT18, GALNT16, ART3, SNCA and WNT7A, implying their potential association with milk protein and fat traits. In addition, 95 CNVRs were overlapped with 75 known QTLs that are associated with milk protein and fat traits of dairy cattle (Cattle QTLdb). In conclusion, based on NGS of 8 Holstein bulls with extremely high and low EBVs for milk PP and FP, we identified a total of 14,821 CNVs, 487 differential CNVRs between groups, and 10 genes, which were suggested as promising candidate genes for milk protein and fat traits.

Prematurity, ventricular septal defect and dysmorphisms are independent predictors of pathogenic copy number variants: a retrospective study on array-CGH results and phenotypical features of 293 children with neurodevelopmental disorders and/or multiple congenital anomalies.

PubMed

Maini, I; Ivanovski, I; Djuric, O; Caraffi, S G; Errichiello, E; Marinelli, M; Franchi, F; Bizzarri, V; Rosato, S; Pollazzon, M; Gelmini, C; Malacarne, M; Fusco, C; Gargano, G; Bernasconi, S; Zuffardi, O; Garavelli, L

2018-03-09

Since 2010, array-CGH (aCGH) has been the first-tier test in the diagnostic approach of children with neurodevelopmental disorders (NDD) or multiple congenital anomalies (MCA) of unknown origin. Its broad application led to the detection of numerous variants of uncertain clinical significance (VOUS). How to appropriately interpret aCGH results represents a challenge for the clinician. We present a retrospective study on 293 patients with age range 1 month - 29 years (median 7 years) with NDD and/or MCA and/or dysmorphisms, investigated through aCGH between 2005 and 2016. The aim of the study was to analyze clinical and molecular cytogenetic data in order to identify what elements could be useful to interpret unknown or poorly described aberrations. Comparison of phenotype and cytogenetic characteristics through univariate analysis and multivariate logistic regression was performed. Copy number variations (CNVs) with a frequency < 1% were detected in 225 patients of the total sample, while 68 patients presented only variants with higher frequency (heterozygous deletions or amplification) and were considered to have negative aCGH. Proved pathogenic CNVs were detected in 70 patients (20.6%). Delayed psychomotor development, intellectual disability, intrauterine growth retardation (IUGR), prematurity, congenital heart disease, cerebral malformations and dysmorphisms correlated to reported pathogenic CNVs. Prematurity, ventricular septal defect and dysmorphisms remained significant predictors of pathogenic CNVs in the multivariate logistic model whereas abnormal EEG and limb dysmorphisms were mainly detected in the group with likely pathogenic VOUS. A flow-chart regarding the care for patients with NDD and/or MCA and/or dysmorphisms and the interpretation of aCGH has been made on the basis of the data inferred from this study and literature. Our work contributes to make the investigative process of CNVs more informative and suggests possible directions in aCGH interpretation and phenotype correlation.

Copy number variations of GSTT1 and GSTM1, colorectal cancer risk and possible effect modification of cigarette smoking and menopausal hormone therapy.

PubMed

Rudolph, Anja; Hein, Rebecca; Hoffmeister, Michael; Försti, Asta; Hemminki, Kari; Risch, Angela; Brenner, Hermann; Chang-Claude, Jenny

2012-09-01

Copy number variations (CNVs) of the glutathione-S-transferase θ-1 (GSTT1) and glutathione-S-transferase μ-1 (GSTM1) gene loci can lead to complete lack of enzyme and have been associated with colorectal cancer (CRC) risk. As GSTs are involved in the detoxification of xenobiotics, CNVs may modify CRC risk associated with smoking exposure and menopausal hormone therapy (MHT) use. We investigated CRC risk associated with GSTT1 and GSTM1 CNVs and their interaction with smoking in 1,796 cases and 1,806 age-, sex- and residence-matched controls from a German population-based case-control study (DACHS). The interaction with MHT was assessed in the subset of 684 postmenopausal female cases and 681 controls. Trimodular genotypes (0/0, 1/0 and 1/1) were determined with relative quantification based on multiplex real-time polymerase chain reaction. The associations with CRC risk as well as possible effect modifications were evaluated using conditional logistic regression analysis. CNVs of GSTT1 and GSTM1 were not significantly associated with CRC risk. Compared to the 1/1 genotype, odds ratios (ORs) for the 0/1 genotype and the 0/0 genotype were 0.89 [95% confidence interval (CI): 0.77-1.04] and 0.97 (95% CI: 0.80-1.18) for GSTT1, and 0.99 (95% CI: 0.78-1.27) and 1.03 (95% CI: 0.81-1.31) for GSTM1. Compared to the non-null genotype, ORs for the null-genotype were 1.04 (95% CI: 0.87-1.23) for GSTT1 and 1.03 (95% CI: 0.91-1.18) for GSTM1. No significant interaction with smoking and MHT use was observed. Our study does not provide evidence for a strong association between CRC risk and CNVs of GSTT1 or GSTM1 or for an effect modification of smoking or MHT use. Copyright © 2012 UICC.

Copy number variations of E2F1: a new genetic risk factor for testicular cancer.

PubMed

Rocca, Maria Santa; Di Nisio, Andrea; Marchiori, Arianna; Ghezzi, Marco; Opocher, Giuseppe; Foresta, Carlo; Ferlin, Alberto

2017-03-01

Testicular germ cell tumor (TGCT) is one of the most heritable forms of cancer. In last years, many evidence suggested that constitutional genetic factors, mainly single nucleotide polymorphisms, can increase its risk. However, the possible contribution of copy number variations (CNVs) in TGCT susceptibility has not been substantially addressed. Indeed, an increasing number of studies have focused on the effect of CNVs on gene expression and on the role of these structural genetic variations as risk factors for different forms of cancer. E2F1 is a transcription factor that plays an important role in regulating cell growth, differentiation, apoptosis and response to DNA damage. Therefore, deficiency or overexpression of this protein might significantly influence fundamental biological processes involved in cancer development and progression, including TGCT. We analyzed E2F1 CNVs in 261 cases with TGCT and 165 controls. We found no CNVs in controls, but 17/261 (6.5%) cases showed duplications in E2F1 Blot analysis demonstrated higher E2F1 expression in testicular samples of TGCT cases with three copies of the gene. Furthermore, we observed higher phosphorylation of Akt and mTOR in samples with E2F1 duplication. Interestingly, normal, non-tumoral testicular tissue in patient with E2F1 duplication showed lower expression of E2F1 and lower AKT/mTOR phosphorylation with respect to adjacent tumor tissue. Furthermore, increased expression of E2F1 obtained in vitro in NTERA-2 testicular cell line induced increased AKT/mTOR phosphorylation. This study suggests for the first time an involvement of E2F1 CNVs in TGCT susceptibility and supports previous preliminary data on the importance of AKT/mTOR signaling pathway in this cancer. © 2017 Society for Endocrinology.

Activation of the classical pathway of complement by binding of bovine lactoferrin to unencapsulated Streptococcus agalactiae.

PubMed Central

Rainard, P

1993-01-01

The ability of lactoferrin (Lf) bound to Streptococcus agalactiae to interfere with the deposition of complement components on the bacterial surface was investigated by enzyme-linked immunosorbent assay (ELISA). By using a strain of S. agalactiae which activates the alternative pathway of complement in the absence of antibodies, it was found that pretreatment of bacteria with Lf shortened the lag phase preceding the deposition of C3 on bacteria. The kinetics of C3 deposition was comparable to that obtained by adding antibodies against S. agalactiae to agammaglobulinaemic precolostral calf serum (PCS) heated at 56 degrees for 3 min to inactivate the alternative pathway. Accelerated C3 deposition did not occur in the absence of Ca2+ ions. Deposition of C4 on bacteria occurred only when either antibodies or Lf were added to PCS. These results demonstrate that the interaction of lactoferrin with bacteria activated the classical pathway of complement in the absence of antibodies. The binding of purified C1q to bacteria was promoted in a dose-dependent manner by Lf, suggesting that recruitment of classical pathway of complement resulted from the interaction of C1q with Lf adsorbed to the bacterial surface. Phagocytosis of bacteria opsonized with heated PCS (at 56 degrees for 3 min) and Lf was comparable to that occurring in the presence of heated PCS and antibodies. In conclusion, Lf was able to substitute for antibodies in order to activate the classical pathway of complement and to opsonize unencapsulated S. agalactiae efficiently. PMID:8406591

Characterization of shark complement factor I gene(s): genomic analysis of a novel shark-specific sequence.

PubMed

Shin, Dong-Ho; Webb, Barbara M; Nakao, Miki; Smith, Sylvia L

2009-07-01

Complement factor I is a crucial regulator of mammalian complement activity. Very little is known of complement regulators in non-mammalian species. We isolated and sequenced four highly similar complement factor I cDNAs from the liver of the nurse shark (Ginglymostoma cirratum), designated as GcIf-1, GcIf-2, GcIf-3 and GcIf-4 (previously referred to as nsFI-a, -b, -c and -d) which encode 689, 673, 673 and 657 amino acid residues, respectively. They share 95% (

Characterization of shark complement factor I gene(s): genomic analysis of a novel shark-specific sequence

PubMed Central

Shin, Dong-Ho; Webb, Barbara M.; Nakao, Miki; Smith, Sylvia L.

2009-01-01

Complement factor I is a crucial regulator of mammalian complement activity. Very little is known of complement regulators in non-mammalian species. We isolated and sequenced four highly similar complement factor I cDNAs from the liver of the nurse shark (Ginglymostoma cirratum), designated as GcIf-1, GcIf-2, GcIf-3 and GcIf-4 (previously referred to as nsFI-a, -b, -c and –d) which encode 689, 673, 673 and 657 amino acid residues, respectively. They share 95% (≤) amino acid identities with each other, 35.4 ~ 39.6% and 62.8 ~ 65.9% with factor I of mammals and banded houndshark (Triakis scyllium), respectively. The modular structure of the GcIf is similar to that of mammals with one notable exception, the presence of a novel shark-specific sequence between the leader peptide (LP) and the factor I membrane attack complex (FIMAC) domain. The cDNA sequences differ only in the size and composition of the shark-specific region (SSR). Sequence analysis of each SSR has identified within the region two novel short sequences (SS1 and SS2) and three repeat sequences (RS1, 2 and 3). Genomic analysis has revealed the existence of three introns between the leader peptide and the FIMAC domain, tentatively designated intron 1, intron 2, and intron 3 which span 4067, 2293 and 2082 bp, respectively. Southern blot analysis suggests the presence of a single gene copy for each cDNA type. Phylogenetic analysis suggests that complement factor I of cartilaginous fish diverged prior to the emergence of mammals. All four GcIf cDNA species are expressed in four different tissues and the liver is the main tissue in which expression level of all four is high. This suggests that the expression of GcIf isotypes is tissue-dependent. PMID:19423168

Susceptibility of pathogenic and nonpathogenic Naegleria ssp

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whiteman, L.Y.

1988-01-01

The susceptibility of four species of Naegleria amoebae to complement-mediated lysis was determined. The amoebicidal activity of normal human serum (NHS) and normal guinea pig serum (NGPS) for Naegleria amoebae was measured by an in vitro cytotoxicity assay. Release of radioactivity from amoebae labeled with {sup 3}H-uridine and visual observation with a compound microscope were used as indices of lysis. Susceptibility or resistance to complement-mediated lysis in vitro correlated with the in vivo pathogenic potential. Nonpathogenic Naegleria amoebae were lysed at a faster rate and at higher cell concentrations than were pathogenic amoebae. Electrophoretic analysis of NHS incubated with pathogenicmore » or nonpathogenic Naegleria spp. demonstrated that amoebae activate the complement cascade resulting in the production of C3 and C5 complement cleavage products. Treatment with papain or trypsin for 1 h, but not with sialidase, increase the susceptibility of highly pathogenic, mouse-passaged N. fowleri to lysis. Treatment with actinomycin D, cycloheximide or various protease inhibitors for 4 h did not increase susceptibility to lysis. Neither a repair process involving de novo protein synthesis nor a complement-inactivating protease appear to account for the increase resistance of N. fowleri amoebae to complement-mediated lysis. A binding study with {sup 125}I radiolabeled C9 indicated that the terminal complement component does not remain stably bound to the membrane of pathogenic amoebae.« less

Chromosomal microarray analysis of Bulgarian patients with epilepsy and intellectual disability.

PubMed

Peycheva, Valentina; Kamenarova, Kunka; Ivanova, Neviana; Stamatov, Dimitar; Avdjieva-Tzavella, Daniela; Alexandrova, Iliana; Zhelyazkova, Sashka; Pacheva, Iliana; Dimova, Petya; Ivanov, Ivan; Litvinenko, Ivan; Bozhinova, Veneta; Tournev, Ivailo; Simeonov, Emil; Mitev, Vanyo; Jordanova, Albena; Kaneva, Radka

2018-08-15

High resolution chromosomal microarray analysis (CMA) has facilitated the identification of small chromosomal rearrangements throughout the genome, associated with various neurodevelopmental phenotypes, including ID/DD. Recently, it became evident that intellectual disability (ID)/developmental delay (DD) can occur with associated co-morbidities like epileptic seizures, autism and additional congenital anomalies. These observations require whole genome approach in order to detect the genetic causes of these complex disorders. In this study, we examined 92 patients of Bulgarian origin at age between 1 and 22 years with ID, generalized epilepsy, autistic signs and congenital anomalies. CMA was carried out using SurePrint G3 Human CGH Microarray Kit, 4 × 180 K and SurePrint G3 Unrestricted CGH ISCA v2, 4 × 180 K oligo platforms. Referral indications for selection of the patients were the presence of generalized refractory seizures disorders and co-morbid ID. Clearly pathogenic copy number variations (CNVs) were detected in eight patients (8.7%) from our cohort. Additionally, possibly pathogenic rearrangements of unclear clinical significance were detected in six individuals (6.5%), which make for an overall diagnostic yield of 15.2% among our cohort of patients. We report here the patients with clearly pathogenic CNVs, discuss the potential causality of the possibly pathogenic CNVs and make genotype - phenotype correlations. One novel possibly pathogenic heterozygous deletion in 15q22.31 region was detected in a case with ID/DD. Additionally, whole APBA2 gene duplication in 15q13.1 was found in three generations of a family with epilepsy, ID and psychiatric abnormalities. The results from this study allow us to define the genetic diagnosis in a subset of Bulgarian patients and improve the genetic counseling of the affected families. To our knowledge, this is the first aCGH evaluation of a Bulgarian cohort of children with epilepsy and ID so far. Copyright © 2018 Elsevier B.V. All rights reserved.

Complement fixation test to C burnetii

MedlinePlus

... complement fixation test; Coxiella burnetii - complement fixation test; C burnetii - complement fixation test ... a specific foreign substance ( antigen ), in this case, C burnetii . Antibodies defend the body against bacteria, viruses, ...

Variola virus immune evasion design: expression of a highly efficient inhibitor of human complement.

PubMed

Rosengard, Ariella M; Liu, Yu; Nie, Zhiping; Jimenez, Robert

2002-06-25

Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans and has no other animal reservoir. Variola causes the contagious disease smallpox, which has a 30-40% mortality rate. Conversely, the prototype orthopoxvirus, vaccinia, causes no disease in immunocompetent humans and was used in the global eradication of smallpox, which ended in 1977. However, the threat of smallpox persists because clandestine stockpiles of variola still exist. Although variola and vaccinia share remarkable DNA homology, the strict human tropism of variola suggests that its proteins are better suited than those of vaccinia to overcome the human immune response. Here, we demonstrate the functional advantage of a variola complement regulatory protein over that of its vaccinia homologue. Because authentic variola proteins are not available for study, we molecularly engineered and characterized the smallpox inhibitor of complement enzymes (SPICE), a homologue of a vaccinia virulence factor, vaccinia virus complement control protein (VCP). SPICE is nearly 100-fold more potent than VCP at inactivating human C3b and 6-fold more potent at inactivating C4b. SPICE is also more human complement-specific than is VCP. By inactivating complement components, SPICE serves to inhibit the formation of the C3/C5 convertases necessary for complement-mediated viral clearance. SPICE provides the first evidence that variola proteins are particularly adept at overcoming human immunity, and the decreased function of VCP suggests one reason why the vaccinia virus vaccine was associated with relatively low mortality. Disabling SPICE may be therapeutically useful if smallpox reemerges.

Variola virus immune evasion design: Expression of a highly efficient inhibitor of human complement

PubMed Central

Rosengard, Ariella M.; Liu, Yu; Nie, Zhiping; Jimenez, Robert

2002-01-01

Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans and has no other animal reservoir. Variola causes the contagious disease smallpox, which has a 30–40% mortality rate. Conversely, the prototype orthopoxvirus, vaccinia, causes no disease in immunocompetent humans and was used in the global eradication of smallpox, which ended in 1977. However, the threat of smallpox persists because clandestine stockpiles of variola still exist. Although variola and vaccinia share remarkable DNA homology, the strict human tropism of variola suggests that its proteins are better suited than those of vaccinia to overcome the human immune response. Here, we demonstrate the functional advantage of a variola complement regulatory protein over that of its vaccinia homologue. Because authentic variola proteins are not available for study, we molecularly engineered and characterized the smallpox inhibitor of complement enzymes (SPICE), a homologue of a vaccinia virulence factor, vaccinia virus complement control protein (VCP). SPICE is nearly 100-fold more potent than VCP at inactivating human C3b and 6-fold more potent at inactivating C4b. SPICE is also more human complement-specific than is VCP. By inactivating complement components, SPICE serves to inhibit the formation of the C3/C5 convertases necessary for complement-mediated viral clearance. SPICE provides the first evidence that variola proteins are particularly adept at overcoming human immunity, and the decreased function of VCP suggests one reason why the vaccinia virus vaccine was associated with relatively low mortality. Disabling SPICE may be therapeutically useful if smallpox reemerges. PMID:12034872

Immune functions of the garment workers.

PubMed

Sultana, R; Ferdous, K J; Hossain, M; Zahid, M S H; Islam, L N

2012-10-01

Occupational exposure to cotton dust, fibers, metal fumes and different chemicals used in the aparrel manufacturing industries cause a wide range of physical and psychological health problems in the garment workers that may also affect their immune function. To assess the immune system function in garment workers. A total of 45 workers of a garment factory, and 41 control subjects, not exposed to the garment working environment were enrolled in this study. In the study subjects, the complement system function was assessed as bactericidal activity on Escherichia coli DH5α cells using the standard plate count method. Serum complement components C3 and C4 were measured by immunoprecipitation, and IgG was measured by immunonephelometry. The bactericidal activity of serum complement in the garment workers (range: 93.5%-99.9%) was significantly (p<0.01) lower than that in the controls (range: 98.6%-100%). The heat-inactivated serum of the workers showed a significantly enhanced bactericidal activity. In the garment workers, the mean levels of complement C3, and C4 were 1.75 and 0.26 g/L, respectively that were close to those of the controls. The mean IgG level in the garment workers was 13.5 g/L that was significantly (p<0.001) higher than that in the controls. Working in a garment factory may affect the immune system.

The two sides of complement C3d: evolution of electrostatics in a link between innate and adaptive immunity.

PubMed

Kieslich, Chris A; Morikis, Dimitrios

2012-01-01

The interaction between complement fragment C3d and complement receptor 2 (CR2) is a key aspect of complement immune system activation, and is a component in a link between innate and adaptive immunities. The complement immune system is an ancient mechanism for defense, and can be found in species that have been on Earth for the last 600 million years. However, the link between the complement system and adaptive immunity, which is formed through the association of the B-cell co-receptor complex, including the C3d-CR2 interaction, is a much more recent adaptation. Human C3d and CR2 have net charges of -1 and +7 respectively, and are believed to have evolved favoring the role of electrostatics in their functions. To investigate the role of electrostatics in the function and evolution of human C3d and CR2, we have applied electrostatic similarity methods to identify regions of evolutionarily conserved electrostatic potential based on 24 homologues of complement C3d and 4 homologues of CR2. We also examine the effects of structural perturbation, as introduced through molecular dynamics and mutations, on spatial distributions of electrostatic potential to identify perturbation resistant regions, generated by so-called electrostatic "hot-spots". Distributions of electrostatic similarity based on families of perturbed structures illustrate the presence of electrostatic "hot-spots" at the two functional sites of C3d, while the surface of CR2 lacks electrostatic "hot-spots" despite its excessively positive nature. We propose that the electrostatic "hot-spots" of C3d have evolved to optimize its dual-functionality (covalently attaching to pathogen surfaces and interaction with CR2), which are both necessary for the formation B-cell co-receptor complexes. Comparison of the perturbation resistance of the electrostatic character of the homologues of C3d suggests that there was an emergence of a new role of electrostatics, and a transition in the function of C3d, after the divergence of jawless fish.

The Two Sides of Complement C3d: Evolution of Electrostatics in a Link between Innate and Adaptive Immunity

PubMed Central

Kieslich, Chris A.; Morikis, Dimitrios

2012-01-01

The interaction between complement fragment C3d and complement receptor 2 (CR2) is a key aspect of complement immune system activation, and is a component in a link between innate and adaptive immunities. The complement immune system is an ancient mechanism for defense, and can be found in species that have been on Earth for the last 600 million years. However, the link between the complement system and adaptive immunity, which is formed through the association of the B-cell co-receptor complex, including the C3d-CR2 interaction, is a much more recent adaptation. Human C3d and CR2 have net charges of −1 and +7 respectively, and are believed to have evolved favoring the role of electrostatics in their functions. To investigate the role of electrostatics in the function and evolution of human C3d and CR2, we have applied electrostatic similarity methods to identify regions of evolutionarily conserved electrostatic potential based on 24 homologues of complement C3d and 4 homologues of CR2. We also examine the effects of structural perturbation, as introduced through molecular dynamics and mutations, on spatial distributions of electrostatic potential to identify perturbation resistant regions, generated by so-called electrostatic “hot-spots”. Distributions of electrostatic similarity based on families of perturbed structures illustrate the presence of electrostatic “hot-spots” at the two functional sites of C3d, while the surface of CR2 lacks electrostatic “hot-spots” despite its excessively positive nature. We propose that the electrostatic “hot-spots” of C3d have evolved to optimize its dual-functionality (covalently attaching to pathogen surfaces and interaction with CR2), which are both necessary for the formation B-cell co-receptor complexes. Comparison of the perturbation resistance of the electrostatic character of the homologues of C3d suggests that there was an emergence of a new role of electrostatics, and a transition in the function of C3d, after the divergence of jawless fish. PMID:23300422

Transient structural variations have strong effects on quantitative traits and reproductive isolation in fission yeast

PubMed Central

Jeffares, Daniel C.; Jolly, Clemency; Hoti, Mimoza; Speed, Doug; Shaw, Liam; Rallis, Charalampos; Balloux, Francois; Dessimoz, Christophe; Bähler, Jürg; Sedlazeck, Fritz J.

2017-01-01

Large structural variations (SVs) within genomes are more challenging to identify than smaller genetic variants but may substantially contribute to phenotypic diversity and evolution. We analyse the effects of SVs on gene expression, quantitative traits and intrinsic reproductive isolation in the yeast Schizosaccharomyces pombe. We establish a high-quality curated catalogue of SVs in the genomes of a worldwide library of S. pombe strains, including duplications, deletions, inversions and translocations. We show that copy number variants (CNVs) show a variety of genetic signals consistent with rapid turnover. These transient CNVs produce stoichiometric effects on gene expression both within and outside the duplicated regions. CNVs make substantial contributions to quantitative traits, most notably intracellular amino acid concentrations, growth under stress and sugar utilization in winemaking, whereas rearrangements are strongly associated with reproductive isolation. Collectively, these findings have broad implications for evolution and for our understanding of quantitative traits including complex human diseases. PMID:28117401

Copy number variations and genetic admixtures in three Xinjiang ethnic minority groups

PubMed Central

Lou, Haiyi; Li, Shilin; Jin, Wenfei; Fu, Ruiqing; Lu, Dongsheng; Pan, Xinwei; Zhou, Huaigu; Ping, Yuan; Jin, Li; Xu, Shuhua

2015-01-01

Xinjiang is geographically located in central Asia, and it has played an important historical role in connecting eastern Eurasian (EEA) and western Eurasian (WEA) people. However, human population genomic studies in this region have been largely underrepresented, especially with respect to studies of copy number variations (CNVs). Here we constructed the first CNV map of the three major ethnic minority groups, the Uyghur, Kazakh and Kirgiz, using Affymetrix Genome-Wide Human SNP Array 6.0. We systematically compared the properties of CNVs we identified in the three groups with the data from representatives of EEA and WEA. The analyses indicated a typical genetic admixture pattern in all three groups with ancestries from both EEA and WEA. We also identified several CNV regions showing significant deviation of allele frequency from the expected genome-wide distribution, which might be associated with population-specific phenotypes. Our study provides the first genome-wide perspective on the CNVs of three major Xinjiang ethnic minority groups and has implications for both evolutionary and medical studies. PMID:25026903

An Organismal CNV Mutator Phenotype Restricted to Early Human Development.

PubMed

Liu, Pengfei; Yuan, Bo; Carvalho, Claudia M B; Wuster, Arthur; Walter, Klaudia; Zhang, Ling; Gambin, Tomasz; Chong, Zechen; Campbell, Ian M; Coban Akdemir, Zeynep; Gelowani, Violet; Writzl, Karin; Bacino, Carlos A; Lindsay, Sarah J; Withers, Marjorie; Gonzaga-Jauregui, Claudia; Wiszniewska, Joanna; Scull, Jennifer; Stankiewicz, Paweł; Jhangiani, Shalini N; Muzny, Donna M; Zhang, Feng; Chen, Ken; Gibbs, Richard A; Rautenstrauss, Bernd; Cheung, Sau Wai; Smith, Janice; Breman, Amy; Shaw, Chad A; Patel, Ankita; Hurles, Matthew E; Lupski, James R

2017-02-23

De novo copy number variants (dnCNVs) arising at multiple loci in a personal genome have usually been considered to reflect cancer somatic genomic instabilities. We describe a multiple dnCNV (MdnCNV) phenomenon in which individuals with genomic disorders carry five to ten constitutional dnCNVs. These CNVs originate from independent formation incidences, are predominantly tandem duplications or complex gains, exhibit breakpoint junction features reminiscent of replicative repair, and show increased de novo point mutations flanking the rearrangement junctions. The active CNV mutation shower appears to be restricted to a transient perizygotic period. We propose that a defect in the CNV formation process is responsible for the "CNV-mutator state," and this state is dampened after early embryogenesis. The constitutional MdnCNV phenomenon resembles chromosomal instability in various cancers. Investigations of this phenomenon may provide unique access to understanding genomic disorders, structural variant mutagenesis, human evolution, and cancer biology. Copyright © 2017 Elsevier Inc. All rights reserved.

Copy number variations and genetic admixtures in three Xinjiang ethnic minority groups.

PubMed

Lou, Haiyi; Li, Shilin; Jin, Wenfei; Fu, Ruiqing; Lu, Dongsheng; Pan, Xinwei; Zhou, Huaigu; Ping, Yuan; Jin, Li; Xu, Shuhua

2015-04-01

Xinjiang is geographically located in central Asia, and it has played an important historical role in connecting eastern Eurasian (EEA) and western Eurasian (WEA) people. However, human population genomic studies in this region have been largely underrepresented, especially with respect to studies of copy number variations (CNVs). Here we constructed the first CNV map of the three major ethnic minority groups, the Uyghur, Kazakh and Kirgiz, using Affymetrix Genome-Wide Human SNP Array 6.0. We systematically compared the properties of CNVs we identified in the three groups with the data from representatives of EEA and WEA. The analyses indicated a typical genetic admixture pattern in all three groups with ancestries from both EEA and WEA. We also identified several CNV regions showing significant deviation of allele frequency from the expected genome-wide distribution, which might be associated with population-specific phenotypes. Our study provides the first genome-wide perspective on the CNVs of three major Xinjiang ethnic minority groups and has implications for both evolutionary and medical studies.

Increased CNV-Region deletions in mild cognitive impairment (MCI) and Alzheimer's disease (AD) subjects in the ADNI sample

PubMed Central

Guffanti, Guia; Torri, Federica; Rasmussen, Jerod; Clark, Andrew P.; Lakatos, Anita; Turner, Jessica A.; Fallon, James H.; Saykin, Andrew J.; Weiner, Michael; Vawter, Marquis P.; Knowles, James A.; Potkin, Steven G.; Macciardi, Fabio

2014-01-01

We investigated the genome-wide distribution of CNVs in the Alzheimer's disease (AD) Neuroimaging Initiative (ADNI) sample (146 with AD, 313 with Mild Cognitive Impairment (MCI), and 181 controls). Comparison of single CNVs between cases (MCI and AD) and controls shows overrepresentation of large heterozygous deletions in cases (p-value < 0.0001). The analysis of CNV-Regions identifies 44 copy number variable loci of heterozygous deletions, with more CNV-Regions among affected than controls (p = 0.005). Seven of the 44 CNV-Regions are nominally significant for association with cognitive impairment. We validated and confirmed our main findings with genome re-sequencing of selected patients and controls. The functional pathway analysis of the genes putatively affected by deletions of CNV-Regions reveals enrichment of genes implicated in axonal guidance, cell–cell adhesion, neuronal morphogenesis and differentiation. Our findings support the role of CNVs in AD, and suggest an association between large deletions and the development of cognitive impairment PMID:23583670

Copy number variation in subjects with major depressive disorder who attempted suicide.

PubMed

Perlis, Roy H; Ruderfer, Douglas; Hamilton, Steven P; Ernst, Carl

2012-01-01

Suicide is one of the top ten leading causes of death in North America and represents a major public health burden, particularly for people with Major Depressive disorder (MD). Many studies have suggested that suicidal behavior runs in families, however, identification of genomic loci that drive this efffect remain to be identified. Using subjects collected as part of STAR D, we genotyped 189 subjects with MD with history of a suicide attempt and 1073 subjects with Major Depressive disorder that had never attempted suicide. Copy Number Variants (CNVs) were called in Birdsuite and analyzed in PLINK. We found a set of CNVs present in the suicide attempter group that were not present in in the non-attempter group including in SNTG2 and MACROD2 - two brain expressed genes previously linked to psychopathology; however, these results failed to reach genome-wide signifigance. These data suggest potential CNVs to be investigated further in relation to suicide attempts in MD using large sample sizes.

Associations of common copy number variants in glutathione S-transferase mu 1 and D-dopachrome tautomerase-like protein genes with risk of schizophrenia in a Japanese population.

PubMed

Nakamura, Toru; Ohnuma, Tohru; Hanzawa, Ryo; Takebayashi, Yuto; Takeda, Mayu; Nishimon, Shohei; Sannohe, Takahiro; Katsuta, Narimasa; Higashiyama, Ryoko; Shibata, Nobuto; Arai, Heii

2015-10-01

Oxidative-stress, genetic regions of interest (1p13 and 22q11), and common copy number variations (CNVs) may play roles in the pathophysiology of schizophrenia. In the present study, we confirmed associations between schizophrenia and the common CNVs in the glutathione (GSH)-related genes GSTT1, DDTL, and GSTM1 using quantitative real-time polymerase chain reaction analyses of 620 patients with schizophrenia and in 622 controls. No significant differences in GSTT1 copy number distributions were found between patient groups. However, frequencies of characterized CNVs and assumed gain alleles of DDTL and GSTM1 were significantly higher in patients with schizophrenia. In agreement with a previous report, the present data indicate that gains in the CNV alleles DDTL and GSTM1 are genetic risk factors in Japanese patients with schizophrenia, and suggest involvement of micro-inflammation and oxidative stress in the pathophysiology of schizophrenia. © 2015 Wiley Periodicals, Inc.

Comprehensive approach to study complement C4 in systemic lupus erythematosus: Gene polymorphisms, protein levels and functional activity.

PubMed

Tsang-A-Sjoe, M W P; Bultink, I E M; Korswagen, L A; van der Horst, A; Rensink, I; de Boer, M; Hamann, D; Voskuyl, A E; Wouters, D

2017-12-01

Genetic variation of the genes encoding complement component C4 is strongly associated with systemic lupus erythematosus (SLE), a chronic multi-organ auto-immune disease. This study examined C4 and its isotypes on a genetic, protein, and functional level in 140 SLE patients and 104 healthy controls. Gene copy number (GCN) variation, silencing CT-insertion, and the retroviral HERV-K(C4) insertion) were analyzed with multiplex ligation-dependent probe amplification. Increased susceptibility to SLE was found for low GCN (≪2) of C4A. Serositis was the only clinical manifestation associated with low C4A GCN. One additional novel silencing mutation in the C4A gene was found by Sanger sequencing. This mutation causes a premature stop codon in exon 11. Protein concentrations of C4 isoforms C4A and C4B were determined with ELISA and were significantly lower in SLE patients compared to healthy controls. To study C4 isotypes on a functional level, a new C4 assay was developed, which distinguishes C4A from C4B by its binding capacity to amino or hydroxyl groups, respectively. This assay showed high correlation with ELISA and detected crossing over of Rodgers and Chido antigens in 3.2% (8/244) of individuals. The binding capacity of available C4 to its substrates was unaffected in SLE. Our study provides, for the first time, a complete overview of C4 in SLE from genetic variation to binding capacity using a novel test. As this test detects crossing over of Rodgers and Chido antigens, it will allow for more accurate measurement of C4 in future studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Eco-geographical diversification of bitter taste receptor genes (TAS2Rs) among subspecies of chimpanzees (Pan troglodytes).

PubMed

Hayakawa, Takashi; Sugawara, Tohru; Go, Yasuhiro; Udono, Toshifumi; Hirai, Hirohisa; Imai, Hiroo

2012-01-01

Chimpanzees (Pan troglodytes) have region-specific difference in dietary repertoires from East to West across tropical Africa. Such differences may result from different genetic backgrounds in addition to cultural variations. We analyzed the sequences of all bitter taste receptor genes (cTAS2Rs) in a total of 59 chimpanzees, including 4 putative subspecies. We identified genetic variations including single-nucleotide variations (SNVs), insertions and deletions (indels), gene-conversion variations, and copy-number variations (CNVs) in cTAS2Rs. Approximately two-thirds of all cTAS2R haplotypes in the amino acid sequence were unique to each subspecies. We analyzed the evolutionary backgrounds of natural selection behind such diversification. Our previous study concluded that diversification of cTAS2Rs in western chimpanzees (P. t. verus) may have resulted from balancing selection. In contrast, the present study found that purifying selection dominates as the evolutionary form of diversification of the so-called human cluster of cTAS2Rs in eastern chimpanzees (P. t. schweinfurthii) and that the other cTAS2Rs were under no obvious selection as a whole. Such marked diversification of cTAS2Rs with different evolutionary backgrounds among subspecies of chimpanzees probably reflects their subspecies-specific dietary repertoires.

Eco-Geographical Diversification of Bitter Taste Receptor Genes (TAS2Rs) among Subspecies of Chimpanzees (Pan troglodytes)

PubMed Central

Hayakawa, Takashi; Sugawara, Tohru; Go, Yasuhiro; Udono, Toshifumi; Hirai, Hirohisa; Imai, Hiroo

2012-01-01

Chimpanzees (Pan troglodytes) have region-specific difference in dietary repertoires from East to West across tropical Africa. Such differences may result from different genetic backgrounds in addition to cultural variations. We analyzed the sequences of all bitter taste receptor genes (cTAS2Rs) in a total of 59 chimpanzees, including 4 putative subspecies. We identified genetic variations including single-nucleotide variations (SNVs), insertions and deletions (indels), gene-conversion variations, and copy-number variations (CNVs) in cTAS2Rs. Approximately two-thirds of all cTAS2R haplotypes in the amino acid sequence were unique to each subspecies. We analyzed the evolutionary backgrounds of natural selection behind such diversification. Our previous study concluded that diversification of cTAS2Rs in western chimpanzees (P. t. verus) may have resulted from balancing selection. In contrast, the present study found that purifying selection dominates as the evolutionary form of diversification of the so-called human cluster of cTAS2Rs in eastern chimpanzees (P. t. schweinfurthii) and that the other cTAS2Rs were under no obvious selection as a whole. Such marked diversification of cTAS2Rs with different evolutionary backgrounds among subspecies of chimpanzees probably reflects their subspecies-specific dietary repertoires. PMID:22916235

Genome wide analysis of rare copy number variations in alcohol abuse or dependence.

PubMed

Rodríguez-López, Julio; Flórez, Gerardo; Blanco, Vanessa; Pereiro, César; Fernández, José Manuel; Fariñas, Emilio; Estévez, Valentín; Gómez-Trigo, Jesús; Gurriarán, Xaquín; Calvo, Raquel; Sáiz, Pilar; Vázquez, Fernando Lino; Arrojo, Manuel; Costas, Javier

2018-06-02

Genetics plays an important role in alcohol abuse/dependence. Its heritability has been estimated as 45-65%. Rare copy number variations (CNVs) have been confirmed as relevant genetic factors in other neuropsychiatric disorders, such as autism spectrum disorders, schizophrenia, epilepsy, or Tourette syndrome. In the present study, we analyzed the role of rare CNVs affecting exons of coding genes in a sample from Northwest Spain genotyped using the Illumina Infinium PsychArray Beadchip. After rigorous genotyping quality control procedure, 712 patients with alcohol abuse or dependence and 804 controls were used for CNV detection. CNV calling was performed using PennCNV and cnvPartition, and analyses were restricted to CNVs of at least 100 kb and including at least 10 single nucleotide polymorphisms. Logistic regression was used to test for the effect of CNV as well as number of genes affected by CNVs on case/control status, after adjustment for demographic and experimental covariates. We have found an excess of deletions (p = 0.008) and genes affected by deletions (p = 0.017) in cases. This effect was restricted to the 14.8% of affected genes that are intolerant to loss-of-function mutations (gene count p = 0.009). The importance of this subset of genes is emerging in other psychiatric disorders of neurodevelopmental origin, suggesting that disturbance in neurodevelopment mediated by genetic alterations may be a risk factor for alcohol use disorder. Copyright © 2018 Elsevier Ltd. All rights reserved.

Phenotypic Association Analyses With Copy Number Variation in Recurrent Depressive Disorder.

PubMed

Rucker, James J H; Tansey, Katherine E; Rivera, Margarita; Pinto, Dalila; Cohen-Woods, Sarah; Uher, Rudolf; Aitchison, Katherine J; Craddock, Nick; Owen, Michael J; Jones, Lisa; Jones, Ian; Korszun, Ania; Barnes, Michael R; Preisig, Martin; Mors, Ole; Maier, Wolfgang; Rice, John; Rietschel, Marcella; Holsboer, Florian; Farmer, Anne E; Craig, Ian W; Scherer, Stephen W; McGuffin, Peter; Breen, Gerome

2016-02-15

Defining the molecular genomic basis of the likelihood of developing depressive disorder is a considerable challenge. We previously associated rare, exonic deletion copy number variants (CNV) with recurrent depressive disorder (RDD). Sex chromosome abnormalities also have been observed to co-occur with RDD. In this reanalysis of our RDD dataset (N = 3106 cases; 459 screened control samples and 2699 population control samples), we further investigated the role of larger CNVs and chromosomal abnormalities in RDD and performed association analyses with clinical data derived from this dataset. We found an enrichment of Turner's syndrome among cases of depression compared with the frequency observed in a large population sample (N = 34,910) of live-born infants collected in Denmark (two-sided p = .023, odds ratio = 7.76 [95% confidence interval = 1.79-33.6]), a case of diploid/triploid mosaicism, and several cases of uniparental isodisomy. In contrast to our previous analysis, large deletion CNVs were no more frequent in cases than control samples, although deletion CNVs in cases contained more genes than control samples (two-sided p = .0002). After statistical correction for multiple comparisons, our data do not support a substantial role for CNVs in RDD, although (as has been observed in similar samples) occasional cases may harbor large variants with etiological significance. Genetic pleiotropy and sample heterogeneity suggest that very large sample sizes are required to study conclusively the role of genetic variation in mood disorders. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Comparative genomics in chicken and Pekin duck using FISH mapping and microarray analysis

PubMed Central

2009-01-01

Background The availability of the complete chicken (Gallus gallus) genome sequence as well as a large number of chicken probes for fluorescent in-situ hybridization (FISH) and microarray resources facilitate comparative genomic studies between chicken and other bird species. In a previous study, we provided a comprehensive cytogenetic map for the turkey (Meleagris gallopavo) and the first analysis of copy number variants (CNVs) in birds. Here, we extend this approach to the Pekin duck (Anas platyrhynchos), an obvious target for comparative genomic studies due to its agricultural importance and resistance to avian flu. Results We provide a detailed molecular cytogenetic map of the duck genome through FISH assignment of 155 chicken clones. We identified one inter- and six intrachromosomal rearrangements between chicken and duck macrochromosomes and demonstrated conserved synteny among all microchromosomes analysed. Array comparative genomic hybridisation revealed 32 CNVs, of which 5 overlap previously designated "hotspot" regions between chicken and turkey. Conclusion Our results suggest extensive conservation of avian genomes across 90 million years of evolution in both macro- and microchromosomes. The data on CNVs between chicken and duck extends previous analyses in chicken and turkey and supports the hypotheses that avian genomes contain fewer CNVs than mammalian genomes and that genomes of evolutionarily distant species share regions of copy number variation ("CNV hotspots"). Our results will expedite duck genomics, assist marker development and highlight areas of interest for future evolutionary and functional studies. PMID:19656363

De Novo Chromosome Copy Number Variation in Fanconi Anemia-Associated Hematopoietic Defects

DTIC Science & Technology

2014-08-01

protein, and not FANCA , is required for the suppression of spontaneous de novo CNV. These findings support a model whereby the FANCD2 protein, possibly...cancer susceptibility gene products BRCA1 and BRCA2 function cooperatively in the FA-BRCA pathway to repair damaged DNA. Recent studies have...the FA-BRCA pathway in the suppression of spontaneous de novo CNVs Task 1. Correction of FA-A, FA-C, and FA-D2 hTERT cells with pLenti6.2/V5- FANCA

Anti-C1q Antibodies in Systemic Lupus Erythematosus

PubMed Central

ORBAI, ANA-MARIA; TRUEDSSON, LENNART; STURFELT, GUNNAR; NIVED, OLA; FANG, HONG; ALARCÓN, GRACIELA S.; GORDON, CAROLINE; MERRILL, JOAN T.; FORTIN, PAUL R.; BRUCE, IAN N.; ISENBERG, DAVID A.; WALLACE, DANIEL J.; RAMSEY-GOLDMAN, ROSALIND; BAE, SANG-CHEOL; HANLY, JOHN G.; SANCHEZ-GUERRERO, JORGE; CLARKE, ANN E.; ARANOW, CYNTHIA B.; MANZI, SUSAN; UROWITZ, MURRAY B.; GLADMAN, DAFNA D.; KALUNIAN, KENNETH C.; COSTNER, MELISSA I.; WERTH, VICTORIA P.; ZOMA, ASAD; BERNATSKY, SASHA; RUIZ-IRASTORZA, GUILLERMO; KHAMASHTA, MUNTHER A.; JACOBSEN, SOREN; BUYON, JILL P.; MADDISON, PETER; DOOLEY, MARY ANNE; VAN VOLLENHOVEN, RONALD F.; GINZLER, ELLEN; STOLL, THOMAS; PESCHKEN, CHRISTINE; JORIZZO, JOSEPH L.; CALLEN, JEFFREY P.; LIM, S. SAM; FESSLER, BARRI J.; INANC, MURAT; KAMEN, DIANE L.; RAHMAN, ANISUR; STEINSSON, KRISTJAN; FRANKS, ANDREW G.; SIGLER, LISA; HAMEED, SUHAIL; PHAM, NEENA; BREY, ROBIN; WEISMAN, MICHAEL H.; MCGWIN, GERALD; MAGDER, LAURENCE S.; PETRI, MICHELLE

2014-01-01

Objective Anti-C1q has been associated with systemic lupus erythematosus (SLE) and lupus nephritis in previous studies. We studied anti-C1q specificity for SLE (vs. rheumatic disease controls) and the association with SLE manifestations in an international multi-center study. Methods Information and blood samples were obtained in a cross-sectional study from patients with SLE (n=308) and other rheumatologic diseases (n=389) from 25 clinical sites (84% female, 68% Caucasian, 17% African descent, 8% Asian, 7% other). IgG anti-C1q against the collagen-like region was measured by ELISA. Results Prevalence of anti-C1q was 28% (86/308) in patients with SLE and 13% (49/389) in controls (OR=2.7, 95% CI: 1.8-4, p<0.001). Anti-C1q was associated with proteinuria (OR=3.0, 95% CI: 1.7-5.1, p<0.001), red cell casts (OR=2.6, 95% CI: 1.2-5.4, p=0.015), anti-dsDNA (OR=3.4, 95% CI: 1.9-6.1, p<0.001) and anti-Smith (OR=2.8, 95% CI: 1.5-5.0, p=0.01). Anti-C1q was independently associated with renal involvement after adjustment for demographics, ANA, anti-dsDNA and low complement (OR=2.3, 95% CI: 1.3-4.2, p<0.01). Simultaneously positive anti-C1q, anti-dsDNA and low complement was strongly associated with renal involvement (OR=14.9, 95% CI: 5.8-38.4, p<0.01). Conclusions Anti-C1q was more common in patients with SLE and those of Asian race/ethnicity. We confirmed a significant association of anti-C1q with renal involvement, independent of demographics and other serologies. Anti-C1q in combination with anti-dsDNA and low complement was the strongest serological association with renal involvement. These data support the usefulness of anti-C1q in SLE, especially in lupus nephritis. PMID:25124676

Low serum immunglobulin G (IgG) during nephrosis is a predictor of urinary tract infection (UTI) in children with nephrotic syndrome.

PubMed

Afroz, S; Roy, D K; Khan, A H

2013-04-01

Low serum level of IgG, complement C3 and C4 in nephrotic syndrome children may cause increased susceptibility to infection. Serum level of IgG and complements in nephrotic children (NS) with UTI has been analyzed in this cross sectional study. It was carried out in the department of Pediatric nephrology, National Institute of Kidney Diseases & Urology (NIKDU), Dhaka, Bangladesh. The study subjects were followed up prospectively for one year to see and compare the frequency of relapse of NS and UTI. Patients were selected in a nonrandom purposive technique. Nephrotic syndrome children with initial attack between 1-12 year of age were included over a period of one year. The patients were grouped into Group I - UTI positive and Group II - UTI negative depending on urine culture positivity and colony count >10⁵ CFU/ml. Serum IgG and complements C3, C4 levels were done in both groups during nephrosis and were compared. A total of 101 children M: F 1.7:1, mean age 5.96±3.2 years were included in this study. Group I, n=45 vs. Group II, n=56. The mean serum level of IgG was low in Group I (549.91±210.71 vs. 728.64±235.81mg/dl, p<0.001). Serum IgG level less than 700mg/dl was found in 37 vs. 23 children {x² (¹) 17.52 p<0.001, OR=6.63}. Mean serum complement C3 level was also low in Group I (123.09±40.52 vs. 143.38±37.06mg/dl, p<0.05). But complement C3 and C4 level do not carry any risk of developing UTI in nephrotic children. Higher number of children in Group II were at remission (n=24) during follow up, while frequent relapsers were high in Group I (n=22). Increased frequency of UTI attack (88 episodes) was found in Group I children compared to none in Group II during follow up. So low serum level of IgG in children with NS during nephrosis can predict UTI with an odds ratio of 6.63 as well as relapse. Serum level of C3, C4 do not associated with any risk of development of UTI in NS children.

Four subunits that are shared by the three classes of RNA polymerase are functionally interchangeable between Homo sapiens and Saccharomyces cerevisiae.

PubMed Central

Shpakovski, G V; Acker, J; Wintzerith, M; Lacroix, J F; Thuriaux, P; Vigneron, M

1995-01-01

Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10 alpha, Hs10 beta, Hs8, and Hs6, respectively), homologous to the ABC10 alpha, ABC10 beta, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10 alpha, Sc10 beta, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Hs10 alpha and the corresponding Sp10 alpha of Schizosaccharomyces pombe can complement an S. cerevisiae mutant (rpc10-delta::HIS3) defective in Sc10 alpha. The peptide sequences are highly conserved in their carboxy-terminal halves, with an invariant motif CX2CX12RCX2CGXR corresponding to a canonical zinc-binding domain. Hs10 beta, Sc10 beta, and the N subunit of archaeal RNA polymerase are homologous. An invariant CX2CGXnCCR motif presumably forms an atypical zinc-binding domain. Hs10 beta, but not the archaeal subunit, complemented an S. cerevisiae mutant (rpb10-delta 1::HIS3) lacking Sc10 beta. Hs8 complemented a yeast mutant (rpb8-delta 1::LYS2) defective in the corresponding Sc8 subunit, although with a strong thermosensitive phenotype. Interspecific complementation also occurred with Hs6 and with the corresponding Dm6 cDNA of Drosophila melanogaster. Hs6 cDNA and the Sp6 cDNA of S. pombe are dosage-dependent suppressors of rpo21-4, a mutation generating a slowly growing yeast defective in the largest subunit of RNA polymerase II. Finally, a doubly chimeric S. cerevisiae strain bearing the Sp6 cDNA and the human Hs10 beta cDNA was also viable. No interspecific complementation was observed for the human hRPB25 (Hs5) homolog of the yeast ABC27 (Sc5) subunit. PMID:7651387

A C3(H20) recycling pathway is a component of the intracellular complement system

PubMed Central

Elvington, Michelle; Bertram, Paula; Atkinson, John P.

2017-01-01

An intracellular complement system (ICS) has recently been described in immune and nonimmune human cells. This system can be activated in a convertase-independent manner from intracellular stores of the complement component C3. The source of these stores has not been rigorously investigated. In the present study, Western blotting identified a band corresponding to C3 in freshly isolated human peripheral blood cells that was absent in corresponding cell lines. One difference between native cells and cell lines was the time absent from a fluid-phase complement source; therefore, we hypothesized that loading C3 from plasma was a route of establishing intracellular C3 stores. We found that many types of human cells specifically internalized C3(H2O), the hydrolytic product of C3, and not native C3, from the extracellular milieu. Uptake was rapid, saturable, and sensitive to competition with unlabeled C3(H2O), indicating a specific mechanism of loading. Under steady-state conditions, approximately 80% of incorporated C3(H2O) was returned to the extracellular space. These studies identify an ICS recycling pathway for C3(H2O). The loaded C3(H2O) represents a source of C3a, and its uptake altered the cytokine profile of activated CD4+ T cells. Importantly, these results indicate that the impact of soluble plasma factors should be considered when performing in vitro studies assessing cellular immune function. PMID:28192370

Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease

PubMed Central

1992-01-01

Serum mannose-binding protein (MBP) is a C-type lectin that binds to terminal mannose and N-acetylglucosamine moieties present on surfaces of certain pathogens and activates the classical complement pathway. In the present study, we describe the mechanism underlying the activation triggered by MBP. The human serum MBP fraction was obtained by sequential affinity chromatography on mannan-Sepharose, anti-IgM- Sepharose and anti-MBP-Sepharose in the presence of calcium ions. This fraction contained a C1s-like serine protease as assessed by C4 consumption. The C1s-like serine protease, designated MBP-associated serine protease (MASP), was separated from MBP by rechromatography on anti-MBP-Sepharose in the presence of ethylenediaminetetraacetic acid. MASP exhibited both C4- and C2-consuming activities. The molecular mass of MASP was estimated to be 83 kD with two polypeptides of heavy (66 kD) and light (L) (31 kD) chains linked by disulfide bonds. The serine residue responsible for protease activity is located on the L chain. Reconstitution experiments using MASP and MBP revealed that combination of the two components restores C4- and C2-activating capacity on mannan. Based on analyses of molecular size, antigenicity, and 11 NH2- terminal amino acid sequences of the L chain, we conclude that MASP is a novel protein different from C1r or C1s. Our findings are not in accord with a proposed mechanism by which MBP utilizes the C1r2-C1s2 complex to initiate the classical complement pathway. PMID:1460414

Dialysis in rats with acute renal failure: evaluation of three different dialyzer membranes.

PubMed

Kränzlin, B; Gretz, N; Kirschfink, M; Mujais, S K

1996-11-01

Exposure to complement-activating cellulosic dialysis membranes has been claimed to adversely affect the course of acute renal failure (ARF). To test this hypothesis, male Sprague-Dawley rats were allocated to 2 groups: in Group 1, ARF was induced by bilateral renal artery clamping whereas in Group 2, animals underwent a sham procedure. In each group, rats were further allocated to undergo hemodialysis with either a Cuprophan, a Hemophan, or a polyacrylonitrile minidialyzer on Days 4 and 8 after surgery, or no dialysis. Renal function was measured by inulin clearance on the days after dialysis. Additionally, total complement activity (CH50) was estimated on Days 1, 2, 4, and 8, and complement factor C3 was detected immunohistochemically. The degree of renal failure and the rate of recovery of renal function were similar in all the ARF groups irrespective of whether they had undergone dialysis or not, or of the type of the dialysis membrane. Furthermore, there were no significant differences in the course of CH50 or in the amount and distribution of complement factor C3 in the kidney tissue between the rats of Groups 1 and 2. Our findings refute the hypothesis that in ischemic ARF exposure to complement-activating cellulosic dialysis membranes impairs the recovery of renal function in rats.

Triatoma infestans Calreticulin: Gene Cloning and Expression of a Main Domain That Interacts with the Host Complement System.

PubMed

Weinberger, Katherine; Collazo, Norberto; Aguillón, Juan Carlos; Molina, María Carmen; Rosas, Carlos; Peña, Jaime; Pizarro, Javier; Maldonado, Ismael; Cattan, Pedro E; Apt, Werner; Ferreira, Arturo

2017-02-08

Triatoma infestans is an important hematophagous vector of Chagas disease, a neglected chronic illness affecting approximately 6 million people in Latin America. Hematophagous insects possess several molecules in their saliva that counteract host defensive responses. Calreticulin (CRT), a multifunctional protein secreted in saliva, contributes to the feeding process in some insects. Human CRT (HuCRT) and Trypanosoma cruzi CRT (TcCRT) inhibit the classical pathway of complement activation, mainly by interacting through their central S domain with complement component C1. In previous studies, we have detected CRT in salivary gland extracts from T. infestans We have called this molecule TiCRT. Given that the S domain is responsible for C1 binding, we have tested its role in the classical pathway of complement activation in vertebrate blood. We have cloned and characterized the complete nucleotide sequence of CRT from T. infestans , and expressed its S domain. As expected, this S domain binds to human C1 and, as a consequence, it inhibits the classical pathway of complement, at its earliest stage of activation, namely the generation of C4b. Possibly, the presence of TiCRT in the salivary gland represents an evolutionary adaptation in hematophagous insects to control a potential activation of complement proteins, present in the massive blood meal that they ingest, with deleterious consequences at least on the anterior digestive tract of these insects. © The American Society of Tropical Medicine and Hygiene.

Autocrine Complement Inhibits IL10-Dependent T-cell-Mediated Antitumor Immunity to Promote Tumor Progression.

PubMed

Wang, Yu; Sun, Sheng-Nan; Liu, Qing; Yu, Yang-Yang; Guo, Jian; Wang, Kun; Xing, Bao-Cai; Zheng, Qing-Feng; Campa, Michael J; Patz, Edward F; Li, Shi-You; He, You-Wen

2016-09-01

In contrast to its inhibitory effects on many cells, IL10 activates CD8(+) tumor-infiltrating lymphocytes (TIL) and enhances their antitumor activity. However, CD8(+) TILs do not routinely express IL10, as autocrine complement C3 inhibits IL10 production through complement receptors C3aR and C5aR. CD8(+) TILs from C3-deficient mice, however, express IL10 and exhibit enhanced effector function. C3-deficient mice are resistant to tumor development in a T-cell- and IL10-dependent manner; human TILs expanded with IL2 plus IL10 increase the killing of primary tumors in vitro compared with IL2-treated TILs. Complement-mediated inhibition of antitumor immunity is independent of the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) immune checkpoint pathway. Our findings suggest that complement receptors C3aR and C5aR expressed on CD8(+) TILs represent a novel class of immune checkpoints that could be targeted for tumor immunotherapy. Moreover, incorporation of IL10 in the expansion of TILs and in gene-engineered T cells for adoptive cell therapy enhances their antitumor efficacy. Our data suggest novel strategies to enhance immunotherapies: a combined blockade of complement signaling by antagonists to C3aR, C5aR, and anti-PD-1 to enhance anti-PD-1 efficacy; a targeted IL10 delivery to CD8(+) TILs using anti-PD-1-IL10 or anti-CTLA4-IL10 fusion proteins; and the addition of IL10 in TIL expansion for adoptive cellular therapy. Cancer Discov; 6(9); 1022-35. ©2016 AACR.See related commentary by Peng et al., p. 953This article is highlighted in the In This Issue feature, p. 932. ©2016 American Association for Cancer Research.

Relationship among antineutrophil cytoplasmic antibody, blood urea nitrogen and complement in patients with eosinophilic granulomatosis polyangiitis (Churg-Strauss syndrome).

PubMed

Kawakami, Tamihiro; Kimura, Satoko; Takeuchi, Sora; Soma, Yoshinao

2013-07-01

Eosinophilic granulomatosis with polyangiitis (EGPA), also known as Churg-Strauss syndrome, is an antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis characterized by a history of asthma, hypereosinophilia. The prevalence of ANCA in EGPA is less common than in other ANCA-associated vasculitis. Increasing evidence of complement activation in the pathogenesis of ANCA-associated vasculitis has been provided by studies in animal models. We examined EGPA patients with cutaneous manifestations as an initial sign and investigated the correlations among clinical, serological and histopathological findings. We focused on differences among ANCA, blood urea nitrogen and complement levels such as complement 3 (C3), C4 and total complement hemolytic activity (CH50). We retrospectively investigated the records of 22 patients (11 male and 11 female) with EGPA admitted to our hospital from 1997-2012. Ten of the 22 patients (46%) were positive for serum myeloperoxidase (MPO)-ANCA. In contrast, all the patients were negative for serum proteinase 3 ANCA. There was a significantly positive correlation between serum CH50 and C4 levels in patients with EGPA. Serum blood urea nitrogen (BUN) levels differed significantly between MPO-ANCA-positive and -negative patients. Serum CH50 levels were higher in MPO-ANCA-positive patients compared to negative patients. Serum BUN levels were higher in elevated CH50 patients compared to normal and low CH50-negative patients. We propose that positive findings for MPO-ANCA with CH50 high activity may be a risk factor for developing renal insufficiency. Assuming there are correlations between the presence of ANCA and complements, earlier diagnosis based on initial efficacious treatment for EGPA. © 2013 Japanese Dermatological Association.

Molecular and expression analysis of complement component C5 in the nurse shark (Ginglymostoma cirratum) and its predicted functional role.

PubMed

Graham, Matthew; Shin, Dong-Ho; Smith, Sylvia L

2009-07-01

We present the complete cDNA sequence of shark (Ginglymostoma cirratum) pro-C5 and its molecular characterization with a descriptive analysis of the structural elements necessary for its potential functional role as a potent mediator of inflammation (fragment C5a) and initiator molecule (fragment C5b) for the assembly of the membrane attack complex (MAC) upon activation by C5 convertase. In mammals the three complement activation cascades, the classical, alternative and lectin pathways, converge at the activation of C3, a pivotal complement protein. It is, however, the subsequent activation of the next complement component, C5, which is the focal point at which the initiation of the terminal lytic pathway takes place and involves the stepwise assembly of the MAC. The effector cytolytic function of complement occurs with the insertion of MAC into target membranes causing dough-nut like holes and cell leakage. The lytic activity of shark complement results in structurally similar holes in target membranes suggesting the assembly of a shark MAC that likely involves a functional analogue of C5. The composition of shark MAC remains unresolved and to date conclusive evidence has been lacking for shark C5. The gene has not been cloned nor has the serum protein been characterized for any elasmobranch species. This report is the first to confirm the presence of C5 homologue in the shark. GcC5 is remarkably similar to human C5 in overall structure and domain arrangement. The GcC5 cDNA measured 5160-bp with 5' and 3' UTRs of 35 bp and 79 bp, respectively. Structural analysis of the derived protein sequence predicts a molecule that is a two-chain structure which lacks a thiolester bond and contains a C5 convertase cleavage site indicating that activation will generate two peptides, akin to C5b and C5a. The putative GcC5 molecule also contains the C-terminal C345C/Netrin module that characterizes C3, C4 and C5. Multiple alignment of deduced amino acid sequences shows that GcC5 shares more amino acid identities/similarities with mammals than that with bony fish. We conclude that at the time of emergence of sharks the elaborate mosaic structure of C5 had already evolved.

Molecular and expression analysis of complement component C5 in the nurse shark (Ginglymostoma cirratum) and its predicted functional role

PubMed Central

Graham, Matthew; Shin, Dong-Ho; Smith, Sylvia L.

2009-01-01

We present the complete cDNA sequence of shark (Ginglymostoma cirratum) pro-C5 and its molecular characterization with a descriptive analysis of the structural elements necessary for its potential functional role as a potent mediator of inflammation (fragment C5a) and initiator molecule (fragment C5b) for the assembly of the membrane attack complex (MAC) upon activation by C5 convertase. In mammals the three complement activation cascades, the classical, alternative and lectin pathways, converge at the activation of C3, a pivotal complement protein. It is, however, the subsequent activation of the next complement component, C5, which is the focal point at which the initiation of the terminal lytic pathway takes place and involves the stepwise assembly of the MAC. The effector cytolytic function of complement occurs with the insertion of MAC into target membranes causing dough-nut like holes and cell leakage. The lytic activity of shark complement results in structurally similar holes in target membranes suggesting the assembly of a shark MAC that likely involves a functional analogue of C5. The composition of shark MAC remains unresolved and to date conclusive evidence has been lacking for shark C5. The gene has not been cloned nor has the serum protein been characterized for any elasmobranch species. This report is the first to confirm the presence of C5 homologue in the shark. GcC5 is remarkably similar to human C5 in overall structure and domain arrangement. The GcC5 cDNA measured 5160-bp with 5′ and 3′ UTRs of 35bp and 79bp, respectively. Structural analysis of the derived protein sequence predicts a molecule that is a two-chain structure which lacks a thiolester bond and contains a C5 convertase cleavage site indicating that activation will generate two peptides, akin to C5b and C5a. The putative GcC5 molecule also contains the C-terminal C345C/Netrin module that characterizes C3, C4 and C5. Multiple alignment of deduced amino acid sequences show that GcC5 shares more amino acid identities/similarities with mammals than that with bony fish. We conclude that at the time of emergence of sharks the elaborate mosaic structure of C5 had already evolved. PMID:19410004

Sex matters: Systemic complement activity of female C57BL/6J and BALB/cJ mice is limited by serum terminal pathway components.

PubMed

Kotimaa, Juha; Klar-Mohammad, Ngaisah; Gueler, Faikah; Schilders, Geurt; Jansen, Aswin; Rutjes, Helma; Daha, Mohamed R; van Kooten, Cees

2016-08-01

Experimental mouse models have been extensively used to elucidate the role of the complement system in different diseases and injuries. Contribution of gender has revealed an intriguing gender specific difference; female mice often show protection against most complement driven injuries such as ischemia/reperfusion injury, graft rejection and sepsis. Interestingly, early studies to the mouse complement system revealed that female mice have very low total complement activity (CH50), which is related to androgen regulation of hepatic complement synthesis. Here, our aim was to understand at which level the female specific differences in mouse complement resides. We have used recently developed complement assays to study the functional activities of female and male mice at the level of C3 and C9 activation, and furthermore assayed key complement factor levels in serum of age-matched female and male C57BL/6 mice. Our results show that the female mice have normal complement cascade functionality at the level of C3 activation, which was supported by determinations of early complement factors. However, all pathways are strongly reduced at the level of C9 activation, suggesting a terminal pathway specific difference. This was in line with C6 and C9 measurements, showing strongly decreased levels in females. Furthermore, similar gender differences were also found in BALB/cJ mice, but not in CD-1 mice. Our results clearly demonstrate that the complement system in females of frequently used mouse strains is restricted by the terminal pathway components and that the perceived female specific protection against experimental disease and injury might be in part explained by the inability promote inflammation through C5b-9. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Molecular characterization of the alpha subunit of complement component C8 (GcC8α) in the nurse shark (Ginglymostoma cirratum)

PubMed Central

Aybar, Lydia; Shin, Dong-Ho; Smith, Sylvia L.

2009-01-01

Target cell lysis by complement is achieved by the assembly and insertion of the membrane attack complex (MAC) composed of glycoproteins C5b through C9. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. Mammalian C8 is composed of α, β, and γ subunits. The subunit structure of shark C8 is not known. This report describes a 2341 nucleotide sequence that translates into a polypeptide of 589 amino acid residues, orthologue to mammalian C8α and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8γ-binding cysteine is conserved in the perforin-like domain. Hydrophobicity profile indicates the presence of hydrophobic residues essential for membrane insertion. It shares 41.1% and 47.4 % identity with human and Xenopus C8α respectively. Southern blot analysis showed GcC8α exists as a single copy gene expressed in most tissues except the spleen with the liver being the main site of synthesis. Phylogenetic analysis places it in a clade with C8α orthologs and as a sister taxa to the Xenopus. PMID:19524681

Copy number variation plays an important role in clinical epilepsy

PubMed Central

Olson, Heather; Shen, Yiping; Avallone, Jennifer; Sheidley, Beth R.; Pinsky, Rebecca; Bergin, Ann M.; Berry, Gerard T.; Duffy, Frank H.; Eksioglu, Yaman; Harris, David J.; Hisama, Fuki M.; Ho, Eugenia; Irons, Mira; Jacobsen, Christina M.; James, Philip; Kothare, Sanjeev; Khwaja, Omar; Lipton, Jonathan; Loddenkemper, Tobias; Markowitz, Jennifer; Maski, Kiran; Megerian, J. Thomas; Neilan, Edward; Raffalli, Peter C.; Robbins, Michael; Roberts, Amy; Roe, Eugene; Rollins, Caitlin; Sahin, Mustafa; Sarco, Dean; Schonwald, Alison; Smith, Sharon E.; Soul, Janet; Stoler, Joan M.; Takeoka, Masanori; Tan, Wen-Han; Torres, Alcy R.; Tsai, Peter; Urion, David K.; Weissman, Laura; Wolff, Robert; Wu, Bai-Lin; Miller, David T.; Poduri, Annapurna

2015-01-01

Objective To evaluate the role of copy number abnormalities detectable by chromosomal microarray (CMA) testing in patients with epilepsy at a tertiary care center. Methods We identified patients with ICD-9 codes for epilepsy or seizures and clinical CMA testing performed between October 2006 and February 2011 at Boston Children’s Hospital. We reviewed medical records and included patients meeting criteria for epilepsy. We phenotypically characterized patients with epilepsy-associated abnormalities on CMA. Results Of 973 patients who had CMA and ICD-9 codes for epilepsy or seizures, 805 patients satisfied criteria for epilepsy. We observed 437 copy number variants (CNVs) in 323 patients (1–4 per patient), including 185 (42%) deletions and 252 (58%) duplications. Forty (9%) were confirmed de novo, 186 (43%) were inherited, and parental data were unavailable for 211 (48%). Excluding full chromosome trisomies, CNV size ranged from 18 kb to 142 Mb, and 34% were over 500 kb. In at least 40 cases (5%), the epilepsy phenotype was explained by a CNV, including 29 patients with epilepsy-associated syndromes and 11 with likely disease-associated CNVs involving epilepsy genes or “hotspots.” We observed numerous recurrent CNVs including 10 involving loss or gain of Xp22.31, a region described in patients with and without epilepsy. Interpretation Copy number abnormalities play an important role in patients with epilepsy. Given that the diagnostic yield of CMA for epilepsy patients is similar to the yield in autism spectrum disorders and in prenatal diagnosis, for which published guidelines recommend testing with CMA, we recommend the implementation of CMA in the evaluation of unexplained epilepsy. PMID:24811917

21 CFR 866.5260 - Complement C3b inactivator immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in the...

21 CFR 866.5260 - Complement C3b inactivator immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in the...

21 CFR 866.5260 - Complement C3b inactivator immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in the...

21 CFR 866.5260 - Complement C3b inactivator immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in the...

BK-virus nephropathy and simultaneous C4d positive staining in renal allografts.

PubMed

Honsová, E; Lodererová, A; Viklický, O; Boucek, P

2005-10-01

The role of antibodies in rejection of transplanted kidneys was the subject of debate at the last two Banff meetings and in medical journals. Diffuse C4d positive staining of peritubular capillaries (PTCs) was recognized as a marker of antibody-mediated rejection and this morphological feature was included in the updated Banff schema. At the same time polyomavirus infection of the renal allografts has been reported more frequently and is emerging as an important cause of renal allograft dysfunction and graft loss. At the present time, BK-virus nephropathy (BKN) represents the most common viral disease affecting renal allografts. BKN was identified in 6 patients in 12 biopsies and 2 graft nephrectomy specimens of 1115 biopsies between September 2000 and December 2003. Definite virus identification was done by immunohistochemistry. The reason for graft nephrectomies was graft failure due to BKN in a recipient after kidney-pancreas transplantation with good function of his pancreas graft and the necessity of continuing immunosuppression. Detection of C4d deposits was performed by immunofluorescence or by immunohistochemistry. In graftectomy samples C4d detection was performed by immunohistochemistry and retrospectively in all cases of BKN. Focal C4d positive PTCs and BKN were found simultaneously in 9 of 12 needle biopsies and in both graft nephrectomy samples. Detection of C4d by immunohistochemistry disclosed focal C4d positive staining in kidney tissue but diffuse in the sites where BK-virus inclusions in tubular epithelial cells were found. The complement system is part of the host defense response and is crucial to our natural ability to ward off infection. In cases of BKN, virus likely gains access to the bloodstream through injured tubular walls and via PTCs. Vascular endothelium in the PTCs represents a potential target antigen for alloresponse, and simultaneously possibly represents an imprint of complement activation or complement production in the places with BK-virus infection.

Association between copy number variation losses and alcohol dependence across African American and European American ethnic groups.

PubMed

Ulloa, Alvaro E; Chen, Jiayu; Vergara, Victor M; Calhoun, Vince; Liu, Jingyu

2014-05-01

Copy number variations (CNVs) are structural genetic mutations consisting of segmental gains or losses in DNA sequence. Although CNVs contribute substantially to genomic variation, few genetic and imaging studies report association of CNVs with alcohol dependence (AD). Our purpose is to find evidence of this association across ethnic populations and genders. This work is the first AD-CNV study across ethnic groups and the first to include the African American (AA) population. This study considers 2 CNV data sets, one for discovery (2,345 samples) and the other for validation (239 samples), both including subjects with AD and healthy controls of European and African ancestry. Our analysis assesses the association between AD and CNV losses across ethnic groups and gender by examining the effect of overall losses across the whole genome, collective losses within individual cytogenetic bands, and specific losses in CNV regions. Results from the discovery data set showed an association between CNV losses within 16q12.2 and AD diagnosis (p = 4.53 × 10(-3) ). An overlapping CNV region from the validation data set exhibited the same direction of effect with respect to AD (p = 0.051). This CNV region affects the genes CES1p1 and CES1, which are members of the carboxylesterase (CES) family. The enzyme encoded by CES1 is a major liver enzyme that typically catalyzes the decomposition of ester into alcohol and carboxylic acid and is involved in drug or xenobiotics, fatty acid, and cholesterol metabolisms. In addition, the most significantly associated CNV region was located at 9p21.2 (p = 1.9 × 10(-3) ) in our discovery data set. Although not observed in the validation data set, probably due to small sample size, this result might hold potential connection to AD given its connection with neuronal death. In contrast, we did not find any association between AD and the overall total losses or the collective losses within individual cytogenetic bands. Overall, our study provides evidence that the specific CNVs at 16q12.2 contribute to the development of alcoholism in AA and European American populations. Copyright © 2014 by the Research Society on Alcoholism.

Genome-wide screening identifies a KCNIP1 copy number variant as a genetic predictor for atrial fibrillation

PubMed Central

Tsai, Chia-Ti; Hsieh, Chia-Shan; Chang, Sheng-Nan; Chuang, Eric Y.; Ueng, Kwo-Chang; Tsai, Chin-Feng; Lin, Tsung-Hsien; Wu, Cho-Kai; Lee, Jen-Kuang; Lin, Lian-Yu; Wang, Yi-Chih; Yu, Chih-Chieh; Lai, Ling-Ping; Tseng, Chuen-Den; Hwang, Juey-Jen; Chiang, Fu-Tien; Lin, Jiunn-Lee

2016-01-01

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Previous genome-wide association studies had identified single-nucleotide polymorphisms in several genomic regions to be associated with AF. In human genome, copy number variations (CNVs) are known to contribute to disease susceptibility. Using a genome-wide multistage approach to identify AF susceptibility CNVs, we here show a common 4,470-bp diallelic CNV in the first intron of potassium interacting channel 1 gene (KCNIP1) is strongly associated with AF in Taiwanese populations (odds ratio=2.27 for insertion allele; P=6.23 × 10−24). KCNIP1 insertion is associated with higher KCNIP1 mRNA expression. KCNIP1-encoded protein potassium interacting channel 1 (KCHIP1) is physically associated with potassium Kv channels and modulates atrial transient outward current in cardiac myocytes. Overexpression of KCNIP1 results in inducible AF in zebrafish. In conclusions, a common CNV in KCNIP1 gene is a genetic predictor of AF risk possibly pointing to a functional pathway. PMID:26831368

Distinct polymer architecture mediates switching of complement activation pathways at the nanosphere-serum interface: implications for stealth nanoparticle engineering.

PubMed

Hamad, Islam; Al-Hanbali, Othman; Hunter, A Christy; Rutt, Kenneth J; Andresen, Thomas L; Moghimi, S Moein

2010-11-23

Nanoparticles with surface projected polyethyleneoxide (PEO) chains in "mushroom-brush" and "brush" configurations display stealth properties in systemic circulation and have numerous applications in site-specific targeting for controlled drug delivery and release as well as diagnostic imaging. We report on the "structure-activity" relationship pertaining to surface-immobilized PEO of various configurations on model nanoparticles, and the initiation of complement cascade, which is the most ancient component of innate human immunity, and its activation may induce clinically significant adverse reactions in some individuals. Conformational states of surface-projected PEO chains, arising from the block copolymer poloxamine 908 adsorption, on polystyrene nanoparticles trigger complement activation differently. Alteration of copolymer architecture on nanospheres from mushroom to brush configuration not only switches complement activation from C1q-dependent classical to lectin pathway but also reduces the level of generated complement activation products C4d, Bb, C5a, and SC5b-9. Also, changes in adsorbed polymer configuration trigger alternative pathway activation differently and through different initiators. Notably, the role for properdin-mediated activation of alternative pathway was only restricted to particles displaying PEO chains in a transition mushroom-brush configuration. Since nanoparticle-mediated complement activation is of clinical concern, our findings provide a rational basis for improved surface engineering and design of immunologically safer stealth and targetable nanosystems with polymers for use in clinical medicine.

Chromosomal contacts connect loci associated with autism, BMI and head circumference phenotypes

PubMed Central

Loviglio, M N; Leleu, M; Männik, K; Passeggeri, M; Giannuzzi, G; van der Werf, I; Waszak, S M; Zazhytska, M; Roberts-Caldeira, I; Gheldof, N; Migliavacca, E; Alfaiz, A A; Hippolyte, L; Maillard, A M; Loviglio, Maria Nicla; Männik, Katrin; van der Werf, Ilse; Giannuzzi, Giuliana; Zazhytska, Marianna; Gheldof, Nele; Migliavacca, Eugenia; Alfaiz, Ali A; Roberts-Caldeira, Inês; Hippolyte, Loyse; Maillard, Anne M; Ferrarini, Alessandra; Butschi, Florence Niel; Conrad, Bernard; Addor, Marie-Claude; Belfiore, Marco; Roetzer, Katharina; Dijck, Anke Van; Blaumeiser, Bettina; Kooy, Frank; Roelens, Filip; Dheedene, Annelies; Chiaie, Barbara Delle; Menten, Björn; Oostra, Ann; Caberg, Jean-Hubert; Carter, Melissa; Kellam, Barbara; Stavropoulos, Dimitri J; Marshall, Christian; Scherer, Stephen W; Weksberg, Rosanna; Cytrynbaum, Cheryl; Bassett, Anne; Lowther, Chelsea; Gillis, Jane; MacKay, Sara; Bache, Iben; Ousager, Lilian B; Smerdel, Maja Patricia; Graakjaer, Jesper; Kjaergaard, Susanne; Metspalu, Andres; Mathieu, Michele; Bonneau, Dominique; Guichet, Agnes; Parent, Philippe; Férec, Claude; Gerard, Marion; Plessis, Ghislaine; Lespinasse, James; Masurel, Alice; Marle, Nathalie; Faivre, Laurence; Callier, Patrick; Layet, Valerie; Meur, Nathalie Le; Le Goff, Céline; Duban-Bedu, Bénédicte; Sukno, Sylvie; Boute, Odile; Andrieux, Joris; Blanchet, Patricia; Geneviève, David; Puechberty, Jacques; Schneider, Anouck; Leheup, Bruno; Jonveaux, Philippe; Mercier, Sandra; David, Albert; Le Caignec, Cédric; de Pontual, Loic; Pipiras, Eva; Jacquette, Aurelia; Keren, Boris; Gilbert-Dussardier, Brigitte; Bilan, Frederic; Goldenberg, Alice; Chambon, Pascal; Toutain, Annick; Till, Marianne; Sanlaville, Damien; Leube, Barbara; Royer-Pokora, Brigitte; Grabe, Hans Jörgen; Schmidt, Carsten Oliver; Schurmann, Claudia; Homuth, Georg; Thorleifsson, Gudmar; Thorsteinsdottir, Unnur; Bernardini, Laura; Novelli, Antonio; Micale, Lucia; Merla, Giuseppe; Zollino, Marcella; Mari, Francesca; Rizzo, Caterina Lo; Renieri, Alessandra; Silengo, Margherita; Vulto-van Silfhout, Anneke T; Schouten, Meyke; Pfundt, Rolph; de Leeuw, Nicole; Vansenne, Fleur; Maas, Saskia M; Barge-Schaapveld, Daniela QCM; Knegt, Alida C; Stadheim, Barbro; Rodningen, Olaug; Houge, Gunnar; Price, Sue; Hawkes, Lara; Campbell, Carolyn; Kini, Usha; Vogt, Julie; Walters, Robin; Blakemore, Alexandra; Gusella, James F; Shen, Yiping; Scott, Daryl; Bacino, Carlos A; Tsuchiya, Karen; Ladda, Roger; Sell, Susan; Asamoah, Alexander; Hamati, Aline I; Rosenfeld, Jill A; Shaffer, Lisa G; Mitchell, Elyse; Hodge, Jennelle C; Beckmann, Jacques S; Jacquemont, Sébastien; Reymond, Alexandre; Reymond, Alexandre; Ewans, Lisa J; Mowat, David; Walker, Jan; Amor, David J; Esch, Hilde Van; Leroy, Patricia; Caberg, Jean-Hubert; Bamforth, John-Steven; Babu, Deepti; Till, Marianne; Sanlaville, Damien; Geneviève, David; Puechberty, Jacques; Isidor, Bertrand; DiDonato, Nataliya; Hackmann, Karl; Passeggeri, Marzia; Haeringen, Arie van; Rosenfeld, Jill A; Shaffer, Lisa G; Smith, Rosemarie; Ellingwood, Sara; Farber, Darren M; Puri, Vinay; Zadeh, Neda; Weaver, David D; Miller, Mandy; Wilks, Timothy; Jorgez, Carolina J; Lafayette, DeeDee; Jacquemont, Sébastien; Van Dijck, A; Kooy, R F; Sanlaville, D; Rosenfeld, J A; Shaffer, L G; Andrieux, J; Marshall, C; Scherer, S W; Shen, Y; Gusella, J F; Thorsteinsdottir, U; Thorleifsson, G; Dermitzakis, E T; Deplancke, B; Beckmann, J S; Rougemont, J; Jacquemont, S; Reymond, A

2017-01-01

Copy number variants (CNVs) are major contributors to genomic imbalance disorders. Phenotyping of 137 unrelated deletion and reciprocal duplication carriers of the distal 16p11.2 220 kb BP2-BP3 interval showed that these rearrangements are associated with autism spectrum disorders and mirror phenotypes of obesity/underweight and macrocephaly/microcephaly. Such phenotypes were previously associated with rearrangements of the non-overlapping proximal 16p11.2 600 kb BP4-BP5 interval. These two CNV-prone regions at 16p11.2 are reciprocally engaged in complex chromatin looping, as successfully confirmed by 4C-seq, fluorescence in situ hybridization and Hi-C, as well as coordinated expression and regulation of encompassed genes. We observed that genes differentially expressed in 16p11.2 BP4-BP5 CNV carriers are concomitantly modified in their chromatin interactions, suggesting that disruption of chromatin interplays could participate in the observed phenotypes. We also identified cis- and trans-acting chromatin contacts to other genomic regions previously associated with analogous phenotypes. For example, we uncovered that individuals with reciprocal rearrangements of the trans-contacted 2p15 locus similarly display mirror phenotypes on head circumference and weight. Our results indicate that chromosomal contacts’ maps could uncover functionally and clinically related genes. PMID:27240531

Increased C3 production in human monocytes after stimulation with Candida albicans is suppressed by granulocyte-macrophage colony-stimulating factor.

PubMed Central

Høgåsen, A K; Abrahamsen, T G

1993-01-01

Activation of the complement system is an important part of host resistance against fungal infections. When human monocytes, cultured for 2 days or more, were treated in vitro with Candida albicans for 24 h, an enhancement of their biosynthesis of the complement components C3 and factor B was found. However, when C. albicans was administered to freshly isolated monocytes, a consistent stimulation of factor B biosynthesis occurred, while the C3 production was increased in about 50% of the donors. C. albicans also induced the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) from the cultured cells, apparently in larger amounts in the donors in whom no stimulation of C3 production was found. An antibody to GM-CSF administered with the yeast at the initiation of the monocyte culture caused an increase in the C3 production. Furthermore, when monocytes were treated with recombinant human GM-CSF either at the same time as or 4 days prior to the addition of C. albicans, the increase in C3 production was suppressed or neutralized, while factor B biosynthesis was unaffected. Taken together, these results indicate that monocytes respond to C. albicans with an increased production of complement factors. This may be an important mechanism both for opsonization of the fungus and for initiation of an inflammatory reaction. At an inflammatory site, this complement response may be suppressed by locally produced GM-CSF. PMID:8478067

Neutrophil extracellular traps can activate alternative complement pathways.

PubMed

Wang, H; Wang, C; Zhao, M-H; Chen, M

2015-09-01

The interaction between neutrophils and activation of alternative complement pathway plays a pivotal role in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). ANCAs activate primed neutrophils to release neutrophil extracellular traps (NETs), which have recently gathered increasing attention in the development of AAV. The relationship between NETs and alternative complement pathway has not been elucidated. The current study aimed to investigate the relationship between NETs and alternative complement pathway. Detection of components of alternative complement pathway on NETs in vitro was assessed by immunostain and confocal microscopy. Complement deposition on NETs were detected after incubation with magnesium salt ethyleneglycol tetraacetic acid (Mg-EGTA)-treated human serum. After incubation of serum with supernatants enriched in ANCA-induced NETs, levels of complement components in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Complement factor B (Bb) and properdin deposited on NETs in vitro. The deposition of C3b and C5b-9 on NETs incubated with heat-inactivated normal human serum (Hi-NHS) or EGTA-treated Hi-NHS (Mg-EGTA-Hi-NHS) were significantly less than that on NETs incubated with NHS or EGTA-treated NHS (Mg-EGTA-NHS). NETs induced by ANCA could activate the alternative complement cascade in the serum. In the presence of EGTA, C3a, C5a and SC5b-9 concentration decreased from 800·42 ± 244·81 ng/ml, 7·68 ± 1·50 ng/ml, 382·15 ± 159·75 ng/ml in the supernatants enriched in ANCA induced NETs to 479·07 ± 156·2 ng/ml, 4·86 ± 1·26 ng/ml, 212·65 ± 44·40 ng/ml in the supernatants of DNase I-degraded NETs (P < 0·001, P = 0·008, P < 0·001, respectively). NETs could activate the alternative complement pathway, and might thus participate in the pathogenesis of AAV. © 2015 British Society for Immunology.

Characterization of the GDP-D-mannose biosynthesis pathway in Coxiella burnetii: the initial steps for GDP-β-D-virenose biosynthesis.

PubMed

Narasaki, Craig T; Mertens, Katja; Samuel, James E

2011-01-01

Coxiella burnetii, the etiologic agent of human Q fever, is a gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars β-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-β-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames (CBU0671, CBU0294 and CBU0689) annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is critical to fully understand the biosynthesic pathway of GDP-β-D-virenose and LPS structure in C. burnetii.

Toll-Like Receptor 4 Stimulation before or after Streptococcus pneumoniae Induced Sepsis Improves Survival and Is Dependent on T-Cells

PubMed Central

Martin, Edward N.; Scheld, W. Michael

2014-01-01

Introduction Endotoxin tolerance improves outcomes from gram negative sepsis but the underlying mechanism is not known. We determined if endotoxin tolerance before or after pneumococcal sepsis improved survival and the role of lymphocytes in this protection. Methods Mice received lipopolysaccharide (LPS) or vehicle before or after a lethal dose of Streptococcus pneumoniae. Survival, quantitative bacteriology, liver function, and cytokine concentrations were measured. We confirmed the necessity of Toll-like receptor 4 (TLR4) for endotoxin tolerance using C3H/HeN (TLR4 replete) and C3H/HeJ (TLR4 deficient) mice. The role of complement was investigated through A/J mice deficient in C5 complement. CBA/CaHN-Btkxid//J mice with dysfunctional B cells and Rag-1 knockout (KO) mice deficient in T and B cells delineated the role of lymphocytes. Results Endotoxin tolerance improved survival from pneumococcal sepsis in mice with TLR4 that received LPS pretreatment or posttreatment. Survival was associated with reduced bacterial burden and serum cytokine concentrations. Death was associated with abnormal liver function and blood glucose concentrations. Endotoxin tolerance improved survival in A/J and CBA/CaHN-Btkxid//J mice but not Rag-1 KO mice. Conclusions TLR4 stimulation before or after S. pneumoniae infection improved survival and was dependent on T-cells but did not require an intact complement cascade or functional B cells. PMID:24465843

Exome sequencing and arrayCGH detection of gene sequence and copy number variation between ILS and ISS mouse strains.

PubMed

Dumas, Laura; Dickens, C Michael; Anderson, Nathan; Davis, Jonathan; Bennett, Beth; Radcliffe, Richard A; Sikela, James M

2014-06-01

It has been well documented that genetic factors can influence predisposition to develop alcoholism. While the underlying genomic changes may be of several types, two of the most common and disease associated are copy number variations (CNVs) and sequence alterations of protein coding regions. The goal of this study was to identify CNVs and single-nucleotide polymorphisms that occur in gene coding regions that may play a role in influencing the risk of an individual developing alcoholism. Toward this end, two mouse strains were used that have been selectively bred based on their differential sensitivity to alcohol: the Inbred long sleep (ILS) and Inbred short sleep (ISS) mouse strains. Differences in initial response to alcohol have been linked to risk for alcoholism, and the ILS/ISS strains are used to investigate the genetics of initial sensitivity to alcohol. Array comparative genomic hybridization (arrayCGH) and exome sequencing were conducted to identify CNVs and gene coding sequence differences, respectively, between ILS and ISS mice. Mouse arrayCGH was performed using catalog Agilent 1 × 244 k mouse arrays. Subsequently, exome sequencing was carried out using an Illumina HiSeq 2000 instrument. ArrayCGH detected 74 CNVs that were strain-specific (38 ILS/36 ISS), including several ISS-specific deletions that contained genes implicated in brain function and neurotransmitter release. Among several interesting coding variations detected by exome sequencing was the gain of a premature stop codon in the alpha-amylase 2B (AMY2B) gene specifically in the ILS strain. In total, exome sequencing detected 2,597 and 1,768 strain-specific exonic gene variants in the ILS and ISS mice, respectively. This study represents the most comprehensive and detailed genomic comparison of ILS and ISS mouse strains to date. The two complementary genome-wide approaches identified strain-specific CNVs and gene coding sequence variations that should provide strong candidates to contribute to the alcohol-related phenotypic differences associated with these strains.

HLA-DQA1 and PLA2R1 polymorphisms and risk of idiopathic membranous nephropathy.

PubMed

Bullich, Gemma; Ballarín, José; Oliver, Artur; Ayasreh, Nadia; Silva, Irene; Santín, Sheila; Díaz-Encarnación, Montserrat M; Torra, Roser; Ars, Elisabet

2014-02-01

Single nucleotide polymorphisms (SNPs) within HLA complex class II HLA-DQ α-chain 1 (HLA-DQA1) and M-type phospholipase A2 receptor (PLA2R1) genes were identified as strong risk factors for idiopathic membranous nephropathy (IMN) development in a recent genome-wide association study. Copy number variants (CNVs) within the Fc gamma receptor III (FCGR3) locus have been associated with several autoimmune diseases, but their role in IMN has not been studied. This study aimed to validate the association of HLA-DQA1 and PLA2R1 risk alleles with IMN in a Spanish cohort, test the putative association of FCGR3A and FCGR3B CNVs with IMN, and assess the use of these genetic factors to predict the clinical outcome of the disease. A Spanish cohort of 89 IMN patients and 286 matched controls without nephropathy was recruited between October of 2009 and July of 2012. Case-control studies for SNPs within HLA-DQA1 (rs2187668) and PLA2R1 (rs4664308) genes and CNVs for FCGR3A and FCGR3B genes were performed. The contribution of these polymorphisms to predict clinical outcome and renal function decline was analyzed. This study validated the association of these HLA-DQA1 and PLA2R1 SNPs with IMN in a Spanish cohort and its increased risk when combining both risk genotypes. No significant association was found between FCGR3 CNVs and IMN. These results revealed that HLA-DQA1 and PLA2R1 genotype combination adjusted for baseline proteinuria strongly predicted response to immunosuppressive therapy. HLA-DQA1 genotype adjusted for proteinuria was also linked with renal function decline. This study confirms that HLA-DQA1 and PLA2R1 genotypes are risk factors for IMN, whereas no association was identified for FCGR3 CNVs. This study provides, for the first time, evidence of the contribution of these HLA-DQA1 and PLA2R1 polymorphisms in predicting IMN response to immunosuppressors and disease progression. Future studies are needed to validate and identify prognostic markers.

A modified multiplex ligation-dependent probe amplification method for the detection of 22q11.2 copy number variations in patients with congenital heart disease.

PubMed

Zhang, Xiaoqing; Xu, Yuejuan; Liu, Deyuan; Geng, Juan; Chen, Sun; Jiang, Zhengwen; Fu, Qihua; Sun, Kun

2015-05-08

Copy number variations (CNVs) of chromosomal region 22q11.2 are associated with a subset of patients with congenital heart disease (CHD). Accurate and efficient detection of CNV is important for genetic analysis of CHD. The aim of the study was to introduce a novel approach named CNVplex®, a high-throughput analysis technique designed for efficient detection of chromosomal CNVs, and to explore the prevalence of sub-chromosomal imbalances in 22q11.2 loci in patients with CHD from a single institute. We developed a novel technique, CNVplex®, for high-throughput detection of sub-chromosomal copy number aberrations. Modified from the multiplex ligation-dependent probe amplification (MLPA) method, it introduced a lengthening ligation system and four universal primer sets, which simplified the synthesis of probes and significantly improved the flexibility of the experiment. We used 110 samples, which were extensively characterized with chromosomal microarray analysis and MLPA, to validate the performance of the newly developed method. Furthermore, CNVplex® was used to screen for sub-chromosomal imbalances in 22q11.2 loci in 818 CHD patients consecutively enrolled from Shanghai Children's Medical Center. In the methodology development phase, CNVplex® detected all copy number aberrations that were previously identified with both chromosomal microarray analysis and MLPA, demonstrating 100% sensitivity and specificity. In the validation phase, 22q11.2 deletion and 22q11.2 duplication were detected in 39 and 1 of 818 patients with CHD by CNVplex®, respectively. Our data demonstrated that the frequency of 22q11.2 deletion varied among sub-groups of CHD patients. Notably, 22q11.2 deletion was more commonly observed in cases with conotruncal defect (CTD) than in cases with non-CTD (P<0.001). With higher resolution and more probes against selected chromosomal loci, CNVplex® also identified several individuals with small CNVs and alterations in other chromosomes. CNVplex® is sensitive and specific in its detection of CNVs, and it is an alternative to MLPA for batch screening of pathogenetic CNVs in known genomic loci.

Cluster Analysis Identifies Distinct Pathogenetic Patterns in C3 Glomerulopathies/Immune Complex-Mediated Membranoproliferative GN.

PubMed

Iatropoulos, Paraskevas; Daina, Erica; Curreri, Manuela; Piras, Rossella; Valoti, Elisabetta; Mele, Caterina; Bresin, Elena; Gamba, Sara; Alberti, Marta; Breno, Matteo; Perna, Annalisa; Bettoni, Serena; Sabadini, Ettore; Murer, Luisa; Vivarelli, Marina; Noris, Marina; Remuzzi, Giuseppe

2018-01-01

Membranoproliferative GN (MPGN) was recently reclassified as alternative pathway complement-mediated C3 glomerulopathy (C3G) and immune complex-mediated membranoproliferative GN (IC-MPGN). However, genetic and acquired alternative pathway abnormalities are also observed in IC-MPGN. Here, we explored the presence of distinct disease entities characterized by specific pathophysiologic mechanisms. We performed unsupervised hierarchical clustering, a data-driven statistical approach, on histologic, genetic, and clinical data and data regarding serum/plasma complement parameters from 173 patients with C3G/IC-MPGN. This approach divided patients into four clusters, indicating the existence of four different pathogenetic patterns. Specifically, this analysis separated patients with fluid-phase complement activation (clusters 1-3) who had low serum C3 levels and a high prevalence of genetic and acquired alternative pathway abnormalities from patients with solid-phase complement activation (cluster 4) who had normal or mildly altered serum C3, late disease onset, and poor renal survival. In patients with fluid-phase complement activation, those in clusters 1 and 2 had massive activation of the alternative pathway, including activation of the terminal pathway, and the highest prevalence of subendothelial deposits, but those in cluster 2 had additional activation of the classic pathway and the highest prevalence of nephrotic syndrome at disease onset. Patients in cluster 3 had prevalent activation of C3 convertase and highly electron-dense intramembranous deposits. In addition, we provide a simple algorithm to assign patients with C3G/IC-MPGN to specific clusters. These distinct clusters may facilitate clarification of disease etiology, improve risk assessment for ESRD, and pave the way for personalized treatment. Copyright © 2018 by the American Society of Nephrology.

Proteomic analysis in peritoneal dialysis patients with different peritoneal transport characteristics.

PubMed

Wen, Qiong; Zhang, Li; Mao, Hai-Ping; Tang, Xue-Qing; Rong, Rong; Fan, Jin-Jin; Yu, Xue-Qing

2013-08-30

Peritoneal membranes can be categorized as high, high average, low average, and low transporters, based on the removal or transport rate of solutes. In this study, we used proteomic analysis to determine the differences in proteins removed by different types of peritoneal membranes. Peritoneal transport characteristics in patients who received peritoneal dialysis therapy were assessed by a peritoneal equilibration test. Two-dimensional differential gel electrophoresis technology followed by quantitative analysis was performed to study the variation in protein expression from peritoneal dialysis effluents (PDE) among different groups. Proteins were identified by MALDI-TOF-MS/MS analyses. Further validation in PDE or serum was performed utilizing ELISA analysis. Proteomics analysis revealed ten protein spots with significant differences in intensity levels among different groups, including vitamin D-binding protein, complement C3, apolipoprotein-A1, complement factor C4A, haptoglobin, alpha-1 antitrypsin, immunoglobulin kappa light chain, alpha-2-microglobulin, retinol-binding protein 4 and transthyretin. The levels of vitamin D-binding protein, complement C3, and apolipoprotein-A1 in PDE derived from different groups were greatly varied (P<0.05). However, no significant difference was found in the serum levels of these proteins among different groups (P>0.05 for all groups). This study provides a novel overview of the differences in PDE proteomes of four types of peritoneal membranes. Vitamin D-binding protein, complement C3, and apolipoprotein-A1 showed enhanced expression in PDE of patients with high transporter. Copyright © 2013 Elsevier Inc. All rights reserved.

Combined array CGH plus SNP genome analyses in a single assay for optimized clinical testing

PubMed Central

Wiszniewska, Joanna; Bi, Weimin; Shaw, Chad; Stankiewicz, Pawel; Kang, Sung-Hae L; Pursley, Amber N; Lalani, Seema; Hixson, Patricia; Gambin, Tomasz; Tsai, Chun-hui; Bock, Hans-Georg; Descartes, Maria; Probst, Frank J; Scaglia, Fernando; Beaudet, Arthur L; Lupski, James R; Eng, Christine; Wai Cheung, Sau; Bacino, Carlos; Patel, Ankita

2014-01-01

In clinical diagnostics, both array comparative genomic hybridization (array CGH) and single nucleotide polymorphism (SNP) genotyping have proven to be powerful genomic technologies utilized for the evaluation of developmental delay, multiple congenital anomalies, and neuropsychiatric disorders. Differences in the ability to resolve genomic changes between these arrays may constitute an implementation challenge for clinicians: which platform (SNP vs array CGH) might best detect the underlying genetic cause for the disease in the patient? While only SNP arrays enable the detection of copy number neutral regions of absence of heterozygosity (AOH), they have limited ability to detect single-exon copy number variants (CNVs) due to the distribution of SNPs across the genome. To provide comprehensive clinical testing for both CNVs and copy-neutral AOH, we enhanced our custom-designed high-resolution oligonucleotide array that has exon-targeted coverage of 1860 genes with 60 000 SNP probes, referred to as Chromosomal Microarray Analysis – Comprehensive (CMA-COMP). Of the 3240 cases evaluated by this array, clinically significant CNVs were detected in 445 cases including 21 cases with exonic events. In addition, 162 cases (5.0%) showed at least one AOH region >10 Mb. We demonstrate that even though this array has a lower density of SNP probes than other commercially available SNP arrays, it reliably detected AOH events >10 Mb as well as exonic CNVs beyond the detection limitations of SNP genotyping. Thus, combining SNP probes and exon-targeted array CGH into one platform provides clinically useful genetic screening in an efficient manner. PMID:23695279

Analysis of genome-wide copy number variations in Chinese indigenous and western pig breeds by 60 K SNP genotyping arrays.

PubMed

Wang, Yanan; Tang, Zhonglin; Sun, Yaqi; Wang, Hongyang; Wang, Chao; Yu, Shaobo; Liu, Jing; Zhang, Yu; Fan, Bin; Li, Kui; Liu, Bang

2014-01-01

Copy number variations (CNVs) represent a substantial source of structural variants in mammals and contribute to both normal phenotypic variability and disease susceptibility. Although low-resolution CNV maps are produced in many domestic animals, and several reports have been published about the CNVs of porcine genome, the differences between Chinese and western pigs still remain to be elucidated. In this study, we used Porcine SNP60 BeadChip and PennCNV algorithm to perform a genome-wide CNV detection in 302 individuals from six Chinese indigenous breeds (Tongcheng, Laiwu, Luchuan, Bama, Wuzhishan and Ningxiang pigs), three western breeds (Yorkshire, Landrace and Duroc) and one hybrid (Tongcheng×Duroc). A total of 348 CNV Regions (CNVRs) across genome were identified, covering 150.49 Mb of the pig genome or 6.14% of the autosomal genome sequence. In these CNVRs, 213 CNVRs were found to exist only in the six Chinese indigenous breeds, and 60 CNVRs only in the three western breeds. The characters of CNVs in four Chinese normal size breeds (Luchuan, Tongcheng and Laiwu pigs) and two minipig breeds (Bama and Wuzhishan pigs) were also analyzed in this study. Functional annotation suggested that these CNVRs possess a great variety of molecular function and may play important roles in phenotypic and production traits between Chinese and western breeds. Our results are important complementary to the CNV map in pig genome, which provide new information about the diversity of Chinese and western pig breeds, and facilitate further research on porcine genome CNVs.

Analysis of Genome-Wide Copy Number Variations in Chinese Indigenous and Western Pig Breeds by 60 K SNP Genotyping Arrays

PubMed Central

Sun, Yaqi; Wang, Hongyang; Wang, Chao; Yu, Shaobo; Liu, Jing; Zhang, Yu; Fan, Bin; Li, Kui; Liu, Bang

2014-01-01

Copy number variations (CNVs) represent a substantial source of structural variants in mammals and contribute to both normal phenotypic variability and disease susceptibility. Although low-resolution CNV maps are produced in many domestic animals, and several reports have been published about the CNVs of porcine genome, the differences between Chinese and western pigs still remain to be elucidated. In this study, we used Porcine SNP60 BeadChip and PennCNV algorithm to perform a genome-wide CNV detection in 302 individuals from six Chinese indigenous breeds (Tongcheng, Laiwu, Luchuan, Bama, Wuzhishan and Ningxiang pigs), three western breeds (Yorkshire, Landrace and Duroc) and one hybrid (Tongcheng×Duroc). A total of 348 CNV Regions (CNVRs) across genome were identified, covering 150.49 Mb of the pig genome or 6.14% of the autosomal genome sequence. In these CNVRs, 213 CNVRs were found to exist only in the six Chinese indigenous breeds, and 60 CNVRs only in the three western breeds. The characters of CNVs in four Chinese normal size breeds (Luchuan, Tongcheng and Laiwu pigs) and two minipig breeds (Bama and Wuzhishan pigs) were also analyzed in this study. Functional annotation suggested that these CNVRs possess a great variety of molecular function and may play important roles in phenotypic and production traits between Chinese and western breeds. Our results are important complementary to the CNV map in pig genome, which provide new information about the diversity of Chinese and western pig breeds, and facilitate further research on porcine genome CNVs. PMID:25198154

NAHR-mediated copy-number variants in a clinical population: mechanistic insights into both genomic disorders and Mendelizing traits.

PubMed

Dittwald, Piotr; Gambin, Tomasz; Szafranski, Przemyslaw; Li, Jian; Amato, Stephen; Divon, Michael Y; Rodríguez Rojas, Lisa Ximena; Elton, Lindsay E; Scott, Daryl A; Schaaf, Christian P; Torres-Martinez, Wilfredo; Stevens, Abby K; Rosenfeld, Jill A; Agadi, Satish; Francis, David; Kang, Sung-Hae L; Breman, Amy; Lalani, Seema R; Bacino, Carlos A; Bi, Weimin; Milosavljevic, Aleksandar; Beaudet, Arthur L; Patel, Ankita; Shaw, Chad A; Lupski, James R; Gambin, Anna; Cheung, Sau Wai; Stankiewicz, Pawel

2013-09-01

We delineated and analyzed directly oriented paralogous low-copy repeats (DP-LCRs) in the most recent version of the human haploid reference genome. The computationally defined DP-LCRs were cross-referenced with our chromosomal microarray analysis (CMA) database of 25,144 patients subjected to genome-wide assays. This computationally guided approach to the empirically derived large data set allowed us to investigate genomic rearrangement relative frequencies and identify new loci for recurrent nonallelic homologous recombination (NAHR)-mediated copy-number variants (CNVs). The most commonly observed recurrent CNVs were NPHP1 duplications (233), CHRNA7 duplications (175), and 22q11.21 deletions (DiGeorge/velocardiofacial syndrome, 166). In the ∼25% of CMA cases for which parental studies were available, we identified 190 de novo recurrent CNVs. In this group, the most frequently observed events were deletions of 22q11.21 (48), 16p11.2 (autism, 34), and 7q11.23 (Williams-Beuren syndrome, 11). Several features of DP-LCRs, including length, distance between NAHR substrate elements, DNA sequence identity (fraction matching), GC content, and concentration of the homologous recombination (HR) hot spot motif 5'-CCNCCNTNNCCNC-3', correlate with the frequencies of the recurrent CNVs events. Four novel adjacent DP-LCR-flanked and NAHR-prone regions, involving 2q12.2q13, were elucidated in association with novel genomic disorders. Our study quantitates genome architectural features responsible for NAHR-mediated genomic instability and further elucidates the role of NAHR in human disease.

Detection of genome-wide copy number variants in myeloid malignancies using next-generation sequencing.

PubMed

Shen, Wei; Paxton, Christian N; Szankasi, Philippe; Longhurst, Maria; Schumacher, Jonathan A; Frizzell, Kimberly A; Sorrells, Shelly M; Clayton, Adam L; Jattani, Rakhi P; Patel, Jay L; Toydemir, Reha; Kelley, Todd W; Xu, Xinjie

2018-04-01

Genetic abnormalities, including copy number variants (CNV), copy number neutral loss of heterozygosity (CN-LOH) and gene mutations, underlie the pathogenesis of myeloid malignancies and serve as important diagnostic, prognostic and/or therapeutic markers. Currently, multiple testing strategies are required for comprehensive genetic testing in myeloid malignancies. The aim of this proof-of-principle study was to investigate the feasibility of combining detection of genome-wide large CNVs, CN-LOH and targeted gene mutations into a single assay using next-generation sequencing (NGS). For genome-wide CNV detection, we designed a single nucleotide polymorphism (SNP) sequencing backbone with 22 762 SNP regions evenly distributed across the entire genome. For targeted mutation detection, 62 frequently mutated genes in myeloid malignancies were targeted. We combined this SNP sequencing backbone with a targeted mutation panel, and sequenced 9 healthy individuals and 16 patients with myeloid malignancies using NGS. We detected 52 somatic CNVs, 11 instances of CN-LOH and 39 oncogenic mutations in the 16 patients with myeloid malignancies, and none in the 9 healthy individuals. All CNVs and CN-LOH were confirmed by SNP microarray analysis. We describe a genome-wide SNP sequencing backbone which allows for sensitive detection of genome-wide CNVs and CN-LOH using NGS. This proof-of-principle study has demonstrated that this strategy can provide more comprehensive genetic profiling for patients with myeloid malignancies using a single assay. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Intratumor heterogeneity predicts metastasis of triple-negative breast cancer.

PubMed

Yang, Fang; Wang, Yucai; Li, Quan; Cao, Lulu; Sun, Zijia; Jin, Juan; Fang, Hehui; Zhu, Aiyu; Li, Yan; Zhang, Wenwen; Wang, Yanru; Xie, Hui; Gustafsson, Jan-Åke; Wang, Shui; Guan, Xiaoxiang

2017-09-01

Even with the identical clinicopathological features, the ability for metastasis is vastly different among triple-negative breast cancer (TNBC) patients. Intratumor heterogeneity (ITH), which is common in breast cancer, may be a key mechanism leading to the tumor progression. In this study, we studied whether a quantitative genetic definition of ITH can predict clinical outcomes in patients with TNBC. We quantified ITH by calculating Shannon index, a measure of diversity in a population, based on Myc, epidermal growth factor receptor/centromeric probe 7 (EGFR/CEP7) and cyclin D1/centromeric probe 11 (CCND1/CEP11) copy number variations (CNVs) in 300 cells at three different locations of a tumor. Among 75 TNBC patients, those who developed metastasis had significantly higher ITH, that is Shannon indices of EGFR/CEP7 and CCND1/CEP11 CNVs. Higher Shannon indices of EGFR/CEP7 and CCND1/CEP11 CNVs were significantly associated with the development of metastasis and were predictive of significantly worse metastasis-free survival (MFS). Regional heterogeneity, defined as the difference in copy numbers of Myc, EGFR or CCND1 at different locations, was found in 52 patients. However, the presence of regional heterogeneity did not correlate with metastasis or MFS. Our findings demonstrate that higher ITH of EGFR/CEP7 and CCND1/CEP11 CNVs is predictive of metastasis and is associated with significantly worse MFS in TNBC patients, suggesting that ITH is a very promising novel prognostic factor in TNBC. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Rare copy number variants in neuropsychiatric disorders: Specific phenotype or not?

PubMed

Van Den Bossche, Maarten J; Johnstone, Mandy; Strazisar, Mojca; Pickard, Benjamin S; Goossens, Dirk; Lenaerts, An-Sofie; De Zutter, Sonia; Nordin, Annelie; Norrback, Karl-Fredrik; Mendlewicz, Julien; Souery, Daniel; De Rijk, Peter; Sabbe, Bernard G; Adolfsson, Rolf; Blackwood, Douglas; Del-Favero, Jurgen

2012-10-01

From a number of genome-wide association studies it was shown that de novo and/or rare copy number variants (CNVs) are found at an increased frequency in neuropsychiatric diseases. In this study we examined the prevalence of CNVs in six genomic regions (1q21.1, 2p16.3, 3q29, 15q11.2, 15q13.3, and 16p11.2) previously implicated in neuropsychiatric diseases. Hereto, a cohort of four neuropsychiatric disorders (schizophrenia, bipolar disorder, major depressive disorder, and intellectual disability) and control individuals from three different populations was used in combination with Multilpex Amplicon Quantifiaction (MAQ) assays, capable of high resolution (kb range) and custom-tailored CNV detection. Our results confirm the etiological candidacy of the six selected CNV regions for neuropsychiatric diseases. It is possible that CNVs in these regions can result in disturbed brain development and in this way lead to an increased susceptibility for different neuropsychiatric disorders, dependent on additional genetic and environmental factors. Our results also suggest that the neurodevelopmental component is larger in the etiology of schizophrenia and intellectual disability than in mood disorders. Finally, our data suggest that deletions are in general more pathogenic than duplications. Given the high frequency of the examined CNVs (1-2%) in patients of different neuropsychiatric disorders, screening of large cohorts with an affordable and feasible method like the MAQ assays used in this study is likely to result in important progress in unraveling the genetic factors leading to an increased susceptibility for several psychiatric disorders. 2012 Wiley Periodicals, Inc

Interallelic complementation of mutations in propionic acidemia by microinjection of mutant cDNAs into fibroblasts of affected patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Loyer, M.; Leclerc, D.; Gravel, R.A.

1994-09-01

Propionic acidemia is a rare autosomal recessive disorder resulting from defects of the {alpha} or {beta} subunit of biotin-dependent propionyl-CoA carboxylase (PCC). Mutations are assigned to defects of the PCCA ({alpha} subunit) or PCCB ({beta} subunit) gene through complementation studies after somatic fusion of patient cell lines. About two-thirds of patients with {beta} subunit defects (complementation group pccBC) show interallelic complementation in cell fusion experiments (subgroups pccB and pccC), monitored by the PCC-dependent metabolisms of {sup 14}C-propionate. Most patient cell lines are heteroallelic for two different mutations, leaving ambiguous the identity of the mutation participating in interallelic complementation. To identifymore » the complementing mutations, we have expressed {beta}-subunit cDNAs containing individual mutations by microinjection of the cDNAs in recipient cells from patients with {beta} subunit defects. Correction of the PCC defect was monitored by autoradiography of {sup 14}C-propionate incorporation. In some experiments, cDNAs were co-injected with a plasmid expressing the E. coli lacZ gene as a positive control for successful injection. Two mutations from the pccB subgroup showed complementation when injected into pccC cells; dupKICK140-143 and Pro228Leu. Similarly, two mutations from the pccC subgroup complemented after injection into pccB cells; {Delta}Ile408 and Arg410Trp. No mutation complemented with mutation of the pccBC group which are classified as non-complementing in cell fusion experiments. The results show that the complementing pccB mutations are found in the N-terminal half of the {beta} subunit, while the complementing pccC mutations cluxter at a site in the C-terminal half. The latter site is a candidate for the propionyl-CoA binding site based on sequence identity with a region of transcarboxylase from Propionibacterium shermanii.« less

Validation of a multi-analyte panel with cell-bound complement activation products for systemic lupus erythematosus.

PubMed

Dervieux, Thierry; Conklin, John; Ligayon, Jo-Anne; Wolover, Leilani; O'Malley, Tyler; Alexander, Roberta Vezza; Weinstein, Arthur; Ibarra, Claudia A

2017-07-01

We describe the analytical validation of an assay panel intended to assist clinicians with the diagnosis of systemic lupus erythematosus (SLE). The multi-analyte panel includes quantitative assessment of complement activation and measurement of autoantibodies. The levels of the complement split product C4d bound to erythrocytes (EC4d) and B-lymphocytes (BC4d) (expressed as mean fluorescence intensity [MFI]) are measured by quantitative flow cytometry, while autoantibodies (inclusive of antinuclear and anti-double stranded DNA antibodies) are determined by immunoassays. Results of the multi-analyte panel are reported as positive or negative based on a 2-tiered index score. Post-phlebotomy stability of EC4d and BC4d in EDTA-anticoagulated blood is determined using specimens collected from patients with SLE and normal donors. Three-level C4 coated positive beads are run daily as controls. Analytical validity is reported using intra-day and inter-day coefficient of variation (CV). EC4d and BC4d are stable for 2days at ambient temperature and for 4days at 4°C post-phlebotomy. Median intra-day and inter-day CV range from 2.9% to 7.8% (n=30) and 7.3% to 12.4% (n=66), respectively. The 2-tiered index score is reproducible over 4 consecutive daysupon storage of blood at 4°C. A total of 2,888 three-level quality control data were collected from 6 flow cytometers with an overall failure rate below 3%. Median EC4d level is 6 net MFI (Interquartile [IQ] range 4-9 net MFI) and median BC4d is 18 net MFI (IQ range 13-27 net MFI) among 86,852 specimens submitted for testing. The incidence of 2-tiered positive test results is 13.4%. We have established the analytical validity of a multi-analyte assay panel for SLE. Copyright © 2017 Elsevier B.V. All rights reserved.

Streptococcus pneumoniae PspC Subgroup Prevalence in Invasive Disease and Differences in Contribution to Complement Evasion.

PubMed

van der Maten, Erika; van den Broek, Bryan; de Jonge, Marien I; Rensen, Kim J W; Eleveld, Marc J; Zomer, Aldert L; Cremers, Amelieke J H; Ferwerda, Gerben; de Groot, Ronald; Langereis, Jeroen D; van der Flier, Michiel

2018-04-01

The pneumococcal capsular serotype is an important determinant of complement resistance and invasive disease potential, but other virulence factors have also been found to contribute. Pneumococcal surface protein C (PspC), a highly variable virulence protein that binds complement factor H to evade C3 opsonization, is divided into two subgroups: choline-bound subgroup I and LPxTG-anchored subgroup II. The prevalence of different PspC subgroups in invasive pneumococcal disease (IPD) and functional differences in complement evasion are unknown. The prevalence of PspC subgroups in IPD isolates was determined in a collection of 349 sequenced strains of Streptococcus pneumoniae isolated from adult patients. pspC deletion mutants and isogenic pspC switch mutants were constructed to study differences in factor H binding and complement evasion in relation to capsule thickness. Subgroup I pspC was far more prevalent in IPD isolates than subgroup II pspC The presence of capsule was associated with a greater ability of bound factor H to reduce complement opsonization. Pneumococcal subgroup I PspC bound significantly more factor H and showed more effective complement evasion than subgroup II PspC in isogenic encapsulated pneumococci. We conclude that variation in the PspC subgroups, independent of capsule serotypes, affects pneumococcal factor H binding and its ability to evade complement deposition. Copyright © 2018 American Society for Microbiology.

Identification of C1q as a Binding Protein for Advanced Glycation End Products.

PubMed

Chikazawa, Miho; Shibata, Takahiro; Hatasa, Yukinori; Hirose, Sayumi; Otaki, Natsuki; Nakashima, Fumie; Ito, Mika; Machida, Sachiko; Maruyama, Shoichi; Uchida, Koji

2016-01-26

Advanced glycation end products (AGEs) make up a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with the free amino groups of proteins. The abundance of AGEs in a variety of age-related diseases, including diabetic complications and atherosclerosis, and their pathophysiological effects suggest the existence of innate defense mechanisms. Here we examined the presence of serum proteins that are capable of binding glycated bovine serum albumin (AGEs-BSA), prepared upon incubation of BSA with dehydroascorbate, and identified complement component C1q subcomponent subunit A as a novel AGE-binding protein in human serum. A molecular interaction analysis showed the specific binding of C1q to the AGEs-BSA. In addition, we identified DNA-binding regions of C1q, including a collagen-like domain, as the AGE-binding site and established that the amount of positive charge on the binding site was the determining factor. C1q indeed recognized several other modified proteins, including acylated proteins, suggesting that the binding specificity of C1q might be ascribed, at least in part, to the electronegative potential of the ligand proteins. We also observed that C1q was involved in the AGEs-BSA-activated deposition of complement proteins, C3b and C4b. In addition, the AGEs-BSA mediated the proteolytic cleavage of complement protein 5 to release C5a. These findings provide the first evidence of AGEs as a new ligand recognized by C1q, stimulating the C1q-dependent classical complement pathway.

Kikuchi-Fujimoto disease (histiocytic necrotizing lymphadenitis): report of a case with other autoimmune manifestations

PubMed Central

Merriman, Richard C.; Stone, Marvin J.

2007-01-01

Kikuchi-Fujimoto disease (KFD), or histiocytic necrotizing lymphadenitis, is a benign and self-limited disease that mainly affects young women. Patients present with localized lymphadenopathy, fever, and leukopenia in up to half of the cases. KFD can occur in association with systemic lupus erythematosus. We present the case of a patient with KFD and systemic lupus erythematosus, as well as relapsing polychondritis. This patient had persistently low C4 complement levels, so she was evaluated for a genetic defect in complement production and was found to have two “null” C4 alleles. We believe that this may have contributed to the development of her diseases. PMID:17431451

Association between anti-beta2 glycoprotein I antibodies and renal glomerular C4d deposition in lupus nephritis patients with glomerular microthrombosis: a prospective study of 155 cases.

PubMed

Shen, Y; Chen, X-W; Sun, C-Y; Dai, M; Yan, Y-C; Yang, C-D

2010-09-01

Glomerular microthrombosis (GMT) is a common vascular change in patients with lupus nephritis (LN). The mechanism underlying GMT is still unknown. In our previous study, we found that the level of IgG anti-beta2 glycoprotein I (beta2GPI) antibodies was higher in the LN-GMT group than in the LN-non-GMT group, which indicated that anti-beta2GPI antibodies may play a role in GMT formation. Many studies have demonstrated that the activation of the classical complement pathway may play a critical role in fetal loss and aPL-induced thrombosis formation. To investigate whether complement activation plays a role in GMT formation and to evaluate its relationship with aPL, we prospectively investigated deposition of C4d in 155 renal biopsy specimens of LN patients. The results revealed a strong relationship between the intensity of glomerular C4d staining and the presence of microthrombi (p < 0.001). The detection rate of IgG anti-beta2GPI antibodies was higher in the LN-GMT group than in the LN-non-GMT group (p < 0.05). Further, the intensity of glomerular C4d staining was significantly related with IgG anti-beta2GPI antibodies (p < 0.05). The results of our study suggest that anti-beta2GPI antibodies may play a role in GMT formation, and this process might involve complement activation.

Complement Depletion Protects Lupus-prone Mice from Ischemia-reperfusion-initiated Organ Injury

DTIC Science & Technology

2012-10-25

injury, we sought to evaluate whether complement inhibition mitigates organ damage. We found that complement deple- tion with cobra venom factor... venom factor and C5a receptor antagonist were able to protect mice from local tissue damage, treatment with C5a receptor antagonist was not able to...Complement depletion or blockage of the complement pathway using molecules such as cobra venom factor (CVF) (24, 33) and C5a receptor antagonists (C5aRA

Homologous species restriction of the complement-mediated killing of nucleated cells.

PubMed Central

Yamamoto, H; Blaas, P; Nicholson-Weller, A; Hänsch, G M

1990-01-01

The homologous restriction of complement (C) lysis is attributed to membrane proteins: decay-accelerating factor (DAF), C8 binding protein (C8bp) and P18/CD59. Since these proteins are also expressed on peripheral blood cells, species restriction was tested for in the complement-mediated killing of antibody-coated human leucocytes by human or rabbit complement. Killing was more efficient when rabbit complement was used. Preincubation of cells with an antibody to DAF abolished the difference. When C1-7 sites were first attached to the cells and either rabbit or human C8, C9 were added, the killing of monocytes and lymphocytes was equally efficient; only in polymorphonuclear neutrophils was a higher efficiency of rabbit C8, C9 seen. Thus, in contrast to haemolysis, restriction occurred predominantly at the C3 level and the action of the terminal complement components was not inhibited. Since C8bp isolated from peripheral blood cells showed essentially similar characteristics as the erythrocyte-derived C8bp, the failure of C8bp to inhibit the action of the terminal components on nucleated cells might reflect differences of the complement membrane interactions between erythrocytes or nucleated cells, respectively. Images Figure 5 PMID:1697561

21 CFR 866.5260 - Complement C3b inactivator immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5260 Complement C3b inactivator immunological test system. (a) Identification. A complement... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Complement C3b inactivator immunological test...

The C8-binding protein of human erythrocytes: interaction with the components of the complement-attack phase.

PubMed Central

Schönermark, S; Filsinger, S; Berger, B; Hänsch, G M

1988-01-01

C8-binding protein is an intrinsic membrane protein of the human erythrocyte. It inhibits the complement (C5b-9)-mediated lysis in a species-restricting manner. In the present study we incorporated C8bp, isolated from human erythrocytes, into sheep erythrocytes (SRBC). SRBC, normally sensitive to lysis by human C5b-9, became insensitive to lysis. Furthermore, we found that C8bp is incorporated into the membrane-attack complex C5b-9, most probably by interacting with C8, since C8bp has an affinity for C8, particularly for the C8 alpha-gamma-subunit. Antibodies to C8bp react with the C8 alpha-subunits and with C9, pointing to the possibility of a partial homology between these proteins. Images Figure 4 Figure 6 Figure 7 PMID:3366469

Synergy between the classical and alternative pathways of complement is essential for conferring effective protection against the pandemic influenza A(H1N1) 2009 virus infection

PubMed Central

Rattan, Ajitanuj; Pawar, Shailesh D.; Nawadkar, Renuka; Kulkarni, Neeraja

2017-01-01

The pandemic influenza A(H1N1) 2009 virus caused significant morbidity and mortality worldwide thus necessitating the need to understand the host factors that influence its control. Previously, the complement system has been shown to provide protection during the seasonal influenza virus infection, however, the role of individual complement pathways is not yet clear. Here, we have dissected the role of intact complement as well as of its individual activation pathways during the pandemic influenza virus infection using mouse strains deficient in various complement components. We show that the virus infection in C3-/- mice results in increased viral load and 100% mortality, which can be reversed by adoptive transfer of naïve wild-type (WT) splenocytes, purified splenic B cells, or passive transfer of immune sera from WT, but not C3-/- mice. Blocking of C3a and/or C5a receptor signaling in WT mice using receptor antagonists and use of C3aR-/- and C5aR-/- mice showed significant mortality after blocking/ablation of C3aR, with little or no effect after blocking/ablation of C5aR. Intriguingly, deficiency of C4 and FB in mice resulted in only partial mortality (24%-32%) suggesting a necessary cross-talk between the classical/lectin and alternative pathways for providing effective protection. In vitro virus neutralization experiments performed to probe the cross-talk between the various pathways indicated that activation of the classical and alternative pathways in concert, owing to coating of viral surface by antibodies, is needed for its efficient neutralization. Examination of the virus-specific complement-binding antibodies in virus positive subjects showed that their levels vary among individuals. Together these results indicate that cooperation between the classical and alternative pathways not only result in efficient direct neutralization of the pandemic influenza virus, but also lead to the optimum generation of C3a, which when sensed by the immune cells along with the antigen culminates in generation of effective protective immune responses. PMID:28301559

Identification of copy number variations and translocations in cancer cells from Hi-C data.

PubMed

Chakraborty, Abhijit; Ay, Ferhat

2017-10-18

Eukaryotic chromosomes adapt a complex and highly dynamic three-dimensional (3D) structure, which profoundly affects different cellular functions and outcomes including changes in epigenetic landscape and in gene expression. Making the scenario even more complex, cancer cells harbor chromosomal abnormalities (e.g., copy number variations (CNVs) and translocations) altering their genomes both at the sequence level and at the level of 3D organization. High-throughput chromosome conformation capture techniques (e.g., Hi-C), which are originally developed for decoding the 3D structure of the chromatin, provide a great opportunity to simultaneously identify the locations of genomic rearrangements and to investigate the 3D genome organization in cancer cells. Even though Hi-C data has been used for validating known rearrangements, computational methods that can distinguish rearrangement signals from the inherent biases of Hi-C data and from the actual 3D conformation of chromatin, and can precisely detect rearrangement locations de novo have been missing. In this work, we characterize how intra and inter-chromosomal Hi-C contacts are distributed for normal and rearranged chromosomes to devise a new set of algorithms (i) to identify genomic segments that correspond to CNV regions such as amplifications and deletions (HiCnv), (Nurtdinov et al.) to call inter-chromosomal translocations and their boundaries (HiCtrans) from Hi-C experiments, and (iii) to simulate Hi-C data from genomes with desired rearrangements and abnormalities (AveSim) in order to select optimal parameters for and to benchmark the accuracy of our methods. Our results on 10 different cancer cell lines with Hi-C data show that we identify a total number of 105 amplifications and 45 deletions together with 90 translocations, whereas we identify virtually no such events for two karyotypically normal cell lines. Our CNV predictions correlate very well with whole genome sequencing (WGS) data among chromosomes with CNV events for a breast cancer cell line (r=0.89) and capture most of the CNVs we simulate using Avesim. For HiCtrans predictions, we report evidence from the literature for 30 out of 90 translocations for eight of our cancer cell lines. Furthermore, we show that our tools identify and correctly classify relatively understudied rearrangements such as double minutes (DMs) and homogeneously staining regions (HSRs). Considering the inherent limitations of existing techniques for karyotyping (i.e., missing balanced rearrangements and those near repetitive regions), the accurate identification of CNVs and translocations in a cost-effective and high-throughput setting is still a challenge. Our results show that the set of tools we develop effectively utilize moderately sequenced Hi-C libraries (100-300 million reads) to identify known and de novo chromosomal rearrangements/abnormalities in well-established cancer cell lines. With the decrease in required number of cells and the increase in attainable resolution, we believe that our framework will pave the way towards comprehensive mapping of genomic rearrangements in primary cells from cancer patients using Hi-C. CNV calling: https://github.com/ay-lab/HiCnvTranslocation calling: https://github.com/ay-lab/HiCtransHi-C simulation: https://github.com/ay-lab/AveSim. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Molecular characterization of the alpha subunit of complement component C8 (GcC8alpha) in the nurse shark (Ginglymostoma cirratum).

PubMed

Aybar, Lydia; Shin, Dong-Ho; Smith, Sylvia L

2009-09-01

Target cell lysis by complement is achieved by the assembly and insertion of the membrane attack complex (MAC) composed of glycoproteins C5b through C9. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. Mammalian C8 is composed of alpha, beta, and gamma subunits. The subunit structure of shark C8 is not known. This report describes a 2341 nucleotide sequence that translates into a polypeptide of 589 amino acid residues, orthologue to mammalian C8alpha and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8gamma-binding cysteine is conserved in the perforin-like domain. Hydrophobicity profile indicates the presence of hydrophobic residues essential for membrane insertion. It shares 41.1% and 47.4% identity with human and Xenopus C8alpha respectively. Southern blot analysis showed GcC8alpha exists as a single copy gene expressed in most tissues except the spleen with the liver being the main site of synthesis. Phylogenetic analysis places it in a clade with C8alpha orthologs and as a sister taxa to the Xenopus. 2009 Elsevier Ltd.

A non-retinoid antagonist of Retinol-Binding Protein 4 rescues phenotype in a model of Stargardt disease without inhibiting the visual cycle.

PubMed

Racz, Boglarka; Varadi, Andras; Kong, Jian; Allikmets, Rando; Pearson, Paul G; Johnson, Graham; Cioffi, Christopher L; Petrukhin, Konstantin

2018-06-05

A primary pathological defect in the heritable eye disorder Stargardt disease is excessive accumulation of cytotoxic lipofuscin bisretinoids in the retina. Age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) matches the age-dependent increase in the incidence of the atrophic (dry) form of age-related macular degeneration (AMD) and therefore may be one of several pathogenic factors contributing to AMD progression. Lipofuscin bisretinoid synthesis in the retina depends on the influx of serum retinol from the circulation into the RPE. Formation of the tertiary retinol-binding protein 4 (RBP4)-transthyretin-retinol complex in the serum is required for this influx. Herein, we report the pharmacological effects of the non-retinoid RBP4 antagonist, BPN-14136. BPN-14136 dosing in the Abca4-/- mouse model of increased lipofuscinogenesis significantly reduced serum RBP4 levels and inhibited bisretinoid synthesis, and this inhibition correlated with a partial reduction in visual cycle retinoids such as retinaldehydes serving as bisretinoid precursors. BPN-14136 administration at doses inducing maximal serum RBP4 reduction did not produce changes in the rate of the visual cycle, consistent with minimal changes in dark adaptation. Abca4-/- mice exhibited dysregulation of the complement system in the retina, and BPN-14136 administration normalized the retinal levels of proinflammatory complement cascade components such as complement factors D and H, C-reactive protein, and C3. We conclude that BPN-14136 has several beneficial characteristics, combining inhibition of bisretinoid synthesis and reduction in retinaldehydes with normalization of the retinal complement system. BPN-14136, or a similar compound, may be a promising drug candidate to manage Stargardt disease and dry AMD. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Structure analysis of a heteropolysaccharide from Taraxacum mongolicum Hand.-Mazz. and anticomplementary activity of its sulfated derivatives.

PubMed

Chen, MiaoMiao; Wu, Jianjun; Shi, Songshan; Chen, Yonglin; Wang, Huijun; Fan, Hongwei; Wang, Shunchun

2016-11-05

A homogenous water-soluble polysaccharide, DPSW-A, with a deduced chemical structure was extracted from the herb Taraxacum mongolicum Hand.-Mazz. Moreover, 80.813-kDa DPSW-A is composed of three types of monosaccharide, namely rhamnose, arabinose, and galactose, at a molar ratio of 1.0:10.7:11.9. The main chain of DPSW-A contains Terminal-Galp, 1,3-Galp, 1,6-Galp, 1,3,6-Galp, and 1,2,4-Rhap; the branched chain contains Terminal-Araf, 1,5-Araf, and 1,3,5-Araf. The sulfated derivatives prepared from DPSW-A showed inhibitory effects on complement activation through the classical pathway (CH50: Sul-DPSW-A, 3.94±0.43μg/mL; heparin, 104.40±3.82μg/mL) and alternative pathway (AP50: Sul-DPSW-A, 42.76±0.46μg/mL; heparin, 43.42±0.22μg/mL). Mechanism studies indicated that Sul-DPSW-A inhibited complement activation by blocking C1q, C1r, C1s, and C9, but not C2, C3, C4, and C5. In addition, Sul-DPSW-A displayed limited anticoagulant effects. These results suggest that Sul-DPSW-A prepared from DPSW-A is valuable for treating diseases caused by excessive complement system activation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Complement C1q formation of immune complexes with milk caseins and wheat glutens in schizophrenia

PubMed Central

Severance, Emily G.; Gressitt, Kristin; Halling, Meredith; Stallings, Cassie R.; Origoni, Andrea E.; Vaughan, Crystal; Khushalani, Sunil; Alaedini, Armin; Dupont, Didier; Dickerson, Faith B.; Yolken, Robert H.

2012-01-01

Immune system factors including complement pathway activation are increasingly linked to the etiology and pathophysiology of schizophrenia. Complement protein, C1q, binds to and helps to clear immune complexes composed of immunoglobulins coupled to antigens. The antigenic stimuli for C1q activation in schizophrenia are not known. Food sensitivities characterized by elevated IgG antibodies to bovine milk caseins and wheat glutens have been reported in individuals with schizophrenia. Here, we examined the extent to which these food products might comprise the antigen component of complement C1q immune complexes in individuals with recent onset schizophrenia (n=38), non-recent onset schizophrenia (n=61) and non-psychiatric controls (n=63). C1q seropositivity was significantly associated with both schizophrenia groups (recent onset, odds ratio (OR)=8.02, p≤0.008; non-recent onset, OR=3.15, p≤0.03) compared to controls (logistic regression models corrected for age, sex, race and smoking status). Casein- and/or gluten-IgG binding to C1q was significantly elevated in the non-recent onset group compared to controls (OR=4.36, p≤0.01). Significant amounts of C1q-casein/gluten-related immune complexes and C1q correlations with a marker for gastrointestinal inflammation in non-recent onset schizophrenia suggests a heightened rate of food antigens in the systemic circulation, perhaps via a disease-associated altered intestinal permeability. In individuals who are in the early stages of disease onset, C1q activation may reflect the formation of immune complexes with non-casein- or non-gluten-related antigens, the presence of C1q autoantibodies, and/or a dissociated state of immune complex components. In conclusion, complement activation may be a useful biomarker to diagnose schizophrenia early during the course of the disease. Future prospective studies should evaluate the impacts of casein- and gluten-free diets on C1q activation in schizophrenia. PMID:22801085

21 CFR 866.5250 - Complement C2 inhibitor (inactivator) immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... the reagents used to measure by immunochemical techniques the complement C1 inhibitor (a plasma protein) in serum. Complement C1 inhibitor occurs normally in plasma and blocks the action of the C1...

21 CFR 866.5250 - Complement C2 inhibitor (inactivator) immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... the reagents used to measure by immunochemical techniques the complement C1 inhibitor (a plasma protein) in serum. Complement C1 inhibitor occurs normally in plasma and blocks the action of the C1...

21 CFR 866.5250 - Complement C2 inhibitor (inactivator) immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... the reagents used to measure by immunochemical techniques the complement C1 inhibitor (a plasma protein) in serum. Complement C1 inhibitor occurs normally in plasma and blocks the action of the C1...

21 CFR 866.5250 - Complement C 2 inhibitor (inactivator) immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... the reagents used to measure by immunochemical techniques the complement C1 inhibitor (a plasma protein) in serum. Complement C1 inhibitor occurs normally in plasma and blocks the action of the C1...

21 CFR 866.5250 - Complement C 2 inhibitor (inactivator) immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... the reagents used to measure by immunochemical techniques the complement C1 inhibitor (a plasma protein) in serum. Complement C1 inhibitor occurs normally in plasma and blocks the action of the C1...

Induction of passive Heymann nephritis in complement component 6-deficient PVG rats.

PubMed

Spicer, S Timothy; Tran, Giang T; Killingsworth, Murray C; Carter, Nicole; Power, David A; Paizis, Kathy; Boyd, Rochelle; Hodgkinson, Suzanne J; Hall, Bruce M

2007-07-01

Passive Heymann nephritis (PHN), a model of human membranous nephritis, is induced in susceptible rat strains by injection of heterologous antisera to rat renal tubular Ag extract. PHN is currently considered the archetypal complement-dependent form of nephritis, with the proteinuria resulting from sublytic glomerular epithelial cell injury induced by the complement membrane attack complex (MAC) of C5b-9. This study examined whether C6 and MAC are essential to the development of proteinuria in PHN by comparing the effect of injection of anti-Fx1A antisera into PVG rats deficient in C6 (PVG/C6(-)) and normal PVG rats (PVG/c). PVG/c and PVG/C6(-) rats developed similar levels of proteinuria at 3, 7, 14, and 28 days following injection of antisera. Isolated whole glomeruli showed similar deposition of rat Ig and C3 staining in PVG/c and PVG/C6(-) rats. C9 deposition was abundant in PVG/c but was not detected in PVG/C6(-) glomeruli, indicating C5b-9/MAC had not formed in PVG/C6(-) rats. There was also no difference in the glomerular cellular infiltrate of T cells and macrophages nor the size of glomerular basement membrane deposits measured on electron micrographs. To examine whether T cells effect injury, rats were depleted of CD8+ T cells which did not affect proteinuria in the early heterologous phase but prevented the increase in proteinuria associated with the later autologous phase. These studies showed proteinuria in PHN occurs without MAC and that other mechanisms, such as immune complex size, early complement components, CD4+ and CD8+ T cells, disrupt glomerular integrity and lead to proteinuria.

Clinical features of patients with homozygous complement C4A or C4B deficiency.

PubMed

Liesmaa, Inka; Paakkanen, Riitta; Järvinen, Asko; Valtonen, Ville; Lokki, Marja-Liisa

2018-01-01

Homozygous deficiencies of complement C4A or C4B are detected in 1-10% of populations. In genome-wide association studies C4 deficiencies are missed because the genetic variation of C4 is complex. There are no studies where the clinical presentation of these patients is analyzed. This study was aimed to characterize the clinical features of patients with homozygous C4A or C4B deficiency. Thirty-two patients with no functional C4A, 87 patients with no C4B and 120 with normal amount of C4 genes were included. C4A and C4B numbers were assessed with genomic quantitative real-time PCR. Medical history was studied retrospectively from patients' files. Novel associations between homozygous C4A deficiency and lymphoma, coeliac disease and sarcoidosis were detected. These conditions were present in 12.5%, (4/32 in patients vs. 0.8%, 1/120, in controls, OR = 17.00, 95%CI = 1.83-158.04, p = 0.007), 12.5% (4/32 in patients vs. 0%, 0/120 in controls, OR = 1.14, 95%CI = 1.00-1.30, p = 0.002) and 12.5%, respectively (4/32 in patients vs. 2.5%, 3/120 in controls, OR = 5.571, 95%CI = 1.79-2.32, p = 0.036). In addition, C4A and C4B deficiencies were both associated with adverse drug reactions leading to drug discontinuation (34.4%, 11/32 in C4A-deficient patients vs. 14.2%, 17/120 in controls, OR = 3.174, 95%CI = 1.30-7.74, p = 0.009 and 28.7%, 25/87 in C4B-deficient patients, OR = 2.44, 95%CI = 1.22-4.88, p = 0.010). This reported cohort of homozygous deficiencies of C4A or C4B suggests that C4 deficiencies may have various unrecorded disease associations. C4 gene should be considered as a candidate gene in studying these selected disease associations.

Identification of a human non-interferon lymphokine activating monocyte complement biosynthesis.

PubMed Central

Drouet, C; Reboul, A; Colomb, M

1989-01-01

A monocyte-stimulating activity produced by mitogen-induced mononuclear cells has been defined by its ability to enhance the synthesis in vitro of complement C1 subcomponents, C2 and C3. A lymphokine responsible for this activity was purified from culture supernatants of peripheral blood mononuclear cells activated by staphylococcal enterotoxin A. From 0.5 litre of supernatant the purification procedure [(NH4)2SO4 precipitation, phenyl-Sepharose chromatography and preparative electrofocusing] yielded about 100 pmol of purified lymphokine. Its pI is 7.9 and its Mr, estimated by SDS/polyacrylamide-gel electrophoresis, is 14,600, 27,000 and 56,000, the high-Mr species representing oligomeric forms of the Mr-14,600 molecule. Its amino acid analysis reveals a high percentage of hydrophobic amino acids (34%); the absence of histidine residues suggests that it is a novel monocyte-activating lymphokine. It enhances C1r and C1s biosynthesis at a pretranslational level. From its structure and activity this lymphokine appears different from gamma-interferon. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:2481436

C4d Deposition and Cellular Infiltrates as Markers of Acute Rejection in Rat Models of Orthotopic Lung Transplantation

PubMed Central

Murata, Kazunori; Iwata, Takekazu; Nakashima, Shinji; Fox-Talbot, Karen; Qian, Zhiping; Wilkes, David S.; Baldwin, William M.

2008-01-01

Background C4d is a useful marker of antibody-mediated rejection in cardiac and renal transplants, but clinical studies examining correlations between circulating alloantibodies, C4d deposition, and rejection in lung transplants have yielded conflicting results. Methods We studied circulating alloantibody levels and C4d deposition in two rat models of lung transplantation: Brown Norway (BN) to Wistar-Kyoto (WKY) and PVG.R8 to PVG.1U lung allografts. The availability of C6 deficient (C6−) and C6 sufficient (C6+) PVG 1U rats allowed evaluation of the effects of the terminal complement components on graft injury and C4d deposition. Results The lung allografts had histologic features resembling human posttransplant capillaritis, characterized by neutrophilic infiltration of alveoli, edema, and hemorrhage. Immunoperoxidase stains on cross sections of allografts showed intense, diffuse, C4d deposition in a continuous linear pattern on the vascular endothelium. C4d deposits were found in both BN to WKY and PVG R8 to 1U allografts, whereas no staining was detectable in WKY to WKY isografts or native lungs. Complement deposition was associated with vascular disruption in C6−, but not in C6+ recipients. The presence of circulating donor-specific alloantibodies was verified by flow cytometry. Cell-specific staining revealed perivascular accumulation of macrophages and T lymphocytes whereas neutrophils were sequestered in the intravascular and alveolar capillary compartments. Conclusions The deposition of C4d on vascular endothelium as well as the coincident presence of alloantibodies is consistent with previous findings in antibody-mediated rejection of renal and cardiac transplants. Furthermore, the histological features of our allografts support the concept that posttransplant capillaritis is a form of humoral rejection. PMID:18622289

Evaluation of copy-number variants as modifiers of breast and ovarian cancer risk for BRCA1 pathogenic variant carriers

PubMed Central

Walker, Logan C; Marquart, Louise; Pearson, John F; Wiggins, George A R; O'Mara, Tracy A; Parsons, Michael T; Barrowdale, Daniel; McGuffog, Lesley; Dennis, Joe; Benitez, Javier; Slavin, Thomas P; Radice, Paolo; Frost, Debra; Godwin, Andrew K; Meindl, Alfons; Schmutzler, Rita Katharina; Isaacs, Claudine; Peshkin, Beth N; Caldes, Trinidad; Hogervorst, Frans BL; Lazaro, Conxi; Jakubowska, Anna; Montagna, Marco; Chen, Xiaoqing; Offit, Kenneth; Hulick, Peter J; Andrulis, Irene L; Lindblom, Annika; Nussbaum, Robert L; Nathanson, Katherine L; Chenevix-Trench, Georgia; Antoniou, Antonis C; Couch, Fergus J; Spurdle, Amanda B

2017-01-01

Genome-wide studies of patients carrying pathogenic variants (mutations) in BRCA1 or BRCA2 have reported strong associations between single-nucleotide polymorphisms (SNPs) and cancer risk. To conduct the first genome-wide association analysis of copy-number variants (CNVs) with breast or ovarian cancer risk in a cohort of 2500 BRCA1 pathogenic variant carriers, CNV discovery was performed using multiple calling algorithms and Illumina 610k SNP array data from a previously published genome-wide association study. Our analysis, which focused on functionally disruptive genomic deletions overlapping gene regions, identified a number of loci associated with risk of breast or ovarian cancer for BRCA1 pathogenic variant carriers. Despite only including putative deletions called by at least two or more algorithms, detection of selected CNVs by ancillary molecular technologies only confirmed 40% of predicted common (>1% allele frequency) variants. These include four loci that were associated (unadjusted P<0.05) with breast cancer (GTF2H2, ZNF385B, NAALADL2 and PSG5), and two loci associated with ovarian cancer (CYP2A7 and OR2A1). An interesting finding from this study was an association of a validated CNV deletion at the CYP2A7 locus (19q13.2) with decreased ovarian cancer risk (relative risk=0.50, P=0.007). Genomic analysis found this deletion coincides with a region displaying strong regulatory potential in ovarian tissue, but not in breast epithelial cells. This study highlighted the need to verify CNVs in vitro, but also provides evidence that experimentally validated CNVs (with plausible biological consequences) can modify risk of breast or ovarian cancer in BRCA1 pathogenic variant carriers. PMID:28145423

Contingent negative variation (CNV) and erotic preference in self-declared homosexuals and in child sex offenders.

PubMed

Howard, R C; Longmore, F J; Mason, P A; Martin, J L

1994-10-01

Contingent negative variation (CNV) was recorded bilaterally from central electrodes using a "match/mismatch" paradigm in (Study 1) samples of heterosexual men (N = 6), gay men (N = 10) and lesbian women (N = 14) and (Study 2) in samples of child sex offenders (N = 34) and heterosexual control men (N = 19). Sexual orientation was assessed using the Multidimensional Scale of Sexuality (MSS) and the Human Sexuality Questionnaire (HSQ). Separate CNV averages were formed for each condition of stimulation: for Study 1, slides of adult male and female nudes; for Study 2, slides of child, pubescent and adult male and female nudes. Penile plethysmographic (PPG) data were also obtained from 15 of the child sex offender sample while they viewed stimuli of the same categories as were used in the CNV recording. On the basis of their PPG responses to children, child sex offenders were classified as either "pedophiles" or "non-pedophiles". In Study 1 significant Group x Sex (of slide) and Group x Electrode interactions indicated that: (i) heterosexual men (but neither homosexual group) showed significantly larger CNVs to female than to male slides; (ii) both homosexual groups showed significantly asymmetrical (R > L) CNVs. In Study 2, controls showed significantly greater CNVs to adult females than to both adult males and female children. Child sex offenders showed no significant differences in CNV to male and female slides for any age. "Non-pedophiles" showed significantly larger CNVs to female adults than to female children, but "pedophiles" did not. It is concluded that CNV has promise as a measure of both deviant and non-deviant sexual preference.

Rare Genome-Wide Copy Number Variation and Expression of Schizophrenia in 22q11.2 Deletion Syndrome.

PubMed

Bassett, Anne S; Lowther, Chelsea; Merico, Daniele; Costain, Gregory; Chow, Eva W C; van Amelsvoort, Therese; McDonald-McGinn, Donna; Gur, Raquel E; Swillen, Ann; Van den Bree, Marianne; Murphy, Kieran; Gothelf, Doron; Bearden, Carrie E; Eliez, Stephan; Kates, Wendy; Philip, Nicole; Sashi, Vandana; Campbell, Linda; Vorstman, Jacob; Cubells, Joseph; Repetto, Gabriela M; Simon, Tony; Boot, Erik; Heung, Tracy; Evers, Rens; Vingerhoets, Claudia; van Duin, Esther; Zackai, Elaine; Vergaelen, Elfi; Devriendt, Koen; Vermeesch, Joris R; Owen, Michael; Murphy, Clodagh; Michaelovosky, Elena; Kushan, Leila; Schneider, Maude; Fremont, Wanda; Busa, Tiffany; Hooper, Stephen; McCabe, Kathryn; Duijff, Sasja; Isaev, Karin; Pellecchia, Giovanna; Wei, John; Gazzellone, Matthew J; Scherer, Stephen W; Emanuel, Beverly S; Guo, Tingwei; Morrow, Bernice E; Marshall, Christian R

2017-11-01

Chromosome 22q11.2 deletion syndrome (22q11.2DS) is associated with a more than 20-fold increased risk for developing schizophrenia. The aim of this study was to identify additional genetic factors (i.e., "second hits") that may contribute to schizophrenia expression. Through an international consortium, the authors obtained DNA samples from 329 psychiatrically phenotyped subjects with 22q11.2DS. Using a high-resolution microarray platform and established methods to assess copy number variation (CNV), the authors compared the genome-wide burden of rare autosomal CNV, outside of the 22q11.2 deletion region, between two groups: a schizophrenia group and those with no psychotic disorder at age ≥25 years. The authors assessed whether genes overlapped by rare CNVs were overrepresented in functional pathways relevant to schizophrenia. Rare CNVs overlapping one or more protein-coding genes revealed significant between-group differences. For rare exonic duplications, six of 19 gene sets tested were enriched in the schizophrenia group; genes associated with abnormal nervous system phenotypes remained significant in a stepwise logistic regression model and showed significant interactions with 22q11.2 deletion region genes in a connectivity analysis. For rare exonic deletions, the schizophrenia group had, on average, more genes overlapped. The additional rare CNVs implicated known (e.g., GRM7, 15q13.3, 16p12.2) and novel schizophrenia risk genes and loci. The results suggest that additional rare CNVs overlapping genes outside of the 22q11.2 deletion region contribute to schizophrenia risk in 22q11.2DS, supporting a multigenic hypothesis for schizophrenia. The findings have implications for understanding expression of psychotic illness and herald the importance of whole-genome sequencing to appreciate the overall genomic architecture of schizophrenia.

Exploiting sequence similarity to validate the sensitivity of SNP arrays in detecting fine-scaled copy number variations.

PubMed

Wong, Gerard; Leckie, Christopher; Gorringe, Kylie L; Haviv, Izhak; Campbell, Ian G; Kowalczyk, Adam

2010-04-15

High-density single nucleotide polymorphism (SNP) genotyping arrays are efficient and cost effective platforms for the detection of copy number variation (CNV). To ensure accuracy in probe synthesis and to minimize production costs, short oligonucleotide probe sequences are used. The use of short probe sequences limits the specificity of binding targets in the human genome. The specificity of these short probeset sequences has yet to be fully analysed against a normal reference human genome. Sequence similarity can artificially elevate or suppress copy number measurements, and hence reduce the reliability of affected probe readings. For the purpose of detecting narrow CNVs reliably down to the width of a single probeset, sequence similarity is an important issue that needs to be addressed. We surveyed the Affymetrix Human Mapping SNP arrays for probeset sequence similarity against the reference human genome. Utilizing sequence similarity results, we identified a collection of fine-scaled putative CNVs between gender from autosomal probesets whose sequence matches various loci on the sex chromosomes. To detect these variations, we utilized our statistical approach, Detecting REcurrent Copy number change using rank-order Statistics (DRECS), and showed that its performance was superior and more stable than the t-test in detecting CNVs. Through the application of DRECS on the HapMap population datasets with multi-matching probesets filtered, we identified biologically relevant SNPs in aberrant regions across populations with known association to physical traits, such as height, covered by the span of a single probe. This provided empirical confirmation of the existence of naturally occurring narrow CNVs as well as the sensitivity of the Affymetrix SNP array technology in detecting them. The MATLAB implementation of DRECS is available at http://ww2.cs.mu.oz.au/ approximately gwong/DRECS/index.html.

Evaluation of the X-Linked High-Grade Myopia Locus (MYP1) with Cone Dysfunction and Color Vision Deficiencies

PubMed Central

Metlapally, Ravikanth; Michaelides, Michel; Bulusu, Anuradha; Li, Yi-Ju; Schwartz, Marianne; Rosenberg, Thomas; Hunt, David M.; Moore, Anthony T.; Züchner, Stephan; Rickman, Catherine Bowes; Young, Terri L.

2014-01-01

Purpose X-linked high myopia with mild cone dysfunction and color vision defects has been mapped to chromosome Xq28 (MYP1 locus). CXorf2/TEX28 is a nested, intercalated gene within the red-green opsin cone pigment gene tandem array on Xq28. The authors investigated whether TEX28 gene alterations were associated with the Xq28-linked myopia phenotype. Genomic DNA from five pedigrees (with high myopia and either protanopia or deuteranopia) that mapped to Xq28 were screened for TEX28 copy number variations (CNVs) and sequence variants. Methods To examine for CNVs, ultra-high resolution array-comparative genomic hybridization (array-CGH) assays were performed comparing the subject genomic DNA with control samples (two pairs from two pedigrees). Opsin or TEX28 gene-targeted quantitative real-time gene expression assays (comparative CT method) were performed to validate the array-CGH findings. All exons of TEX28, including intron/exon boundaries, were amplified and sequenced using standard techniques. Results Array-CGH findings revealed predicted duplications in affected patient samples. Although only three copies of TEX28 were previously reported within the opsin array, quantitative real-time analysis of the TEX28 targeted assay of affected male or carrier female individuals in these pedigrees revealed either fewer (one) or more (four or five) copies than did related and control unaffected individuals. Sequence analysis of TEX28 did not reveal any variants associated with the disease status. Conclusions CNVs have been proposed to play a role in disease inheritance and susceptibility as they affect gene dosage. TEX28 gene CNVs appear to be associated with the MYP1 X-linked myopia phenotypes. PMID:19098318

Biological relevance of CNV calling methods using familial relatedness including monozygotic twins.

PubMed

Castellani, Christina A; Melka, Melkaye G; Wishart, Andrea E; Locke, M Elizabeth O; Awamleh, Zain; O'Reilly, Richard L; Singh, Shiva M

2014-04-21

Studies involving the analysis of structural variation including Copy Number Variation (CNV) have recently exploded in the literature. Furthermore, CNVs have been associated with a number of complex diseases and neurodevelopmental disorders. Common methods for CNV detection use SNP, CNV, or CGH arrays, where the signal intensities of consecutive probes are used to define the number of copies associated with a given genomic region. These practices pose a number of challenges that interfere with the ability of available methods to accurately call CNVs. It has, therefore, become necessary to develop experimental protocols to test the reliability of CNV calling methods from microarray data so that researchers can properly discriminate biologically relevant data from noise. We have developed a workflow for the integration of data from multiple CNV calling algorithms using the same array results. It uses four CNV calling programs: PennCNV (PC), Affymetrix® Genotyping Console™ (AGC), Partek® Genomics Suite™ (PGS) and Golden Helix SVS™ (GH) to analyze CEL files from the Affymetrix® Human SNP 6.0 Array™. To assess the relative suitability of each program, we used individuals of known genetic relationships. We found significant differences in CNV calls obtained by different CNV calling programs. Although the programs showed variable patterns of CNVs in the same individuals, their distribution in individuals of different degrees of genetic relatedness has allowed us to offer two suggestions. The first involves the use of multiple algorithms for the detection of the largest possible number of CNVs, and the second suggests the use of PennCNV over all other methods when the use of only one software program is desirable.

Cell-free DNA fragment-size distribution analysis for non-invasive prenatal CNV prediction.

PubMed

Arbabi, Aryan; Rampášek, Ladislav; Brudno, Michael

2016-06-01

Non-invasive detection of aneuploidies in a fetal genome through analysis of cell-free DNA circulating in the maternal plasma is becoming a routine clinical test. Such tests, which rely on analyzing the read coverage or the allelic ratios at single-nucleotide polymorphism (SNP) loci, are not sensitive enough for smaller sub-chromosomal abnormalities due to sequencing biases and paucity of SNPs in a genome. We have developed an alternative framework for identifying sub-chromosomal copy number variations in a fetal genome. This framework relies on the size distribution of fragments in a sample, as fetal-origin fragments tend to be smaller than those of maternal origin. By analyzing the local distribution of the cell-free DNA fragment sizes in each region, our method allows for the identification of sub-megabase CNVs, even in the absence of SNP positions. To evaluate the accuracy of our method, we used a plasma sample with the fetal fraction of 13%, down-sampled it to samples with coverage of 10X-40X and simulated samples with CNVs based on it. Our method had a perfect accuracy (both specificity and sensitivity) for detecting 5 Mb CNVs, and after reducing the fetal fraction (to 11%, 9% and 7%), it could correctly identify 98.82-100% of the 5 Mb CNVs and had a true-negative rate of 95.29-99.76%. Our source code is available on GitHub at https://github.com/compbio-UofT/FSDA CONTACT: : brudno@cs.toronto.edu. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Eight further individuals with intellectual disability and epilepsy carrying bi-allelic CNTNAP2 aberrations allow delineation of the mutational and phenotypic spectrum.

PubMed

Smogavec, Mateja; Cleall, Alison; Hoyer, Juliane; Lederer, Damien; Nassogne, Marie-Cécile; Palmer, Elizabeth E; Deprez, Marie; Benoit, Valérie; Maystadt, Isabelle; Noakes, Charlotte; Leal, Alejandro; Shaw, Marie; Gecz, Jozef; Raymond, Lucy; Reis, André; Shears, Deborah; Brockmann, Knut; Zweier, Christiane

2016-12-01

Heterozygous copy number variants (CNVs) or sequence variants in the contactin-associated protein 2 gene CNTNAP2 have been discussed as risk factors for a wide spectrum of neurodevelopmental and neuropsychiatric disorders. Bi-allelic aberrations in this gene are causative for an autosomal-recessive disorder with epilepsy, severe intellectual disability (ID) and cortical dysplasia (CDFES). As the number of reported individuals is still limited, we aimed at a further characterisation of the full mutational and clinical spectrum. Targeted sequencing, chromosomal microarray analysis or multigene panel sequencing was performed in individuals with severe ID and epilepsy. We identified homozygous mutations, compound heterozygous CNVs or CNVs and mutations in CNTNAP2 in eight individuals from six unrelated families. All aberrations were inherited from healthy, heterozygous parents and are predicted to be deleterious for protein function. Epilepsy occurred in all affected individuals with onset in the first 3.5 years of life. Further common aspects were ID (severe in 6/8), regression of speech development (5/8) and behavioural anomalies (7/8). Interestingly, cognitive impairment in one of two affected brothers was, in comparison, relatively mild with good speech and simple writing abilities. Cortical dysplasia that was previously reported in CDFES was not present in MRIs of six individuals and only suspected in one. By identifying novel homozygous or compound heterozygous, deleterious CNVs and mutations in eight individuals from six unrelated families with moderate-to-severe ID, early onset epilepsy and behavioural anomalies, we considerably broaden the mutational and clinical spectrum associated with bi-allelic aberrations in CNTNAP2. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Complement component 4 copy number variation and CYP21A2 genotype associations in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

PubMed

Chen, Wuyan; Xu, Zhi; Nishitani, Miki; Van Ryzin, Carol; McDonnell, Nazli B; Merke, Deborah P

2012-12-01

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder of cortisol biosynthesis caused by CYP21A2 mutations. An increase in gene copy number variation (CNV) exists at the CYP21A2 locus. CNV of C4, a neighboring gene that encodes complement component 4, is associated with autoimmune disease susceptibility. In this study, we performed comprehensive genetic analysis of the RP-C4-CYP21-TNX (RCCX) region in 127 unrelated 21-OHD patients (100 classic, 27 nonclassic). C4 copy number was determined by Southern blot. C4 CNV and serum C4 levels were evaluated in relation to CYP21A2 mutations and relevant phenotypes. We found that the most common CYP21A2 mutation associated with the nonclassic form of CAH, V281L, was associated with high C4 copy number (p = 7.13 × 10(-16)). Large CYP21A2 deletion, a common mutation associated with the classic form of CAH, was associated with low C4 copy number (p = 1.61 × 10(-14)). Monomodular RCCX with a short C4 gene, a risk factor for autoimmune disease, was significantly less frequent in CAH patients compared to population estimates (2.8 vs. 10.6 %; p = 1.08 × 10(-4)). In conclusion, CAH patients have increased C4 CNV, with mutation-specific associations that may be protective for autoimmune disease. The study of CYP21A2 in relation to neighboring genes provides insight into the genetics of CNV hotspots, an important determinant of human health.

Complement therapeutics in inflammatory diseases: promising drug candidates for C3-targeted intervention.

PubMed

Mastellos, D C; Ricklin, D; Hajishengallis, E; Hajishengallis, G; Lambris, J D

2016-02-01

There is increasing appreciation that complement dysregulation lies at the heart of numerous immune-mediated and inflammatory disorders. Complement inhibitors are therefore being evaluated as new therapeutic options in various clinical translation programs and the first clinically approved complement-targeted drugs have profoundly impacted the management of certain complement-mediated diseases. Among the many members of the intricate protein network of complement, the central component C3 represents a 'hot-spot' for complement-targeted therapeutic intervention. C3 modulates both innate and adaptive immune responses and is linked to diverse immunomodulatory systems and biological processes that affect human pathophysiology. Compelling evidence from preclinical disease models has shown that C3 interception may offer multiple benefits over existing therapies or even reveal novel therapeutic avenues in disorders that are not commonly regarded as complement-driven, such as periodontal disease. Using the clinically developed compstatin family of C3 inhibitors and periodontitis as illustrative examples, this review highlights emerging therapeutic concepts and developments in the design of C3-targeted drug candidates as novel immunotherapeutics for oral and systemic inflammatory diseases. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Defining the Complement Biomarker Profile of C3 Glomerulopathy

PubMed Central

Zhang, Yuzhou; Nester, Carla M.; Martin, Bertha; Skjoedt, Mikkel-Ole; Meyer, Nicole C.; Shao, Dingwu; Borsa, Nicolò; Palarasah, Yaseelan

2014-01-01

Background and objectives C3 glomerulopathy (C3G) applies to a group of renal diseases defined by a specific renal biopsy finding: a dominant pattern of C3 fragment deposition on immunofluorescence. The primary pathogenic mechanism involves abnormal control of the alternative complement pathway, although a full description of the disease spectrum remains to be determined. This study sought to validate and define the association of complement dysregulation with C3G and to determine whether specific complement pathway abnormalities could inform disease definition. Design, setting, participants, & measurements This study included 34 patients with C3G (17 with C3 glomerulonephritis [C3GN] and 17 with dense deposit disease [DDD]) diagnosed between 2008 and 2013 selected from the C3G Registry. Control samples (n=100) were recruited from regional blood drives. Nineteen complement biomarkers were assayed on all samples. Results were compared between C3G disease categories and with normal controls. Results Assessment of the alternative complement pathway showed that compared with controls, patients with C3G had lower levels of serum C3 (P<0.001 for both DDD and C3GN) and factor B (P<0.001 for both DDD and C3GN) as well as higher levels of complement breakdown products including C3d (P<0.001 for both DDD and C3GN) and Bb (P<0.001 for both DDD and C3GN). A comparison of terminal complement pathway proteins showed that although C5 levels were significantly suppressed (P<0.001 for both DDD and C3GN) its breakdown product C5a was significantly higher only in patients with C3GN (P<0.05). Of the other terminal pathway components (C6–C9), the only significant difference was in C7 levels between patients with C3GN and controls (P<0.01). Soluble C5b-9 was elevated in both diseases but only the difference between patients with C3GN and controls reached statistical significance (P<0.001). Levels of C3 nephritic factor activity were qualitatively higher in patients with DDD compared with patients with C3GN. Conclusions Complement biomarkers are significantly abnormal in patients with C3G compared with controls. These data substantiate the link between complement dysregulation and C3G and identify C3G interdisease differences. PMID:25341722

A bayesian analysis for identifying DNA copy number variations using a compound poisson process.

PubMed

Chen, Jie; Yiğiter, Ayten; Wang, Yu-Ping; Deng, Hong-Wen

2010-01-01

To study chromosomal aberrations that may lead to cancer formation or genetic diseases, the array-based Comparative Genomic Hybridization (aCGH) technique is often used for detecting DNA copy number variants (CNVs). Various methods have been developed for gaining CNVs information based on aCGH data. However, most of these methods make use of the log-intensity ratios in aCGH data without taking advantage of other information such as the DNA probe (e.g., biomarker) positions/distances contained in the data. Motivated by the specific features of aCGH data, we developed a novel method that takes into account the estimation of a change point or locus of the CNV in aCGH data with its associated biomarker position on the chromosome using a compound Poisson process. We used a Bayesian approach to derive the posterior probability for the estimation of the CNV locus. To detect loci of multiple CNVs in the data, a sliding window process combined with our derived Bayesian posterior probability was proposed. To evaluate the performance of the method in the estimation of the CNV locus, we first performed simulation studies. Finally, we applied our approach to real data from aCGH experiments, demonstrating its applicability.

Population-genetic properties of differentiated copy number variations in cattle.

PubMed

Xu, Lingyang; Hou, Yali; Bickhart, Derek M; Zhou, Yang; Hay, El Hamidi Abdel; Song, Jiuzhou; Sonstegard, Tad S; Van Tassell, Curtis P; Liu, George E

2016-03-23

While single nucleotide polymorphism (SNP) is typically the variant of choice for population genetics, copy number variation (CNV) which comprises insertion, deletion and duplication of genomic sequence, is an informative type of genetic variation. CNVs have been shown to be both common in mammals and important for understanding the relationship between genotype and phenotype. However, CNV differentiation, selection and its population genetic properties are not well understood across diverse populations. We performed a population genetics survey based on CNVs derived from the BovineHD SNP array data of eight distinct cattle breeds. We generated high resolution results that show geographical patterns of variations and genome-wide admixture proportions within and among breeds. Similar to the previous SNP-based studies, our CNV-based results displayed a strong correlation of population structure and geographical location. By conducting three pairwise comparisons among European taurine, African taurine, and indicine groups, we further identified 78 unique CNV regions that were highly differentiated, some of which might be due to selection. These CNV regions overlapped with genes involved in traits related to parasite resistance, immunity response, body size, fertility, and milk production. Our results characterize CNV diversity among cattle populations and provide a list of lineage-differentiated CNVs.

Identifying CNVs in 15q11q13 and 16p11.2 of Patients with Seizures Increases the Rates of Detecting Pathogenic Changes

PubMed Central

Vianna, Gabrielle S.; Freitas, Mariana L.; Oliveira, Valdirene T.de; Pietra, Rafaella X.; Gonçalves, Michele da S.; Rocha, Patrícia P.O.; Monteiro, Rejane A.C.; Ferreira, Luana C.A.; Xavier, Rosana R.; Carvalho, Andréia M.; Lima, Patrícia R. de M.; Monteiro, Maria Augusta N.P.; Mateo, Elvis C.; Giannetti, Juliana G.; César, Giovana da C.; Lima, Joziele de S.; Medeiros, Paula F.V.; Jehee, Fernanda S.

2016-01-01

Chromosomal changes are frequently observed in patients with syndromic seizures. Understanding the genetic etiology of this pathology is crucial for the guidance and genetic counseling of families as well as for the establishment of appropriate treatment. A combination of MLPA kits was used to identify pathogenic CNVs in a group of 70 syndromic patients with seizures. Initially, a screening was performed for subtelomeric changes (MLPA P036 and P070 kits) and for the regions most frequently related to microdeletion/microduplication syndromes (MLPA P064). Subsequently, the MLPA P343 was used to identify alterations in the 15q11q13, 16p11.2, and 22q13 regions. Screening with MLPA P343 allowed a 10-15.7% increase in the detection rate of CNVs reinforcing the importance of investigating changes in 15q11q13 and 16p11.2 in syndromic patients with seizures. We also demonstrated that the MLPA technique is an alternative with a great diagnostic potential, and we proposed its use as part of the initial assessment of syndromic patients with seizures. PMID:27920636

Whole genome comparative studies between chicken and turkey and their implications for avian genome evolution

PubMed Central

Griffin, Darren K; Robertson, Lindsay B; Tempest, Helen G; Vignal, Alain; Fillon, Valérie; Crooijmans, Richard PMA; Groenen, Martien AM; Deryusheva, Svetlana; Gaginskaya, Elena; Carré, Wilfrid; Waddington, David; Talbot, Richard; Völker, Martin; Masabanda, Julio S; Burt, Dave W

2008-01-01

Background Comparative genomics is a powerful means of establishing inter-specific relationships between gene function/location and allows insight into genomic rearrangements, conservation and evolutionary phylogeny. The availability of the complete sequence of the chicken genome has initiated the development of detailed genomic information in other birds including turkey, an agriculturally important species where mapping has hitherto focused on linkage with limited physical information. No molecular study has yet examined conservation of avian microchromosomes, nor differences in copy number variants (CNVs) between birds. Results We present a detailed comparative cytogenetic map between chicken and turkey based on reciprocal chromosome painting and mapping of 338 chicken BACs to turkey metaphases. Two inter-chromosomal changes (both involving centromeres) and three pericentric inversions have been identified between chicken and turkey; and array CGH identified 16 inter-specific CNVs. Conclusion This is the first study to combine the modalities of zoo-FISH and array CGH between different avian species. The first insight into the conservation of microchromosomes, the first comparative cytogenetic map of any bird and the first appraisal of CNVs between birds is provided. Results suggest that avian genomes have remained relatively stable during evolution compared to mammalian equivalents. PMID:18410676

HLA-DQA1 and PLA2R1 Polymorphisms and Risk of Idiopathic Membranous Nephropathy

PubMed Central

Bullich, Gemma; Ballarín, José; Oliver, Artur; Ayasreh, Nadia; Silva, Irene; Santín, Sheila; Díaz-Encarnación, Montserrat M.; Torra, Roser

2014-01-01

Summary Background and objectives Single nucleotide polymorphisms (SNPs) within HLA complex class II HLA-DQ α-chain 1 (HLA-DQA1) and M-type phospholipase A2 receptor (PLA2R1) genes were identified as strong risk factors for idiopathic membranous nephropathy (IMN) development in a recent genome-wide association study. Copy number variants (CNVs) within the Fc gamma receptor III (FCGR3) locus have been associated with several autoimmune diseases, but their role in IMN has not been studied. This study aimed to validate the association of HLA-DQA1 and PLA2R1 risk alleles with IMN in a Spanish cohort, test the putative association of FCGR3A and FCGR3B CNVs with IMN, and assess the use of these genetic factors to predict the clinical outcome of the disease. Design, settings, participants, & measurements A Spanish cohort of 89 IMN patients and 286 matched controls without nephropathy was recruited between October of 2009 and July of 2012. Case-control studies for SNPs within HLA-DQA1 (rs2187668) and PLA2R1 (rs4664308) genes and CNVs for FCGR3A and FCGR3B genes were performed. The contribution of these polymorphisms to predict clinical outcome and renal function decline was analyzed. Results This study validated the association of these HLA-DQA1 and PLA2R1 SNPs with IMN in a Spanish cohort and its increased risk when combining both risk genotypes. No significant association was found between FCGR3 CNVs and IMN. These results revealed that HLA-DQA1 and PLA2R1 genotype combination adjusted for baseline proteinuria strongly predicted response to immunosuppressive therapy. HLA-DQA1 genotype adjusted for proteinuria was also linked with renal function decline. Conclusion This study confirms that HLA-DQA1 and PLA2R1 genotypes are risk factors for IMN, whereas no association was identified for FCGR3 CNVs. This study provides, for the first time, evidence of the contribution of these HLA-DQA1 and PLA2R1 polymorphisms in predicting IMN response to immunosuppressors and disease progression. Future studies are needed to validate and identify prognostic markers. PMID:24262501

Complement mRNA in the mammalian brain: responses to Alzheimer's disease and experimental brain lesioning.

PubMed

Johnson, S A; Lampert-Etchells, M; Pasinetti, G M; Rozovsky, I; Finch, C E

1992-01-01

This study describes evidence in the adult human and rat brain for mRNAs that encode two complement (C) proteins, C1qB and C4. C proteins are important effectors of humoral immunity and inflammation in peripheral tissues but have not been considered as normally present in brain. Previous immunocytochemical studies showed that C proteins are associated with plaques, tangles, and dystrophic neurites in Alzheimer's disease (AD), but their source is unknown. Combined immunocytochemistry and in situ hybridization techniques show C4 mRNA in pyramidal neurons and C1qB mRNA in microglia. Primary rat neuron cultures also show C1qB mRNA. In the cortex from AD brains, there were two- to threefold increases of C1qB mRNA and C4 mRNA, and increased C1qB mRNA prevalence was in part associated with microglia. As a model for AD, we examined entorhinal cortex perforant path transection in the rat brain, which caused rapid increases of C1qB mRNA in the ipsilateral, but not contralateral, hippocampus and entorhinal cortex. The role of brain-derived acute and chronic C induction during AD and experimental lesions can now be considered in relation to functions of C proteins that pertain to cell degeneration and/or cell preservation and synaptic plasticity.

Clinical Performance of an Ultrahigh Resolution Chromosomal Microarray Optimized for Neurodevelopmental Disorders.

PubMed

Ho, Karen S; Twede, Hope; Vanzo, Rena; Harward, Erin; Hensel, Charles H; Martin, Megan M; Page, Stephanie; Peiffer, Andreas; Mowery-Rushton, Patricia; Serrano, Moises; Wassman, E Robert

2016-01-01

Copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) significantly contribute to the etiology of neurodevelopmental disorders, such as developmental delay (DD), intellectual disability (ID), and autism spectrum disorder (ASD). This study summarizes the results of 3.5 years of CMA testing by a CLIA-certified clinical testing laboratory 5487 patients with neurodevelopmental conditions were clinically evaluated for rare copy number variants using a 2.8-million probe custom CMA optimized for the detection of CNVs associated with neurodevelopmental disorders. We report an overall detection rate of 29.4% in our neurodevelopmental cohort, which rises to nearly 33% when cases with DD/ID and/or MCA only are considered. The detection rate for the ASD cohort is also significant, at 25%. Additionally, we find that detection rate and pathogenic yield of CMA vary significantly depending on the primary indications for testing, the age of the individuals tested, and the specialty of the ordering doctor. We also report a significant difference between the detection rate on the ultrahigh resolution optimized array in comparison to the array from which it originated. This increase in detection can significantly contribute to the efficient and effective medical management of neurodevelopmental conditions in the clinic.

A genome-wide detection of copy number variation using SNP genotyping arrays in Beijing-You chickens.

PubMed

Zhou, Wei; Liu, Ranran; Zhang, Jingjing; Zheng, Maiqing; Li, Peng; Chang, Guobin; Wen, Jie; Zhao, Guiping

2014-10-01

Copy number variation (CNV) has been recently examined in many species and is recognized as being a source of genetic variability, especially for disease-related phenotypes. In this study, the PennCNV software, a genome-wide CNV detection system based on the 60 K SNP BeadChip was used on a total sample size of 1,310 Beijing-You chickens (a Chinese local breed). After quality control, 137 high confidence CNVRs covering 27.31 Mb of the chicken genome and corresponding to 2.61 % of the whole chicken genome. Within these regions, 131 known genes or coding sequences were involved. Q-PCR was applied to verify some of the genes related to disease development. Results showed that copy number of genes such as, phosphatidylinositol-5-phosphate 4-kinase II alpha, PHD finger protein 14, RHACD8 (a CD8α- like messenger RNA), MHC B-G, zinc finger protein, sarcosine dehydrogenase and ficolin 2 varied between individual chickens, which also supports the reliability of chip-detection of the CNVs. As one source of genomic variation, CNVs may provide new insight into the relationship between the genome and phenotypic characteristics.

Identification of C3b-Binding Small-Molecule Complement Inhibitors Using Cheminformatics.

PubMed

Garcia, Brandon L; Skaff, D Andrew; Chatterjee, Arindam; Hanning, Anders; Walker, John K; Wyckoff, Gerald J; Geisbrecht, Brian V

2017-05-01

The complement system is an elegantly regulated biochemical cascade formed by the collective molecular recognition properties and proteolytic activities of more than two dozen membrane-bound or serum proteins. Complement plays diverse roles in human physiology, such as acting as a sentry against invading microorganisms, priming of the adaptive immune response, and removal of immune complexes. However, dysregulation of complement can serve as a trigger for a wide range of human diseases, which include autoimmune, inflammatory, and degenerative conditions. Despite several potential advantages of modulating complement with small-molecule inhibitors, small-molecule drugs are highly underrepresented in the current complement-directed therapeutics pipeline. In this study, we have employed a cheminformatics drug discovery approach based on the extensive structural and functional knowledge available for the central proteolytic fragment of the cascade, C3b. Using parallel in silico screening methodologies, we identified 45 small molecules that putatively bind C3b near ligand-guided functional hot spots. Surface plasmon resonance experiments resulted in the validation of seven dose-dependent C3b-binding compounds. Competition-based biochemical assays demonstrated the ability of several C3b-binding compounds to interfere with binding of the original C3b ligand that guided their discovery. In vitro assays of complement function identified a single complement inhibitory compound, termed cmp-5, and mechanistic studies of the cmp-5 inhibitory mode revealed it acts at the level of C5 activation. This study has led to the identification of a promising new class of C3b-binding small-molecule complement inhibitors and, to our knowledge, provides the first demonstration of cheminformatics-based, complement-directed drug discovery. Copyright © 2017 by The American Association of Immunologists, Inc.

Identification of C3b-binding Small Molecule Complement Inhibitors Using Cheminformatics

PubMed Central

Garcia, Brandon L.; Skaff, D. Andrew; Chatterjee, Arindam; Hanning, Anders; Walker, John K.; Wyckoff, Gerald J.; Geisbrecht, Brian V.

2017-01-01

The complement system is an elegantly regulated biochemical cascade formed by the collective molecular recognition properties and proteolytic activities of over two dozen membrane-bound or serum proteins. Complement plays diverse roles in human physiology which include acting as a sentry against invading microorganisms, priming of the adaptive immune response, and removal of immune complexes. However, dysregulation of complement can serve as a trigger for a wide range of human diseases which include autoimmune, inflammatory, and degenerative conditions. Despite several potential advantages of modulating complement with small molecule inhibitors, small molecule drugs are highly underrepresented in the current complement-directed therapeutics pipeline. In this study we have employed a cheminformatics drug discovery approach based on the extensive structural and functional knowledge available for the central proteolytic fragment of the cascade, C3b. Using parallel in silico screening methodologies we identified 45 small molecules which putatively bind C3b near ligand-guided functional hot-spots. Surface plasmon resonance experiments resulted in the validation of seven dose-dependent C3b-binding compounds. Competition-based biochemical assays demonstrated the ability of several C3b-binding compounds to interfere with binding of the original C3b ligand which guided their discovery. In vitro assays of complement function identified a single complement inhibitory compound, termed cmp-5, and mechanistic studies of the cmp-5 inhibitory mode revealed it acts at the level of C5 activation. This study has led to the identification of a promising new class of C3b-binding small molecule complement inhibitors, and to our knowledge, provides the first demonstration of cheminformatics-based complement-directed drug discovery. PMID:28298523

Complement anaphylatoxins as immune regulators in cancer.

PubMed

Sayegh, Eli T; Bloch, Orin; Parsa, Andrew T

2014-08-01

The role of the complement system in innate immunity is well characterized. However, a recent body of research implicates the complement anaphylatoxins C3a and C5a as insidious propagators of tumor growth and progression. It is now recognized that certain tumors elaborate C3a and C5a and that complement, as a mediator of chronic inflammation and regulator of immune function, may in fact foster rather than defend against tumor growth. A putative mechanism for this function is complement-mediated suppression of immune effector cells responsible for immunosurveillance within the tumor microenvironment. This paradigm accords with models of immune dysregulation, such as autoimmunity and infectious disease, which have defined a pathophysiological role for abnormal complement signaling. Several types of immune cells express the cognate receptors for the complement anaphylatoxins, C3aR and C5aR, and demonstrate functional modulation in response to complement stimulation. In turn, impairment of antitumor immunity has been intimately tied to tumor progression in animal models of cancer. In this article, the literature was systematically reviewed to identify studies that have characterized the effects of the complement anaphylatoxins on the composition and function of immune cells within the tumor microenvironment. The search identified six studies based upon models of lymphoma and ovarian, cervical, lung, breast, and mammary cancer, which collectively support the paradigm of complement as an immune regulator in the tumor microenvironment. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.