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Sample records for functional promoter variant

  1. Two Functional Lupus-Associated BLK Promoter Variants Control Cell-Type- and Developmental-Stage-Specific Transcription

    PubMed Central

    Guthridge, Joel M.; Lu, Rufei; Sun, Harry; Sun, Celi; Wiley, Graham B.; Dominguez, Nicolas; Macwana, Susan R.; Lessard, Christopher J.; Kim-Howard, Xana; Cobb, Beth L.; Kaufman, Kenneth M.; Kelly, Jennifer A.; Langefeld, Carl D.; Adler, Adam J.; Harley, Isaac T.W.; Merrill, Joan T.; Gilkeson, Gary S.; Kamen, Diane L.; Niewold, Timothy B.; Brown, Elizabeth E.; Edberg, Jeffery C.; Petri, Michelle A.; Ramsey-Goldman, Rosalind; Reveille, John D.; Vilá, Luis M.; Kimberly, Robert P.; Freedman, Barry I.; Stevens, Anne M.; Boackle, Susan A.; Criswell, Lindsey A.; Vyse, Tim J.; Behrens, Timothy W.; Jacob, Chaim O.; Alarcón-Riquelme, Marta E.; Sivils, Kathy L.; Choi, Jiyoung; Joo, Young Bin; Bang, So-Young; Lee, Hye-Soon; Bae, Sang-Cheol; Shen, Nan; Qian, Xiaoxia; Tsao, Betty P.; Scofield, R. Hal; Harley, John B.; Webb, Carol F.; Wakeland, Edward K.; James, Judith A.; Nath, Swapan K.; Graham, Robert R.; Gaffney, Patrick M.

    2014-01-01

    Efforts to identify lupus-associated causal variants in the FAM167A/BLK locus on 8p21 are hampered by highly associated noncausal variants. In this report, we used a trans-population mapping and sequencing strategy to identify a common variant (rs922483) in the proximal BLK promoter and a tri-allelic variant (rs1382568) in the upstream alternative BLK promoter as putative causal variants for association with systemic lupus erythematosus. The risk allele (T) at rs922483 reduced proximal promoter activity and modulated alternative promoter usage. Allelic differences at rs1382568 resulted in altered promoter activity in B progenitor cell lines. Thus, our results demonstrated that both lupus-associated functional variants contribute to the autoimmune disease association by modulating transcription of BLK in B cells and thus potentially altering immune responses. PMID:24702955

  2. Genetic and Functional Sequence Variants of the SIRT3 Gene Promoter in Myocardial Infarction

    PubMed Central

    Yin, Xiaoyun; Pang, Shuchao; Huang, Jian; Cui, Yinghua; Yan, Bo

    2016-01-01

    Coronary artery disease (CAD), including myocardial infarction (MI), is a common complex disease that is caused by atherosclerosis. Although a large number of genetic variants have been associated with CAD, only 10% of CAD cases could be explained. It has been proposed that low frequent and rare genetic variants may be main causes for CAD. SIRT3, a mitochondrial deacetylase, plays important roles in mitochondrial function and metabolism. Lack of SIRT3 in experimental animal leads to several age-related diseases, including cardiovascular diseases. Therefore, SIRT3 gene variants may contribute to the MI development. In this study, SIRT3 gene promoter was genetically and functionally analyzed in large cohorts of MI patients (n = 319) and ethnic-matched controls (n = 322). Total twenty-three DNA sequence variants (DSVs) were identified, including 10 single-nucleotide polymorphisms (SNPs). Six novel heterozygous DSVs, g.237307A>G, g.237270G>A, g.237023_25del, g.236653C>A, g.236628G>C, g.236557T>C, and two SNPs g.237030C>T (rs12293349) and g.237022C>G (rs369344513), were identified in nine MI patients, but in none of controls. Three SNPs, g.236473C>T (rs11246029), g.236380_81ins (rs71019893) and g.236370C>G (rs185277566), were more significantly frequent in MI patients than controls (P<0.05). These DSVs and SNPs, except g.236557T>C, significantly decreased the transcriptional activity of the SIRT3 gene promoter in cultured HEK-293 cells and H9c2 cells. Therefore, these DSVs identified in MI patients may change SIRT3 level by affecting the transcriptional activity of SIRT3 gene promoter, contributing to the MI development as a risk factor. PMID:27078640

  3. GADD45a Promoter Regulation by a Functional Genetic Variant Associated with Acute Lung Injury

    PubMed Central

    Mitra, Sumegha; Wade, Michael S.; Sun, Xiaoguang; Moldobaeva, Nurgul; Flores, Carlos; Ma, Shwu-Fan; Zhang, Wei

    2014-01-01

    Rationale Growth arrest DNA damage inducible alpha (GADD45a) is a stress-induced gene we have shown to participate in the pathophysiology of ventilator-induced lung injury (VILI) via regulation of mechanical stress-induced Akt ubiquitination and phosphorylation. The regulation of GADD45a expression by mechanical stress and its relationship with acute lung injury (ALI) susceptibility and severity, however, remains unknown. Objectives We examined mechanical stress-dependent regulatory elements (MSRE) in the GADD45a promoter and the contribution of promoter polymorphisms in GADD45a expression and ALI susceptibility. Methods and Results Initial studies in GADD45a knockout and heterozygous mice confirmed the relationship of GADD45a gene dose to VILI severity. Human lung endothelial cells (EC) transfected with a luciferase vector containing the full length GADD45a promoter sequence (−771 to +223) demonstrated a >4 fold increase in GADD45a expression in response to 18% cyclic stretch (CS, 4 h) compared to static controls while specific promoter regions harboring CS-dependent MSRE were identified using vectors containing serial deletion constructs of the GADD45a promoter. In silico analyses of GADD45a promoter region (−371 to −133) revealed a potential binding site for specificity protein 1 (SP1), a finding supported by confirmed SP1 binding with the GADD45a promoter and by the significant attenuation of CS-dependent GADD45a promoter activity in response to SP1 silencing. Separately, case-control association studies revealed a significant association of a GADD45a promoter SNP at −589 (rs581000, G>C) with reduced ALI susceptibility. Subsequently, we found allelic variation of this SNP is associated with both differential GADD45a expression in mechanically stressed EC (18% CS, 4 h) and differential binding site of interferon regulatory factor 7 (IRF7) at this site. Conclusion These results strongly support a functional role for GADD45a in ALI/VILI and identify a

  4. Variants of the CNTNAP2 5' promoter as risk factors for autism spectrum disorders: a genetic and functional approach.

    PubMed

    Chiocchetti, A G; Kopp, M; Waltes, R; Haslinger, D; Duketis, E; Jarczok, T A; Poustka, F; Voran, A; Graab, U; Meyer, J; Klauck, S M; Fulda, S; Freitag, C M

    2015-07-01

    Contactin-associated protein-like 2 gene (CNTNAP2), a member of the Neurexin gene superfamily, is one of the best-replicated risk genes for autism spectrum disorders (ASD). ASD are predominately genetically determined neurodevelopmental disorders characterized by impairments of language development, social interaction and communication, as well as stereotyped behavior and interests. Although CNTNAP2 expression levels were proposed to alter ASD risk, no study to date has focused on its 5' promoter. Here, we directly sequenced the CNTNAP2 5' promoter region of 236 German families with one child with ASD and detected four novel variants. Furthermore, we genotyped the three most frequent variants (rs150447075, rs34712024, rs71781329) in an additional sample of 356 families and found nominal association of rs34712024G with ASD and rs71781329GCG[7] with language development. The four novel and the three known minor alleles of the identified variants were predicted to alter transcription factor binding sites (TFBS). At the functional level, the respective sequences spanning these seven variants were bound by nuclear factors. In a luciferase promoter assay, the respective minor alleles showed cell line-specific and differentiation stage-dependent effects at the level of promoter activation. The novel potential rare risk-variant M2, a G>A mutation -215 base pairs 5' of the transcriptional start site, significantly reduced promoter efficiency in HEK293T and in undifferentiated and differentiated neuroblastoid SH-SY5Y cells. This variant was transmitted to a patient with autistic disorder. The under-transmitted, protective minor G allele of the common variant rs34712024, in contrast, increased transcriptional activity. These results lead to the conclusion that the pathomechanism of CNTNAP2 promoter variants on ASD risk is mediated by their effect on TFBSs, and thus confirm the hypothesis that a reduced CNTNAP2 level during neuronal development increases liability for ASD

  5. Autism-Associated Promoter Variant in MET Impacts Functional and Structural Brain Networks

    PubMed Central

    Rudie, J. D.; Hernandez, L. M.; Brown, J. A.; Beck-Pancer, D.; Colich, N. L.; Gorrindo, P.; Thompson, P. M.; Geschwind, D. H.; Bookheimer, S. Y.; Levitt, P.; Dapretto, M.

    2012-01-01

    SUMMARY As genes that confer increased risk for autism spectrum disorder (ASD) are identified, a crucial next step is to determine how these risk factors impact brain structure and function and contribute to disorder heterogeneity. With three converging lines of evidence, we show that a common, functional ASD risk variant in the Met Receptor Tyrosine Kinase (MET) gene is a potent modulator of key social brain circuitry in children and adolescents with and without ASD. MET risk genotype predicted atypical fMRI activation and deactivation patterns to social stimuli (i.e., emotional faces), as well as reduced functional and structural connectivity in temporo-parietal regions known to have high MET expression, particularly within the default mode network. Notably, these effects were more pronounced in individuals with ASD. These findings highlight how genetic stratification may reduce heterogeneity and help elucidate the biological basis of complex neuropsychiatric disorders such as ASD. PMID:22958829

  6. Functional differences exist between TNFα promoters encoding the common -237G SNP and the rarer HLA-B*5701-linked A variant.

    PubMed

    Simpson, Peter D; Moysi, Eirini; Wicks, Kate; Sudan, Kritika; Rowland-Jones, Sarah L; McMichael, Andrew J; Knight, Julian; Gillespie, Geraldine M

    2012-01-01

    A large body of functional and epidemiological evidence have previously illustrated the impact of specific MHC class I subtypes on clinical outcome during HIV-1 infection, and these observations have recently been re-iterated in genome wide association studies (GWAS). Yet because of the complexities surrounding GWAS-based approaches and the lack of knowledge relating to the identity of rarer single nucleotide polymorphism (SNP) variants, it has proved difficult to discover independent causal variants associated with favourable immune control. This is especially true of the candidate variants within the HLA region where many of the recently proposed disease influencing SNPs appear to reflect linkage with 'protective' MHC class I alleles. Yet causal MHC-linked SNPs may exist but remain overlooked owing to the complexities associated with their identification. Here we focus on the ancestral TNFα promoter -237A variant (rs361525), shown historically to be in complete linkage disequilibrium with the 'protective' HLA-B*5701 allele. Many of the ancestral SNPs within the extended TNFα promoter have been associated with both autoimmune conditions and disease outcomes, however, the direct role of these variants on TNFα expression remains controversial. Yet, because of the important role played by TNFα in HIV-1 infection, and given the proximity of the -237 SNP to the core promoter, its location within a putative repressor region previously characterized in mice, and its disruption of a methylation-susceptible CpG dinucleotide motif, we chose to carefully evaluate its impact on TNFα production. Using a variety of approaches we now demonstrate that carriage of the A SNP is associated with lower TNFα production, via a mechanism not readily explained by promoter methylation nor the binding of transcription factors or repressors. We propose that the -237A variant could represent a minor causal SNP that additionally contributes to the HLA-B*5701-mediated 'protective' effect

  7. Functional Variant in Complement C3 Gene Promoter and Genetic Susceptibility to Temporal Lobe Epilepsy and Febrile Seizures

    PubMed Central

    Perroud, Nader; Ponsole-Lenfant, Magali; Cillario, Jennifer; Roll, Patrice; Roeckel-Trevisiol, Nathalie; Crespel, Ariel; Balzar, Jorg; Schlachter, Kurt; Gruber-Sedlmayr, Ursula; Pataraia, Ekaterina; Baumgartner, Christoph; Zimprich, Alexander; Zimprich, Fritz; Malafosse, Alain; Szepetowski, Pierre

    2010-01-01

    Background Human mesial temporal lobe epilepsies (MTLE) represent the most frequent form of partial epilepsies and are frequently preceded by febrile seizures (FS) in infancy and early childhood. Genetic associations of several complement genes including its central component C3 with disorders of the central nervous system, and the existence of C3 dysregulation in the epilepsies and in the MTLE particularly, make it the C3 gene a good candidate for human MTLE. Methodology/Principal Findings A case-control association study of the C3 gene was performed in a first series of 122 patients with MTLE and 196 controls. Four haplotypes (HAP1 to 4) comprising GF100472, a newly discovered dinucleotide repeat polymorphism [(CA)8 to (CA)15] in the C3 promoter region showed significant association after Bonferroni correction, in the subgroup of MTLE patients having a personal history of FS (MTLE-FS+). Replication analysis in independent patients and controls confirmed that the rare HAP4 haplotype comprising the minimal length allele of GF100472 [(CA)8], protected against MTLE-FS+. A fifth haplotype (HAP5) with medium-size (CA)11 allele of GF100472 displayed four times higher frequency in controls than in the first cohort of MTLE-FS+ and showed a protective effect against FS through a high statistical significance in an independent population of 97 pure FS. Consistently, (CA)11 allele by its own protected against pure FS in a second group of 148 FS patients. Reporter gene assays showed that GF100472 significantly influenced C3 promoter activity (the higher the number of repeats, the lower the transcriptional activity). Taken together, the consistent genetic data and the functional analysis presented here indicate that a newly-identified and functional polymorphism in the promoter of the complement C3 gene might participate in the genetic susceptibility to human MTLE with a history of FS, and to pure FS. Conclusions/Significance The present study provides important data

  8. A Functional Variant in the Stearoyl-CoA Desaturase Gene Promoter Enhances Fatty Acid Desaturation in Pork

    PubMed Central

    Estany, Joan; Ros-Freixedes, Roger; Tor, Marc; Pena, Ramona N.

    2014-01-01

    There is growing public concern about reducing saturated fat intake. Stearoyl-CoA desaturase (SCD) is the lipogenic enzyme responsible for the biosynthesis of oleic acid (18∶1) by desaturating stearic acid (18∶0). Here we describe a total of 18 mutations in the promoter and 3′ non-coding region of the pig SCD gene and provide evidence that allele T at AY487830:g.2228T>C in the promoter region enhances fat desaturation (the ratio 18∶1/18∶0 in muscle increases from 3.78 to 4.43 in opposite homozygotes) without affecting fat content (18∶0+18∶1, intramuscular fat content, and backfat thickness). No mutations that could affect the functionality of the protein were found in the coding region. First, we proved in a purebred Duroc line that the C-T-A haplotype of the 3 single nucleotide polymorphisms (SNPs) (g.2108C>T; g.2228T>C; g.2281A>G) of the promoter region was additively associated to enhanced 18∶1/18∶0 both in muscle and subcutaneous fat, but not in liver. We show that this association was consistent over a 10-year period of overlapping generations and, in line with these results, that the C-T-A haplotype displayed greater SCD mRNA expression in muscle. The effect of this haplotype was validated both internally, by comparing opposite homozygote siblings, and externally, by using experimental Duroc-based crossbreds. Second, the g.2281A>G and the g.2108C>T SNPs were excluded as causative mutations using new and previously published data, restricting the causality to g.2228T>C SNP, the last source of genetic variation within the haplotype. This mutation is positioned in the core sequence of several putative transcription factor binding sites, so that there are several plausible mechanisms by which allele T enhances 18∶1/18∶0 and, consequently, the proportion of monounsaturated to saturated fat. PMID:24465944

  9. Structural characteristics of two wheat histone H2A genes encoding distinct types of variants and functional differences in their promoter activity.

    PubMed

    Huh, G H; Nakayama, T; Meshi, T; Iwabuchi, M

    1997-03-01

    To investigate the regulation of plant histone H2A gene expression, we isolated two H2A genes (TH254 and TH274) from wheat, which encode two variants of H2A. Both genes had an intron in the coding region. In the promoters, some characteristic sequences, such as Oct and Nona motifs, which are conserved among plant histone genes, were located in a short region (about 120 bp) upstream from the putative TATA box. Transient expression analyses of promoter activity with H2A-GUS fusion genes using tobacco protoplasts revealed novel types of positive cis-acting sequences in the TH254 promoter: a direct repeat of a 13 bp sequence (AGTTACATTATTG) and a stretch composed of an AT-rich sequence (ATATAGAAAATTAAAA) and a G-box (CACGTG). Quantitative S1 assay of the mRNA amounts from the TH254/GUS and TH274/GUS chimeric genes in stably transformed and cell cycle-synchronized tobacco cell lines showed that the promoters of both genes contained at least one cis-acting element responsible for S phase-specific expression. Histochemical analysis of transgenic tobacco plants carrying the chimeric genes showed that the promoters of the two H2A genes were active in developing seedlings and flower organs but were regulated in a different manner. PMID:9106503

  10. A splicing variant of Merlin promotes metastasis in hepatocellular carcinoma

    PubMed Central

    Luo, Zai-Li; Cheng, Shu-Qun; Shi, Jie; Zhang, Hui-Lu; Zhang, Cun-Zhen; Chen, Hai-Yang; Qiu, Bi-Jun; Tang, Liang; Hu, Cong-Li; Wang, Hong-Yang; Li, Zhong

    2015-01-01

    Merlin, which is encoded by the tumour suppressor gene Nf2, plays a crucial role in tumorigenesis and metastasis. However, little is known about the functional importance of Merlin splicing forms. In this study, we show that Merlin is present at low levels in human hepatocellular carcinoma (HCC), particularly in metastatic tumours, where it is associated with a poor prognosis. Surprisingly, a splicing variant of Merlin that lacks exons 2, 3 and 4 (Δ2–4Merlin) is amplified in HCC and portal vein tumour thrombus (PVTT) specimens and in the CSQT2 cell line derived from PVTT. Our studies show that Δ2–4Merlin interferes with the capacity of wild-type Merlin to bind β-catenin and ERM, and it is expressed in the cytoplasm rather than at the cell surface. Furthermore, Δ2–4Merlin overexpression increases the expression levels of β-catenin and stemness-related genes, induces the epithelium–mesenchymal-transition phenotype promoting cell migration in vitro and the formation of lung metastasis in vivo. Our results indicate that the Δ2–4Merlin variant disrupts the normal function of Merlin and promotes tumour metastasis. PMID:26443326

  11. Exome-based Variant Detection in Core Promoters

    PubMed Central

    Kim, Yeong C.; Cui, Jian; Luo, Jiangtao; Xiao, Fengxia; Downs, Bradley; Wang, San Ming

    2016-01-01

    Core promoter controls the initiation of transcription. Core promoter sequence change can disrupt transcriptional regulation, lead to impairment of gene expression and ultimately diseases. Therefore, comprehensive characterization of core promoters is essential to understand normal and abnormal gene expression in biomedical studies. Here we report the development of EVDC (Exome-based Variant Detection in Core promoters) method for genome-scale analysis of core-promoter sequence variation. This method is based on the fact that exome sequences contain the sequences not only from coding exons but also from non-coding region including core promoters generated by random fragmentation in exome sequencing process. Using exome data from three cell types of CD4+ T cells, CD19+ B cells and neutrophils of a single individual, we characterized the features of core promoter-mapped exome sequences, and analysed core-promoter variation in this individual genome. We also compared the core promoters between YRI (Yoruba in Ibadan, Nigeria) and the CEU (Utah residents of European decedent) populations using the exome data generated by the 1000 Genome project, and observed much higher variation in YRI population than in CEU population. Our study demonstrates that the EVDC method provides a simple but powerful means for genome-wile de novo characterization of core promoter sequence variation. PMID:27464681

  12. Exome-based Variant Detection in Core Promoters.

    PubMed

    Kim, Yeong C; Cui, Jian; Luo, Jiangtao; Xiao, Fengxia; Downs, Bradley; Wang, San Ming

    2016-01-01

    Core promoter controls the initiation of transcription. Core promoter sequence change can disrupt transcriptional regulation, lead to impairment of gene expression and ultimately diseases. Therefore, comprehensive characterization of core promoters is essential to understand normal and abnormal gene expression in biomedical studies. Here we report the development of EVDC (Exome-based Variant Detection in Core promoters) method for genome-scale analysis of core-promoter sequence variation. This method is based on the fact that exome sequences contain the sequences not only from coding exons but also from non-coding region including core promoters generated by random fragmentation in exome sequencing process. Using exome data from three cell types of CD4+ T cells, CD19+ B cells and neutrophils of a single individual, we characterized the features of core promoter-mapped exome sequences, and analysed core-promoter variation in this individual genome. We also compared the core promoters between YRI (Yoruba in Ibadan, Nigeria) and the CEU (Utah residents of European decedent) populations using the exome data generated by the 1000 Genome project, and observed much higher variation in YRI population than in CEU population. Our study demonstrates that the EVDC method provides a simple but powerful means for genome-wile de novo characterization of core promoter sequence variation. PMID:27464681

  13. Understanding the Pathogenicity of Noncoding Mismatch Repair Gene Promoter Variants in Lynch Syndrome.

    PubMed

    Liu, Qing; Thompson, Bryony A; Ward, Robyn L; Hesson, Luke B; Sloane, Mathew A

    2016-05-01

    Lynch syndrome is the most common familial cancer condition that mainly predisposes to tumors of the colon and endometrium. Cancer susceptibility is caused by the autosomal dominant inheritance of a loss-of-function mutation or epimutation in one of the DNA mismatch repair (MMR) genes. Cancer risk assessment is often possible with nonsynonymous coding region mutations, but in many cases patients present with DNA sequence changes within noncoding regions, including the promoters, of MMR genes. The pathogenic role of promoter variants, and hence clinical significance, is unclear and this hinders the clinical management of carriers. In this review, we provide an overview of the classification of MMR gene variants, outline the laboratory assays and online resources that can be used to assess the causality of promoter variants in Lynch syndrome, and highlight some of the practical challenges of demonstrating the pathogenicity of these variants. In conclusion, we propose a guide that could be integrated into the current InSiGHT classification scheme to help determine if a MMR gene promoter variant is pathogenic. PMID:26888055

  14. COMT gene locus: new functional variants.

    PubMed

    Meloto, Carolina B; Segall, Samantha K; Smith, Shad; Parisien, Marc; Shabalina, Svetlana A; Rizzatti-Barbosa, Célia M; Gauthier, Josée; Tsao, Douglas; Convertino, Marino; Piltonen, Marjo H; Slade, Gary Dmitri; Fillingim, Roger B; Greenspan, Joel D; Ohrbach, Richard; Knott, Charles; Maixner, William; Zaykin, Dmitri; Dokholyan, Nikolay V; Reenilä, Ilkka; Männistö, Pekka T; Diatchenko, Luda

    2015-10-01

    Catechol-O-methyltransferase (COMT) metabolizes catecholaminergic neurotransmitters. Numerous studies have linked COMT to pivotal brain functions such as mood, cognition, response to stress, and pain. Both nociception and risk of clinical pain have been associated with COMT genetic variants, and this association was shown to be mediated through adrenergic pathways. Here, we show that association studies between COMT polymorphic markers and pain phenotypes in 2 independent cohorts identified a functional marker, rs165774, situated in the 3' untranslated region of a newfound splice variant, (a)-COMT. Sequence comparisons showed that the (a)-COMT transcript is highly conserved in primates, and deep sequencing data demonstrated that (a)-COMT is expressed across several human tissues, including the brain. In silico analyses showed that the (a)-COMT enzyme features a distinct C-terminus structure, capable of stabilizing substrates in its active site. In vitro experiments demonstrated not only that (a)-COMT is catalytically active but also that it displays unique substrate specificity, exhibiting enzymatic activity with dopamine but not epinephrine. They also established that the pain-protective A allele of rs165774 coincides with lower COMT activity, suggesting contribution to decreased pain sensitivity through increased dopaminergic rather than decreased adrenergic tone, characteristic of reference isoforms. Our results provide evidence for an essential role of the (a)-COMT isoform in nociceptive signaling and suggest that genetic variations in (a)-COMT isoforms may contribute to individual variability in pain phenotypes. PMID:26207649

  15. Functional Promoter Variant rs2868371 of HSPB1 Is Associated With Risk of Radiation Pneumonitis After Chemoradiation for Non-Small Cell Lung Cancer

    SciTech Connect

    Pang, Qingsong; Wei, Qingyi; Xu, Ting; Yuan, Xianglin; Lopez Guerra, Jose Luis; Levy, Lawrence B.; Liu, Zhensheng; Gomez, Daniel R.; Zhuang, Yan; Wang, Li-E.; Mohan, Radhe; Komaki, Ritsuko; Liao, Zhongxing

    2013-04-01

    Purpose: To date, no biomarkers have been found to predict, before treatment, which patients will develop radiation pneumonitis (RP), a potentially fatal toxicity, after chemoradiation for lung cancer. We investigated potential associations between single nucleotide polymorphisms (SNPs) in HSPB1 and risk of RP after chemoradiation for non-small cell lung cancer (NSCLC). Methods and Materials: Subjects were patients with NSCLC treated with chemoradiation at 1 institution. The training data set comprised 146 patients treated from 1999 to July 2004; the validation data set was 125 patients treated from August 2004 to March 2010. We genotyped 2 functional SNPs of HSPB1 (rs2868370 and rs2868371) from all patients. We used Kaplan-Meier analysis to assess the risk of grade ≥2 or ≥3 RP in both data sets and a parametric log-logistic survival model to evaluate the association of HSPB1 genotypes with that risk. Results: Grade ≥3 RP was experienced by 13% of those with CG/GG and 29% of those with CC genotype of HSPB1 rs2868371 in the training data set (P=.028); corresponding rates in the validation data set were 2% CG/GG and 14% CC (P=.02). Univariate and multivariate analysis confirmed the association of CC of HSPB1 rs2868371 with higher risk of grade ≥3 RP than CG/GG after adjustment for sex, age, performance status, and lung mean dose. This association was validated both in the validation data set and with Harrell's C statistic. Conclusions: The CC genotype of HSPB1 rs2868371 was associated with severe RP after chemoradiation for NSCLC.

  16. Functional annotation of non-coding sequence variants

    PubMed Central

    Ritchie, Graham R. S.; Dunham, Ian; Zeggini, Eleftheria; Flicek, Paul

    2016-01-01

    Identifying functionally relevant variants against the background of ubiquitous genetic variation is a major challenge in human genetics. For variants that fall in protein-coding regions our understanding of the genetic code and splicing allow us to identify likely candidates, but interpreting variants that fall outside of genic regions is more difficult. Here we present a new tool, GWAVA, which supports prioritisation of non-coding variants by integrating a range of annotations. PMID:24487584

  17. Human Dopamine β-Hydroxylase Promoter Variant Alters Transcription in Chromaffin Cells, Enzyme Secretion, and Blood Pressure

    PubMed Central

    Chen, Yuqing; Zhang, Kuixing; Wen, Gen; Rao, Fangwen; Sanchez, Amber P.; Wang, Lei; Rodriguez-Flores, Juan L.; Mahata, Manjula; Mahata, Sushil K.; Waalen, Jill; Ziegler, Michael G.; Hamilton, Bruce A.

    2011-01-01

    Background Dopamine β-hydroxylase (DBH) plays an indispensable role in catecholamine synthesis by converting dopamine into norepinephrine. Here, we characterized a DBH promoter polymorphism (C-2073T; rs1989787; minor allele frequency ∼16%) that influences not only gene transcription but also enzyme secretion and blood pressure (BP) in vivo. Methods Plasma DBH activity was measured spectrophotometrically. DBH genetic effects on BP were tested in subjects with the most extreme BP values in a large primary care population. Functional effects of promoter variants were studied by site-directed mutagenesis in DBH promoter haplotype/luciferase reporter plasmids transfected into chromaffin cells. Sequence motifs were predicted from position weight matrices, and endogenous transcription factor binding was probed by Chromatin ImmunoPrecipitation (ChIP). Results The T-allele of common promoter variant C-2073T was contained in a promoter haplotype that associated with plasma DBH activity, a trait also predicted by that variant itself. Promoter haplotypes including C-2073T predicted BP in the population, and the effect was also referable to C-2073T itself. Computationally, C-2073 disrupted a predicted match for transcription factor c-FOS. Site-directed mutagenesis at C-2073T altered not only basal promoter activity, but also transactivation by c-FOS, as well as the chromaffin cell secretory stimuli nicotine or pituitary adenylate cyclase-activating polypeptide (PACAP). Endogenous c-FOS bound to the motif in chromatin. Conclusion These results suggest that DBH promoter variant C-2073T is functional in vivo: this promoter variant seems to initiate a cascade of transcriptional and biochemical changes including augmented DBH secretion, eventuating in elevation of basal BP, and hence cardiovascular risk. The observations suggest new strategies for probing the pathophysiology, risk, and treatment of hypertension. American Journal of Hypertension, advance online publication 2

  18. A disease-associated PTPN22 variant promotes systemic autoimmunity in murine models

    PubMed Central

    Dai, Xuezhi; James, Richard G.; Habib, Tania; Singh, Swati; Jackson, Shaun; Khim, Socheath; Moon, Randall T.; Liggitt, Denny; Wolf-Yadlin, Alejandro; Buckner, Jane H.; Rawlings, David J.

    2013-01-01

    Multiple autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, Graves disease, and systemic lupus erythematosus, are associated with an allelic variant of protein tyrosine phosphatase nonreceptor 22 (PTPN22), which encodes the protein LYP. To model the human disease-linked variant LYP-R620W, we generated knockin mice expressing the analogous mutation, R619W, in the murine ortholog PEST domain phosphatase (PEP). In contrast with a previous report, we found that this variant exhibits normal protein stability, but significantly alters lymphocyte function. Aged knockin mice exhibited effector T cell expansion and transitional, germinal center, and age-related B cell expansion as well as the development of autoantibodies and systemic autoimmunity. Further, PEP-R619W affected B cell selection and B lineage–restricted variant expression and was sufficient to promote autoimmunity. Consistent with these features, PEP-R619W lymphocytes were hyperresponsive to antigen-receptor engagement with a distinct profile of tyrosine-phosphorylated substrates. Thus, PEP-R619W uniquely modulates T and B cell homeostasis, leading to a loss in tolerance and autoimmunity. PMID:23619366

  19. The personal genome browser: visualizing functions of genetic variants

    PubMed Central

    Juan, Liran; Teng, Mingxiang; Zang, Tianyi; Hao, Yafeng; Wang, Zhenxing; Yan, Chengwu; Liu, Yongzhuang; Li, Jie; Zhang, Tianjiao; Wang, Yadong

    2014-01-01

    Advances in high-throughput sequencing technologies have brought us into the individual genome era. Projects such as the 1000 Genomes Project have led the individual genome sequencing to become more and more popular. How to visualize, analyse and annotate individual genomes with knowledge bases to support genome studies and personalized healthcare is still a big challenge. The Personal Genome Browser (PGB) is developed to provide comprehensive functional annotation and visualization for individual genomes based on the genetic–molecular–phenotypic model. Investigators can easily view individual genetic variants, such as single nucleotide variants (SNVs), INDELs and structural variations (SVs), as well as genomic features and phenotypes associated to the individual genetic variants. The PGB especially highlights potential functional variants using the PGB built-in method or SIFT/PolyPhen2 scores. Moreover, the functional risks of genes could be evaluated by scanning individual genetic variants on the whole genome, a chromosome, or a cytoband based on functional implications of the variants. Investigators can then navigate to high risk genes on the scanned individual genome. The PGB accepts Variant Call Format (VCF) and Genetic Variation Format (GVF) files as the input. The functional annotation of input individual genome variants can be visualized in real time by well-defined symbols and shapes. The PGB is available at http://www.pgbrowser.org/. PMID:24799434

  20. The personal genome browser: visualizing functions of genetic variants.

    PubMed

    Juan, Liran; Teng, Mingxiang; Zang, Tianyi; Hao, Yafeng; Wang, Zhenxing; Yan, Chengwu; Liu, Yongzhuang; Li, Jie; Zhang, Tianjiao; Wang, Yadong

    2014-07-01

    Advances in high-throughput sequencing technologies have brought us into the individual genome era. Projects such as the 1000 Genomes Project have led the individual genome sequencing to become more and more popular. How to visualize, analyse and annotate individual genomes with knowledge bases to support genome studies and personalized healthcare is still a big challenge. The Personal Genome Browser (PGB) is developed to provide comprehensive functional annotation and visualization for individual genomes based on the genetic-molecular-phenotypic model. Investigators can easily view individual genetic variants, such as single nucleotide variants (SNVs), INDELs and structural variations (SVs), as well as genomic features and phenotypes associated to the individual genetic variants. The PGB especially highlights potential functional variants using the PGB built-in method or SIFT/PolyPhen2 scores. Moreover, the functional risks of genes could be evaluated by scanning individual genetic variants on the whole genome, a chromosome, or a cytoband based on functional implications of the variants. Investigators can then navigate to high risk genes on the scanned individual genome. The PGB accepts Variant Call Format (VCF) and Genetic Variation Format (GVF) files as the input. The functional annotation of input individual genome variants can be visualized in real time by well-defined symbols and shapes. The PGB is available at http://www.pgbrowser.org/. PMID:24799434

  1. A Rb1 promoter variant with reduced activity contributes to osteosarcoma susceptibility in irradiated mice

    PubMed Central

    2014-01-01

    Background Syndromic forms of osteosarcoma (OS) account for less than 10% of all recorded cases of this malignancy. An individual OS predisposition is also possible by the inheritance of low penetrance alleles of tumor susceptibility genes, usually without evidence of a syndromic condition. Genetic variants involved in such a non-syndromic form of tumor predisposition are difficult to identify, given the low incidence of osteosarcoma cases and the genetic heterogeneity of patients. We recently mapped a major OS susceptibility QTL to mouse chromosome 14 by comparing alpha-radiation induced osteosarcoma in mouse strains which differ in their tumor susceptibility. Methods Tumor-specific allelic losses in murine osteosacoma were mapped along chromosome 14 using microsatellite markers and SNP allelotyping. Candidate gene search in the mapped interval was refined using PosMed data mining and mRNA expression analysis in normal osteoblasts. A strain-specific promoter variant in Rb1 was tested for its influence on mRNA expression using reporter assay. Results A common Rb1 allele derived from the BALB/cHeNhg strain was identified as the major determinant of radiation-induced OS risk at this locus. Increased OS-risk is linked with a hexanucleotide deletion in the promoter region which is predicted to change WT1 and SP1 transcription factor-binding sites. Both in-vitro reporter and in-vivo expression assays confirmed an approx. 1.5 fold reduced gene expression by this promoter variant. Concordantly, the 50% reduction in Rb1 expression in mice bearing a conditional hemizygous Rb1 deletion causes a significant rise of OS incidence following alpha-irradiation. Conclusion This is the first experimental demonstration of a functional and genetic link between reduced Rb1 expression from a common promoter variant and increased tumor risk after radiation exposure. We propose that a reduced Rb1 expression by common variants in regulatory regions can modify the risk for a malignant

  2. Fine-Mapping of Common Genetic Variants Associated with Colorectal Tumor Risk Identified Potential Functional Variants

    PubMed Central

    Gala, Manish; Abecasis, Goncalo; Bezieau, Stephane; Brenner, Hermann; Butterbach, Katja; Caan, Bette J.; Carlson, Christopher S.; Casey, Graham; Chang-Claude, Jenny; Conti, David V.; Curtis, Keith R.; Duggan, David; Gallinger, Steven; Haile, Robert W.; Harrison, Tabitha A.; Hayes, Richard B.; Hoffmeister, Michael; Hopper, John L.; Hudson, Thomas J.; Jenkins, Mark A.; Küry, Sébastien; Le Marchand, Loic; Leal, Suzanne M.; Newcomb, Polly A.; Nickerson, Deborah A.; Potter, John D.; Schoen, Robert E.; Schumacher, Fredrick R.; Seminara, Daniela; Slattery, Martha L.; Hsu, Li; Chan, Andrew T.; White, Emily; Berndt, Sonja I.; Peters, Ulrike

    2016-01-01

    Genome-wide association studies (GWAS) have identified many common single nucleotide polymorphisms (SNPs) associated with colorectal cancer risk. These SNPs may tag correlated variants with biological importance. Fine-mapping around GWAS loci can facilitate detection of functional candidates and additional independent risk variants. We analyzed 11,900 cases and 14,311 controls in the Genetics and Epidemiology of Colorectal Cancer Consortium and the Colon Cancer Family Registry. To fine-map genomic regions containing all known common risk variants, we imputed high-density genetic data from the 1000 Genomes Project. We tested single-variant associations with colorectal tumor risk for all variants spanning genomic regions 250-kb upstream or downstream of 31 GWAS-identified SNPs (index SNPs). We queried the University of California, Santa Cruz Genome Browser to examine evidence for biological function. Index SNPs did not show the strongest association signals with colorectal tumor risk in their respective genomic regions. Bioinformatics analysis of SNPs showing smaller P-values in each region revealed 21 functional candidates in 12 loci (5q31.1, 8q24, 11q13.4, 11q23, 12p13.32, 12q24.21, 14q22.2, 15q13, 18q21, 19q13.1, 20p12.3, and 20q13.33). We did not observe evidence of additional independent association signals in GWAS-identified regions. Our results support the utility of integrating data from comprehensive fine-mapping with expanding publicly available genomic databases to help clarify GWAS associations and identify functional candidates that warrant more onerous laboratory follow-up. Such efforts may aid the eventual discovery of disease-causing variant(s). PMID:27379672

  3. Fine-Mapping of Common Genetic Variants Associated with Colorectal Tumor Risk Identified Potential Functional Variants.

    PubMed

    Du, Mengmeng; Jiao, Shuo; Bien, Stephanie A; Gala, Manish; Abecasis, Goncalo; Bezieau, Stephane; Brenner, Hermann; Butterbach, Katja; Caan, Bette J; Carlson, Christopher S; Casey, Graham; Chang-Claude, Jenny; Conti, David V; Curtis, Keith R; Duggan, David; Gallinger, Steven; Haile, Robert W; Harrison, Tabitha A; Hayes, Richard B; Hoffmeister, Michael; Hopper, John L; Hudson, Thomas J; Jenkins, Mark A; Küry, Sébastien; Le Marchand, Loic; Leal, Suzanne M; Newcomb, Polly A; Nickerson, Deborah A; Potter, John D; Schoen, Robert E; Schumacher, Fredrick R; Seminara, Daniela; Slattery, Martha L; Hsu, Li; Chan, Andrew T; White, Emily; Berndt, Sonja I; Peters, Ulrike

    2016-01-01

    Genome-wide association studies (GWAS) have identified many common single nucleotide polymorphisms (SNPs) associated with colorectal cancer risk. These SNPs may tag correlated variants with biological importance. Fine-mapping around GWAS loci can facilitate detection of functional candidates and additional independent risk variants. We analyzed 11,900 cases and 14,311 controls in the Genetics and Epidemiology of Colorectal Cancer Consortium and the Colon Cancer Family Registry. To fine-map genomic regions containing all known common risk variants, we imputed high-density genetic data from the 1000 Genomes Project. We tested single-variant associations with colorectal tumor risk for all variants spanning genomic regions 250-kb upstream or downstream of 31 GWAS-identified SNPs (index SNPs). We queried the University of California, Santa Cruz Genome Browser to examine evidence for biological function. Index SNPs did not show the strongest association signals with colorectal tumor risk in their respective genomic regions. Bioinformatics analysis of SNPs showing smaller P-values in each region revealed 21 functional candidates in 12 loci (5q31.1, 8q24, 11q13.4, 11q23, 12p13.32, 12q24.21, 14q22.2, 15q13, 18q21, 19q13.1, 20p12.3, and 20q13.33). We did not observe evidence of additional independent association signals in GWAS-identified regions. Our results support the utility of integrating data from comprehensive fine-mapping with expanding publicly available genomic databases to help clarify GWAS associations and identify functional candidates that warrant more onerous laboratory follow-up. Such efforts may aid the eventual discovery of disease-causing variant(s). PMID:27379672

  4. Linear motifs confer functional diversity onto splice variants

    PubMed Central

    Weatheritt, Robert J.; Davey, Norman E.; Gibson, Toby J.

    2012-01-01

    The pre-translational modification of messenger ribonucleic acids (mRNAs) by alternative promoter usage and alternative splicing is an important source of pleiotropy. Despite intensive efforts, our understanding of the functional implications of this dynamically created diversity is still incomplete. Using the available knowledge of interaction modules, particularly within intrinsically disordered regions (IDRs), we analysed the occurrences of protein modules within alternative exons. We find that regions removed or included by pre-translational variation are enriched in linear motifs suggesting that the removal or inclusion of exons containing these interaction modules is an important regulatory mechanism. In particular, we observe that PDZ-, PTB-, SH2- and WW-domain binding motifs are more likely to occur within alternative exons. We also determine that regions removed or included by alternative promoter usage are enriched in IDRs suggesting that protein isoform diversity is tightly coupled to the modulation of IDRs. This study, therefore, demonstrates that short linear motifs are key components for establishing protein diversity between splice variants. PMID:22638587

  5. A TNF Variant that Associates with Susceptibility to Musculoskeletal Disease Modulates Thyroid Hormone Receptor Binding to Control Promoter Activation

    PubMed Central

    Kiss-Toth, Endre; Harlock, Edward; Lath, Darren; Quertermous, Thomas; Wilkinson, J. Mark

    2013-01-01

    Tumor necrosis factor (TNF) is a powerful pro-inflammatory cytokine and immuno-regulatory molecule, and modulates susceptibility to musculoskeletal diseases. Several meta-analyses and replicated association studies have implicated the minor ‘A’ variant within the TNF promoter single nucleotide polymorphism (SNP) rs361525 (-238A/G) as a risk allele in joint related disorders, including psoriatic and juvenile idiopathic arthritis, and osteolysis after joint arthroplasty. Here we characterized the effect of this variant on TNF promoter function. A transcriptional reporter, encoding the -238A variant of the TNF promoter, resulted in 2.2 to 2.8 times greater transcriptional activation versus the ‘G’ variant in murine macrophages when stimulated with pro-inflammatory stimuli. Bioinformatic analysis predicted a putative binding site for thyroid hormone receptor (TR) for the -238A but not the -238G allele. Overexpression of TR-α induced promoter expression 1.8-fold in the presence of the ‘A’ allele only. TR-α expression both potentiated and sensitized the -238A response to LPS or a titanium particulate stimulus, whilst siRNA knockdown of either THRA or THRB impaired transcriptional activation for the -238A variant only. This effect was independent of receptor-ligand binding of triiodothyronine. Immunohistochemical analysis of osteolysis interface membranes from patients undergoing revision surgery confirmed expression of TR-α within osteoclast nuclei at the resorption surface. The ‘A’ allele at rs361525 confers increased transcriptional activation of the TNF promoter and influences susceptibility to several arthritic conditions. This effect is modulated, at least in part, by binding of TR, which both sensitizes and potentiates transcriptional activation of the ‘A’ variant independent of its endogenous ligand. PMID:24069456

  6. Variants on the promoter region of PTEN affect breast cancer progression and patient survival

    PubMed Central

    2011-01-01

    Introduction The PTEN gene, a regulator of the phosphatidylinositol-3-kinase (PI3K)/Akt oncogenic pathway, is mutated in various cancers and its expression has been associated with tumor progression in a dose-dependent fashion. We investigated the effect of germline variation in the promoter region of the PTEN gene on clinical characteristics and survival in breast cancer. Methods We screened the promoter region of the PTEN gene for germline variation in 330 familial breast cancer cases and further determined the genotypes of three detected PTEN promoter polymorphisms -903GA, -975GC, and -1026CA in a total of 2,412 breast cancer patients to evaluate the effects of the variants on tumor characteristics and disease outcome. We compared the gene expression profiles in breast cancers of 10 variant carriers and 10 matched non-carriers and performed further survival analyses based on the differentially expressed genes. Results All three promoter variants associated with worse prognosis. The Cox's regression hazard ratio for 10-year breast cancer specific survival in multivariate analysis was 2.01 (95% CI 1.17 to 3.46) P = 0.0119, and for 5-year breast cancer death or distant metastasis free survival 1.79 (95% CI 1.03 to 3.11) P = 0.0381 for the variant carriers, indicating PTEN promoter variants as an independent prognostic factor. The breast tumors from the promoter variant carriers exhibited a similar gene expression signature of 160 differentially expressed genes compared to matched non-carrier tumors. The signature further stratified patients into two groups with different recurrence free survival in independent breast cancer gene expression data sets. Conclusions Inherited variation in the PTEN promoter region affects the tumor progression and gene expression profile in breast cancer. Further studies are warranted to establish PTEN promoter variants as clinical markers for prognosis in breast cancer. PMID:22171747

  7. Promoter Variants of the ADAM10 Gene and Their Roles in Temporal Lobe Epilepsy

    PubMed Central

    Tao, Hua; Zhao, Jianghao; Zhou, Xu; Ma, Zhonghua; Chen, Ying; Sun, Fuhai; Cui, Lili; Zhou, Haihong; Cai, Yujie; Chen, Yanyan; Zhao, Shu; Yao, Lifen; Zhao, Bin; Li, Keshen

    2016-01-01

    Previous evidence has indicated that downregulated ADAM10 gives rise to epileptic seizures in Alzheimer’s disease, and this study investigated the association of ADAM10 with temporal lobe epilepsy (TLE) from a genetic perspective. A total of 496 TLE patients and 528 healthy individuals were enrolled and genotyped for ADAM10 promoter variants (rs653765 G > A and rs514049 A > C). The alleles, genotypes, and haplotypes were then compared with clarify the association of these variants with TLE and their impacts upon age at onset, initial seizure types before treatments, and responses to drug treatments. In cohorts I, II, and I + II, the frequencies of the A allele and AA genotype at rs514049 were consistently increased in the cases compared with the controls (p = 0.020 and p = 0.009; p = 0.008 and p = 0.009; p = 0.000 and p = 0.000; q = 0.003 and q = 0.002, respectively). In contrast, the frequency of the AC haplotype (rs653765–rs514049) decreased in cohorts I + II (p = 0.013). Further analyses of the TLE patients indicated that the AA genotype functioned as a predisposing factor to drug-resistant TLE and the AC haplotype as a protective factor against generalized tonic–clonic seizures (GTCS) and drug-resistant TLE. This study is the first to demonstrate an association of the ADAM10 promoter variants with TLE. In particular, the AA genotype and AC haplotype showed their effects upon GTCS and drug-resistant TLE. PMID:27445971

  8. Epigenomic functional characterization of genetic susceptibility variants in systemic vasculitis.

    PubMed

    Sawalha, Amr H; Dozmorov, Mikhail G

    2016-02-01

    Systemic vasculitides are poorly understood inflammatory diseases of the blood vessels that are frequently associated with significant organ damage. Genetic risk variants contribute to the susceptibility of vasculitis, but functional consequences of these genetic variants are largely unknown. Most genetic risk variants in immune-mediated diseases, including systemic vasculitis, are localized to non-coding genetic regions suggesting they might increase disease risk by influencing regulatory elements within the genome. Long range regulatory interactions pose an additional obstacle in localizing functional consequences associated with risk variants to specific genes or cell types. We used cell-type specific enrichment patterns of histone changes that mark poised, primed, and active enhancers, and DNase hypersensitivity to identify specific immune cells mediating genetic risk in vasculitis. Our data suggest that genetic risk variants in ANCA-associated vasculitis are significantly enriched in enhancer elements in Th17 cells, supporting a role for Th17 cells in this disease. Primed and active enhancer elements in B cells can be potentially affected by genetic risk variants associated with Kawasaki disease. Genetic risk in Behçet's disease and Takayasu arteritis might affect enhancer elements in multiple cell types, possibly explained by influencing enhancers in hematopoietic stem cells. Interestingly, our analyses indicate a role for B cells in Kawasaki disease, Behçet's disease, and Takayasu arteritis, and suggest that further work to characterize the involvement of B cells in these diseases is warranted. PMID:26492816

  9. Diverse Functional Properties of Wilson Disease ATP7B Variants

    PubMed Central

    Huster, Dominik; Kühne, Angelika; Bhattacharjee, Ashima; Raines, Lily; Jantsch, Vanessa; Noe, Johannes; Schirrmeister, Wiebke; Sommerer, Ines; Sabri, Osama; Berr, Frieder; Mössner, Joachim; Stieger, Bruno; Caca, Karel; Lutsenko, Svetlana

    2012-01-01

    BACKGROUND & AIMS Wilson disease is a severe disorder of copper metabolism caused by mutations in ATP7B, which encodes a copper-transporting adenosine triphosphatase. The disease presents with a variable phenotype that complicates the diagnostic process and treatment. Little is known about the mechanisms that contribute to the different phenotypes of the disease. METHODS We analyzed 28 variants of ATP7B from patients with Wilson disease that affected different functional domains; the gene products were expressed using the baculovirus expression system in Sf9 cells. Protein function was analyzed by measuring catalytic activity and copper (64Cu) transport into vesicles. We studied intracellular localization of variants of ATP7B that had measurable transport activities and were tagged with green fluorescent protein in mammalian cells using confocal laser scanning microscopy. RESULTS Properties of ATP7B variants with pathogenic amino-acid substitution varied greatly even if substitutions were in the same functional domain. Some variants had complete loss of catalytic and transport activity, whereas others lost transport activity but retained phosphor-intermediate formation or had partial losses of activity. In mammalian cells, transport-competent variants differed in stability and subcellular localization. CONCLUSIONS Variants in ATP7B associated with Wilson disease disrupt the protein’s transport activity, result in its mislocalization, and reduce its stability. Single assays are insufficient to accurately predict the effects of ATP7B variants the function of its product and development of Wilson disease. These findings will contribute to our understanding of genotype–phenotype correlation and mechanisms of disease pathogenesis. PMID:22240481

  10. Radon Exposure, IL-6 Promoter Variants, and Lung Squamous Cell Carcinoma in Former Uranium Miners

    PubMed Central

    Leng, Shuguang; Thomas, Cynthia L.; Snider, Amanda M.; Picchi, Maria A.; Chen, Wenshu; Willis, Derall G.; Carr, Teara G.; Krzeminski, Jacek; Desai, Dhimant; Shantu, Amin; Lin, Yong; Jacobson, Marty R.; Belinsky, Steven A.

    2015-01-01

    Background: High radon exposure is a risk factor for squamous cell carcinoma, a major lung cancer histology observed in former uranium miners. Radon exposure can cause oxidative stress, leading to pulmonary inflammation. Interleukin-6 (IL-6) is a pro-carcinogenic inflammatory cytokine that plays a pivotal role in lung cancer development. Objectives: We assessed whether single nucleotide polymorphisms (SNPs) in the IL6 promoter are associated with lung cancer in former uranium miners with high occupational exposure to radon gas. Methods: Genetic associations were assessed in a case–control study of former uranium miners (242 cases and 336 controls). A replication study was performed using data from the Gene Environment Association Studies (GENEVA) Genome Wide Association Study (GWAS) of Lung Cancer and Smoking. Functional relevance of the SNPs was characterized using in vitro approaches. Results: We found that rs1800797 was associated with squamous cell carcinoma in miners and with a shorter time between the midpoint of the period of substantial exposure and diagnosis among the cases. Furthermore, rs1800797 was also associated with lung cancer among never smokers in the GENEVA dataset. Functional studies identified that the risk allele was associated with increased basal IL-6 mRNA level and greater promoter activity. Furthermore, fibroblasts with the risk allele showed greater induction of IL-6 secretion by hydrogen peroxide or benzo[a]pyrene diolepoxide treatments. Conclusions: An IL6 promoter variant was associated with lung cancer in uranium miners and never smokers in two external study populations. The associations are strongly supported by the functional relevance that the IL6 promoter SNP affects basal expression and carcinogen-induced IL-6 secretion. Citation: Leng S, Thomas CL, Snider AM, Picchi MA, Chen W, Willis DG, Carr TG, Krzeminski J, Desai D, Shantu A, Lin Y, Jacobson MR, Belinsky SA. 2016. Radon exposure, IL-6 promoter variants, and lung squamous

  11. Identification of four variants in the tryptophan hydroxylase promoter and association to behavior.

    PubMed

    Rotondo, A; Schuebel, K; Bergen, A; Aragon, R; Virkkunen, M; Linnoila, M; Goldman, D; Nielsen, D

    1999-07-01

    One of the most replicated findings in biological psychiatry is the observation of lower 5-hydroxyindoleacetic acid concentrations, the major metabolite of serotonin, in the brain and cerebrospinal fluid of subjects with impulsive aggression. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of serotonin, however functional variants have not been reported from the coding sequence of this gene. Therefore, we screened the human TPH promoter (TPH-P) for genetic variants which could modulate TPH gene transcription. The TPH-P (2093 nucleotides) was screened for sequence variation by SSCP analysis of 260 individuals from Finnish, Italian, American Caucasian, and American Indian populations. Four common polymorphisms were identified: -7180T>G, -7065C>T, -6526A>G, and -5806G>T (designated as nucleotides upstream of the translation start site). In the Finns, the four polymorphisms had a minor allele frequency of 0.40 and in this population linkage disequilibrium between the four loci was complete. In the other populations the minor allele frequencies ranged from 0.40 to 0.45. TPH -6526A>G genotype was determined in 167 unrelated Finnish offenders and 153 controls previously studied for the TPH IVS7+779C>A polymorphism. A significant association was observed between -6526A>G and suicidality in the offenders. TPH -6526A>G and the previously reported intron seven polymorphism, TPH IVS7+779C>A, exhibited a normalised linkage disequilibrium of 0.89 in Finns. Normalized linkage disequilibrium was reduced in other populations, being 0.49 and 0.21 in Italians and American Indians, respectively. In conclusion, four TPH-P variants were identified which can be used for haplotype-based analysis to localize functional TPH alleles influencing behavior. PMID:10483053

  12. Functional compensation among HMGN variants modulates the DNase I hypersensitive sites at enhancers

    PubMed Central

    Deng, Tao; Zhu, Z. Iris; Zhang, Shaofei; Postnikov, Yuri; Huang, Di; Horsch, Marion; Furusawa, Takashi; Beckers, Johannes; Rozman, Jan; Klingenspor, Martin; Amarie, Oana; Graw, Jochen; Rathkolb, Birgit; Wolf, Eckhard; Adler, Thure; Busch, Dirk H.; Gailus-Durner, Valérie; Fuchs, Helmut; Hrabě de Angelis, Martin; van der Velde, Arjan; Tessarollo, Lino; Ovcherenko, Ivan; Landsman, David; Bustin, Michael

    2015-01-01

    DNase I hypersensitive sites (DHSs) are a hallmark of chromatin regions containing regulatory DNA such as enhancers and promoters; however, the factors affecting the establishment and maintenance of these sites are not fully understood. We now show that HMGN1 and HMGN2, nucleosome-binding proteins that are ubiquitously expressed in vertebrate cells, maintain the DHS landscape of mouse embryonic fibroblasts (MEFs) synergistically. Loss of one of these HMGN variants led to a compensatory increase of binding of the remaining variant. Genome-wide mapping of the DHSs in Hmgn1−/−, Hmgn2−/−, and Hmgn1−/−n2−/− MEFs reveals that loss of both, but not a single HMGN variant, leads to significant remodeling of the DHS landscape, especially at enhancer regions marked by H3K4me1 and H3K27ac. Loss of HMGN variants affects the induced expression of stress-responsive genes in MEFs, the transcription profiles of several mouse tissues, and leads to altered phenotypes that are not seen in mice lacking only one variant. We conclude that the compensatory binding of HMGN variants to chromatin maintains the DHS landscape, and the transcription fidelity and is necessary to retain wild-type phenotypes. Our study provides insight into mechanisms that maintain regulatory sites in chromatin and into functional compensation among nucleosome binding architectural proteins. PMID:26156321

  13. Expression Pattern, Regulation, and Functions of Methionine Adenosyltransferase 2β Splicing Variants in Hepatoma Cells

    PubMed Central

    YANG, HEPING; ARA, AINHOA IGLESIAS; MAGILNICK, NATHANIEL; XIA, MENG; RAMANI, KOMAL; CHEN, HUI; LEE, TAUNIA D.; MATO, JOSÉ M.; LU, SHELLY C.

    2008-01-01

    Background & Aims Methionine adenosyltransferase (MAT) catalyzes S-adenosylmethionine biosynthesis. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, while a third gene (MAT2β) encodes for a regulatory subunit that modulates the activity of MAT2A-encoded isoenzyme. We uncovered multiple splicing variants while characterizing its 5′-flanking region. The aims of our current study are to examine the expression pattern, regulation, and functions of the 2 major variants: V1 and V2. Methods Studies were conducted using RNA from normal human tissues, resected hepatocellular carcinoma specimens, and cell lines. Gene expression, promoter and nuclear binding activities, growth, and apoptosis were measured by routine assays. Results MAT2β is expressed in most but not all tissues, and the 2 variants are differentially expressed. The messenger RNA levels of both variants are markedly increased in hepatocellular carcinoma. Tumor necrosis factor (TNF)-α, which induces MAT2A in HepG2 cells, also induced V1 (but not V2) expression. TNF-α induced the promoter activity of MAT2β V1, likely via nuclear factor κB and activator protein 1. Both variants regulate growth, but only V1 regulates apoptosis. Reduced expression of V1 led to c-Jun-N-terminal kinase (JNK) activation, apoptosis, and sensitized HepG2 cells to TNF-α–induced apoptosis, while overexpression of V1 was protective. However, blocking JNK1 or JNK2 activation did not prevent apoptosis induced by V1 knockdown. V1 (but not V2) knockdown also leads to apoptosis in a colon cancer cell line, suggesting these variants play similar roles in many cell types. Conclusions Different variants of MAT2β regulate growth and death, which broadens their importance in biology. PMID:18045590

  14. Functional characterisation of ADIPOQ variants using individuals recruited by genotype.

    PubMed

    Lee, Benjamin P; Lloyd-Laney, Henry O; Locke, Jonathan M; McCulloch, Laura J; Knight, Bridget; Yaghootkar, Hanieh; Cory, Giles; Kos, Katarina; Frayling, Timothy M; Harries, Lorna W

    2016-06-15

    Four non-coding GWAS variants in or near the ADIPOQ gene (rs17300539, rs17366653, rs3821799 and rs56354395) together explain 4% of the variation in circulating adiponectin. The functional basis for this is unknown. We tested the effect of these variants on ADIPOQ transcription, splicing and stability respectively in adipose tissue samples from participants recruited by rs17366653 genotype. Transcripts carrying rs17300539 demonstrated a 17% increase in expression (p = 0.001). Variant rs17366653 was associated with disruption of ADIPOQ splicing leading to a 7 fold increase in levels of a non-functional transcript (p = 0.002). Transcripts carrying rs56354395 demonstrated a 59% decrease in expression (p = <0.0001). No effects of rs3821799 genotype on expression was observed. Association between variation in the ADIPOQ gene and serum adiponectin may arise from effects on mRNA transcription, splicing or stability. These studies illustrate the utility of recruit-by-genotype studies in relevant human tissues in functional interpretation of GWAS signals. PMID:26996131

  15. Molecular prioritization strategies to identify functional genetic variants in the cardiovascular disease-associated expression QTL Vanin-1

    PubMed Central

    Kaskow, Belinda J; Diepeveen, Luke A; Michael Proffitt, J; Rea, Alexander J; Ulgiati, Daniela; Blangero, John; Moses, Eric K; Abraham, Lawrence J

    2014-01-01

    There is now good evidence that non-coding sequence variants are involved in the heritability of many common complex traits. The current ‘gold standard' approach for assessing functionality is the in vitro reporter gene assay to assess allelic differences in transcriptional activity, usually followed by electrophoretic mobility shift assays to assess allelic differences in transcription factor binding. Although widely used, these assays have inherent limitations, including the lack of endogenous chromatin context. Here we present a more contemporary approach to assessing functionality of non-coding sequence variation within the Vanin-1 (VNN1) promoter. By combining ‘gold standard' assays with in vivo assessments of chromatin accessibility, we greatly increase our confidence in the statistically assigned functional relevance. The standard assays revealed the −137 single nucleotide variant to be functional but the −587 variant to have no functional relevance. However, our in vivo tests show an allelic difference in chromatin accessibility surrounding the −587 variant supporting strong functional potential at both sites. Our approach advances the identification of functional variants by providing strong in vivo biological evidence for function. PMID:24045843

  16. Surfactant Protein-C Promoter Variants Associated with Neonatal Respiratory Distress Syndrome Reduce Transcription

    PubMed Central

    Wambach, Jennifer A.; Yang, Ping; Wegner, Daniel J.; An, Ping; Hackett, Brian P.; Cole, F. S.; Hamvas, Aaron

    2010-01-01

    Dominant mutations in coding regions of the surfactant protein-C gene (SFTPC) cause respiratory distress syndrome (RDS) in infants. However, the contribution of variants in noncoding regions of SFTPC to pulmonary phenotypes is unknown. Using a case-control group of infants ≥34 weeks gestation (n=538), we used complete resequencing of SFTPC and its promoter, genotyping, and logistic regression to identify 80 single nucleotide polymorphisms (SNPs). Three promoter SNPs were statistically associated with neonatal RDS among European descent infants. To assess the transcriptional effects of these three promoter SNPs, we selectively mutated the SFTPC promoter and performed transient transfection using MLE-15 cells and a firefly luciferase reporter vector. Each promoter SNP decreased SFTPC transcription. The combination of two variants in high linkage dysequilibrium also decreased SFTPC transcription. In silico evaluation of transcription factor binding demonstrated that the rare allele at g.-1167 disrupts a SOX (SRY-related high mobility group box) consensus motif and introduces a GATA-1 site, at g.-2385 removes a MZF-1 (myeloid zinc finger) binding site, and at g.-1647 removes a potential methylation site. This combined statistical, in vitro, and in silico approach suggests that reduced SFTPC transcription contributes to the genetic risk for neonatal RDS in developmentally susceptible infants. PMID:20539253

  17. Identification and Functional Characterization of Novel Genetic Variations in the OCTN1 Promoter

    PubMed Central

    Park, Hyo Jin

    2014-01-01

    Human organic cation/carnitine transporter 1 (OCTN1) plays an important role in the transport of drugs and endogenous substances. It is known that a missense variant of OCTN1 is significantly associated with Crohn's disease susceptibility. This study was performed to identify genetic variants of the OCTN1 promoter in Korean individuals and to determine their functional effects. First, the promoter region of OCTN1 was directly sequenced using genomic DNA samples from 48 healthy Koreans. OCTN1 promoter activity was then measured using a luciferase reporter assay in HCT-116 cells. Seven variants of the OCTN1 promoter were identified, two of which were novel. There were also four major OCTN1 promoter haplotypes. Three haplotypes (H1, H3, and H4) showed decreased transcriptional activity, which was reduced by 22.9%, 23.0%, and 44.6%, respectively (p<0.001), compared with the reference haplotype (H2). Transcription factor binding site analyses and gel shift assays revealed that NF-Y could bind to the region containing g.-1875T>A, a variant present in H3, and that the binding affinity of NF-Y was higher for the g.-1875T allele than for the g.-1875A allele. NF-Y could also repress OCTN1 transcription. These data suggest that three OCTN1 promoter haplotypes could regulate OCTN1 transcription. To our knowledge, this is the first study to identify functional variants of the OCTN1 promoter. PMID:24757380

  18. Functional Prostacyclin Synthase Promoter Polymorphisms. Impact in Pulmonary Arterial Hypertension

    PubMed Central

    Cornelius, Amber R.; Lu, Xiao; Conklin, David S.; Del Rosario, Mark J.; Lowe, Anita M.; Elos, Mihret T.; Fettig, Lynsey M.; Wong, Randall E.; Hara, Naoko; Cogan, Joy D.; Phillips, John A.; Taylor, Matthew R.; Graham, Brian B.; Tuder, Rubin M.; Loyd, James E.; Geraci, Mark W.

    2014-01-01

    Rationale: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary artery pressure, vascular remodeling, and ultimately right ventricular heart failure. PAH can have a genetic component (heritable PAH), most often through mutations of bone morphogenetic protein receptor 2, and idiopathic and associated forms. Heritable PAH is not completely penetrant within families, with approximately 20% concurrence of inactivating bone morphogenetic protein receptor 2 mutations and delayed onset of PAH disease. Because one of the treatment options is using prostacyclin analogs, we hypothesized that prostacyclin synthase promoter sequence variants associated with increased mRNA expression may play a protective role in the bone morphogenetic protein receptor 2 unaffected carriers. Objectives: To characterize the range of prostacyclin synthase promoter variants and assess their transcriptional activities in PAH-relevant cell types. To determine the distribution of prostacyclin synthase promoter variants in PAH, unaffected carriers in heritable PAH families, and control populations. Methods: Polymerase chain reaction approaches were used to genotype prostacyclin synthase promoter variants in more than 300 individuals. Prostacyclin synthase promoter haplotypes’ transcriptional activities were determined with luciferase reporter assays. Measurements and Main Results: We identified a comprehensive set of prostacyclin synthase promoter variants and tested their transcriptional activities in PAH-relevant cell types. We demonstrated differences of prostacyclin synthase promoter activities dependent on their haplotype. Conclusions: Prostacyclin synthase promoter sequence variants exhibit a range of transcriptional activities. We discovered a significant bias for more active prostacyclin synthase promoter variants in unaffected carriers as compared with affected patients with PAH. PMID:24605778

  19. Mitochondrial DNA Polymerase POLG1 Disease Mutations and Germline Variants Promote Tumorigenic Properties

    PubMed Central

    Singh, Bhupendra; Owens, Kjerstin M.; Bajpai, Prachi; Desouki, Mohamed Mokhtar; Srinivasasainagendra, Vinodh; Tiwari, Hemant K.; Singh, Keshav K.

    2015-01-01

    Germline mutations in mitochondrial DNA polymerase gamma (POLG1) induce mitochondrial DNA (mtDNA) mutations, depletion, and decrease oxidative phosphorylation. Earlier, we identified somatic mutations in POLG1 and the contribution of these mutations in human cancer. However, a role for germline variations in POLG1 in human cancers is unknown. In this study, we examined a role for disease associated germline variants of POLG1, POLG1 gene expression, copy number variation and regulation in human cancers. We analyzed the mutations, expression and copy number variation in POLG1 in several cancer databases and validated the analyses in primary breast tumors and breast cancer cell lines. We discovered 5-aza-2'-deoxycytidine led epigenetic regulation of POLG1, mtDNA-encoded genes and increased mitochondrial respiration. We conducted comprehensive race based bioinformatics analyses of POLG1 gene in more than 33,000 European-Americans and 5,000 African-Americans. We identified a mitochondrial disease causing missense variation in polymerase domain of POLG1 protein at amino acid 1143 (E1143G) to be 25 times more prevalent in European-Americans (allele frequency 0.03777) when compared to African-American (allele frequency 0.00151) population. We identified T251I and P587L missense variations in exonuclease and linker region of POLG1 also to be more prevalent in European-Americans. Expression of these variants increased glucose consumption, decreased ATP production and increased matrigel invasion. Interestingly, conditional expression of these variants revealed that matrigel invasion properties conferred by these germline variants were reversible suggesting a role of epigenetic regulators. Indeed, we identified a set of miRNA whose expression was reversible after variant expression was turned off. Together, our studies demonstrate altered genetic and epigenetic regulation of POLG1 in human cancers and suggest a role for POLG1 germline variants in promoting tumorigenic

  20. Expression of P. falciparum var Genes Involves Exchange of the Histone Variant H2A.Z at the Promoter

    PubMed Central

    Petter, Michaela; Lee, Chin Chin; Byrne, Timothy J.; Boysen, Katja E.; Volz, Jennifer; Ralph, Stuart A.; Cowman, Alan F.; Brown, Graham V.; Duffy, Michael F.

    2011-01-01

    Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are

  1. Identification of novel functional sequence variants in the gene for peptidase inhibitor 3

    PubMed Central

    Chowdhury, Mahboob A; Kuivaniemi, Helena; Romero, Roberto; Edwin, Samuel; Chaiworapongsa, Tinnakorn; Tromp, Gerard

    2006-01-01

    Background Peptidase inhibitor 3 (PI3) inhibits neutrophil elastase and proteinase-3, and has a potential role in skin and lung diseases as well as in cancer. Genome-wide expression profiling of chorioamniotic membranes revealed decreased expression of PI3 in women with preterm premature rupture of membranes. To elucidate the molecular mechanisms contributing to the decreased expression in amniotic membranes, the PI3 gene was searched for sequence variations and the functional significance of the identified promoter variants was studied. Methods Single nucleotide polymorphisms (SNPs) were identified by direct sequencing of PCR products spanning a region from 1,173 bp upstream to 1,266 bp downstream of the translation start site. Fourteen SNPs were genotyped from 112 and nine SNPs from 24 unrelated individuals. Putative transcription factor binding sites as detected by in silico search were verified by electrophoretic mobility shift assay (EMSA) using nuclear extract from Hela and amnion cell nuclear extract. Deviation from Hardy-Weinberg equilibrium (HWE) was tested by χ2 goodness-of-fit test. Haplotypes were estimated using expectation maximization (EM) algorithm. Results Twenty-three sequence variations were identified by direct sequencing of polymerase chain reaction (PCR) products covering 2,439 nt of the PI3 gene (-1,173 nt of promoter sequences and all three exons). Analysis of 112 unrelated individuals showed that 20 variants had minor allele frequencies (MAF) ranging from 0.02 to 0.46 representing "true polymorphisms", while three had MAF ≤ 0.01. Eleven variants were in the promoter region; several putative transcription factor binding sites were found at these sites by database searches. Differential binding of transcription factors was demonstrated at two polymorphic sites by electrophoretic mobility shift assays, both in amniotic and HeLa cell nuclear extracts. Differential binding of the transcription factor GATA1 at -689C>G site was confirmed by a

  2. Functional Inducible Nitric Oxide Synthase Gene Variants Associate With Hypertension

    PubMed Central

    Nikkari, Seppo T.; Määttä, Kirsi M.; Kunnas, Tarja A.

    2015-01-01

    Abstract Increased inducible nitric oxide synthase (iNOS) activity and expression has been associated with hypertension, but less is known whether the 2 known functional polymorphic sites in the iNOS gene (g.–1026 C/A (rs2779249), g.2087 G/A (rs2297518)) affect susceptibility to hypertension. The objective of this study was to investigate the association between the genetic variants of iNOS and diagnosed hypertension in a Finnish cohort. This study included 320 hypertensive cases and 439 healthy controls. All participants were 50-year-old men and women and the data were collected from the Tampere adult population cardiovascular risk study (TAMRISK). DNA was extracted from buccal swabs and iNOS single nucleotide polymorphisms (SNPs) were analyzed using KASP genotyping PCR. Data analysis was done by logistic regression. At the age of 50 years, the SNP rs2779249 (C/A) associated significantly with hypertension (P = 0.009); specifically, subjects carrying the A-allele had higher risk of hypertension compared to those carrying the CC genotype (OR = 1.47; CI = 1.08–2.01; P = 0.015). In addition, a 15-year follow-up period (35, 40, and 45 years) of the same individuals showed that carriers of the A-allele had more often hypertension in all of the studied age-groups. The highest risk for developing hypertension was obtained among 35-year-old subjects (odds ratio [OR] 3.83; confidence interval [CI] = 1.20–12.27; P = 0.024). Those carrying variant A had also significantly higher readings of both systolic (P = 0.047) and diastolic (P = 0.048) blood pressure during the follow-up. No significant associations between rs2297518 (G/A) variants alone and hypertension were found. However, haplotype analysis of rs2779249 and rs2297518 revealed that individuals having haplotype H3 which combines both A alleles (CA–GA, 19.7% of individuals) was more commonly found in the hypertensive group than in the normotensive group (OR = 2.01; CI = 1

  3. Filtering genetic variants and placing informative priors based on putative biological function.

    PubMed

    Friedrichs, Stefanie; Malzahn, Dörthe; Pugh, Elizabeth W; Almeida, Marcio; Liu, Xiao Qing; Bailey, Julia N

    2016-01-01

    High-density genetic marker data, especially sequence data, imply an immense multiple testing burden. This can be ameliorated by filtering genetic variants, exploiting or accounting for correlations between variants, jointly testing variants, and by incorporating informative priors. Priors can be based on biological knowledge or predicted variant function, or even be used to integrate gene expression or other omics data. Based on Genetic Analysis Workshop (GAW) 19 data, this article discusses diversity and usefulness of functional variant scores provided, for example, by PolyPhen2, SIFT, or RegulomeDB annotations. Incorporating functional scores into variant filters or weights and adjusting the significance level for correlations between variants yielded significant associations with blood pressure traits in a large family study of Mexican Americans (GAW19 data set). Marker rs218966 in gene PHF14 and rs9836027 in MAP4 significantly associated with hypertension; additionally, rare variants in SNUPN significantly associated with systolic blood pressure. Variant weights strongly influenced the power of kernel methods and burden tests. Apart from variant weights in test statistics, prior weights may also be used when combining test statistics or to informatively weight p values while controlling false discovery rate (FDR). Indeed, power improved when gene expression data for FDR-controlled informative weighting of association test p values of genes was used. Finally, approaches exploiting variant correlations included identity-by-descent mapping and the optimal strategy for joint testing rare and common variants, which was observed to depend on linkage disequilibrium structure. PMID:26866982

  4. The Asian-American E6 Variant Protein of Human Papillomavirus 16 Alone Is Sufficient To Promote Immortalization, Transformation, and Migration of Primary Human Foreskin Keratinocytes

    PubMed Central

    Niccoli, Sarah; Abraham, Suraj; Richard, Christina

    2012-01-01

    We examined how well the human papillomavirus (HPV) E6 oncogene can function in the absence of the E7 oncogene during the carcinogenic process in human keratinocytes using a common HPV variant strongly associated with cervical cancer: the Asian-American E6 variant (AAE6). This E6 variant is 20 times more frequently detected in cervical cancer than the prototype European E6 variant, as evidenced by independent epidemiological data. Using cell culture and cell-based functional assays, we assessed how this variant can perform crucial carcinogenesis steps compared to the prototype E6 variant. The ability to immortalize and transform primary human foreskin keratinocytes (PHFKs) to acquire resilient phenotypes and the ability to promote cell migration were evaluated. The immortalization capability was assayed based on population doublings, number of passages, surpassing mortality stages 1 and 2, human telomerase reverse transcriptase (hTERT) expression, and the ability to overcome G1 arrest via p53 degradation. Transformation and migration efficiency were analyzed using a combination of functional cell-based assays. We observed that either AAE6 or prototype E6 proteins alone were sufficient to immortalize PHFKs, although AAE6 was more potent in doing so. The AAE6 variant protein alone pushed PHFKs through transformation and significantly increased their migration ability over that of the E6 prototype. Our findings are in line with epidemiological data that the AA variant of HPV16 confers an increased risk over the European prototype for cervical cancer, as evidenced by a superior immortalization, transformation, and metastatic potential. PMID:22951839

  5. Gene-environment interaction between the MMP9 C-1562T promoter variant and cigarette smoke in the pathogenesis of chronic obstructive pulmonary disease.

    PubMed

    Stankovic, Marija; Kojic, Snezana; Djordjevic, Valentina; Tomovic, Andrija; Nagorni-Obradovic, Ljudmila; Petrovic-Stanojevic, Natasa; Mitic-Milikic, Marija; Radojkovic, Dragica

    2016-07-01

    The aetiology of chronic obstructive pulmonary disease (COPD) is complex. While cigarette smoking is a well-established cause of COPD, a myriad of assessed genetic factors has given conflicting data. Since gene-environment interactions are thought to be implicated in aetiopathogenesis of COPD, we aimed to examine the matrix metalloproteinase (MMP) 9 C-1562T (rs3918242) functional variant and cigarette smoke in the pathogenesis of this disease. The distribution of the MMP9 C-1562T variant was analyzed in COPD patients and controls with normal pulmonary function from Serbia. Interaction between the C-1562T genetic variant and cigarette smoking was assessed using a case-control model. The response of the C-1562T promoter variant to cigarette smoke condensate (CSC) exposure was examined using a dual luciferase reporter assay. The frequency of T allele carriers was higher in the COPD group than in smoker controls (38.4% vs. 20%; OR = 2.7, P = 0.027). Interaction between the T allele and cigarette smoking was identified in COPD occurrence (OR = 4.38, P = 0.005) and severity (P = 0.001). A functional analysis of the C-1562T variant demonstrated a dose-dependent and allele-specific response (P < 0.01) to CSC. Significantly higher MMP9 promoter activity following CSC exposure was found for the promoter harboring the T allele compared to the promoter harboring the C allele (P < 0.05). Our study is the first to reveal an interaction between the MMP9-1562T allele and cigarette smoke in COPD, emphasising gene-environment interactions as a possible cause of lung damage in the pathogenesis of COPD. Environ. Mol. Mutagen. 57:447-454, 2016. © 2016 Wiley Periodicals, Inc. PMID:27270564

  6. SPINK1 Promoter Variants Are Associated with Prostate Cancer Predisposing Alterations in Benign Prostatic Hyperplasia Patients

    PubMed Central

    WINCHESTER, DANYELLE; RICKS-SANTI, LUISEL; MASON, TSHELA; ABBAS, MUNEER; COPELAND, ROBERT L.; BEYENE, DESTA; JINGWI, EMMANUEL Y.; DUNSTON, GEORGIA M.; KANAAN, YASMINE M.

    2015-01-01

    Background/Aim Several studies reported that patients with benign prostatic hyperplasia (BPH) experienced a 10% increased incidence of prostate cancer (PCa) after the first 5 years of diagnosis. We investigated the association between single nucleotide polymorphisms (SNPs) in the promoter of Serine Protease Inhibitor Kazal Type 1 (SPINK1) and the increased risk of BPH and PCa. Materials and Methods We genotyped three SNPs in a cases-control study, including BPH and PCa cases. Multiple logistic regression models were applied to analyze clinical and genotypic data. Results We found an inverse association between SNP rs10035432 and BPH under the log-additive (p=0.007) model. No association was found between these SNPs and PCa risk. However, we observed a possible association between rs1432982 and lower-grade PCa (p=0.05) under the recessive model. Conclusion SPINK1 promoter variants are likely to be associated with the risk of BPH. PMID:26124326

  7. First functional polymorphism in CFTR promoter that results in decreased transcriptional activity and Sp1/USF binding

    SciTech Connect

    Taulan, M. Lopez, E.; Guittard, C.; Rene, C.; Baux, D.; Altieri, J.P.; DesGeorges, M.; Claustres, M.; Romey, M.C.

    2007-09-28

    Growing evidences show that functionally relevant polymorphisms in various promoters alter both transcriptional activity and affinities of existing protein-DNA interactions, and thus influence disease progression in humans. We previously reported the -94G>T CFTR promoter variant in a female CF patient in whom any known disease-causing mutation has been detected. To investigate whether the -94G>T could be a regulatory variant, we have proceeded to in silico analyses and functional studies including EMSA and reporter gene assays. Our data indicate that the promoter variant decreases basal CFTR transcriptional activity in different epithelial cells and alters binding affinities of both Sp1 and USF nuclear proteins to the CFTR promoter. The present report provides evidence for the first functional polymorphism that negatively affects the CFTR transcriptional activity and demonstrates a cooperative role of Sp1 and USF transcription factors in transactivation of the CFTR gene promoter.

  8. Inheritance of rare functional GCKR variants and their contribution to triglyceride levels in families

    PubMed Central

    Rees, Matthew G.; Raimondo, Anne; Wang, Jian; Ban, Matthew R.; Davis, Mindy I.; Barrett, Amy; Ranft, Jessica; Jagdhuhn, David; Waterstradt, Rica; Baltrusch, Simone; Simeonov, Anton; Collins, Francis S.; Hegele, Robert A.; Gloyn, Anna L.

    2014-01-01

    Significant resources have been invested in sequencing studies to investigate the role of rare variants in complex disease etiology. However, the diagnostic interpretation of individual rare variants remains a major challenge, and may require accurate variant functional classification and the collection of large numbers of variant carriers. Utilizing sequence data from 458 individuals with hypertriglyceridemia and 333 controls with normal plasma triglyceride levels, we investigated these issues using GCKR, encoding glucokinase regulatory protein. Eighteen rare non-synonymous GCKR variants identified in these 791 individuals were comprehensively characterized by a range of biochemical and cell biological assays, including a novel high-throughput-screening-based approach capable of measuring all variant proteins simultaneously. Functionally deleterious variants were collectively associated with hypertriglyceridemia, but a range of in silico prediction algorithms showed little consistency between algorithms and poor agreement with functional data. We extended our study by obtaining sequence data on family members; however, functional variants did not co-segregate with triglyceride levels. Therefore, despite evidence for their collective functional and clinical relevance, our results emphasize the low predictive value of rare GCKR variants in individuals and the complex heritability of lipid traits. PMID:24879641

  9. Functional Analysis of Missense Variants in the Putative Breast Cancer Susceptibility Gene XRCC2.

    PubMed

    Hilbers, Florentine S; Luijsterburg, Martijn S; Wiegant, Wouter W; Meijers, Caro M; Völker-Albert, Moritz; Boonen, Rick A; van Asperen, Christi J; Devilee, Peter; van Attikum, Haico

    2016-09-01

    XRCC2 genetic variants have been associated with breast cancer susceptibility. However, association studies have been complicated because XRCC2 variants are extremely rare and consist mainly of amino acid substitutions whose grouping is sensitive to misclassification by the predictive algorithms. We therefore functionally characterized variants in XRCC2 by testing their ability to restore XRCC2-DNA repair deficient phenotypes using a cDNA-based complementation approach. While the protein-truncating variants p.Leu117fs, p.Arg215*, and p.Cys217* were unable to restore XRCC2 deficiency, 19 out of 23 missense variants showed no or just a minor (<25%) reduction in XRCC2 function. The remaining four (p.Cys120Tyr, p.Arg91Trp, p.Leu133Pro, and p.Ile95Leu) had a moderate effect. Overall, measured functional effects correlated poorly with those predicted by in silico analysis. After regrouping variants from published case-control studies based on the functional effect found in this study and reanalysis of the prevalence data, there was no longer evidence for an association with breast cancer. This suggests that if breast cancer susceptibility alleles of XRCC2 exist, they are likely restricted to protein-truncating variants and a minority of missense changes. Our study emphasizes the use of functional analyses of missense variants to support variant classification in association studies. PMID:27233470

  10. Targeted resequencing of regulatory regions at schizophrenia risk loci: Role of rare functional variants at chromatin repressive states.

    PubMed

    González-Peñas, Javier; Amigo, Jorge; Santomé, Luis; Sobrino, Beatriz; Brenlla, Julio; Agra, Santiago; Paz, Eduardo; Páramo, Mario; Carracedo, Ángel; Arrojo, Manuel; Costas, Javier

    2016-07-01

    There is mounting evidence that regulatory variation plays an important role in genetic risk for schizophrenia. Here, we specifically search for regulatory variants at risk by sequencing promoter regions of twenty-three genes implied in schizophrenia by copy number variant or genome-wide association studies. After strict quality control, a total of 55,206bp per sample were analyzed in 526 schizophrenia cases and 516 controls from Galicia, NW Spain, using the Applied Biosystems SOLiD System. Variants were filtered based on frequency from public databases, chromatin states from the RoadMap Epigenomics Consortium at tissues relevant for schizophrenia, such as fetal brain, mid-frontal lobe, and angular gyrus, and prediction of functionality from RegulomeDB. The proportion of rare variants at polycomb repressive chromatin state at relevant tissues was higher in cases than in controls. The proportion of rare variants with predicted regulatory role was significantly higher in cases than in controls (P=0.0028, OR=1.93, 95% C.I.=1.23-3.04). Combination of information from both sources led to the identification of an excess of carriers of rare variants with predicted regulatory role located at polycomb repressive chromatin state at relevant tissues in cases versus controls (P=0.0016, OR=19.34, 95% C.I.=2.45-2495.26). The variants are located at two genes affected by the 17q12 copy number variant, LHX1 and HNF1B. These data strongly suggest that a specific epigenetic mechanism, chromatin remodeling by histone modification during early development, may be impaired in a subset of schizophrenia patients, in agreement with previous data. PMID:27066855

  11. Functional variants of sphingosine-1-phosphate receptor 1 gene associate with asthma susceptibility

    PubMed Central

    Sun, Xiaoguang; Ma, Shwu-Fan; Wade, Michael S.; Flores, Carlos; Pino-Yanes, Maria; Moitra, Jaideep; Ober, Carole; Kittles, Rick; Husain, Aliya N.; Ford, Jean G.; Garcia, Joe G. N.

    2012-01-01

    Background The genetic mechanisms underlying asthma remain unclear. Increased permeability of the microvasculature is a feature of asthma and the sphingosine-1-phosphate receptor, S1PR1, is an essential participant regulating lung vascular integrity and responses to lung inflammation. Objective We explored the contribution of polymorphisms in the S1PR1 gene (S1PR1) to asthma susceptibility. Methods A combination of gene re-sequencing for SNP discovery, case-control association, functional evaluation of associated SNPs, and protein immunochemistry studies was utilized. Results Immunohistochemistry studies demonstrated significantly decreased S1PR1 protein expression in pulmonary vessels in asthmatic lungs compared to non-asthmatic individuals (p<0.05). Direct DNA sequencing of 27 multiethnic samples identified 39 S1PR1 variants (18 novel SNPs). Association studies were performed based on genotyping results from cosmopolitan tagging SNPs in three case-control cohorts from Chicago and New York totaling 1061 subjects (502 cases and 559 controls). Promoter SNP rs2038366 (−1557G/T) was found to be associated with asthma (p=0.03) in European Americans. In African Americans, an association was found for both asthma and severe asthma for intronic SNP rs3753194 (c.−164+170A/G) (p=0.006 and p=0.040, respectively) and for promoter SNP rs59317557 (−532C/G) with severe asthma (p=0.028). Consistent with predicted in silico functionality, alleles of promoter SNPs rs2038366 (−1557G/T) and rs59317557 (−532C/G) influenced the activity of a luciferase S1PR1 reporter vector in transfected endothelial cells exposed to growth factors (EGF, PDGF, VEGF) known to be increased in asthmatic airways. Conclusion These data provide strong support for a role for S1PR1 gene variants in asthma susceptibility and severity. Clinical Implications Our results indicate S1PR1 is a novel asthma candidate gene and an attractive target for future therapeutic strategies. Capsule summary This study

  12. Better prediction of functional effects for sequence variants

    PubMed Central

    2015-01-01

    Elucidating the effects of naturally occurring genetic variation is one of the major challenges for personalized health and personalized medicine. Here, we introduce SNAP2, a novel neural network based classifier that improves over the state-of-the-art in distinguishing between effect and neutral variants. Our method's improved performance results from screening many potentially relevant protein features and from refining our development data sets. Cross-validated on >100k experimentally annotated variants, SNAP2 significantly outperformed other methods, attaining a two-state accuracy (effect/neutral) of 83%. SNAP2 also outperformed combinations of other methods. Performance increased for human variants but much more so for other organisms. Our method's carefully calibrated reliability index informs selection of variants for experimental follow up, with the most strongly predicted half of all effect variants predicted at over 96% accuracy. As expected, the evolutionary information from automatically generated multiple sequence alignments gave the strongest signal for the prediction. However, we also optimized our new method to perform surprisingly well even without alignments. This feature reduces prediction runtime by over two orders of magnitude, enables cross-genome comparisons, and renders our new method as the best solution for the 10-20% of sequence orphans. SNAP2 is available at: https://rostlab.org/services/snap2web Definitions used Delta, input feature that results from computing the difference feature scores for native amino acid and feature scores for variant amino acid; nsSNP, non-synoymous SNP; PMD, Protein Mutant Database; SNAP, Screening for non-acceptable polymorphisms; SNP, single nucleotide polymorphism; variant, any amino acid changing sequence variant. PMID:26110438

  13. A Computational Framework Discovers New Copy Number Variants with Functional Importance

    PubMed Central

    Banerjee, Samprit; Oldridge, Derek; Poptsova, Maria; Hussain, Wasay M.; Chakravarty, Dimple; Demichelis, Francesca

    2011-01-01

    Structural variants which cause changes in copy numbers constitute an important component of genomic variability. They account for 0.7% of genomic differences in two individual genomes, of which copy number variants (CNVs) are the largest component. A recent population-based CNV study revealed the need of better characterization of CNVs, especially the small ones (<500 bp).We propose a three step computational framework (Identification of germline Changes in Copy Number or IgC2N) to discover and genotype germline CNVs. First, we detect candidate CNV loci by combining information across multiple samples without imposing restrictions to the number of coverage markers or to the variant size. Secondly, we fine tune the detection of rare variants and infer the putative copy number classes for each locus. Last, for each variant we combine the relative distance between consecutive copy number classes with genetic information in a novel attempt to estimate the reference model bias. This computational approach is applied to genome-wide data from 1250 HapMap individuals. Novel variants were discovered and characterized in terms of size, minor allele frequency, type of polymorphism (gains, losses or both), and mechanism of formation. Using data generated for a subset of individuals by a 42 million marker platform, we validated the majority of the variants with the highest validation rate (66.7%) was for variants of size larger than 1 kb. Finally, we queried transcriptomic data from 129 individuals determined by RNA-sequencing as further validation and to assess the functional role of the new variants. We investigated the possible enrichment for variant's regulatory effect and found that smaller variants (<1 Kb) are more likely to regulate gene transcript than larger variants (p-value = 2.04e-08). Our results support the validity of the computational framework to detect novel variants relevant to disease susceptibility studies and provide evidence of the importance of

  14. Splice variants and promoter methylation status of the Bovine Vasa Homology (Bvh) gene may be involved in bull spermatogenesis

    PubMed Central

    2013-01-01

    Background Vasa is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. Bovine vasa homology (Bvh) of Bos taurus has been reported, however, its function in bovine testicular tissue remains obscure. This study aimed to reveal the functions of Bvh and to determine whether Bvh is a candidate gene in the regulation of spermatogenesis in bovine, and to illustrate whether its transcription is regulated by alternative splicing and DNA methylation. Results Here we report the molecular characterization, alternative splicing pattern, expression and promoter methylation status of Bvh. The full-length coding region of Bvh was 2190 bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. Bvh is expressed only in the ovary and testis of adult cattle. Two splice variants were identified and termed Bvh-V4 (2112 bp and 703 aa) and Bvh-V45 (2040 bp and 679 aa). In male cattle, full-length Bvh (Bvh-FL), Bvh-V4 and Bvh-V45 are exclusively expressed in the testes in the ratio of 2.2:1.6:1, respectively. Real-time PCR revealed significantly reduced mRNA expression of Bvh-FL, Bvh-V4 and Bvh-V45 in testes of cattle-yak hybrids, with meiotic arrest compared with cattle and yaks with normal spermatogenesis (P < 0.01). The promoter methylation level of Bvh in the testes of cattle-yak hybrids was significantly greater than in cattle and yaks (P < 0.01). Conclusion In the present study, Bvh was isolated and characterized. These data suggest that Bvh functions in bovine spermatogenesis, and that transcription of the gene in testes were regulated by alternative splice and promoter methylation. PMID:23815438

  15. Promoting Motor Function by Exercising the Brain

    PubMed Central

    Perrey, Stephane

    2013-01-01

    Exercise represents a behavioral intervention that enhances brain health and motor function. The increase in cerebral blood volume in response to physical activity may be responsible for improving brain function. Among the various neuroimaging techniques used to monitor brain hemodynamic response during exercise, functional near-infrared spectroscopy could facilitate the measurement of task-related cortical responses noninvasively and is relatively robust with regard to the subjects’ motion. Although the components of optimal exercise interventions have not been determined, evidence from animal and human studies suggests that aerobic exercise with sufficiently high intensity has neuroprotective properties and promotes motor function. This review provides an insight into the effect of physical activity (based on endurance and resistance exercises) on brain function for producing movement. Since most progress in the study of brain function has come from patients with neurological disorders (e.g., stroke and Parkinson’s patients), this review presents some findings emphasizing training paradigms for restoring motor function. PMID:24961309

  16. Promoting motor function by exercising the brain.

    PubMed

    Perrey, Stephane

    2013-01-01

    Exercise represents a behavioral intervention that enhances brain health and motor function. The increase in cerebral blood volume in response to physical activity may be responsible for improving brain function. Among the various neuroimaging techniques used to monitor brain hemodynamic response during exercise, functional near-infrared spectroscopy could facilitate the measurement of task-related cortical responses noninvasively and is relatively robust with regard to the subjects' motion. Although the components of optimal exercise interventions have not been determined, evidence from animal and human studies suggests that aerobic exercise with sufficiently high intensity has neuroprotective properties and promotes motor function. This review provides an insight into the effect of physical activity (based on endurance and resistance exercises) on brain function for producing movement. Since most progress in the study of brain function has come from patients with neurological disorders (e.g., stroke and Parkinson's patients), this review presents some findings emphasizing training paradigms for restoring motor function. PMID:24961309

  17. Schizophrenia risk variants affecting microRNA function and site-specific regulation of NT5C2 by miR-206.

    PubMed

    Hauberg, Mads Engel; Holm-Nielsen, Marie Hebsgaard; Mattheisen, Manuel; Askou, Anne Louise; Grove, Jakob; Børglum, Anders Dupont; Corydon, Thomas Juhl

    2016-09-01

    Despite the identification of numerous schizophrenia-associated genetic variants, few have been examined functionally to identify and characterize the causal variants. To mitigate this, we aimed at identifying functional variants affecting miRNA function. Using data from a large-scale genome-wide association study of schizophrenia, we looked for schizophrenia risk variants altering either miRNA binding sites, miRNA genes, promoters for miRNA genes, or variants that were expression quantitative trait loci (eQTLs) for miRNA genes. We hereby identified several potentially functional variants relating to miRNA function with our top finding being a schizophrenia protective allele that disrupts miR-206׳s binding to NT5C2 thus leading to increased expression of this gene. A subsequent experimental follow-up of the variant using a luciferase-based reporter assay confirmed that the allele disrupts the binding. Our study therefore suggests that miR-206 may contribute to schizophrenia risk through allele-dependent regulation of the genome-wide significant gene NT5C2. PMID:27424800

  18. A Naturally-Occurring Transcript Variant of MARCO Reveals the SRCR Domain is Critical for Function

    PubMed Central

    Novakowski, Kyle E.; Huynh, Angela; Han, SeongJun; Dorrington, Michael G.; Yin, Charles; Tu, Zhongyuan; Pelka, Peter; Whyte, Peter; Guarné, Alba; Sakamoto, Kaori; Bowdish, Dawn M.E.

    2016-01-01

    Macrophage receptor with collagenous structure (MARCO) is a Class A Scavenger Receptor (cA-SR) that recognizes and phagocytoses of a wide variety of pathogens. Most cA-SRs that contain a C-terminal Scavenger Receptor Cysteine Rich (SRCR) domain use the proximal collagenous domain to bind ligands. In contrast, for the role of the SRCR domain of MARCO in phagocytosis, adhesion and pro-inflammatory signalling is less clear. The discovery of a naturally-occurring transcript variant lacking the SRCR domain, MARCOII, provided the opportunity to study the role of the SRCR domain of MARCO. We tested whether the SRCR domain is required for ligand binding, promoting downstream signalling, and enhancing cellular adhesion. Unlike cells expressing full-length MARCO, ligand binding was abolished in MARCOII-expressing cells. Furthermore, co-expression of MARCO and MARCOII impaired phagocytic function, indicating that MARCOII acts as a dominant negative variant. Unlike MARCO, expression of MARCOII did not enhance Toll-Like Receptor 2 (TLR2)-mediated pro-inflammatory signalling in response to bacterial stimulation. MARCO-expressing cells were more adherent and exhibited a dendritic-like phenotype, while MARCOII-expressing cells were less adherent and did not exhibit changes in morphology. These data suggest the SRCR domain of MARCO is the key domain in modulating ligand binding, enhancing downstream pro-inflammatory signalling, and MARCO-mediated cellular adhesion. PMID:26888252

  19. In Silico Functional Pathway Annotation of 86 Established Prostate Cancer Risk Variants

    PubMed Central

    Loo, Lenora W. M.; Fong, Aaron Y. W.; Cheng, Iona; Le Marchand, Loïc

    2015-01-01

    Heritability is one of the strongest risk factors of prostate cancer, emphasizing the importance of the genetic contribution towards prostate cancer risk. To date, 86 established prostate cancer risk variants have been identified by genome-wide association studies (GWAS). To determine if these risk variants are located near genes that interact together in biological networks or pathways contributing to prostate cancer initiation or progression, we generated gene sets based on proximity to the 86 prostate cancer risk variants. We took two approaches to generate gene lists. The first strategy included all immediate flanking genes, up- and downstream of the risk variant, regardless of distance from the index variant, and the second strategy included genes closest to the index GWAS marker and to variants in high LD (r2 ≥0.8 in Europeans) with the index variant, within a 100 kb window up- and downstream. Pathway mapping of the two gene sets supported the importance of the androgen receptor-mediated signaling in prostate cancer biology. In addition, the hedgehog and Wnt/β-catenin signaling pathways were identified in pathway mapping for the flanking gene set. We also used the HaploReg resource to examine the 86 risk loci and variants high LD (r2 ≥0.8) for functional elements. We found that there was a 12.8 fold (p = 2.9 x 10-4) enrichment for enhancer motifs in a stem cell line and a 4.4 fold (p = 1.1 x 10-3) enrichment of DNase hypersensitivity in a prostate adenocarcinoma cell line, indicating that the risk and correlated variants are enriched for transcriptional regulatory motifs. Our pathway-based functional annotation of the prostate cancer risk variants highlights the potential regulatory function that GWAS risk markers, and their highly correlated variants, exert on genes. Our study also shows that these genes may function cooperatively in key signaling pathways in prostate cancer biology. PMID:25658610

  20. Novel intronic promoter in the rat ER alpha gene responsible for the transient transcription of a variant receptor.

    PubMed

    Tiffoche, C; Vaillant, C; Schausi, D; Thieulant, M L

    2001-09-01

    To analyze the molecular origin of an ER variant, the truncated ER product-1, transiently expressed at the proestrus in lactotrope cells, we generated a 2.5-kb sequence of a genomic region upstream and downstream the specific sequence truncated ER product-1. Genomic Southern blot analysis showed that truncated ER product-1 is spliced from a noncoding leader exon localized within the intron 4 of the ER alpha gene. Analysis of the promoter sequence revealed the presence of a major transcriptional start site, a canonical TATA box and putative cis regulatory elements for pituitary specific expression as well as an E-responsive element. In transient transfection, the truncated ER product-1 promoter was transcriptionally the most active in the lactotrope cell lines (MMQ). Analysis of truncated ER product-1 functionality showed that: 1) the protein inhibited ER alpha binding to the E-responsive element in electromobility shift assays, 2) inhibited the E2 binding to ER alpha in binding assays, 3) the truncated ER product-1/ER alpha complex antagonized the transcriptional activity elicited by E2, 4) nuclear localization of green fluorescent protein-ER alpha was altered in Chinese hamster ovary cell lines stably expressing truncated ER product-1. Collectively, these data demonstrated that the protein exerts full dominant negative activity against ER alpha. Moreover, truncated ER product-1/ER alpha complex also repressed the activity of all promoters tested to date, suggesting a general inhibitory effect toward transcription. In conclusion, the data suggest that truncated ER product-1 could regulate estrogen signaling via a specific promoter in lactotrope cells. PMID:11517190

  1. Functional Assessment of Genetic Variants with Outcomes Adapted to Clinical Decision-Making.

    PubMed

    Thouvenot, Pierre; Ben Yamin, Barbara; Fourrière, Lou; Lescure, Aurianne; Boudier, Thomas; Del Nery, Elaine; Chauchereau, Anne; Goldgar, David E; Houdayer, Claude; Stoppa-Lyonnet, Dominique; Nicolas, Alain; Millot, Gaël A

    2016-06-01

    Understanding the medical effect of an ever-growing number of human variants detected is a long term challenge in genetic counseling. Functional assays, based on in vitro or in vivo evaluations of the variant effects, provide essential information, but they require robust statistical validation, as well as adapted outputs, to be implemented in the clinical decision-making process. Here, we assessed 25 pathogenic and 15 neutral missense variants of the BRCA1 breast/ovarian cancer susceptibility gene in four BRCA1 functional assays. Next, we developed a novel approach that refines the variant ranking in these functional assays. Lastly, we developed a computational system that provides a probabilistic classification of variants, adapted to clinical interpretation. Using this system, the best functional assay exhibits a variant classification accuracy estimated at 93%. Additional theoretical simulations highlight the benefit of this ready-to-use system in the classification of variants after functional assessment, which should facilitate the consideration of functional evidences in the decision-making process after genetic testing. Finally, we demonstrate the versatility of the system with the classification of siRNAs tested for human cell growth inhibition in high throughput screening. PMID:27272900

  2. Functional Assessment of Genetic Variants with Outcomes Adapted to Clinical Decision-Making

    PubMed Central

    Thouvenot, Pierre; Ben Yamin, Barbara; Fourrière, Lou; Lescure, Aurianne; Boudier, Thomas; Del Nery, Elaine; Chauchereau, Anne; Goldgar, David E.; Stoppa-Lyonnet, Dominique; Nicolas, Alain; Millot, Gaël A.

    2016-01-01

    Understanding the medical effect of an ever-growing number of human variants detected is a long term challenge in genetic counseling. Functional assays, based on in vitro or in vivo evaluations of the variant effects, provide essential information, but they require robust statistical validation, as well as adapted outputs, to be implemented in the clinical decision-making process. Here, we assessed 25 pathogenic and 15 neutral missense variants of the BRCA1 breast/ovarian cancer susceptibility gene in four BRCA1 functional assays. Next, we developed a novel approach that refines the variant ranking in these functional assays. Lastly, we developed a computational system that provides a probabilistic classification of variants, adapted to clinical interpretation. Using this system, the best functional assay exhibits a variant classification accuracy estimated at 93%. Additional theoretical simulations highlight the benefit of this ready-to-use system in the classification of variants after functional assessment, which should facilitate the consideration of functional evidences in the decision-making process after genetic testing. Finally, we demonstrate the versatility of the system with the classification of siRNAs tested for human cell growth inhibition in high throughput screening. PMID:27272900

  3. Interaction between SLC6A4 promoter variants and childhood trauma on the age at onset of bipolar disorders

    PubMed Central

    Etain, B.; Lajnef, M.; Henrion, A.; Dargél, A.A.; Stertz, L.; Kapczinski, F.; Mathieu, F.; Henry, C.; Gard, S.; Kahn, J. P.; Leboyer, M.; Jamain, S.; Bellivier, F.

    2015-01-01

    Age at onset (AAO) of bipolar disorders (BD) could be influenced both by a repeat length polymorphism (5HTTLPR) in the promoter region of the serotonin transporter gene (SLC6A4) and exposure to childhood trauma. We assessed 308 euthymic patients with BD for the AAO of their first mood episode and childhood trauma. Patients were genotyped for the 5HTTLPR (long/short variant) and the rs25531. Genotypes were classified on functional significance (LL, LS, SS). A sample of 126 Brazilian euthymic patients with BD was used for replication. In the French sample, the correlation between AAO and trauma score was observed only among ‘SS’ homozygotes (p = 0.002) but not among ‘L’ allele carriers. A history of at least one trauma decreased the AAO only in ‘SS’ homozygotes (p = 0.001). These results remained significant after correction using FDR. Regression models suggested an interaction between emotional neglect and ‘SS’ genotype on the AAO (p = 0.009) and no further interaction with other trauma subtypes. Partial replication was obtained in the Brazilian sample, showing an interaction between emotional abuse and ‘LS’ genotype on the AAO (p = 0.02). In conclusion, an effect of childhood trauma on AAO of BD was observed only in patients who carry a specific stress responsiveness-related SLC6A4 promoter genotype. PMID:26542422

  4. Functional analysis of missense variants in the TRESK (KCNK18) K channel.

    PubMed

    Andres-Enguix, Isabelle; Shang, Lijun; Stansfeld, Phillip J; Morahan, Julia M; Sansom, Mark S P; Lafrenière, Ronald G; Roy, Bishakha; Griffiths, Lyn R; Rouleau, Guy A; Ebers, George C; Cader, Zameel M; Tucker, Stephen J

    2012-01-01

    A loss of function mutation in the TRESK K2P potassium channel (KCNK18), has recently been linked with typical familial migraine with aura. We now report the functional characterisation of additional TRESK channel missense variants identified in unrelated patients. Several variants either had no apparent functional effect, or they caused a reduction in channel activity. However, the C110R variant was found to cause a complete loss of TRESK function, yet is present in both sporadic migraine and control cohorts, and no variation in KCNK18 copy number was found. Thus despite the previously identified association between loss of TRESK channel activity and migraine in a large multigenerational pedigree, this finding indicates that a single non-functional TRESK variant is not alone sufficient to cause typical migraine and highlights the genetic complexity of this disorder. PMID:22355750

  5. The WIP1 Oncogene Promotes Progression and Invasion of Aggressive Medulloblastoma Variants

    PubMed Central

    Buss, Meghan C.; Remke, Marc; Lee, Juhyun; Gandhi, Khanjan; Schniederjan, Matthew J.; Kool, Marcel; Northcott, Paul A.; Pfister, Stefan M.; Taylor, Michael D.; Castellino, Robert C.

    2014-01-01

    Recent studies suggest that medulloblastoma, the most common malignant brain tumor of childhood, is comprised of four disease variants. The WIP1 oncogene is overexpressed in Group 3 and 4 tumors, which contain medulloblastomas with the most aggressive clinical behavior. Our data demonstrate increased WIP1 expression in metastatic medulloblastomas, and inferior progression-free and overall survival of patients with WIP1 high-expressing medulloblastoma. Microarray analysis identified up-regulation of genes involved in tumor metastasis, including the G protein-coupled receptor CXCR4, in medulloblastoma cells with high WIP1 expression. Stimulation with the CXCR4 ligand SDF1ααactivated PI-3 kinase signaling, and promoted growth and invasion of WIP1 high-expressing medulloblastoma cells in a p53-dependent manner. When xenografted into the cerebellum of immunodeficient mice, medulloblastoma cells with stable or endogenous high WIP1 expression exhibited strong expression of CXCR4 and activated AKT in primary and invasive tumor cells. WIP1 or CXCR4 knock-down inhibited medulloblastoma growth and invasion. WIP1 knock-down also improved the survival of mice xenografted with WIP1 high-expressing medulloblastoma cells. WIP1 knock-down inhibited cell surface localization of CXCR4 by suppressing expression of the G protein receptor kinase 5, GRK5. Restoration of wild-type GRK5 promoted Ser339 phosphorylation of CXCR4 and inhibited the growth of WIP1-stable medulloblastoma cells. Conversely, GRK5 knock-down inhibited Ser339 phosphorylation of CXCR4, increased cell surface localization of CXCR4, and promoted the growth of medulloblastoma cells with low WIP1 expression. These results demonstrate cross-talk among WIP1, CXCR4, and GRK5, which may be important for the aggressive phenotype of a subclass of medulloblastomas in children. PMID:24632620

  6. Comprehensive splicing functional analysis of DNA variants of the BRCA2 gene by hybrid minigenes

    PubMed Central

    2012-01-01

    Introduction The underlying pathogenic mechanism of a large fraction of DNA variants of disease-causing genes is the disruption of the splicing process. We aimed to investigate the effect on splicing of the BRCA2 variants c.8488-1G > A (exon 20) and c.9026_9030del (exon 23), as well as 41 BRCA2 variants reported in the Breast Cancer Information Core (BIC) mutation database. Methods DNA variants were analyzed with the splicing prediction programs NNSPLICE and Human Splicing Finder. Functional analyses of candidate variants were performed by lymphocyte RT-PCR and/or hybrid minigene assays. Forty-one BIC variants of exons 19, 20, 23 and 24 were bioinformatically selected and generated by PCR-mutagenesis of the wild type minigenes. Results Lymphocyte RT-PCR of c.8488-1G > A showed intron 19 retention and a 12-nucleotide deletion in exon 20, whereas c.9026_9030del did not show any splicing anomaly. Minigene analysis of c.8488-1G > A displayed the aforementioned aberrant isoforms but also exon 20 skipping. We further evaluated the splicing outcomes of 41 variants of four BRCA2 exons by minigene analysis. Eighteen variants presented splicing aberrations. Most variants (78.9%) disrupted the natural splice sites, whereas four altered putative enhancers/silencers and had a weak effect. Fluorescent RT-PCR of minigenes accurately detected 14 RNA isoforms generated by cryptic site usage, exon skipping and intron retention events. Fourteen variants showed total splicing disruptions and were predicted to truncate or eliminate essential domains of BRCA2. Conclusions A relevant proportion of BRCA2 variants are correlated with splicing disruptions, indicating that RNA analysis is a valuable tool to assess the pathogenicity of a particular DNA change. The minigene system is a straightforward and robust approach to detect variants with an impact on splicing and contributes to a better knowledge of this gene expression step. PMID:22632462

  7. Common interleukin-6 promoter variants associate with the more severe forms of distal interphalangeal osteoarthritis

    PubMed Central

    Kämäräinen, Olli-Pekka; Solovieva, Svetlana; Vehmas, Tapio; Luoma, Katariina; Riihimäki, Hilkka; Ala-Kokko, Leena; Männikkö, Minna; Leino-Arjas, Päivi

    2008-01-01

    Introduction The objective of this study was to investigate the relationship of the IL-6 promoter variants G-597A, G-572C and G-174C (rs1800797, rs1800796 and rs1800795, respectively), which have been shown to affect both the transcription and secretion of IL-6, to symptomatic distal interphalangeal (DIP) osteoarthritis (OA). Methods A total of 535 women aged 45 to 63 years were included. Radiographs of both hands were taken and each DIP joint was evaluated (grade 0 to 4) for the presence of OA. Information on symptoms (pain, tenderness) in each joint was collected by using a self-administered questionnaire. Symptomatic DIP OA was defined by the presence of both radiographic findings of grade 2 or more and symptoms in at least two DIP joints, and symmetrical DIP OA by the presence of radiographic findings of grade 2 or more in at least one symmetrical pair of DIP joints. Common polymorphic loci in the IL-6 gene were amplified and the promoter haplotypes were reconstructed from genotype data with the PHASE program. Logistic regression analysis was used to examine the association between the IL-6 genotypes/diplotypes and the DIP OA outcome. Results The G alleles of two promoter single nucleotide polymorphisms (SNPs) G-597A and G-174C were more common among the subjects with symptomatic DIP OA than among those with no disease (P = 0.020 and 0.024, corrected for multiple testing). In addition, the carriage of at least one G allele in these positions increased the risk of disease (P = 0.006 and P = 0.008, respectively). Carrying a haplotype with the G allele in all three promoter SNPs increased the risk of symptomatic DIP OA more than fourfold (odds ratio (OR) 4.45, P = 0.001). Carriage of the G-G diplotype indicated an increased risk of both symmetrical DIP OA (OR 1.52, 95% confidence interval 1.01 to 2.28) and symptomatic DIP OA (OR 3.67, 95% confidence interval 1.50 to 9.00). Conclusion The present study showed that the presence of G alleles at common IL-6

  8. Genetic variants of ApoE and ApoER2 differentially modulate endothelial function

    PubMed Central

    Ulrich, Victoria; Konaniah, Eddy S.; Herz, Joachim; Gerard, Robert D.; Jung, Eunjeong; Yuhanna, Ivan S.; Ahmed, Mohamed; Hui, David Y.; Mineo, Chieko; Shaul, Philip W.

    2014-01-01

    It is poorly understood why there is greater cardiovascular disease risk associated with the apolipoprotein E4 (apoE) allele vs. apoE3, and also greater risk with the LRP8/apolipoprotein E receptor 2 (ApoER2) variant ApoER2-R952Q. Little is known about the function of the apoE–ApoER2 tandem outside of the central nervous system. We now report that in endothelial cells apoE3 binding to ApoER2 stimulates endothelial NO synthase (eNOS) and endothelial cell migration, and it also attenuates monocyte–endothelial cell adhesion. However, apoE4 does not stimulate eNOS or endothelial cell migration or dampen cell adhesion, and alternatively it selectively antagonizes apoE3/ApoER2 actions. The contrasting endothelial actions of apoE4 vs. apoE3 require the N-terminal to C-terminal interaction in apoE4 that distinguishes it structurally from apoE3. Reconstitution experiments further reveal that ApoER2-R952Q is a loss-of-function variant of the receptor in endothelium. Carotid artery reendothelialization is decreased in ApoER2−/− mice, and whereas adenoviral-driven apoE3 expression in wild-type mice has no effect, apoE4 impairs reendothelialization. Moreover, in a model of neointima formation invoked by carotid artery endothelial denudation, ApoER2−/− mice display exaggerated neointima development. Thus, the apoE3/ApoER2 tandem promotes endothelial NO production, endothelial repair, and endothelial anti-inflammatory properties, and it prevents neointima formation. In contrast, apoE4 and ApoER2-R952Q display dominant-negative action and loss of function, respectively. Thus, genetic variants of apoE and ApoER2 impact cardiovascular health by differentially modulating endothelial function. PMID:25197062

  9. Computational approaches to identify functional genetic variants in cancer genomes

    PubMed Central

    Gonzalez-Perez, Abel; Mustonen, Ville; Reva, Boris; Ritchie, Graham R.S.; Creixell, Pau; Karchin, Rachel; Vazquez, Miguel; Fink, J. Lynn; Kassahn, Karin S.; Pearson, John V.; Bader, Gary; Boutros, Paul C.; Muthuswamy, Lakshmi; Ouellette, B.F. Francis; Reimand, Jüri; Linding, Rune; Shibata, Tatsuhiro; Valencia, Alfonso; Butler, Adam; Dronov, Serge; Flicek, Paul; Shannon, Nick B.; Carter, Hannah; Ding, Li; Sander, Chris; Stuart, Josh M.; Stein, Lincoln D.; Lopez-Bigas, Nuria

    2014-01-01

    The International Cancer Genome Consortium (ICGC) aims to catalog genomic abnormalities in tumors from 50 different cancer types. Genome sequencing reveals hundreds to thousands of somatic mutations in each tumor, but only a minority drive tumor progression. We present the result of discussions within the ICGC on how to address the challenge of identifying mutations that contribute to oncogenesis, tumor maintenance or response to therapy, and recommend computational techniques to annotate somatic variants and predict their impact on cancer phenotype. PMID:23900255

  10. Arginine-Glycine Amidinotransferase Deficiency and Functional Characterization of Missense Variants in GATM.

    PubMed

    DesRoches, Caro-Lyne; Bruun, Theodora; Wang, Peixiang; Marshall, Christian R; Mercimek-Mahmutoglu, Saadet

    2016-09-01

    Arginine-glycine amidinotransferase (GATM) deficiency is an autosomal-recessive disorder caused by pathogenic variants in GATM. Clinical features include intellectual disability, hypotonia, and myopathy. Due to normal neurodevelopment in asymptomatic individuals on creatine monotherapy, GATM deficiency is a good candidate for newborn screening. To determine the carrier frequency of GATM deficiency, we performed functional characterization of rare missense variants in GATM reported as heterozygous in the Exome Variant Server database. To assess phenotype and genotype correlation, we developed a clinical severity scoring system. Two patients with mild phenotype had a nonsense missense variant. Severe phenotype was present in patients with missense as well as truncating variants. There seems to be no phenotype and genotype correlation. We cloned a novel GATM transcript. We found seven missense variants retaining 0% of wild-type GATM activity indicating putative pathogenicity. Based on our study results, high Genomic Evolutionary Rate Profiling conservation score, conserved amino acid substitution in species, and low allele frequency in exome databases would be the most sensitive in silico analysis tools to predict pathogenicity of missense variants. We present first study of the functional characterization of missense variants in GATM as well as clinical severity score of patients with GATM deficiency. PMID:27233232

  11. Multiple sclerosis associated genetic variants of CD226 impair regulatory T cell function.

    PubMed

    Piédavent-Salomon, Melanie; Willing, Anne; Engler, Jan Broder; Steinbach, Karin; Bauer, Simone; Eggert, Britta; Ufer, Friederike; Kursawe, Nina; Wehrmann, Sabine; Jäger, Jan; Reinhardt, Stefanie; Friese, Manuel A

    2015-11-01

    Recent association studies have linked numerous genetic variants with an increased risk for multiple sclerosis, although their functional relevance remains largely unknown. Here we investigated phenotypical and functional consequences of a genetic variant in the CD226 gene that, among other autoimmune diseases, predisposes to multiple sclerosis. Phenotypically, effector and regulatory CD4(+) memory T cells of healthy individuals carrying the predisposing CD226 genetic variant showed, in comparison to carriers of the protective variant, reduced surface expression of CD226 and an impaired induction of CD226 after stimulation. This haplotype-dependent reduction in CD226 expression on memory T cells was abrogated in patients with multiple sclerosis, as CD226 expression was comparable to healthy risk haplotype carriers irrespective of genetic variant. Functionally, FOXP3-positive regulatory T cells from healthy carriers of the genetic protective variant showed superior suppressive capacity, which was again abrogated in multiple sclerosis patients. Mimicking the phenotype of human CD226 genetic risk variant carriers, regulatory T cells derived from Cd226-deficient mice showed similarly reduced inhibitory activity, eventually resulting in an exacerbated disease course of experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. Therefore, by combining human and mouse analyses we show that CD226 exhibits an important role in the activation of regulatory T cells, with its genetically imposed dysregulation impairing regulatory T cell function. PMID:26359290

  12. Post-GWAS methodologies for localisation of functional non-coding variants: ANGPTL3

    PubMed Central

    Oldoni, Federico; Palmen, Jutta; Giambartolomei, Claudia; Howard, Philip; Drenos, Fotios; Plagnol, Vincent; Humphries, Steve E.; Talmud, Philippa J.; Smith, Andrew J.P.

    2016-01-01

    Genome-wide association studies have confirmed the involvement of non-coding angiopoietin-like 3 (ANGPTL3) gene variants with coronary artery disease, levels of low-density lipoprotein cholesterol (LDL-C), triglycerides and ANGPTL3 mRNA transcript. Extensive linkage disequilibrium at the locus, however, has hindered efforts to identify the potential functional variants. Using regulatory annotations from ENCODE, combined with functional in vivo assays such as allele-specific formaldehyde-assisted isolation of regulatory elements, statistical approaches including eQTL/lipid colocalisation, and traditional in vitro methodologies including electrophoretic mobility shift assay and luciferase reporter assays, variants affecting the ANGPTL3 regulome were examined. From 253 variants associated with ANGPTL3 mRNA expression, and/or lipid traits, 46 were located within liver regulatory elements and potentially functional. One variant, rs10889352, demonstrated allele-specific effects on DNA-protein interactions, reporter gene expression and chromatin accessibility, in line with effects on LDL-C levels and expression of ANGPTL3 mRNA. The ANGPTL3 gene lies within DOCK7, although the variant is within non-coding regions outside of ANGPTL3, within DOCK7, suggesting complex long-range regulatory effects on gene expression. This study illustrates the power of combining multiple genome-wide datasets with laboratory data to localise functional non-coding variation and provides a model for analysis of regulatory variants from GWAS. PMID:26800306

  13. Post-GWAS methodologies for localisation of functional non-coding variants: ANGPTL3.

    PubMed

    Oldoni, Federico; Palmen, Jutta; Giambartolomei, Claudia; Howard, Philip; Drenos, Fotios; Plagnol, Vincent; Humphries, Steve E; Talmud, Philippa J; Smith, Andrew J P

    2016-03-01

    Genome-wide association studies have confirmed the involvement of non-coding angiopoietin-like 3 (ANGPTL3) gene variants with coronary artery disease, levels of low-density lipoprotein cholesterol (LDL-C), triglycerides and ANGPTL3 mRNA transcript. Extensive linkage disequilibrium at the locus, however, has hindered efforts to identify the potential functional variants. Using regulatory annotations from ENCODE, combined with functional in vivo assays such as allele-specific formaldehyde-assisted isolation of regulatory elements, statistical approaches including eQTL/lipid colocalisation, and traditional in vitro methodologies including electrophoretic mobility shift assay and luciferase reporter assays, variants affecting the ANGPTL3 regulome were examined. From 253 variants associated with ANGPTL3 mRNA expression, and/or lipid traits, 46 were located within liver regulatory elements and potentially functional. One variant, rs10889352, demonstrated allele-specific effects on DNA-protein interactions, reporter gene expression and chromatin accessibility, in line with effects on LDL-C levels and expression of ANGPTL3 mRNA. The ANGPTL3 gene lies within DOCK7, although the variant is within non-coding regions outside of ANGPTL3, within DOCK7, suggesting complex long-range regulatory effects on gene expression. This study illustrates the power of combining multiple genome-wide datasets with laboratory data to localise functional non-coding variation and provides a model for analysis of regulatory variants from GWAS. PMID:26800306

  14. Mannose-Binding Lectin Promoter Polymorphisms and Gene Variants in Pulmonary Tuberculosis Patients from Cantabria (Northern Spain)

    PubMed Central

    Lavín-Alconero, Lucía; Sánchez-Velasco, Pablo; Guerrero-Alonso, M.-Ángeles; Ausín, Fernando; Fariñas, M.-Carmen; Leyva-Cobián, Francisco

    2012-01-01

    Mannose-binding lectin is a central molecule of the innate immune system. Mannose-binding lectin 2 promoter polymorphisms and structural variants have been associated with susceptibility to tuberculosis. However, contradictory results among different populations have been reported, resulting in no convincing evidence of association between mannose-binding lectin 2 and susceptibility to tuberculosis. For this reason, we conducted a study in a well genetically conserved Spanish population in order to shed light on this controversial association. We analysed the six promoter and structural mannose-binding lectin 2 gene variants in 107 patients with pulmonary tuberculosis and 441 healthy controls. Only D variant and HYPD haplotype were significantly more frequents in controls which would indicate that this allele could confer protection against pulmonary tuberculosis, but this difference disappeared after statistical correction. Neither the rest of alleles nor the haplotypes were significantly associated with the disease. These results would indicate that mannose-binding lectin promoter polymorphisms and gene variants would not be associated with an increased risk to pulmonary tuberculosis. Despite the slight trend of the D allele and HYPD haplotype in conferring protection against pulmonary tuberculosis, susceptibility to this disease would probably be due to other genetic factors, at least in our population. PMID:23304495

  15. Mannose-binding lectin promoter polymorphisms and gene variants in pulmonary tuberculosis patients from cantabria (northern Spain).

    PubMed

    Ocejo-Vinyals, J-Gonzalo; Lavín-Alconero, Lucía; Sánchez-Velasco, Pablo; Guerrero-Alonso, M-Ángeles; Ausín, Fernando; Fariñas, M-Carmen; Leyva-Cobián, Francisco

    2012-01-01

    Mannose-binding lectin is a central molecule of the innate immune system. Mannose-binding lectin 2 promoter polymorphisms and structural variants have been associated with susceptibility to tuberculosis. However, contradictory results among different populations have been reported, resulting in no convincing evidence of association between mannose-binding lectin 2 and susceptibility to tuberculosis. For this reason, we conducted a study in a well genetically conserved Spanish population in order to shed light on this controversial association. We analysed the six promoter and structural mannose-binding lectin 2 gene variants in 107 patients with pulmonary tuberculosis and 441 healthy controls. Only D variant and HYPD haplotype were significantly more frequents in controls which would indicate that this allele could confer protection against pulmonary tuberculosis, but this difference disappeared after statistical correction. Neither the rest of alleles nor the haplotypes were significantly associated with the disease. These results would indicate that mannose-binding lectin promoter polymorphisms and gene variants would not be associated with an increased risk to pulmonary tuberculosis. Despite the slight trend of the D allele and HYPD haplotype in conferring protection against pulmonary tuberculosis, susceptibility to this disease would probably be due to other genetic factors, at least in our population. PMID:23304495

  16. Functional characterization of common protein variants in the efflux transporter ABCC11 and identification of T546M as functionally damaging variant.

    PubMed

    Arlanov, R; Lang, T; Jedlitschky, G; Schaeffeler, E; Ishikawa, T; Schwab, M; Nies, A T

    2016-04-01

    Multidrug resistance protein 8 (ABCC11) is an efflux transporter for anionic lipophilic compounds, conferring resistance to antiviral and anticancer agents like 5-fluorouracil (5-FU). ABCC11 missense variants may contribute to variability in drug response but functional consequences, except for the 'earwax variant' c.538G>A, are unknown. Using the 'Screen and Insert' technology, we generated human embryonic kidney 293 cells stably expressing ABCC11 missense variants frequently occurring in different ethnic populations: c.57G>A, c.538G>A, c.950C>A, c.1637C>T, c.1942G>A, c.4032A>G. A series of in silico prediction analyses and in vitro plasma membrane vesicle uptake, immunoblotting and immunolocalization experiments were undertaken to investigate functional consequences. We identified c.1637C>T (T546M), previously associated with 5-FU-related toxicity, as a novel functionally damaging ABCC11 variant exhibiting markedly reduced transport function of 5-FdUMP, the active cytotoxic metabolite of 5-FU. Detailed analysis of 14 subpopulations revealed highest allele frequencies of c.1637C>T in Europeans and Americans (up to 11%) compared with Africans and Asians (up to 3%). PMID:25896536

  17. Retrospective study investigating the prevalence and clinical significance of hepatitis B virus precore and basal core promoter variants

    PubMed Central

    O’Brien, Meaghan; Casselman, Adara; Smart, Gerry; Gretchen, Ainsley; Kaita, Kelly; Kadkhoda, Kamran

    2015-01-01

    BACKGROUND: Hepatitis B virus (HBV) precore (PC) and basal core promoter (BCP) variants are well known; however, their prevalence in North America is unclear, especially among hepatitis B e antigen-negative patients. OBJECTIVE: To investigate the prevalence of PC/BCP mutations and their clinical significance. METHODS: One hundred twenty-eight patients positive for both hepatitis B surface antigen and hepatitis B e antibody were selected, and PC/BCP mutations were identified using a line probe assay. The subjects’ charts were reviewed for race/ethnicity, HBV genotype, HBV viral load, sex, liver enzyme levels, imaging and biopsy results up to 10 years before the study. RESULTS: The prevalence of PC and BCP variants were 47.6% and 62.5%, respectively. Older age was associated with aspartate aminotransferase-to-platelet index ratio (APRI) ≥0.7 (P=0.011) and abnormal imaging/biopsy results (P=0.0008). Although the presence of BCP variant(s) was associated with APRI ≥0.7 (P=0.029), it was not associated with abnormal imaging/biopsy results. The combination of age ≥50 years and the presence of BCP variant(s) was associated with abnormal imaging/biopsy results, suggestive of either cirrhosis or hepatocellular carcinoma (not observed with PC mutation). Neither sex or genotype, or median HBV viral load showed significant influence on any of these outcomes. CONCLUSIONS: The present study suggests that the prevalence of PC and BCP mutations are higher than what has been previously reported. One potential explanation would be increased immigration in the past decade. Considering the potential public health and clinical implications of these variants, long-term multicentre and prospective studies could further unravel the uncertainty around these variants. PMID:26401823

  18. Genetic Load of Loss-of-Function Polymorphic Variants in Great Apes

    PubMed Central

    de Valles-Ibáñez, Guillem; Hernandez-Rodriguez, Jessica; Prado-Martinez, Javier; Luisi, Pierre; Marquès-Bonet, Tomàs; Casals, Ferran

    2016-01-01

    Loss of function (LoF) genetic variants are predicted to disrupt gene function, and are therefore expected to substantially reduce individual’s viability. Knowing the genetic burden of LoF variants in endangered species is of interest for a better understanding of the effects of declining population sizes on species viability. In this study, we have estimated the number of LoF polymorphic variants in six great ape populations, based on whole-genome sequencing data in 79 individuals. Our results show that although the number of functional variants per individual is conditioned by the effective population size, the number of variants with a drastic phenotypic effect is very similar across species. We hypothesize that for those variants with high selection coefficients, differences in effective population size are not important enough to affect the efficiency of natural selection to remove them. We also describe that mostly CpG LoF mutations are shared across species, and an accumulation of LoF variants at olfactory receptor genes in agreement with its pseudogenization in humans and other primate species. PMID:26912403

  19. Genetic Load of Loss-of-Function Polymorphic Variants in Great Apes.

    PubMed

    de Valles-Ibáñez, Guillem; Hernandez-Rodriguez, Jessica; Prado-Martinez, Javier; Luisi, Pierre; Marquès-Bonet, Tomàs; Casals, Ferran

    2016-03-01

    Loss of function (LoF) genetic variants are predicted to disrupt gene function, and are therefore expected to substantially reduce individual's viability. Knowing the genetic burden of LoF variants in endangered species is of interest for a better understanding of the effects of declining population sizes on species viability. In this study, we have estimated the number of LoF polymorphic variants in six great ape populations, based on whole-genome sequencing data in 79 individuals. Our results show that although the number of functional variants per individual is conditioned by the effective population size, the number of variants with a drastic phenotypic effect is very similar across species. We hypothesize that for those variants with high selection coefficients, differences in effective population size are not important enough to affect the efficiency of natural selection to remove them. We also describe that mostly CpG LoF mutations are shared across species, and an accumulation of LoF variants at olfactory receptor genes in agreement with its pseudogenization in humans and other primate species. PMID:26912403

  20. Functional TLR5 genetic variants affect human colorectal cancer survival.

    PubMed

    Klimosch, Sascha N; Försti, Asta; Eckert, Jana; Knezevic, Jelena; Bevier, Melanie; von Schönfels, Witigo; Heits, Nils; Walter, Jessica; Hinz, Sebastian; Lascorz, Jesus; Hampe, Jochen; Hartl, Dominik; Frick, Julia-Stefanie; Hemminki, Kari; Schafmayer, Clemens; Weber, Alexander N R

    2013-12-15

    Toll-like receptors (TLR) are overexpressed on many types of cancer cells, including colorectal cancer cells, but little is known about the functional relevance of these immune regulatory molecules in malignant settings. Here, we report frequent single-nucleotide polymorphisms (SNP) in the flagellin receptor TLR5 and the TLR downstream effector molecules MyD88 and TIRAP that are associated with altered survival in a large cohort of Caucasian patients with colorectal cancer (n = 613). MYD88 rs4988453, a SNP that maps to a promoter region shared with the acetyl coenzyme-A acyl-transferase-1 (ACAA1), was associated with decreased survival of patients with colorectal cancer and altered transcriptional activity of the proximal genes. In the TLR5 gene, rs5744174/F616L was associated with increased survival, whereas rs2072493/N592S was associated with decreased survival. Both rs2072493/N592S and rs5744174/F616L modulated TLR5 signaling in response to flagellin or to different commensal and pathogenic intestinal bacteria. Notably, we observed a reduction in flagellin-induced p38 phosphorylation, CD62L shedding, and elevated expression of interleukin (IL)-6 and IL-1β mRNA in human primary immune cells from TLR5 616LL homozygote carriers, as compared with 616FF carriers. This finding suggested that the well-documented effect of cytokines like IL-6 on colorectal cancer progression might be mediated by TLR5 genotype-dependent flagellin sensing. Our results establish an important link between TLR signaling and human colorectal cancer with relevance for biomarker and therapy development. PMID:24154872

  1. Onco-lncRNA HOTAIR and its functional genetic variants in papillary thyroid carcinoma.

    PubMed

    Zhu, Hui; Lv, Zheng; An, Changming; Shi, Meng; Pan, Wenting; Zhou, Liqing; Yang, Wenjun; Yang, Ming

    2016-01-01

    The role of long noncoding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) and its functional single nucleotide polymorphisms (SNPs) in papillary thyroid carcinoma (PTC) is still largely unclear. Therefore, we investigated the involvement of lncRNA HOTAIR and its three haplotype-tagging SNPs (htSNPs) in PTC. There was higher expression of HOTAIR in PTC tissues compared to normal tissues. A series of gain-loss assays demonstrated that HOTAIR acts as a PTC oncogene via promoting tumorigenic properties of PTC cells. Additionally, the functional HOTAIR rs920778 genetic variant was a PTC susceptibility SNP. Subjects with the HOTAIR rs920778 TT genotype had an odds ratio (OR) of 1.88, 1.25 and 1.61 (P = 6.0 × 10(-6), P = 0.028 and P = 3.2 × 10(-5)) for developing PTC in Shandong, Jiangsu and Jilin case-control sets compared with subjects with the CC genotype. This statistically significant associations were only found between the rs920778 genetic polymorphism and PTC risk in females but not in males. The allele-specific regulation on HOTAIR expression by the rs920778 SNP was confirmed both in vitro and in vivo. Our results demonstrate that functional SNPs influencing lncRNA regulation may explain a part of PTC genetic basis. PMID:27549736

  2. Onco-lncRNA HOTAIR and its functional genetic variants in papillary thyroid carcinoma

    PubMed Central

    Zhu, Hui; Lv, Zheng; An, Changming; Shi, Meng; Pan, Wenting; Zhou, Liqing; Yang, Wenjun; Yang, Ming

    2016-01-01

    The role of long noncoding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) and its functional single nucleotide polymorphisms (SNPs) in papillary thyroid carcinoma (PTC) is still largely unclear. Therefore, we investigated the involvement of lncRNA HOTAIR and its three haplotype-tagging SNPs (htSNPs) in PTC. There was higher expression of HOTAIR in PTC tissues compared to normal tissues. A series of gain-loss assays demonstrated that HOTAIR acts as a PTC oncogene via promoting tumorigenic properties of PTC cells. Additionally, the functional HOTAIR rs920778 genetic variant was a PTC susceptibility SNP. Subjects with the HOTAIR rs920778 TT genotype had an odds ratio (OR) of 1.88, 1.25 and 1.61 (P = 6.0 × 10−6, P = 0.028 and P = 3.2 × 10−5) for developing PTC in Shandong, Jiangsu and Jilin case-control sets compared with subjects with the CC genotype. This statistically significant associations were only found between the rs920778 genetic polymorphism and PTC risk in females but not in males. The allele-specific regulation on HOTAIR expression by the rs920778 SNP was confirmed both in vitro and in vivo. Our results demonstrate that functional SNPs influencing lncRNA regulation may explain a part of PTC genetic basis. PMID:27549736

  3. Cooperation between distinct viral variants promotes growth of H3N2 influenza in cell culture.

    PubMed

    Xue, Katherine S; Hooper, Kathryn A; Ollodart, Anja R; Dingens, Adam S; Bloom, Jesse D

    2016-01-01

    RNA viruses rapidly diversify into quasispecies of related genotypes. This genetic diversity has long been known to facilitate adaptation, but recent studies have suggested that cooperation between variants might also increase population fitness. Here, we demonstrate strong cooperation between two H3N2 influenza variants that differ by a single mutation at residue 151 in neuraminidase, which normally mediates viral exit from host cells. Residue 151 is often annotated as an ambiguous amino acid in sequenced isolates, indicating mixed viral populations. We show that mixed populations grow better than either variant alone in cell culture. Pure populations of either variant generate the other through mutation and then stably maintain a mix of the two genotypes. We suggest that cooperation arises because mixed populations combine one variant's proficiency at cell entry with the other's proficiency at cell exit. Our work demonstrates a specific cooperative interaction between defined variants in a viral quasispecies. PMID:26978794

  4. Overlap between Parkinson disease and Alzheimer disease in ABCA7 functional variants

    PubMed Central

    Nuytemans, Karen; Maldonado, Lizmarie; Ali, Aleena; John-Williams, Krista; Beecham, Gary W.; Martin, Eden; Scott, William K.

    2016-01-01

    Objective: Given their reported function in phagocytosis and clearance of protein aggregates in Alzheimer disease (AD), we hypothesized that variants in ATP-binding cassette transporter A7 (ABCA7) might be involved in Parkinson disease (PD). Methods: ABCA7 variants were identified using whole-exome sequencing (WES) on 396 unrelated patients with PD and 222 healthy controls. In addition, we used the publicly available WES data from the Parkinson's Progression Markers Initiative (444 patients and 153 healthy controls) as a second, independent data set. Results: We observed a higher frequency of loss-of-function (LOF) variants and rare putative highly functional variants (Combined Annotation Dependent Depletion [CADD] >20) in clinically diagnosed patients with PD than in healthy controls in both data sets. Overall, we identified LOF variants in 11 patients and 1 healthy control (odds ratio [OR] 4.94, Fisher exact p = 0.07). Four of these variants have been previously implicated in AD risk (p.E709AfsX86, p.W1214X, p.L1403RfsX7, and rs113809142). In addition, rare variants with CADD >20 were observed in 19 patients vs 3 healthy controls (OR 2.85, Fisher exact p = 0.06). Conclusion: The presence of ABCA7 LOF variants in clinically defined PD suggests that they might be risk factors for neurodegeneration in general, especially those variants hallmarked by protein aggregation. More studies will be needed to evaluate the overall impact of this transporter in neurodegenerative disease. PMID:27066581

  5. A Kaposi's Sarcoma-Associated Herpesvirus MicroRNA and Its Variants Target the Transforming Growth Factor β Pathway To Promote Cell Survival

    PubMed Central

    Lei, Xiufen; Zhu, Ying; Jones, Tiffany; Bai, Zhiqiang; Huang, Yufei

    2012-01-01

    Transforming growth factor β (TGF-β) signaling regulates cell growth and survival. Dysregulation of the TGF-β pathway is common in viral infection and cancer. Latent infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is required for the development of several AIDS-related malignancies, including Kaposi's sarcoma and primary effusion lymphoma (PEL). KSHV encodes more than two dozen microRNAs (miRs) derived from 12 pre-miRs with largely unknown functions. In this study, we show that miR variants processed from pre-miR-K10 are expressed in KSHV-infected PEL cells and endothelial cells, while cellular miR-142-3p and its variant miR-142-3p_-1_5, which share the same seed sequence with miR-K10a_ +1_5, are expressed only in PEL cells and not in uninfected and KSHV-infected TIME cells. KSHV miR-K10 variants inhibit TGF-β signaling by targeting TGF-β type II receptor (TβRII). Computational and reporter mutagenesis analyses identified three functional target sites in the TβRII 3′ untranslated region (3′UTR). Expression of miR-K10 variants is sufficient to inhibit TGF-β-induced cell apoptosis. A suppressor of the miRs sensitizes latent KSHV-infected PEL cells to TGF-β and induces apoptosis. These results indicate that miR-K10 variants manipulate the TGF-β pathway to confer cells with resistance to the growth-inhibitory effect of TGF-β. Thus, KSHV miRs might target the tumor-suppressive TGF-β pathway to promote viral latency and contribute to malignant cellular transformation. PMID:22915806

  6. Functional genetic variants in the vesicular monoamine transporter 1 (VMAT1) modulate emotion processing

    PubMed Central

    Lohoff, Falk W.; Hodge, Rachel; Narasimhan, Sneha; Nall, Aleksandra; Ferraro, Thomas N.; Mickey, Brian J.; Heitzeg, Mary M.; Langenecker, Scott A.; Zubieta, Jon-Kar; Bogdan, Ryan; Nikolova, Yuliya S.; Drabant, Emily; Hariri, Ahmad R.; Bevilacqua, Laura; Goldman, David; Doyle, Glenn A.

    2012-01-01

    SUMMARY Emotional behavior is in part heritable and often disrupted in psychopathology. Identification of specific genetic variants that drive this heritability may provide important new insight into molecular and neurobiological mechanisms involved in emotionality. Our results demonstrate that the presynaptic vesicular monoamine transporter 1 (VMAT1) Thr136Ile (rs1390938) polymorphism is functional in vitro, with the Ile allele leading to increased monoamine transport into presynaptic vesicles. Moreover, we show that the Thr136Ile variant predicts differential responses in emotional brain circuits consistent with its effects in vitro. Lastly, deep sequencing of bipolar disorder (BPD) patients and controls identified several rare novel VMAT1 variants. The variant Phe84Ser was only present in individuals with BPD and leads to marked increase monoamine transport in vitro. Taken together, our data show that VMAT1 polymorphisms influence monoamine signaling, the functional response of emotional brain circuits, and risk for psychopathology. PMID:23337945

  7. Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants?

    PubMed Central

    Lovelock, Paul K; Spurdle, Amanda B; Mok, Myth TS; Farrugia, Daniel J; Lakhani, Sunil R; Healey, Sue; Arnold, Stephen; Buchanan, Daniel; Investigators, kConFab; Couch, Fergus J; Henderson, Beric R; Goldgar, David E; Tavtigian, Sean V; Chenevix-Trench, Georgia; Brown, Melissa A

    2007-01-01

    Introduction Many of the DNA sequence variants identified in the breast cancer susceptibility gene BRCA1 remain unclassified in terms of their potential pathogenicity. Both multifactorial likelihood analysis and functional approaches have been proposed as a means to elucidate likely clinical significance of such variants, but analysis of the comparative value of these methods for classifying all sequence variants has been limited. Methods We have compared the results from multifactorial likelihood analysis with those from several functional analyses for the four BRCA1 sequence variants A1708E, G1738R, R1699Q, and A1708V. Results Our results show that multifactorial likelihood analysis, which incorporates sequence conservation, co-inheritance, segregation, and tumour immunohistochemical analysis, may improve classification of variants. For A1708E, previously shown to be functionally compromised, analysis of oestrogen receptor, cytokeratin 5/6, and cytokeratin 14 tumour expression data significantly strengthened the prediction of pathogenicity, giving a posterior probability of pathogenicity of 99%. For G1738R, shown to be functionally defective in this study, immunohistochemistry analysis confirmed previous findings of inconsistent 'BRCA1-like' phenotypes for the two tumours studied, and the posterior probability for this variant was 96%. The posterior probabilities of R1699Q and A1708V were 54% and 69%, respectively, only moderately suggestive of increased risk. Interestingly, results from functional analyses suggest that both of these variants have only partial functional activity. R1699Q was defective in foci formation in response to DNA damage and displayed intermediate transcriptional transactivation activity but showed no evidence for centrosome amplification. In contrast, A1708V displayed an intermediate transcriptional transactivation activity and a normal foci formation response in response to DNA damage but induced centrosome amplification. Conclusion

  8. Distribution and medical impact of loss-of-function variants in the Finnish founder population.

    PubMed

    Lim, Elaine T; Würtz, Peter; Havulinna, Aki S; Palta, Priit; Tukiainen, Taru; Rehnström, Karola; Esko, Tõnu; Mägi, Reedik; Inouye, Michael; Lappalainen, Tuuli; Chan, Yingleong; Salem, Rany M; Lek, Monkol; Flannick, Jason; Sim, Xueling; Manning, Alisa; Ladenvall, Claes; Bumpstead, Suzannah; Hämäläinen, Eija; Aalto, Kristiina; Maksimow, Mikael; Salmi, Marko; Blankenberg, Stefan; Ardissino, Diego; Shah, Svati; Horne, Benjamin; McPherson, Ruth; Hovingh, Gerald K; Reilly, Muredach P; Watkins, Hugh; Goel, Anuj; Farrall, Martin; Girelli, Domenico; Reiner, Alex P; Stitziel, Nathan O; Kathiresan, Sekar; Gabriel, Stacey; Barrett, Jeffrey C; Lehtimäki, Terho; Laakso, Markku; Groop, Leif; Kaprio, Jaakko; Perola, Markus; McCarthy, Mark I; Boehnke, Michael; Altshuler, David M; Lindgren, Cecilia M; Hirschhorn, Joel N; Metspalu, Andres; Freimer, Nelson B; Zeller, Tanja; Jalkanen, Sirpa; Koskinen, Seppo; Raitakari, Olli; Durbin, Richard; MacArthur, Daniel G; Salomaa, Veikko; Ripatti, Samuli; Daly, Mark J; Palotie, Aarno

    2014-07-01

    Exome sequencing studies in complex diseases are challenged by the allelic heterogeneity, large number and modest effect sizes of associated variants on disease risk and the presence of large numbers of neutral variants, even in phenotypically relevant genes. Isolated populations with recent bottlenecks offer advantages for studying rare variants in complex diseases as they have deleterious variants that are present at higher frequencies as well as a substantial reduction in rare neutral variation. To explore the potential of the Finnish founder population for studying low-frequency (0.5-5%) variants in complex diseases, we compared exome sequence data on 3,000 Finns to the same number of non-Finnish Europeans and discovered that, despite having fewer variable sites overall, the average Finn has more low-frequency loss-of-function variants and complete gene knockouts. We then used several well-characterized Finnish population cohorts to study the phenotypic effects of 83 enriched loss-of-function variants across 60 phenotypes in 36,262 Finns. Using a deep set of quantitative traits collected on these cohorts, we show 5 associations (p<5×10⁻⁸) including splice variants in LPA that lowered plasma lipoprotein(a) levels (P = 1.5×10⁻¹¹⁷). Through accessing the national medical records of these participants, we evaluate the LPA finding via Mendelian randomization and confirm that these splice variants confer protection from cardiovascular disease (OR = 0.84, P = 3×10⁻⁴), demonstrating for the first time the correlation between very low levels of LPA in humans with potential therapeutic implications for cardiovascular diseases. More generally, this study articulates substantial advantages for studying the role of rare variation in complex phenotypes in founder populations like the Finns and by combining a unique population genetic history with data from large population cohorts and centralized research access to National Health Registers. PMID

  9. Genetic and Molecular Functional Characterization of Variants within TNFSF13B, a Positional Candidate Preeclampsia Susceptibility Gene on 13q

    PubMed Central

    Roten, Linda T.; Aas, Per A.; Forsmo, Siri; Klepper, Kjetil; East, Christine E.; Abraham, Lawrence J.; Blangero, John; Brennecke, Shaun P.; Austgulen, Rigmor; Moses, Eric K.

    2010-01-01

    Background Preeclampsia is a serious pregnancy complication, demonstrating a complex pattern of inheritance. The elucidation of genetic liability to preeclampsia remains a major challenge in obstetric medicine. We have adopted a positional cloning approach to identify maternal genetic components, with linkages previously demonstrated to chromosomes 2q, 5q and 13q in an Australian/New Zealand familial cohort. The current study aimed to identify potential functional and structural variants in the positional candidate gene TNFSF13B under the 13q linkage peak and assess their association status with maternal preeclampsia genetic susceptibility. Methodology/Principal Findings The proximal promoter and coding regions of the positional candidate gene TNFSF13B residing within the 13q linkage region was sequenced using 48 proband or founder individuals from Australian/New Zealand families. Ten sequence variants (nine SNPs and one single base insertion) were identified and seven SNPs were successfully genotyped in the total Australian/New Zealand family cohort (74 families/480 individuals). Borderline association to preeclampsia (p = 0.0153) was observed for three rare SNPs (rs16972194, rs16972197 and rs56124946) in strong linkage disequilibrium with each other. Functional evaluation by electrophoretic mobility shift assays showed differential nuclear factor binding to the minor allele of the rs16972194 SNP, residing upstream of the translation start site, making this a putative functional variant. The observed genetic associations were not replicated in a Norwegian case/control cohort (The Nord-Trøndelag Health Study (HUNT2), 851 preeclamptic and 1,440 non-preeclamptic women). Conclusion/Significance TNFSF13B has previously been suggested to contribute to the normal immunological adaption crucial for a successful pregnancy. Our observations support TNFSF13B as a potential novel preeclampsia susceptibility gene. We discuss a possible role for TNFSF13B in preeclampsia

  10. Genetic and functional analysis of the TBX3 gene promoter in indirect inguinal hernia.

    PubMed

    Zhao, Zhongqing; Tian, Wenjun; Wang, Lin; Wang, Haihua; Qin, Xianyun; Xing, Qining; Pang, Shuchao; Yan, Bo

    2015-01-01

    Inguinal hernia is a common developmental disease in children and most cases are indirect inguinal hernia (IIH). Genetic factors have been suggested to play important roles in IIH. Although IIH has been observed in several human syndromes, genetic causes and molecular mechanisms for IIH remain unknown. TBX3 is a member of the T-box family of transcription factors that are essential to the embryonic development. Human studies and animal experiments have demonstrated that TBX3 is required for the development of the heart, limbs, mammary glands and other tissues and organs. TBX3 gene expression has been detected in human fibroblast and tissues of abdominal wall. We speculated that TBX3 may be involved in the IIH formation. Since TBX3 activity is highly dosage-sensitive, a TBX3 gene promoter was genetically and functionally analyzed in IIH patients and ethnic-matched controls in this study. One heterozygous deletion variant (g.4820_4821del) was identified in one IIH patient, but in none of controls. The variant significantly decreased TBX3 gene promoter activities, likely by creating a binding site for sex-determining region Y (SRY), mobility group transcription factor. One heterozygous insertion variant (g.3913_3914ins) was only found in one control, which did not affect TBX3 gene promoter activities. Taken together, TBX3 gene variants may contribute to IIH as a rare risk factor by reducing TBX3 levels. PMID:25455105

  11. Genetic Variants in KLOTHO Associate With Cognitive Function in the Oldest Old Group.

    PubMed

    Mengel-From, Jonas; Soerensen, Mette; Nygaard, Marianne; McGue, Matt; Christensen, Kaare; Christiansen, Lene

    2016-09-01

    Decline in cognitive abilities is a major concern in aging individuals. A potential important factor for functioning of the central nervous system in late-life stages is the KLOTHO (KL) gene. KL is expressed in various organs including the brain and is involved in multiple biological processes, for example, growth factor signaling. In the present study, 19 tagging gene variants in KL were studied in relation to 2 measures of cognitive function, a 5-item cognitive composite score and the Mini Mental State Examination, in 1,480 Danes 92-100 years of age. We found that heterozygotes for the previously reported KL-VS had poorer cognitive function than noncarriers. Two other variants positioned in the 5' end of the gene, rs398655 and rs562020, were associated with better cognitive function independently of KL-VS, and the common haplotype AG was associated with poorer cognition, consistently across two cognitive measures in two cohort strata. The haplotype effect was stronger than that of KL-VS. Two variants, rs2283368 and rs9526984, were the only variants significantly associated with cognitive decline over 7 years. We discuss an age-dependent effect of KL and the possibility that multiple gene variants in KL are important for cognitive function among the oldest old participants. PMID:26405063

  12. Expression and functionality of histone H2A variants in cancer

    PubMed Central

    Monteiro, Fátima Liliana; Baptista, Tiago; Amado, Francisco; Vitorino, Rui; Jerónimo, Carmen; Helguero, Luisa A.

    2014-01-01

    Regulation of gene expression includes the replacement of canonical histones for non-allelic histone variants, as well as their multiple targeting by postranslational modifications. H2A variants are highly conserved between species suggesting they execute important functions that cannot be accomplished by canonical histones. Altered expression of many H2A variants is associated to cancer. MacroH2A variants are enriched in heterocromatic foci and are necessary for chromatin condensation. MacroH2A1.1 and macroH2A1.2 are two mutually exclusive isoforms. MacroH2A1.1 and macroH2A2 inhibit proliferation and are associated with better cancer prognosis; while macroH2A1.2 is associated to cancer progression. H2AX variant functions as a sensor of DNA damage and defines the cellular response towards DNA repair or apoptosis; therefore, screening approaches and therapeutic options targeting H2AX have been proposed. H2A.Z is enriched in euchromatin, acting as a proto-oncogene with established roles in hormone responsive cancers and overexpressed in endocrine-resistant disease. Other H2A family members have also been found altered in cancer, but their function remains unknown. Substantial progress has been made to understand histone H2A variants, their contribution to normal cellular function and to cancer development and progression. Yet, implementation of high resolution mass spectrometry is needed to further our knowledge on highly homologous H2A variants expression and function. PMID:25003966

  13. Functional assays for analysis of variants of uncertain significance in BRCA2

    PubMed Central

    Guidugli, Lucia; Carreira, Aura; Caputo, Sandrine M.; Ehlen, Asa; Galli, Alvaro; Monteiro, Alvaro N.A.; Neuhausen, Susan L.; Hansen, Thomas V.O.; Couch, Fergus J.; Vreeswijk, Maaike P.G.

    2014-01-01

    Missense variants in the BRCA2 gene are routinely detected during clinical screening for pathogenic mutations in patients with a family history of breast and ovarian cancer. These subtle changes frequently remain of unknown clinical significance because of the lack of genetic information that may help establish a direct correlation with cancer predisposition. Therefore alternative ways of predicting the pathogenicity of these variants are urgently needed. Since BRCA2 is a protein involved in important cellular mechanisms such as DNA repair, replication and cell cycle control, functional assays have been developed that exploit these cellular activities to explore the impact of the variants on protein function. In this review we summarize assays developed and currently utilized for studying missense variants in BRCA2. We specifically depict details of each assay, including VUS analyzed, and describe a validation set of (genetically) proven pathogenic and neutral missense variants to serve as a golden standard for the validation of each assay. Guidelines are proposed to enable implementation of laboratory-based methods to assess the impact of the variant on cancer risk. PMID:24323938

  14. Natural cytotoxicity receptor splice variants orchestrate the distinct functions of human natural killer cell subtypes

    PubMed Central

    Siewiera, Johan; Gouilly, Jordi; Hocine, Hocine-Rachid; Cartron, Géraldine; Levy, Claude; Al-Daccak, Reem; Jabrane-Ferrat, Nabila

    2015-01-01

    The natural cytotoxicity receptors NKp46/NCR1, NKp44/NCR2 and NKp30/NCR3 are critical for natural killer (NK) cell functions. Their genes are transcribed into several splice variants whose physiological relevance is not yet fully understood. Here we report that decidua basalis NK (dNK) cells of the pregnant uterine mucosa and peripheral blood NK (pNK) cells, two functionally distinct subsets of the physiological NK cell pool, display differential expression of NKp30/NCR3 and NKp44/NCR2 splice variants. The presence of cytokines that are enriched within the decidual microenvironment is sufficient to convert the splice variant profile of pNK cells into one similar to that of dNK cells. This switch is associated with decreased cytotoxic function and major adaptations to the secretome, hallmarks of the decidual phenotype. Thus, NKp30/NCR3 and NKp44/NCR2 splice variants delineate functionally distinct NK cell subsets. To our knowledge, this is the first conclusive evidence underlining the physiological importance of NCR splice variants. PMID:26666685

  15. Homozygous loss-of-function variants in European cosmopolitan and isolate populations

    PubMed Central

    Kaiser, Vera B.; Svinti, Victoria; Prendergast, James G.; Chau, You-Ying; Campbell, Archie; Patarcic, Inga; Barroso, Inês; Joshi, Peter K.; Hastie, Nicholas D.; Miljkovic, Ana; Taylor, Martin S.; Enroth, Stefan; Memari, Yasin; Kolb-Kokocinski, Anja; Wright, Alan F.; Gyllensten, Ulf; Durbin, Richard; Rudan, Igor; Campbell, Harry; Polašek, Ozren; Johansson, Åsa; Sauer, Sascha; Porteous, David J.; Fraser, Ross M.; Drake, Camilla; Vitart, Veronique; Hayward, Caroline; Semple, Colin A.; Wilson, James F.

    2015-01-01

    Homozygous loss of function (HLOF) variants provide a valuable window on gene function in humans, as well as an inventory of the human genes that are not essential for survival and reproduction. All humans carry at least a few HLOF variants, but the exact number of inactivated genes that can be tolerated is currently unknown—as are the phenotypic effects of losing function for most human genes. Here, we make use of 1432 whole exome sequences from five European populations to expand the catalogue of known human HLOF mutations; after stringent filtering of variants in our dataset, we identify a total of 173 HLOF mutations, 76 (44%) of which have not been observed previously. We find that population isolates are particularly well suited to surveys of novel HLOF genes because individuals in such populations carry extensive runs of homozygosity, which we show are enriched for novel, rare HLOF variants. Further, we make use of extensive phenotypic data to show that most HLOFs, ascertained in population-based samples, appear to have little detectable effect on the phenotype. On the contrary, we document several genes directly implicated in disease that seem to tolerate HLOF variants. Overall HLOF genes are enriched for olfactory receptor function and are expressed in testes more often than expected, consistent with reduced purifying selection and incipient pseudogenisation. PMID:26173456

  16. Phenome-wide association studies (PheWASs) for functional variants

    PubMed Central

    Ye, Zhan; Mayer, John; Ivacic, Lynn; Zhou, Zhiyi; He, Min; Schrodi, Steven J; Page, David; Brilliant, Murray H; Hebbring, Scott J

    2015-01-01

    The genome-wide association study (GWAS) is a powerful approach for studying the genetic complexities of human disease. Unfortunately, GWASs often fail to identify clinically significant associations and describing function can be a challenge. GWAS is a phenotype-to-genotype approach. It is now possible to conduct a converse genotype-to-phenotype approach using extensive electronic medical records to define a phenome. This approach associates a single genetic variant with many phenotypes across the phenome and is called a phenome-wide association study (PheWAS). The majority of PheWASs conducted have focused on variants identified previously by GWASs. This approach has been efficient for rediscovering gene–disease associations while also identifying pleiotropic effects for some single-nucleotide polymorphisms (SNPs). However, the use of SNPs identified by GWAS in a PheWAS is limited by the inherent properties of the GWAS SNPs, including weak effect sizes and difficulty when translating discoveries to function. To address these challenges, we conducted a PheWAS on 105 presumed functional stop-gain and stop-loss variants genotyped on 4235 Marshfield Clinic patients. Associations were validated on an additional 10 640 Marshfield Clinic patients. PheWAS results indicate that a nonsense variant in ARMS2 (rs2736911) is associated with age-related macular degeneration (AMD). These results demonstrate that focusing on functional variants may be an effective approach when conducting a PheWAS. PMID:25074467

  17. STRONG GRAVITATIONAL LENS MODELING WITH SPATIALLY VARIANT POINT-SPREAD FUNCTIONS

    SciTech Connect

    Rogers, Adam; Fiege, Jason D.

    2011-12-10

    Astronomical instruments generally possess spatially variant point-spread functions, which determine the amount by which an image pixel is blurred as a function of position. Several techniques have been devised to handle this variability in the context of the standard image deconvolution problem. We have developed an iterative gravitational lens modeling code called Mirage that determines the parameters of pixelated source intensity distributions for a given lens model. We are able to include the effects of spatially variant point-spread functions using the iterative procedures in this lensing code. In this paper, we discuss the methods to include spatially variant blurring effects and test the results of the algorithm in the context of gravitational lens modeling problems.

  18. Strong Gravitational Lens Modeling with Spatially Variant Point-spread Functions

    NASA Astrophysics Data System (ADS)

    Rogers, Adam; Fiege, Jason D.

    2011-12-01

    Astronomical instruments generally possess spatially variant point-spread functions, which determine the amount by which an image pixel is blurred as a function of position. Several techniques have been devised to handle this variability in the context of the standard image deconvolution problem. We have developed an iterative gravitational lens modeling code called Mirage that determines the parameters of pixelated source intensity distributions for a given lens model. We are able to include the effects of spatially variant point-spread functions using the iterative procedures in this lensing code. In this paper, we discuss the methods to include spatially variant blurring effects and test the results of the algorithm in the context of gravitational lens modeling problems.

  19. Disease associations and altered immune function in CD45 138G variant carriers.

    PubMed

    Boxall, Sally; Stanton, Tara; Hirai, Kouzo; Ward, Victoria; Yasui, Tomoyo; Tahara, Hideki; Tamori, Akihiro; Nishiguchi, Shuhei; Shiomi, Susumu; Ishiko, Osamu; Inaba, Masaaki; Nishizawa, Yoshiki; Dawes, Ritu; Bodmer, Walter; Beverley, Peter C L; Tchilian, Elma Z

    2004-10-15

    The CD45 antigen is a haemopoietic cell specific tyrosine phosphatase essential for antigen receptor mediated signalling in lymphocytes. Expression of different patterns of alternatively spliced CD45 isoforms is associated with distinct functions. We recently identified a polymorphism in exon 6 (A138G) of the gene encoding CD45 (PTPRC) that results in altered CD45 splicing. The 138G allele is present at a high frequency among Japanese (23.7%), with 5.1% individuals homozygous for the G allele. In this study we show that the A138G polymorphism is the cause of altered CD45 isoform expression, promoting splicing towards low molecular weight CD45 isoforms. We further report that the frequency of A138G heterozygotes is significantly reduced in number in cohorts of patients with autoimmune Graves' disease or hepatitis B infection, whereas G138G homozygotes are absent from a cohort of Hashimoto's thyroiditis patients. We also show that 138G individuals exhibit altered cytokine production in vitro and an increased proportion of memory T cells. These data suggest that the 138G variant allele strongly influences these diseases by modulation of immune mechanisms and may have achieved its high frequency as a result of a natural selection probably related to pathogen resistance. PMID:15333587

  20. Splice variants of the SWR1-type nucleosome remodeling factor Domino have distinct functions during Drosophila melanogaster oogenesis.

    PubMed

    Börner, Kenneth; Becker, Peter B

    2016-09-01

    SWR1-type nucleosome remodeling factors replace histone H2A by variants to endow chromatin locally with specialized functionality. In Drosophila melanogaster a single H2A variant, H2A.V, combines functions of mammalian H2A.Z and H2A.X in transcription regulation and the DNA damage response. A major role in H2A.V incorporation for the only SWR1-like enzyme in flies, Domino, is assumed but not well documented in vivo. It is also unclear whether the two alternatively spliced isoforms, DOM-A and DOM-B, have redundant or specialized functions. Loss of both DOM isoforms compromises oogenesis, causing female sterility. We systematically explored roles of the two DOM isoforms during oogenesis using a cell type-specific knockdown approach. Despite their ubiquitous expression, DOM-A and DOM-B have non-redundant functions in germline and soma for egg formation. We show that chromatin incorporation of H2A.V in germline and somatic cells depends on DOM-B, whereas global incorporation in endoreplicating germline nurse cells appears to be independent of DOM. By contrast, DOM-A promotes the removal of H2A.V from stage 5 nurse cells. Remarkably, therefore, the two DOM isoforms have distinct functions in cell type-specific development and H2A.V exchange. PMID:27578180

  1. Structural and Functional Analysis of the ApolipoproteinA-I A164S Variant

    PubMed Central

    Dalla-Riva, Jonathan; Lagerstedt, Jens O.; Petrlova, Jitka

    2015-01-01

    Apolipoprotein A-I (apoA-I) is the main protein involved in the formation of high-density lipoprotein (HDL), it is the principal mediator of the reverse cholesterol transfer (RCT) pathway and provides cardio-protection. In addition to functional wild-type apoA-I, several variants have been shown to associate with hereditary amyloidosis. In this study we have performed biophysical and biochemical analyses of the structure and functional properties of the A164S variant of apoA-I (1:500 in the Danish general population), which is the first known mutation of apoA-I that leads to an increased risk of ischaemic heart disease (IHD), myocardial infarction and mortality without associated low HDL cholesterol levels. Despite the fact that epidemiologically IHD is associated with low plasma levels of HDL, the A164S mutation is linked to normal plasma levels of lipids, HDL and apoA-I, suggesting impaired functionality of this variant. Using biophysical techniques (e.g., circular dichroism spectroscopy and electron microscopy) to determine secondary structure, stability and pro-amyloidogenic property of the lipid free A164S apoA-I variant, our observations suggest similarity in structural properties between apoA-I WT and apoA-I A164S. However, the A164S apoA-I variant exhibits lower binding affinity to lipids but forms similar sized HDL particles to those produced by WT. PMID:26605794

  2. Functional characterization of Ape1 variants identified in the human population

    PubMed Central

    Hadi, Masood Z.; Coleman, Matthew A.; Fidelis, Krzysztof; Mohrenweiser, Harvey W.; Wilson, David M.

    2000-01-01

    Apurinic/apyrimidinic (AP) sites are common mutagenic and cytotoxic DNA lesions. Ape1 is the major human repair enzyme for abasic sites and incises the phosphodiester backbone 5′ to the lesion to initiate a cascade of events aimed at removing the AP moiety and maintaining genetic integrity. Through resequencing of genomic DNA from 128 unrelated individuals, and searching published reports and sequence databases, seven amino acid substitution variants were identified in the repair domain of human Ape1. Functional characterization revealed that three of the variants, L104R, E126D and R237A, exhibited ∼40–60% reductions in specific incision activity. A fourth variant, D283G, is similar to the previously characterized mutant D283A found to exhibit ∼10% repair capacity. The most common substitution (D148E; observed at an allele frequency of 0.38) had no impact on endonuclease and DNA binding activities, nor did a G306A substitution. A G241R variant showed slightly enhanced endonuclease activity relative to wild-type. In total, four of seven substitutions in the repair domain of Ape1 imparted reduced function. These reduced function variants may represent low penetrance human polymorphisms that associate with increased disease susceptibility. PMID:11024165

  3. A new γ-interferon-inducible promoter and splice variants of an anti-angiogenic human tRNA synthetase

    PubMed Central

    Liu, Jianming; Shue, Eveline; Ewalt, Karla L.; Schimmel, Paul

    2004-01-01

    Two forms of human tryptophanyl-tRNA synthetase (TrpRS) are produced in vivo through alternative mRNA splicing. The two forms, full-length TrpRS and mini TrpRS, are catalytically active, but are distinguished by the striking anti-proliferative and anti-angiogenic activity specific to mini TrpRS. Here we describe two new splice variants of human TrpRS mRNA. Their production was strongly regulated by γ-interferon (IFN-γ), an anti-proliferative cytokine known to stimulate the expression of other anti-angiogenic factors. A new IFN-γ-sensitive promoter was demonstrated to drive production of these splice variants. In human endothelial cells, both the newly discovered and a previously reported promoter were shown to respond specifically to IFN-γ and not to other cytokines such as tumor necrosis factor-α, transforming growth factor-β, interleukin-4 or erythropoietin. In addition, both promoters were stimulated by the ‘downstream’ interferon regulatory factor 1 that, in turn, is known to be regulated by the ‘upstream’ signal transducer and activator of transcription 1α subunit. Thus, the tandem promoters provide a dual system to regulate expression and alternative splicing of human TrpRS in vivo. PMID:14757836

  4. Promotion of waterpipe tobacco use, its variants and accessories in young adult newspapers: a content analysis of message portrayal

    PubMed Central

    Sterling, Kymberle L.; Fryer, Craig S.; Majeed, Ban; Duong, Melissa M.

    2015-01-01

    The objective of our study was to identify waterpipe tobacco smoking advertisements and those that promoted a range of products and accessories used to smoke waterpipe tobacco. The content of these advertisements was analyzed to understand the messages portrayed about waterpipe tobacco smoking in young adult (aged 18–30) newspapers. The study methods include monitoring of six newspapers targeting young adults from four major cities in the Southeastern United States over a 6-month period. A total of 87 advertisements were found; 73.5% (64) were distinct and content analyzed. The study results showed that of the advertisements analyzed, 25% advertised waterpipe tobacco smoking, 54.7% featured waterpipe tobacco smoking and other tobacco use, 14.1% featured non-tobacco waterpipe variants (i.e. vaporizers), and 6.3% featured waterpipe apparatus accessories (e.g. charcoal, hoses). The sociability (34%) and sensuality (29.7%) of waterpipe smoking were promoted themes. Alternative to cigarette use messages (3.1%), and harm-reduction messages (17.1%) emphasized that smoking waterpipe tobacco using the featured accessory or waterpipe variant was a healthier experience than cigarette smoking. The study concluded that the messages that promoted waterpipe tobacco smoking to young adults are parallel to those used to promote cigarette use. Tobacco control professionals should continue to monitor young adult newspapers as a source of waterpipe-related advertising. PMID:24957675

  5. Carrier frequency of guanidinoacetate methyltransferase deficiency in the general population by functional characterization of missense variants in the GAMT gene.

    PubMed

    Desroches, Caro-Lyne; Patel, Jaina; Wang, Peixiang; Minassian, Berge; Marshall, Christian R; Salomons, Gajja S; Mercimek-Mahmutoglu, Saadet

    2015-12-01

    Guanidinoacetate methyltransferase (GAMT) deficiency is a neurodegenerative disease. Although no symptomatic patients on treatment achieved normal neurodevelopment, three asymptomatic newborns were reported with normal neurodevelopmental outcome on neonatal treatment. GAMT deficiency is therefore a candidate for newborn screening programs, but there are no studies for the carrier frequency of this disease in the general population. To determine carrier frequency of GAMT deficiency, we studied the variants in the GAMT gene reported in the Exome Variant Server database and performed functional characterization of missense variants. We used previously cloned GAMT transcript variant 1 (7 missense variants) and cloned a novel GAMT transcript variant 2 (5 missense variants). The latter was used in Exome Variant Server database according to recommendations of the Human Genome Variation Society. There were 4 missense variants (1 previously reported and 3 novel) with low GAMT enzyme activity indicating pathogenicity. Additionally, there was one novel frameshift and one novel nonsense variant likely pathogenic. There was no measurable GAMT enzyme activity in the wild type of GAMT transcript variant 2. We concluded that GAMT transcript variant 2 is not involved in GAMT protein synthesis. For this reason, Human Genome Variation Society should use mutation nomenclature according to the coding region of the GAMT transcript variant 1. The carrier frequency of GAMT deficiency was 0.123 % in the general population. As early diagnosis results in normal neurodevelopmental outcome, GAMT deficiency should be included in newborn screening programs to diagnose individuals at the asymptomatic stage of the disease to prevent permanent neurodevelopmental disability. PMID:26003046

  6. Cooperation between distinct viral variants promotes growth of H3N2 influenza in cell culture

    PubMed Central

    Xue, Katherine S; Hooper, Kathryn A; Ollodart, Anja R; Dingens, Adam S; Bloom, Jesse D

    2016-01-01

    RNA viruses rapidly diversify into quasispecies of related genotypes. This genetic diversity has long been known to facilitate adaptation, but recent studies have suggested that cooperation between variants might also increase population fitness. Here, we demonstrate strong cooperation between two H3N2 influenza variants that differ by a single mutation at residue 151 in neuraminidase, which normally mediates viral exit from host cells. Residue 151 is often annotated as an ambiguous amino acid in sequenced isolates, indicating mixed viral populations. We show that mixed populations grow better than either variant alone in cell culture. Pure populations of either variant generate the other through mutation and then stably maintain a mix of the two genotypes. We suggest that cooperation arises because mixed populations combine one variant’s proficiency at cell entry with the other’s proficiency at cell exit. Our work demonstrates a specific cooperative interaction between defined variants in a viral quasispecies. DOI: http://dx.doi.org/10.7554/eLife.13974.001 PMID:26978794

  7. Identification and functional characterization of rare SHANK2 variants in schizophrenia.

    PubMed

    Peykov, S; Berkel, S; Schoen, M; Weiss, K; Degenhardt, F; Strohmaier, J; Weiss, B; Proepper, C; Schratt, G; Nöthen, M M; Boeckers, T M; Rietschel, M; Rappold, G A

    2015-12-01

    Recent genetic data on schizophrenia (SCZ) have suggested that proteins of the postsynaptic density of excitatory synapses have a role in its etiology. Mutations in the three SHANK genes encoding for postsynaptic scaffolding proteins have been shown to represent risk factors for autism spectrum disorders and other neurodevelopmental disorders. To address if SHANK2 variants are associated with SCZ, we sequenced SHANK2 in 481 patients and 659 unaffected individuals. We identified a significant increase in the number of rare (minor allele frequency<1%) SHANK2 missense variants in SCZ individuals (6.9%) compared with controls (3.9%, P=0.039). Four out of fifteen non-synonymous variants identified in the SCZ cohort (S610Y, R958S, P1119T and A1731S) were selected for functional analysis. Overexpression and knockdown-rescue experiments were carried out in cultured primary hippocampal neurons with a major focus on the analysis of morphological changes. Furthermore, the effect on actin polymerization in fibroblast cell lines was investigated. All four variants revealed functional impairment to various degrees, as a consequence of alterations in spine volume and clustering at synapses and an overall loss of presynaptic contacts. The A1731S variant was identified in four unrelated SCZ patients (0.83%) but not in any of the sequenced controls and public databases (P=4.6 × 10(-5)). Patients with the A1731S variant share an early prodromal phase with an insidious onset of psychiatric symptoms. A1731S overexpression strongly decreased the SHANK2-Bassoon-positive synapse number and diminished the F/G-actin ratio. Our results strongly suggest a causative role of rare SHANK2 variants in SCZ and underline the contribution of SHANK2 gene mutations in a variety of neuropsychiatric disorders. PMID:25560758

  8. Loss-of-function variants influence the human serum metabolome.

    PubMed

    Yu, Bing; Li, Alexander H; Metcalf, Ginger A; Muzny, Donna M; Morrison, Alanna C; White, Simon; Mosley, Thomas H; Gibbs, Richard A; Boerwinkle, Eric

    2016-08-01

    The metabolome is a collection of small molecules resulting from multiple cellular and biological processes that can act as biomarkers of disease, and African-Americans exhibit high levels of genetic diversity. Exome sequencing of a sample of deeply phenotyped African-Americans allowed us to analyze the effects of annotated loss-of-function (LoF) mutations on 308 serum metabolites measured by untargeted liquid and gas chromatography coupled with mass spectrometry. In an independent sample, we identified and replicated four genes harboring six LoF mutations that significantly affected five metabolites. These sites were related to a 19 to 45% difference in geometric mean metabolite levels, with an average effect size of 25%. We show that some of the affected metabolites are risk predictors or diagnostic biomarkers of disease and, using the principle of Mendelian randomization, are in the causal pathway of disease. For example, LoF mutations in SLCO1B1 elevate the levels of hexadecanedioate, a fatty acid significantly associated with increased blood pressure levels and risk of incident heart failure in both African-Americans and an independent sample of European-Americans. We show that SLCO1B1 LoF mutations significantly increase the risk of incident heart failure, thus implicating the metabolite in the causal pathway of disease. These results reveal new avenues into gene function and the understanding of disease etiology by integrating -omic technologies into a deeply phenotyped population study. PMID:27602404

  9. Loss-of-function variants influence the human serum metabolome

    PubMed Central

    Yu, Bing; Li, Alexander H.; Metcalf, Ginger A.; Muzny, Donna M.; Morrison, Alanna C.; White, Simon; Mosley, Thomas H.; Gibbs, Richard A.; Boerwinkle, Eric

    2016-01-01

    The metabolome is a collection of small molecules resulting from multiple cellular and biological processes that can act as biomarkers of disease, and African-Americans exhibit high levels of genetic diversity. Exome sequencing of a sample of deeply phenotyped African-Americans allowed us to analyze the effects of annotated loss-of-function (LoF) mutations on 308 serum metabolites measured by untargeted liquid and gas chromatography coupled with mass spectrometry. In an independent sample, we identified and replicated four genes harboring six LoF mutations that significantly affected five metabolites. These sites were related to a 19 to 45% difference in geometric mean metabolite levels, with an average effect size of 25%. We show that some of the affected metabolites are risk predictors or diagnostic biomarkers of disease and, using the principle of Mendelian randomization, are in the causal pathway of disease. For example, LoF mutations in SLCO1B1 elevate the levels of hexadecanedioate, a fatty acid significantly associated with increased blood pressure levels and risk of incident heart failure in both African-Americans and an independent sample of European-Americans. We show that SLCO1B1 LoF mutations significantly increase the risk of incident heart failure, thus implicating the metabolite in the causal pathway of disease. These results reveal new avenues into gene function and the understanding of disease etiology by integrating -omic technologies into a deeply phenotyped population study. PMID:27602404

  10. The -374T/A variant of the rage gene promoter is associated with clinical restenosis after coronary stent placement.

    PubMed

    Falcone, C; Emanuele, E; Buzzi, M P; Ballerini, L; Repetto, A; Canosi, U; Mazzucchelli, I; Schirinzi, S; Sbarsi, I; Boiocchi, C; Cuccia, M

    2007-01-01

    Upregulation of the receptor for advanced glycation end products (RAGE) may play a crucial role in neointimal formation upon vessel injury. The -374T/A variant of the RAGE gene promoter, which has been associated with an altered expression of the cell-surface receptor, could exert a protective effect toward the development of vascular disease. The aim of this study is to determine the impact of this common genetic variant in the occurrence of clinical in-stent restenosis after coronary stent implantation. The -374T/A polymorphism of the RAGE gene promoter was evaluated by PCR-RFLPs in 267 patients with coronary artery disease who underwent coronary stent implantation and a subsequent coronary angiography 6-9 months later for suspected restenosis. In-stent restenosis was assessed by means of quantitative angiography. Carriers of the -374AA genotype showed a significantly reduced risk of developing restenosis after percutaneous transluminal intervention than non-carriers. To determine whether the protective effect of the homozygous AA genotype toward clinical restenosis was independent of potential confounders, we performed multivariable logistic regression analysis. After allowance for clinical and biochemical risk factors and stent length, the AA genotype remained significantly associated with a reduced prevalence of in-stent restenosis. No relation was evident between the RAGE genotype and established cardiovascular risk factors. In conclusion, the -374AA genotype of the RAGE gene promoter could be associated with a reduced risk of in-stent restenosis after coronary stent implantation. PMID:18179750

  11. Identification and functional characterization of four TRPA1 variants in Apolygus lucorum (Meyer-Dür)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As signal integrators that respond to various physical and chemical stimuli, transient receptor potential (TRP) channels fulfil critical functional roles in the sensory systems of both vertebrate and invertebrate organisms. Here, four variants of TRP ankyrin 1 (TRPA1) were identified and cloned from...

  12. Three functional variants of IFN regulatory factor 5 (IRF5) define risk and protective haplotypes for human lupus

    PubMed Central

    Graham, Robert R.; Kyogoku, Chieko; Sigurdsson, Snaevar; Vlasova, Irina A.; Davies, Leela R. L.; Baechler, Emily C.; Plenge, Robert M.; Koeuth, Thearith; Ortmann, Ward A.; Hom, Geoffrey; Bauer, Jason W.; Gillett, Clarence; Burtt, Noel; Cunninghame Graham, Deborah S.; Onofrio, Robert; Petri, Michelle; Gunnarsson, Iva; Svenungsson, Elisabet; Rönnblom, Lars; Nordmark, Gunnel; Gregersen, Peter K.; Moser, Kathy; Gaffney, Patrick M.; Criswell, Lindsey A.; Vyse, Timothy J.; Syvänen, Ann-Christine; Bohjanen, Paul R.; Daly, Mark J.; Behrens, Timothy W.; Altshuler, David

    2007-01-01

    Systematic genome-wide studies to map genomic regions associated with human diseases are becoming more practical. Increasingly, efforts will be focused on the identification of the specific functional variants responsible for the disease. The challenges of identifying causal variants include the need for complete ascertainment of genetic variants and the need to consider the possibility of multiple causal alleles. We recently reported that risk of systemic lupus erythematosus (SLE) is strongly associated with a common SNP in IFN regulatory factor 5 (IRF5), and that this variant altered spicing in a way that might provide a functional explanation for the reproducible association to SLE risk. Here, by resequencing and genotyping in patients with SLE, we find evidence for three functional alleles of IRF5: the previously described exon 1B splice site variant, a 30-bp in-frame insertion/deletion variant of exon 6 that alters a proline-, glutamic acid-, serine- and threonine-rich domain region, and a variant in a conserved polyA+ signal sequence that alters the length of the 3′ UTR and stability of IRF5 mRNAs. Haplotypes of these three variants define at least three distinct levels of risk to SLE. Understanding how combinations of variants influence IRF5 function may offer etiological and therapeutic insights in SLE; more generally, IRF5 and SLE illustrates how multiple common variants of the same gene can together influence risk of common disease. PMID:17412832

  13. Genetic variants in microRNA genes: impact on microRNA expression, function, and disease

    PubMed Central

    Cammaerts, Sophia; Strazisar, Mojca; De Rijk, Peter; Del Favero, Jurgen

    2015-01-01

    MicroRNAs (miRNAs) are important regulators of gene expression and like any other gene, their coding sequences are subject to genetic variation. Variants in miRNA genes can have profound effects on miRNA functionality at all levels, including miRNA transcription, maturation, and target specificity, and as such they can also contribute to disease. The impact of variants in miRNA genes is the focus of the present review. To put these effects into context, we first discuss the requirements of miRNA transcripts for maturation. In the last part an overview of available databases and tools and experimental approaches to investigate miRNA variants related to human disease is presented. PMID:26052338

  14. ADAM19 and HTR4 Variants and Pulmonary Function: The Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Targeted Sequencing Study

    PubMed Central

    London, Stephanie J.; Gao, Wei; Gharib, Sina A.; Hancock, Dana B.; Wilk, Jemma B.; House, John S.; Gibbs, Richard A.; Muzny, Donna M.; Lumley, Thomas; Franceschini, Nora; North, Kari E.; Psaty, Bruce M.; Kovar, Christie L.; Coresh, Josef; Zhou, Yanhua; Heckbert, Susan R.; Brody, Jennifer A.; Morrison, Alanna C.; Dupuis, Josée

    2014-01-01

    Background The pulmonary function measures of forced expiratory volume in one second (FEV1) and its ratio to forced vital capacity (FVC) are used in the diagnosis and monitoring of lung diseases and predict cardiovascular mortality in the general population. Genome wide association studies (GWAS) have identified numerous loci associated with FEV1 and FEV1/FVC but the causal variants remain uncertain. We hypothesized that novel or rare variants poorly tagged by GWAS may explain the significant associations between FEV1/FVC and two genes: ADAM19 and HTR4. Methods and Results We sequenced ADAM19 and its promoter region along with the approximately 21 kb portion of HTR4 harboring GWAS SNPs for pulmonary function and analyzed associations with FEV1/FVC among 3,983 participants of European ancestry from Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE). Meta-analysis of common variants in each region identified statistically significant associations (316 tests, P < 1.58×10−4) with FEV1/FVC for 14 ADAM19 SNPs and 24 HTR4 SNPs. After conditioning on the sentinel GWAS hit in each gene [ADAM19 rs1422795, minor allele frequency (MAF)=0.33 and HTR4 rs11168048, MAF=0.40] one SNP remained statistically significant (ADAM19 rs13155908, MAF = 0.12, P = 1.56×10−4). Analysis of rare variants (MAF < 1%) using Sequence Kernel Association Test did not identify associations with either region. Conclusions Sequencing identified one common variant associated with FEV1/FVC independently of the sentinel ADAM19 GWAS hit and supports the original HTR4 GWAS findings. Rare variants do not appear to underlie GWAS associations with pulmonary function for common variants in ADAM19 and HTR4. PMID:24951661

  15. Functional dissection of the lck proximal promoter.

    PubMed Central

    Allen, J M; Forbush, K A; Perlmutter, R M

    1992-01-01

    The lck gene encodes a protein tyrosine kinase that participates in lymphocyte-specific signal transduction pathways. Previous studies have established that lck transcription is regulated by two distinct promoter elements termed proximal (or 3') and distal (or 5'). The proximal promoter is active almost exclusively in thymocytes and becomes inactive later during T-cell maturation. To dissect the mechanisms responsible for lck gene regulation, we generated transgenic animals bearing 5' truncations in the proximal promoter element. Sequences between -584 and +37 with respect to the proximal promoter transcription start site act to direct tissue-specific and temporally correct transcription of either a tagged version of the lck gene itself or a heterologous reporter sequence (lacZ). This region contains binding sites for at least five distinct nuclear proteins, of which one is found only in cells that support proximal lck promoter activity and a second appears only in nonexpressing cells. Interestingly, the transcribed region of the lck gene contains positive control elements that can substantially boost expression from minimal (-130 bp) proximal promoter constructs. These results provide a basis for the biochemical dissection of transcriptional regulators that act at defined points during T-cell development. Images PMID:1588967

  16. Variant antigenic peptide promotes cytotoxic T lymphocyte adhesion to target cells without cytotoxicity

    PubMed Central

    Shotton, David M.; Attaran, Amir

    1998-01-01

    Timelapse video microscopy has been used to record the motility and dynamic interactions between an H-2Db-restricted murine cytotoxic T lymphocyte clone (F5) and Db-transfected L929 mouse fibroblasts (LDb) presenting normal or variant antigenic peptides from human influenza nucleoprotein. F5 cells will kill LDb target cells presenting specific antigen (peptide NP68: ASNENMDAM) after “browsing” their surfaces for between 8 min and many hours. Cell death is characterized by abrupt cellular rounding followed by zeiosis (vigorous “boiling” of the cytoplasm and blebbing of the plasma membrane) for 10–20 min, with subsequent cessation of all activity. Departure of cytotoxic T lymphocytes from unkilled target cells is rare, whereas serial killing is sometimes observed. In the absence of antigenic peptide, cytotoxic T lymphocytes browse target cells for much shorter periods, and readily leave to encounter other targets, while never causing target cell death. Two variant antigenic peptides, differing in nonamer position 7 or 8, also act as antigens, albeit with lower efficiency. A third variant peptide NP34 (ASNENMETM), which differs from NP68 in both positions and yet still binds Db, does not stimulate F5 cytotoxicity. Nevertheless, timelapse video analysis shows that NP34 leads to a significant modification of cell behavior, by up-regulating F5–LDb adhesive interactions. These data extend recent studies showing that partial agonists may elicit a subset of the T cell responses associated with full antigen stimulation, by demonstrating that TCR interaction with variant peptide antigens can trigger target cell adhesion and surface exploration without activating the signaling pathway that results in cytotoxicity. PMID:9861010

  17. Multiple loss-of-function variants of taste receptors in modern humans

    PubMed Central

    Fujikura, K.

    2015-01-01

    Despite recent advances in the knowledge of interindividual taste differences, the underlying genetic backgrounds have remained to be fully elucidated. Much of the taste variation among different mammalian species can be explained by pseudogenization of taste receptors. Here I investigated whether the most recent disruptions of taste receptor genes segregate with their intact forms in modern humans by analyzing 14 ethnically diverse populations. The results revealed an unprecedented prevalence of 25 segregating loss-of-function (LoF) taste receptor variants, identifying one of the most pronounced cases of functional population diversity in the human genome. LoF variant frequency in taste receptors (2.10%) was considerably higher than the overall LoF frequency in human genome (0.16%). In particular, molecular evolutionary rates of candidate sour (14.7%) and bitter (1.8%) receptors were far higher in humans than those of sweet (0.02%), salty (0.05%), and umami (0.17%) receptors compared with other carnivorous mammals, although not all of the taste receptors were identified. Many LoF variants are population-specific, some of which arose even after population differentiation, not before divergence of the modern and archaic human. I conclude that modern humans might have been losing some sour and bitter receptor genes because of high-frequency LoF variants. PMID:26307445

  18. A rare functional cardioprotective APOC3 variant has risen in frequency in distinct population isolates

    PubMed Central

    Tachmazidou, Ioanna; Dedoussis, George; Southam, Lorraine; Farmaki, Aliki-Eleni; Ritchie, Graham R. S.; Xifara, Dionysia K.; Matchan, Angela; Hatzikotoulas, Konstantinos; Rayner, Nigel W.; Chen, Yuan; Pollin, Toni I.; O’Connell, Jeffrey R.; Yerges-Armstrong, Laura M.; Kiagiadaki, Chrysoula; Panoutsopoulou, Kalliope; Schwartzentruber, Jeremy; Moutsianas, Loukas; Tsafantakis, Emmanouil; Tyler-Smith, Chris; McVean, Gil; Xue, Yali; Zeggini, Eleftheria

    2013-01-01

    Isolated populations can empower the identification of rare variation associated with complex traits through next generation association studies, but the generalizability of such findings remains unknown. Here we genotype 1,267 individuals from a Greek population isolate on the Illumina HumanExome Beadchip, in search of functional coding variants associated with lipids traits. We find genome-wide significant evidence for association between R19X, a functional variant in APOC3, with increased high-density lipoprotein and decreased triglycerides levels. Approximately 3.8% of individuals are heterozygous for this cardioprotective variant, which was previously thought to be private to the Amish founder population. R19X is rare (<0.05% frequency) in outbred European populations. The increased frequency of R19X enables discovery of this lipid traits signal at genome-wide significance in a small sample size. This work exemplifies the value of isolated populations in successfully detecting transferable rare variant associations of high medical relevance. PMID:24343240

  19. Multiple loss-of-function variants of taste receptors in modern humans.

    PubMed

    Fujikura, Kohei

    2015-01-01

    Despite recent advances in the knowledge of interindividual taste differences, the underlying genetic backgrounds have remained to be fully elucidated. Much of the taste variation among different mammalian species can be explained by pseudogenization of taste receptors. Here I investigated whether the most recent disruptions of taste receptor genes segregate with their intact forms in modern humans by analyzing 14 ethnically diverse populations. The results revealed an unprecedented prevalence of 25 segregating loss-of-function (LoF) taste receptor variants, identifying one of the most pronounced cases of functional population diversity in the human genome. LoF variant frequency in taste receptors (2.10%) was considerably higher than the overall LoF frequency in human genome (0.16%). In particular, molecular evolutionary rates of candidate sour (14.7%) and bitter (1.8%) receptors were far higher in humans than those of sweet (0.02%), salty (0.05%), and umami (0.17%) receptors compared with other carnivorous mammals, although not all of the taste receptors were identified. Many LoF variants are population-specific, some of which arose even after population differentiation, not before divergence of the modern and archaic human. I conclude that modern humans might have been losing some sour and bitter receptor genes because of high-frequency LoF variants. PMID:26307445

  20. A truncated splice variant of human lysyl oxidase-like 2 promotes migration and invasion in esophageal squamous cell carcinoma.

    PubMed

    Zou, Hai-Ying; Lv, Guo-Qing; Dai, Li-Hua; Zhan, Xiu-Hui; Jiao, Ji-Wei; Liao, Lian-Di; Zhou, Tai-Mei; Li, Chun-Quan; Wu, Bing-Li; Xu, Li-Yan; Li, En-Min

    2016-06-01

    Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family, which plays an important role in extracellular matrix protein biosynthesis and tumor progression. In the present study, we identified a novel splice variant, LOXL2Δ72, which encodes a peptide having the same N- and C-termini as wild-type LOXL2 (LOXL2WT), but lacks 72 nucleotides encoding 24 amino acids. LOXL2Δ72 had dramatically reduced enzymatic activity, and was no longer secreted. However, LOXL2Δ72 promoted greater cell migration and invasion than LOXL2WT. Furthermore, a dual luciferase reporter assay indicated that LOXL2Δ72 activates distinct signal transduction pathways compared to LOXL2WT, consistent with cDNA microarray data showing different expression levels of cell migration- and invasion-related genes induced following over-expression of each LOXL2 isoform. In particular, LOXL2Δ72 distinctly promoted esophageal squamous cell carcinoma (ESCC) cell migration via up-regulating the C-C motif chemokine ligand 28 (CCL28). Our results suggest that the new LOXL2 splice variant contributes to tumor progression by novel molecular mechanisms different from LOXL2WT. PMID:27063404

  1. Association between promoter region genetic variants of PTH SNPs and serum 25(OH)-vitamin D level

    PubMed Central

    Al-Daghri, Nasser M; Al-Attas, Omar S; Krishnaswamy, Soundararajan; Yakout, Sobhy M; Mohammed, Abdul Khader; Alenad, Amal M; Chrousos, George P; Alokail, Majed S

    2015-01-01

    Parathyroid hormone (PTH) plays a crucial role in calcium metabolism and skeletal development via altering vitamin D level. Besides, hypersecretion of PTH is implicated in the etiology of osteoporosis. In this study, we analyzed association between promoter region sequence variants of PTH gene and circulating 25-hydroxy-vitamin D (25(OH)D) level. Genotypes of PTH SNPs rs1459015, rs10500783 and rs10500784 and circulating serum 25(OH)D level of healthy adults (N=386) of different nationalities living in Riyadh were determined and relation between the different PTH allelic variants and corresponding mean 25(OH)D values were obtained using Analysis of Variance (ANOVA) and Bonferroni post-hoc test for multiple comparisons. We observed a high prevalence of vitamin D deficiency (<50 nmol/l) among all nationals which ranged from 59% among Indians to 82% among Yemeni. Comparison of the means of 25(OH)D levels corresponding to different genotypes of PTH SNPs indicated that the T allele of SNP rs1459015 was associated with higher 25(OH)D level in the Sudanese (P=0.03), while the T allele of SNP rs10500783 was associated with higher 25(OH)D level in Saudis (P=0.03). Analysis of results also indicated that the Sudanese carriers of the CC genotype of SNP rs1459015 had a higher risk of suffering from vitamin D deficiency (P=0.02). In conclusion, our study indicated significant association between specific PTH gene promoter region variants and altered levels of 25(OH)D and vitamin D deficiency among specific nationals. PMID:26339419

  2. A SPECTRAL APPROACH INTEGRATING FUNCTIONAL GENOMIC ANNOTATIONS FOR CODING AND NONCODING VARIANTS

    PubMed Central

    IONITA-LAZA, IULIANA; MCCALLUM, KENNETH; XU, BIN; BUXBAUM, JOSEPH

    2015-01-01

    Over the past few years, substantial effort has been put into the functional annotation of variation in human genome sequence. Such annotations can play a critical role in identifying putatively causal variants among the abundant natural variation that occurs at a locus of interest. The main challenges in using these various annotations include their large numbers, and their diversity. Here we develop an unsupervised approach to integrate these different annotations into one measure of functional importance (Eigen), that, unlike most existing methods, is not based on any labeled training data. We show that the resulting meta-score has better discriminatory ability using disease associated and putatively benign variants from published studies (in both coding and noncoding regions) compared with the recently proposed CADD score. Across varied scenarios, the Eigen score performs generally better than any single individual annotation, representing a powerful single functional score that can be incorporated in fine-mapping studies. PMID:26727659

  3. Transforming growth factor-B1 and matrix metalloproteinase-7 promoter variants induce risk for Helicobacter pylori-associated gastric precancerous lesions.

    PubMed

    Achyut, B R; Ghoshal, Uday C; Moorchung, Nikhil; Mittal, Balraj

    2009-06-01

    The expression of growth factors, proteolytic enzymes, fibrogenic factors, and cytokines is altered in the Helicobacter pylori-infected gastric mucosa. Therefore, we aimed to evaluate the association of functional promoter variants of transforming growth factor (TGF)-B1 and matrix metalloproteinase (MMP)-7 genes with gastritis and gastric precancerous lesions. After upper gastrointestinal endoscopy, a total of 130 rapid urease test-positive patients with nonulcer dyspepsia were examined for H. pylori infection using modified Giemsa stain and IgG anti-CagA ELISA. All patients and 200 asymptomatic controls were genotyped for TGF-B1 (-509 C>T) and MMP-7 (-181 A>G) substitutions using PCR-RFLP. The genotype and allele frequencies of TGF-B1 and MMP-7 polymorphisms did not differ between patients and controls (p > 0.05). However, the CagA-positive patients with TGF-B1 -509 T allele had higher risk for gastric atrophy (p = 0.026, odds ratio [OR] = 2.38) and lymphoid follicle development (p = 0.028, OR = 2.29). In addition, CagA-positive patients carrying MMP-7 -181 G allele had risk for lymphoid follicle formation (p = 0.027, OR = 2.30). Thus, the present study revealed significant association of functional MMP-7 and TGF-B1 gene variants toward susceptibility to H. pylori-induced precancerous gastric lesions. PMID:19317620

  4. VarMod: modelling the functional effects of non-synonymous variants.

    PubMed

    Pappalardo, Morena; Wass, Mark N

    2014-07-01

    Unravelling the genotype-phenotype relationship in humans remains a challenging task in genomics studies. Recent advances in sequencing technologies mean there are now thousands of sequenced human genomes, revealing millions of single nucleotide variants (SNVs). For non-synonymous SNVs present in proteins the difficulties of the problem lie in first identifying those nsSNVs that result in a functional change in the protein among the many non-functional variants and in turn linking this functional change to phenotype. Here we present VarMod (Variant Modeller) a method that utilises both protein sequence and structural features to predict nsSNVs that alter protein function. VarMod develops recent observations that functional nsSNVs are enriched at protein-protein interfaces and protein-ligand binding sites and uses these characteristics to make predictions. In benchmarking on a set of nearly 3000 nsSNVs VarMod performance is comparable to an existing state of the art method. The VarMod web server provides extensive resources to investigate the sequence and structural features associated with the predictions including visualisation of protein models and complexes via an interactive JSmol molecular viewer. VarMod is available for use at http://www.wasslab.org/varmod. PMID:24906884

  5. Reduction of Cellular Expression Levels Is a Common Feature of Functionally Affected Pendrin (SLC26A4) Protein Variants

    PubMed Central

    de Moraes, Vanessa C S; Bernardinelli, Emanuele; Zocal, Nathalia; Fernandez, Jhonathan A; Nofziger, Charity; Castilho, Arthur M; Sartorato, Edi L; Paulmichl, Markus; Dossena, Silvia

    2016-01-01

    Sequence alterations in the pendrin gene (SLC26A4) leading to functionally affected protein variants are frequently involved in the pathogenesis of syndromic and nonsyndromic deafness. Considering the high number of SLC26A4 sequence alterations reported to date, discriminating between functionally affected and unaffected pendrin protein variants is essential in contributing to determine the genetic cause of deafness in a given patient. In addition, identifying molecular features common to the functionally affected protein variants can be extremely useful to design future molecule-directed therapeutic approaches. Here we show the functional and molecular characterization of six previously uncharacterized pendrin protein variants found in a cohort of 58 Brazilian deaf patients. Two variants (p.T193I and p.L445W) were undetectable in the plasma membrane, completely retained in the endoplasmic reticulum and showed no transport function; four (p.P142L, p.G149R, p.C282Y and p.Q413R) showed reduced function and significant, although heterogeneous, expression levels in the plasma membrane. Importantly, total expression levels of all of the functionally affected protein variants were significantly reduced with respect to the wild-type and a fully functional variant (p.R776C), regardless of their subcellular localization. Interestingly, reduction of expression may also reduce the transport activity of variants with an intrinsic gain of function (p.Q413R). As reduction of overall cellular abundance was identified as a common molecular feature of pendrin variants with affected function, the identification of strategies to prevent reduction in expression levels may represent a crucial step of potential future therapeutic interventions aimed at restoring the transport activity of dysfunctional pendrin variants. PMID:26752218

  6. Natural Yeast Promoter Variants Reveal Epistasis in the Generation of Transcriptional-Mediated Noise and Its Potential Benefit in Stressful Conditions

    PubMed Central

    Liu, Jian; Martin-Yken, Hélène; Bigey, Frédéric; Dequin, Sylvie; François, Jean-Marie; Capp, Jean-Pascal

    2015-01-01

    The increase in phenotypic variability through gene expression noise is proposed to be an evolutionary strategy in selective environments. Differences in promoter-mediated noise between Saccharomyces cerevisiae strains could have been selected for thanks to the benefit conferred by gene expression heterogeneity in the stressful conditions, for instance, those experienced by industrial strains. Here, we used a genome-wide approach to identify promoters conferring high noise levels in the industrial wine strain EC1118. Many promoters of genes related to environmental factors were identified, some of them containing genetic variations compared with their counterpart in the laboratory strain S288c. Each variant of eight promoters has been fused to yeast-Enhanced Green Fluorescent Protein and integrated in the genome of both strains. Some industrial variants conferred higher expression associated, as expected, with lower noise, but other variants either increased or decreased expression without modifying variability, so that they might exhibit different levels of transcriptional-mediated noise at equal mean. At different induction conditions giving similar expression for both variants of the CUP1 promoter, we indeed observed higher noise with the industrial variant. Nevertheless, this difference was only observed in the industrial strain, revealing epistasis in the generation of promoter-mediated noise. Moreover, the increased expression variability conferred by this natural yeast promoter variant provided a clear benefit in the face of an environmental stress. Thus, modulation of gene expression noise by a combination of promoter modifications and trans-influences might be a possible adaptation mechanism in yeast. PMID:25762217

  7. DISSECTION OF PLANT PROMOTER FUNCTION IN VIVO

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Combinatorial interactions between MYB and transcription HLH factors are required for the regulation of several important processes in plants. Protein-DNA binding and transient expression experiments established a modular structure for several maize flavonoid biosynthetic gene promoters, in which h...

  8. Identification of Allelic Variants of Pendrin (SLC26A4) with Loss and Gain of Function

    PubMed Central

    Dossena, Silvia; Bizhanova, Aigerim; Nofziger, Charity; Bernardinelli, Emanuele; Ramsauer, Josef; Kopp, Peter; Paulmichl, Markus

    2011-01-01

    Background Pendrin is a multifunctional anion transporter that exchanges chloride and iodide in the thyroid, as well as chloride and bicarbonate in the inner ear, kidney and airways. Loss or reduction in the function of pendrin results in both syndromic (Pendred syndrome) and non-syndromic (non-syndromic enlarged vestibular aqueduct (ns-EVA)) hearing loss. Factors inducing an up-regulation of pendrin in the kidney and the lung may have an impact on the pathogenesis of hypertension, chronic obstructive pulmonary disease (COPD) and asthma. Here we characterize the ion transport activity of wild-type (WT) pendrin and seven of its allelic variants selected among those reported in the single nucleotide polymorphisms data base (dbSNPs), some of which were previously identified in a cohort of individuals with normal hearing or deaf patients belonging to the Spanish population. Methods WT and mutated pendrin allelic variants were functionally characterized in a heterologous over-expression system by means of fluorometric methods evaluating the I−/Cl− and Cl−/OH− exchange and an assay evaluating the efflux of radiolabeled iodide. Results The transport activity of pendrin P70L, P301L and F667C is completely abolished; pendrin V609G and D687Y allelic variants are functionally impaired but retain significant transport. Pendrin F354S activity is indistinguishable from WT, while pendrin V88I and G740S exhibit a gain of function. Conclusion Amino acid substitutions involving a proline always result in a severe loss of function of pendrin. Two hyperfunctional allelic variants (V88I, G740S) have been identified, and they may have a contributing role in the pathogenesis of hypertension, COPD and asthma. PMID:22116359

  9. Candidate genes and functional noncoding variants identified in a canine model of obsessive-compulsive disorder

    PubMed Central

    2014-01-01

    Background Obsessive-compulsive disorder (OCD), a severe mental disease manifested in time-consuming repetition of behaviors, affects 1 to 3% of the human population. While highly heritable, complex genetics has hampered attempts to elucidate OCD etiology. Dogs suffer from naturally occurring compulsive disorders that closely model human OCD, manifested as an excessive repetition of normal canine behaviors that only partially responds to drug therapy. The limited diversity within dog breeds makes identifying underlying genetic factors easier. Results We use genome-wide association of 87 Doberman Pinscher cases and 63 controls to identify genomic loci associated with OCD and sequence these regions in 8 affected dogs from high-risk breeds and 8 breed-matched controls. We find 119 variants in evolutionarily conserved sites that are specific to dogs with OCD. These case-only variants are significantly more common in high OCD risk breeds compared to breeds with no known psychiatric problems. Four genes, all with synaptic function, have the most case-only variation: neuronal cadherin (CDH2), catenin alpha2 (CTNNA2), ataxin-1 (ATXN1), and plasma glutamate carboxypeptidase (PGCP). In the 2 Mb gene desert between the cadherin genes CDH2 and DSC3, we find two different variants found only in dogs with OCD that disrupt the same highly conserved regulatory element. These variants cause significant changes in gene expression in a human neuroblastoma cell line, likely due to disrupted transcription factor binding. Conclusions The limited genetic diversity of dog breeds facilitates identification of genes, functional variants and regulatory pathways underlying complex psychiatric disorders that are mechanistically similar in dogs and humans. PMID:24995881

  10. Engineering hyperactive variants of human deoxyribonuclease I by altering its functional mechanism.

    PubMed

    Pan, C Q; Lazarus, R A

    1997-06-01

    Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been engineered to more effectively degrade double-stranded DNA to lower molecular weight fragments by altering its functional mechanism from the native single-stranded nicking pathway to a much more efficient one which results in increased double-stranded scission. By introducing positively charged amino acids at DNase I positions that can interact favorably with the proximal negatively charged phosphate groups of the DNA, we have created a hyperactive variant with approximately 35-fold higher DNA-degrading activity relative to wild type. This enhancement can be attributed to both a decrease in Km and an increase in Vmax. Furthermore, unlike wild-type DNase I, the hyperactive variants are no longer inhibited by physiological saline. Replacement of the same positions with negatively charged amino acids greatly reduced DNA cleavage activity, consistent with a repulsive effect with the neighboring DNA phosphates. In addition, these variants displayed similar activities toward a small synthetic substrate, p-nitrophenyl phenylphosphonate, suggesting that the difference in DNA cleavage activity is due to the interaction of the engineered charged residues with the DNA phosphate backbone rather than any change in catalytic machinery. Finally, experiments involving the repair of DNase I digested DNA with T4 DNA ligase and the Klenow fragment of DNA polymerase I suggest that single-stranded gaps are introduced by the hyperactive variants. Thus, the increased functional activity of the hyperactive variants may be explained in part by a shift toward a processive DNA nicking mechanism, which leads to a higher frequency of double-stranded breaks. PMID:9184142

  11. Identification of a Functional Risk Variant for Pemphigus Vulgaris in the ST18 Gene

    PubMed Central

    Vodo, Dan; Sarig, Ofer; Ben-Asher, Edna; Olender, Tsviya; Bochner, Ron; Goldberg, Ilan; Nosgorodsky, Judith; Alkelai, Anna; Tatarskyy, Pavel; Peled, Alon; Baum, Sharon; Barzilai, Aviv; Ibrahim, Saleh M.; Zillikens, Detlef; Lancet, Doron; Sprecher, Eli

    2016-01-01

    Pemphigus vulgaris (PV) is a life-threatening autoimmune mucocutaneous blistering disease caused by disruption of intercellular adhesion due to auto-antibodies directed against epithelial components. Treatment is limited to immunosuppressive agents, which are associated with serious adverse effects. The propensity to develop the disease is in part genetically determined. We therefore reasoned that the delineation of PV genetic basis may point to novel therapeutic strategies. Using a genome-wide association approach, we recently found that genetic variants in the vicinity of the ST18 gene confer a significant risk for the disease. Here, using targeted deep sequencing, we identified a PV-associated variant residing within the ST18 promoter region (p<0.0002; odds ratio = 2.03). This variant was found to drive increased gene transcription in a p53/p63-dependent manner, which may explain the fact that ST18 is up-regulated in the skin of PV patients. We then discovered that when overexpressed, ST18 stimulates PV serum-induced secretion of key inflammatory molecules and contributes to PV serum-induced disruption of keratinocyte cell-cell adhesion, two processes previously implicated in the pathogenesis of PV. Thus, the present findings indicate that ST18 may play a direct role in PV and consequently represents a potential target for the treatment of this disease. PMID:27148741

  12. Admixture Mapping in Lupus Identifies Multiple Functional Variants within IFIH1 Associated with Apoptosis, Inflammation, and Autoantibody Production

    PubMed Central

    Looger, Loren L.; Han, Shizhong; Kim-Howard, Xana; Glenn, Stuart; Adler, Adam; Kelly, Jennifer A.; Niewold, Timothy B.; Gilkeson, Gary S.; Brown, Elizabeth E.; Alarcón, Graciela S.; Edberg, Jeffrey C.; Petri, Michelle; Ramsey-Goldman, Rosalind; Reveille, John D.; Vilá, Luis M.; Freedman, Barry I.; Tsao, Betty P.; Criswell, Lindsey A.; Jacob, Chaim O.; Moore, Jason H.; Vyse, Timothy J.; Langefeld, Carl L.; Guthridge, Joel M.; Gaffney, Patrick M.; Moser, Kathy L.; Scofield, R. Hal; Alarcón-Riquelme, Marta E.; Williams, Scott M.; Merrill, Joan T.; James, Judith A.; Kaufman, Kenneth M.; Kimberly, Robert P.; Harley, John B.; Nath, Swapan K.

    2013-01-01

    Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease with a strong genetic component. African-Americans (AA) are at increased risk of SLE, but the genetic basis of this risk is largely unknown. To identify causal variants in SLE loci in AA, we performed admixture mapping followed by fine mapping in AA and European-Americans (EA). Through genome-wide admixture mapping in AA, we identified a strong SLE susceptibility locus at 2q22–24 (LOD = 6.28), and the admixture signal is associated with the European ancestry (ancestry risk ratio ∼1.5). Large-scale genotypic analysis on 19,726 individuals of African and European ancestry revealed three independently associated variants in the IFIH1 gene: an intronic variant, rs13023380 [Pmeta = 5.20×10−14; odds ratio, 95% confidence interval = 0.82 (0.78–0.87)], and two missense variants, rs1990760 (Ala946Thr) [Pmeta = 3.08×10−7; 0.88 (0.84–0.93)] and rs10930046 (Arg460His) [Pdom = 1.16×10−8; 0.70 (0.62–0.79)]. Both missense variants produced dramatic phenotypic changes in apoptosis and inflammation-related gene expression. We experimentally validated function of the intronic SNP by DNA electrophoresis, protein identification, and in vitro protein binding assays. DNA carrying the intronic risk allele rs13023380 showed reduced binding efficiency to a cellular protein complex including nucleolin and lupus autoantigen Ku70/80, and showed reduced transcriptional activity in vivo. Thus, in SLE patients, genetic susceptibility could create a biochemical imbalance that dysregulates nucleolin, Ku70/80, or other nucleic acid regulatory proteins. This could promote antibody hypermutation and auto-antibody generation, further destabilizing the cellular network. Together with molecular modeling, our results establish a distinct role for IFIH1 in apoptosis, inflammation, and autoantibody production, and explain the molecular basis of these three risk alleles for SLE pathogenesis. PMID

  13. Admixture mapping in lupus identifies multiple functional variants within IFIH1 associated with apoptosis, inflammation, and autoantibody production.

    PubMed

    Molineros, Julio E; Maiti, Amit K; Sun, Celi; Looger, Loren L; Han, Shizhong; Kim-Howard, Xana; Glenn, Stuart; Adler, Adam; Kelly, Jennifer A; Niewold, Timothy B; Gilkeson, Gary S; Brown, Elizabeth E; Alarcón, Graciela S; Edberg, Jeffrey C; Petri, Michelle; Ramsey-Goldman, Rosalind; Reveille, John D; Vilá, Luis M; Freedman, Barry I; Tsao, Betty P; Criswell, Lindsey A; Jacob, Chaim O; Moore, Jason H; Vyse, Timothy J; Langefeld, Carl L; Guthridge, Joel M; Gaffney, Patrick M; Moser, Kathy L; Scofield, R Hal; Alarcón-Riquelme, Marta E; Williams, Scott M; Merrill, Joan T; James, Judith A; Kaufman, Kenneth M; Kimberly, Robert P; Harley, John B; Nath, Swapan K

    2013-01-01

    Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease with a strong genetic component. African-Americans (AA) are at increased risk of SLE, but the genetic basis of this risk is largely unknown. To identify causal variants in SLE loci in AA, we performed admixture mapping followed by fine mapping in AA and European-Americans (EA). Through genome-wide admixture mapping in AA, we identified a strong SLE susceptibility locus at 2q22-24 (LOD=6.28), and the admixture signal is associated with the European ancestry (ancestry risk ratio ~1.5). Large-scale genotypic analysis on 19,726 individuals of African and European ancestry revealed three independently associated variants in the IFIH1 gene: an intronic variant, rs13023380 [P(meta) = 5.20×10(-14); odds ratio, 95% confidence interval = 0.82 (0.78-0.87)], and two missense variants, rs1990760 (Ala946Thr) [P(meta) = 3.08×10(-7); 0.88 (0.84-0.93)] and rs10930046 (Arg460His) [P(dom) = 1.16×10(-8); 0.70 (0.62-0.79)]. Both missense variants produced dramatic phenotypic changes in apoptosis and inflammation-related gene expression. We experimentally validated function of the intronic SNP by DNA electrophoresis, protein identification, and in vitro protein binding assays. DNA carrying the intronic risk allele rs13023380 showed reduced binding efficiency to a cellular protein complex including nucleolin and lupus autoantigen Ku70/80, and showed reduced transcriptional activity in vivo. Thus, in SLE patients, genetic susceptibility could create a biochemical imbalance that dysregulates nucleolin, Ku70/80, or other nucleic acid regulatory proteins. This could promote antibody hypermutation and auto-antibody generation, further destabilizing the cellular network. Together with molecular modeling, our results establish a distinct role for IFIH1 in apoptosis, inflammation, and autoantibody production, and explain the molecular basis of these three risk alleles for SLE pathogenesis. PMID:23441136

  14. Human microsomal epoxide hydrolase: genetic polymorphism and functional expression in vitro of amino acid variants

    PubMed Central

    Hassett, Christopher; Aicher, Lauri; Sidhu, Jaspreet S.

    2016-01-01

    Human microsomal epoxide hydrolase (mEH) is a biotransformation enzyme that metabolizes reactive epoxide intermediates to more water-soluble trans-dihydrodiol derivatives. We compared protein-coding sequences from six full-length human mEH DNA clones and assessed potential amino acid variation at seven positions. The prevalence of these variants was assessed in at least 37 unrelated individuals using polymerase chain reaction experiments. Only Tyr/His 113 (exon 3) and His/Arg 139 (exon 4) variants were observed. The genotype frequencies determined for residue 113 alleles indicate that this locus may not be in Hardy – Weinberg equilibrium, whereas frequencies observed for residue 139 alleles were similar to expected values. Nucleotide sequences coding for the variant amino acids were constructed in an mEH cDNA using site-directed mutagenesis, and each was expressed in vitro by transient transfection of COS-1 cells. Epoxide hydrolase mRNA level, catalytic activity, and immunoreactive protein were evaluated for each construct. The results of these analyses demonstrated relatively uniform levels of mEH RNA expression between the constructs. mEH enzymatic activity and immunoreactive protein were strongly correlated, indicating that mEH specific activity was similar for each variant. However, marked differences were noted in the relative amounts of immunoreactive protein and enzymatic activity resulting from the amino acid substitutions. These data suggest that common human mEH amino acid polymorphisms may alter enzymatic function, possibly by modifying protein stability. PMID:7516776

  15. Functional characteristics of the Staphylococcus aureus δ-toxin allelic variant G10S

    PubMed Central

    Cheung, Gordon Y. C.; Yeh, Anthony J.; Kretschmer, Dorothee; Duong, Anthony C.; Tuffuor, Kwame; Fu, Chih-Lung; Joo, Hwang-Soo; Diep, Binh A.; Li, Min; Nakamura, Yuumi; Nunez, Gabriel; Peschel, Andreas; Otto, Michael

    2015-01-01

    Staphylococcus aureus δ-toxin is a member of the phenol-soluble modulin (PSM) peptide family. PSMs have multiple functions in staphylococcal pathogenesis; for example, they lyse red and white blood cells and trigger inflammatory responses. Compared to other PSMs, δ-toxin is usually more strongly expressed but has only moderate cytolytic capacities. The amino acid sequences of S. aureus PSMs are well conserved with two exceptions, one of which is the δ-toxin allelic variant G10S. This variant is a characteristic of the subspecies S. argenteus and S. aureus sequence types ST1 and ST59, the latter representing the most frequent cause of community-associated infections in Asia. δ-toxin G10S and strains expressing that variant from plasmids or the genome had significantly reduced cytolytic and pro-inflammatory capacities, including in a strain background with pronounced production of other PSMs. However, in murine infection models, isogenic strains expressing the two δ-toxin variants did not cause measurable differences in disease severity. Our findings indicate that the widespread G10S allelic variation of the δ-toxin locus has a significant impact on key pathogenesis mechanisms, but more potent members of the PSM peptide family may overshadow that impact in vivo. PMID:26658455

  16. In Silico Characterization of Functional Divergence of Two Cathelicidin Variants in Indian Sheep

    PubMed Central

    Dhaliwal, Kamaljeet K; Arora, Jaspreet S; Mukhopadhyay, Chandra S; Dubey, Prem P

    2015-01-01

    The present work focuses on the in silico characterization of functional divergence of two ovine cathelicidin coding sequence (cds) variants (ie, Cath1 and Cath2) of Indian sheep. Overlapping partial cds of both the cathelicidin variants were cloned in pJet1.2/blunt vector and sequenced. Evolutionary analysis of the Cath2 and Cath1 indicated that the mammalian cathelicidins clustered separately from avian fowlicidins. The avian fowlicidins, which are very different from mammalian cathelicidins (Caths), clearly displayed signatures of purifying selection. The pairwise sequence alignments of translated amino acid sequences of these two sheep cathelicidins showed gaps in the antimicrobial domain of Cath1 variant; however, the amino terminal cathelin regions of both the Caths were conserved. Amino acid sequence analysis of full-length cathelicidins available at public database revealed that Cath1, Cath2, and Cath7 of different ruminant species (including our Cath1 and Cath2 variants) formed individual clads, suggesting that these types have evolved to target specific types of microbes. In silico analysis of Cath1 and Cath2 peptide sequences indicated that the C-terminal antimicrobial peptide domain of Cath2 is more immunogenic than that of the ovine Cath1 due to its higher positive antigenic index, making Cath1 a promising antigen for production of monoclonal antibodies. PMID:26380546

  17. Designed angiopoietin-1 variant, COMP-angiopoietin-1, rescues erectile function through healthy cavernous angiogenesis in a hypercholesterolemic mouse

    PubMed Central

    Ryu, Ji-Kan; Kim, Woo Jean; Koh, Young Jun; Piao, Shuguang; Jin, Hai-Rong; Lee, Sae-Won; Choi, Min Ji; Shin, Hwa-Yean; Kwon, Mi-Hye; Jung, Keehoon; Koh, Gou Young; Suh, Jun-Kyu

    2015-01-01

    Despite the advent of oral phosphodiesterase-5 inhibitors, curative treatment for erectile dysfunction (ED) remains unavailable. Recently, the link between ED and cardiovascular disease was unveiled and the main etiology of ED was found to be vasculogenic. Therefore, neovascularization is a promising strategy for curing ED. Angiopoietin-1 (Ang1) is an angiogenic growth factor that promotes the generation of stable and functional vasculature. Here, we demonstrate that local delivery of the soluble, stable, and potent Ang1 variant, COMP-Ang1 gene or protein, into the penises of hypercholesterolemic mice increases cavernous angiogenesis, eNOS phosphorylation, and cGMP expression, resulting in full recovery of erectile function and cavernous blood flow up to 8 weeks after treatment. COMP-Ang1-induced promotion of cavernous angiogenesis and erectile function was abolished in Nos3-/- mice and in the presence of the NOS inhibitor, L-NAME. COMP-Ang1 also restored the integrity of endothelial cell-cell junction by down-regulating the expression of histone deacetylase 2 in the penis of hypercholesterolemic mice and in primary cultured mouse cavernous endothelial cells. These findings constitute a new paradigm toward curative treatment of both cavernous angiopathy and ED. PMID:25783805

  18. Designed angiopoietin-1 variant, COMP-angiopoietin-1, rescues erectile function through healthy cavernous angiogenesis in a hypercholesterolemic mouse.

    PubMed

    Ryu, Ji-Kan; Kim, Woo Jean; Koh, Young Jun; Piao, Shuguang; Jin, Hai-Rong; Lee, Sae-Won; Choi, Min Ji; Shin, Hwa-Yean; Kwon, Mi-Hye; Jung, Keehoon; Koh, Gou Young; Suh, Jun-Kyu

    2015-01-01

    Despite the advent of oral phosphodiesterase-5 inhibitors, curative treatment for erectile dysfunction (ED) remains unavailable. Recently, the link between ED and cardiovascular disease was unveiled and the main etiology of ED was found to be vasculogenic. Therefore, neovascularization is a promising strategy for curing ED. Angiopoietin-1 (Ang1) is an angiogenic growth factor that promotes the generation of stable and functional vasculature. Here, we demonstrate that local delivery of the soluble, stable, and potent Ang1 variant, COMP-Ang1 gene or protein, into the penises of hypercholesterolemic mice increases cavernous angiogenesis, eNOS phosphorylation, and cGMP expression, resulting in full recovery of erectile function and cavernous blood flow up to 8 weeks after treatment. COMP-Ang1-induced promotion of cavernous angiogenesis and erectile function was abolished in Nos3(-/-) mice and in the presence of the NOS inhibitor, L-NAME. COMP-Ang1 also restored the integrity of endothelial cell-cell junction by down-regulating the expression of histone deacetylase 2 in the penis of hypercholesterolemic mice and in primary cultured mouse cavernous endothelial cells. These findings constitute a new paradigm toward curative treatment of both cavernous angiopathy and ED. PMID:25783805

  19. Many amino acid substitution variants identified in DNA repair genes during human population screenings are predicted to impact protein function

    SciTech Connect

    Xi, T; Jones, I M; Mohrenweiser, H W

    2003-11-03

    Over 520 different amino acid substitution variants have been previously identified in the systematic screening of 91 human DNA repair genes for sequence variation. Two algorithms were employed to predict the impact of these amino acid substitutions on protein activity. Sorting Intolerant From Tolerant (SIFT) classified 226 of 508 variants (44%) as ''Intolerant''. Polymorphism Phenotyping (PolyPhen) classed 165 of 489 amino acid substitutions (34%) as ''Probably or Possibly Damaging''. Another 9-15% of the variants were classed as ''Potentially Intolerant or Damaging''. The results from the two algorithms are highly associated, with concordance in predicted impact observed for {approx}62% of the variants. Twenty one to thirty one percent of the variant proteins are predicted to exhibit reduced activity by both algorithms. These variants occur at slightly lower individual allele frequency than do the variants classified as ''Tolerant'' or ''Benign''. Both algorithms correctly predicted the impact of 26 functionally characterized amino acid substitutions in the APE1 protein on biochemical activity, with one exception. It is concluded that a substantial fraction of the missense variants observed in the general human population are functionally relevant. These variants are expected to be the molecular genetic and biochemical basis for the associations of reduced DNA repair capacity phenotypes with elevated cancer risk.

  20. Associations of Filaggrin Gene Loss-of-Function Variants with Urinary Phthalate Metabolites and Testicular Function in Young Danish Men

    PubMed Central

    Jørgensen, Niels; Meldgaard, Michael; Frederiksen, Hanne; Andersson, Anna-Maria; Menné, Torkil; Johansen, Jeanne Duus; Carlsen, Berit Christina; Stender, Steen; Szecsi, Pal Bela; Skakkebæk, Niels Erik; De Meyts, Ewa Rajpert; Thyssen, Jacob P.

    2014-01-01

    Background: Filaggrin is an epidermal protein that is crucial for skin barrier function. Up to 10% of Europeans and 5% of Asians carry at least one null allele in the filaggrin gene (FLG). Reduced expression of filaggrin in carriers of the null allele is associated with facilitated transfer of allergens across the epidermis. We hypothesized that these individuals may have increased transdermal uptake of endocrine disruptors, including phthalates. Objectives: We investigated urinary excretion of phthalate metabolites and testicular function in young men with and without FLG loss-of-function variants in a cross-sectional study of 861 young men from the general Danish population. Methods: All men were genotyped for FLG R501X, 2282del4, and R2447X loss-of-function variants. We measured urinary concentrations of 14 phthalate metabolites and serum levels of reproductive hormones. We also evaluated semen quality. Results: Sixty-five men (7.5%) carried at least one FLG-null allele. FLG-null carriers had significantly higher urinary concentrations of several phthalate metabolites, including a 33% higher concentration of MnBP (mono-n-butyl phthalate; 95% CI: 16, 51%). FLG-null variants were not significantly associated with reproductive hormones or semen quality parameters. Conclusion: This study provides evidence that carriers of FLG loss-of-function alleles may have higher internal exposure to phthalates, possibly due to increased transepidermal absorption. FLG loss-of-function variants may indicate susceptible populations for which special attention to transepidermal absorption of chemicals and medication may be warranted. Citation: Joensen UN, Jørgensen N, Meldgaard M, Frederiksen H, Andersson AM, Menné T, Johansen JD, Carlsen BC, Stender S, Szecsi PB, Skakkebæk NE, Rajpert-De Meyts E, Thyssen JP. 2014. Associations of filaggrin gene loss-of-function variants with urinary phthalate metabolites and testicular function in young Danish men. Environ Health Perspect 122

  1. Effect of Cys85 on biochemical properties and biological function of human SP-A variants

    PubMed Central

    Wang, Guirong; Myers, Catherine; Mikerov, Anatoly; Floros, Joanna

    2008-01-01

    Four “core” amino acid differences within the collagen-like domain distinguish the human surfactant proteins A1 (SP-A1) variants from the SP-A2 variants. One of these, cysteine 85 that could form intermolecular disulfide bonds, is present in SP-A1 (Cys85) and absent in SP-A2 (Arg85). We hypothesized that residue85 affects both structure and function of SP-A1 and SP-A2 variants. To test this, wild type (WT) variants, 6A2 of SP-A1 and 1A0 of SP-A2, and their mutants (6A2(C85R) and 1A0(R85C)), were generated and studied. We found: 1) Residue85 affected the binding ability to mannose and the oligomerization pattern of SP-As. The 1A0(R85C) and 6A2(C85R) patterns were similar and/or resembled those of WT 6A2 and 1A0, respectively. 2) Both SP-A WT and mutants differentially induced rough LPS and Pseudomonas aeruginosa aggregation in the following order: 1A0 > 6A2 > 6A2(C85R) > 1A0(R85C) for Re-LPS aggregation, and 1A0 > 6A2 = 6A2(C85R) = 1A0(R85C) for bacterial aggregation. 3) SP-A WT and mutants enhanced phagocytosis of P. aeruginosa by rat alveolar macrophages. Their phagocytic index order was: 6A2(C85R) > 1A0 > 6A2 = 1A0(R85C). The activity of mutant 1A0(C85R) was significantly lower from WT 1A0 but similar to 6A2. Compared to WT 6A2, the 6A2(C85R) mutant exhibited a significantly higher activity. These results indicate that SP-A variant/mutant with Arg85 exhibits higher ability to enhance bacterial phagocytosis than that with Cys85. Residue85 plays a important role in the structure and function of SP-A, and is a major factor for the differences between SP-A1 and SPA2 variants. PMID:17580966

  2. Effect of cysteine 85 on biochemical properties and biological function of human surfactant protein A variants.

    PubMed

    Wang, Guirong; Myers, Catherine; Mikerov, Anatoly; Floros, Joanna

    2007-07-17

    Four "core" amino acid differences within the collagen-like domain distinguish the human surfactant protein A1 (SP-A1) variants from the SP-A2 variants. One of these, cysteine 85 that could form intermolecular disulfide bonds, is present in SP-A1 (Cys85) and absent in SP-A2 (Arg85). We hypothesized that residue 85 affects both the structure and function of SP-A1 and SP-A2 variants. To test this, wild-type (WT) variants, 6A2 of SP-A1 and 1A0 of SP-A2, and their mutants (6A2(C85R) and 1A0(R85C)) were generated and studied. We found the following: (1) Residue 85 affected the binding ability to mannose and the oligomerization pattern of SP-As. The 1A0(R85C) and 6A2(C85R) patterns were similar and/or resembled those of WT 6A2 and 1A0, respectively. (2) Both SP-A WT and mutants differentially induced rough LPS and Pseudomonas aeruginosa aggregation in the following order: 1A0 > 6A2 > 6A2(C85R) > 1A0(R85C) for Re-LPS aggregation and 1A0 > 6A2 = 6A2(C85R) = 1A0(R85C) for bacterial aggregation. (3) SP-A WT and mutants enhanced phagocytosis of P. aeruginosa by rat alveolar macrophages. Their phagocytic index order was 6A2(C85R) > 1A0 > 6A2 = 1A0(R85C). The activity of mutant 1A0(C85R) was significantly lower than WT 1A0 but similar to 6A2. Compared to WT 6A2, the 6A2(C85R) mutant exhibited a significantly higher activity. These results indicate that the SP-A variant/mutant with Arg85 exhibits a higher ability to enhance bacterial phagocytosis than that with Cys85. Residue 85 plays an important role in the structure and function of SP-A and is a major factor for the differences between SP-A1 and SP-A2 variants. PMID:17580966

  3. A functional variant of elafin with improved anti-inflammatory activity for pulmonary inflammation.

    PubMed

    Small, Donna M; Zani, Marie-Louise; Quinn, Derek J; Dallet-Choisy, Sandrine; Glasgow, Arlene M A; O'Kane, Cecilia; McAuley, Danny F; McNally, Paul; Weldon, Sinéad; Moreau, Thierry; Taggart, Clifford C

    2015-01-01

    Elafin is a serine protease inhibitor produced by epithelial and immune cells with anti-inflammatory properties. Research has shown that dysregulated protease activity may elicit proteolytic cleavage of elafin, thereby impairing the innate immune function of the protein. The aim of this study was to generate variants of elafin (GG- and QQ-elafin) that exhibit increased protease resistance while retaining the biological properties of wild-type (WT) elafin. Similar to WT-elafin, GG- and QQ-elafin variants retained antiprotease activity and susceptibility to transglutaminase-mediated fibronectin cross-linking. However, in contrast to WT-elafin, GG- and QQ-elafin displayed significantly enhanced resistance to degradation when incubated with bronchoalveolar lavage fluid from patients with cystic fibrosis. Intriguingly, both variants, particularly GG-elafin, demonstrated improved lipopolysaccharide (LPS) neutralization properties in vitro. In addition, GG-elafin showed improved anti-inflammatory activity in a mouse model of LPS-induced acute lung inflammation. Inflammatory cell infiltration into the lung was reduced in lungs of mice treated with GG-elafin, predominantly neutrophilic infiltration. A reduction in MCP-1 levels in GG-elafin treated mice compared to the LPS alone treatment group was also demonstrated. GG-elafin showed increased functionality when compared to WT-elafin and may be of future therapeutic relevance in the treatment of lung diseases characterized by a protease burden. PMID:25189740

  4. Functional Characterization of Human CYP2C9 Allelic Variants in COS-7 Cells

    PubMed Central

    Du, Huihui; Wei, Zhiyun; Yan, Yucai; Xiong, Yuyu; Zhang, Xiaoqing; Shen, Lu; Ruan, Yunfeng; Wu, Xi; Xu, Qingqing; He, Lin; Qin, Shengying

    2016-01-01

    Variability in activity of CYP2C9, which is involved in the metabolism of approximately 15% of current therapeutic drugs, is an important contributor to interindividual differences in drug response. To evaluate the functional alternations of CYP2C9*2, CYP2C9*3, CYP2C9*8, CYP2C9*11 and CYP2C9*31, identified in our previous study in Chinese Han population, allelic variants as well as the wild-type CYP2C9 were transiently expressed in COS-7 cells. Kinetic parameters (Km, Vmax, and Clint) for S-warfarin 7-hydroxylation by these recombinant CYP2C9s were determined. Relative to CYP2C9.1, recombinant CYP2C9.3 and CYP2C9.11 exhibited significantly higher Km values, and all allelic variants showed significantly decreased Vmax and Clint values. Among all allelic variants, catalytic activity of CYP2C9.3 and CYP2C9.11 reduced the most (8.2% and 9.8% of Clint ratio, respectively; P < 0.001). These findings should be useful for predicting the phenotype profiles of CYP2C9 in Chinese Han population, comparing the functional results of these alleles accurately, and finally optimizing pharmacotherapy of drug treatment. PMID:27199745

  5. Identification of a Novel Rat NR2B Subunit Gene Promoter Region Variant and Its Association with Microwave-Induced Neuron Impairment.

    PubMed

    Wang, Li-Feng; Tian, Da-Wei; Li, Hai-Juan; Gao, Ya-Bing; Wang, Chang-Zhen; Zhao, Li; Zuo, Hong-Yan; Dong, Ji; Qiao, Si-Mo; Zou, Yong; Xiong, Lu; Zhou, Hong-Mei; Yang, Yue-Feng; Peng, Rui-Yun; Hu, Xiang-Jun

    2016-05-01

    Microwave radiation has been implicated in cognitive dysfunction and neuronal injury in animal models and in human investigations; however, the mechanism of these effects is unclear. In this study, single nucleotide polymorphism (SNP) sites in the rat GRIN2B promoter region were screened. The associations of these SNPs with microwave-induced rat brain dysfunction and with rat pheochromocytoma-12 (PC12) cell function were investigated. Wistar rats (n = 160) were exposed to microwave radiation (30 mW/cm(2) for 5 min/day, 5 days/week, over a period of 2 months). Screening of the GRIN2B promoter region revealed a stable C-to-T variant at nucleotide position -217 that was not induced by microwave exposure. The learning and memory ability, amino acid contents in the hippocampus and cerebrospinal fluid, and NR2B expression were then investigated in the different genotypes. Following microwave exposure, NR2B protein expression decreased, while the Glu contents in the hippocampus and CSF increased, and memory impairment was observed in the TT genotype but not the CC and CT genotypes. In PC12 cells, the effects of the T allele were more pronounced than those of the C allele on transcription factor binding ability, transcriptional activity, NR2B mRNA, and protein expression. These effects may be related to the detrimental role of the T allele and the protective role of the C allele in rat brain function and PC12 cells exposed to microwave radiation. PMID:25917873

  6. Genome-Wide Functional Annotation of Human Protein-Coding Splice Variants Using Multiple Instance Learning.

    PubMed

    Panwar, Bharat; Menon, Rajasree; Eksi, Ridvan; Li, Hong-Dong; Omenn, Gilbert S; Guan, Yuanfang

    2016-06-01

    The vast majority of human multiexon genes undergo alternative splicing and produce a variety of splice variant transcripts and proteins, which can perform different functions. These protein-coding splice variants (PCSVs) greatly increase the functional diversity of proteins. Most functional annotation algorithms have been developed at the gene level; the lack of isoform-level gold standards is an important intellectual limitation for currently available machine learning algorithms. The accumulation of a large amount of RNA-seq data in the public domain greatly increases our ability to examine the functional annotation of genes at isoform level. In the present study, we used a multiple instance learning (MIL)-based approach for predicting the function of PCSVs. We used transcript-level expression values and gene-level functional associations from the Gene Ontology database. A support vector machine (SVM)-based 5-fold cross-validation technique was applied. Comparatively, genes with multiple PCSVs performed better than single PCSV genes, and performance also improved when more examples were available to train the models. We demonstrated our predictions using literature evidence of ADAM15, LMNA/C, and DMXL2 genes. All predictions have been implemented in a web resource called "IsoFunc", which is freely available for the global scientific community through http://guanlab.ccmb.med.umich.edu/isofunc . PMID:27142340

  7. Systematic Functional Interrogation of Rare Cancer Variants Identifies Oncogenic Alleles | Office of Cancer Genomics

    Cancer.gov

    Cancer genome characterization efforts now provide an initial view of the somatic alterations in primary tumors. However, most point mutations occur at low frequency, and the function of these alleles remains undefined. We have developed a scalable systematic approach to interrogate the function of cancer-associated gene variants. We subjected 474 mutant alleles curated from 5,338 tumors to pooled in vivo tumor formation assays and gene expression profiling. We identified 12 transforming alleles, including two in genes (PIK3CB, POT1) that have not been shown to be tumorigenic.

  8. Genetic and functional analysis of CHEK2 (CHK2) variants in multiethnic cohorts

    PubMed Central

    Bell, Daphne W.; Kim, Sang H.; Godwin, Andrew K.; Schiripo, Taryn A.; Harris, Patricia L.; Haserlat, Sara M.; Wahrer, Doke C.R.; Haiman, Christopher A.; Daly, Mary B.; Niendorf, Kristin B.; Smith, Matthew R.; Sgroi, Dennis C.; Garber, Judy E.; Olopade, Olufunmilayo I.; Marchand, Loic Le; Henderson, Brian E.; Altshuler, David; Haber, Daniel A.; Freedman, Matthew L.

    2011-01-01

    The CHEK2-1100delC mutation is recurrent in the population and is a moderate risk factor for breast cancer. To identify additional CHEK2 mutations potentially contributing to breast cancer susceptibility, we sequenced 248 cases with early-onset disease; functionally characterized new variants and conducted a population-based case–control analysis to evaluate their contribution to breast cancer risk. We identified 1 additional null mutation and 5 missense variants in the germline of cancer patients. In vitro, the CHEK2-H143Y variant resulted in gross protein destabilization, while others had variable suppression of in vitro kinase activity using BRCA1 as a substrate. The germline CHEK2-1100delC mutation was present among 8/1,646 (0.5%) sporadic, 2/400 (0.5%) early-onset and 3/302 (1%) familial breast cancer cases, but undetectable amongst 2,105 multiethnic controls, including 633 from the US. CHEK2-positive breast cancer families also carried a deleterious BRCA1 mutation. 1100delC appears to be the only recurrent CHEK2 mutation associated with a potentially significant contribution to breast cancer risk in the general population. Another recurrent mutation with attenuated in vitro function, CHEK2-P85L, is not associated with increased breast cancer susceptibility, but exhibits a striking difference in frequency across populations with different ancestral histories. These observations illustrate the importance of genotyping ethnically diverse groups when assessing the impact of low-penetrance susceptibility alleles on population risk. Our findings highlight the notion that clinical testing for rare missense mutations within CHEK2 may have limited value in predicting breast cancer risk, but that testing for the 1100delC variant may be valuable in phenotypically- and geographically-selected populations. PMID:17721994

  9. A genetic variant in the LDLR promoter is responsible for part of the LDL-cholesterol variability in primary hypercholesterolemia

    PubMed Central

    2014-01-01

    Background GWAS have consistently revealed that LDLR locus variability influences LDL-cholesterol in general population. Severe LDLR mutations are responsible for familial hypercholesterolemia (FH). However, most primary hypercholesterolemias are polygenic diseases. Although Cis-regulatory regions might be the cause of LDL-cholesterol variability; an extensive analysis of the LDLR distal promoter has not yet been performed. We hypothesized that genetic variants in this region are responsible for the LDLR association with LDL-cholesterol found in GWAS. Methods Four-hundred seventy-seven unrelated subjects with polygenic hypercholesterolemia (PH) and without causative FH-mutations and 525 normolipemic subjects were selected. A 3103 pb from LDLR (-625 to +2468) was sequenced in 125 subjects with PH. All subjects were genotyped for 4 SNPs (rs17242346, rs17242739, rs17248720 and rs17249120) predicted to be potentially involved in transcription regulation by in silico analysis. EMSA and luciferase assays were carried out for the rs17248720 variant. Multivariable linear regression analysis using LDL-cholesterol levels as the dependent variable were done in order to find out the variables that were independently associated with LDL-cholesterol. Results The sequencing of the 125 PH subjects did not show variants with minor allele frequency ≥ 10%. The T-allele from g.3131C > T (rs17248720) had frequencies of 9% (PH) and 16.4% (normolipemic), p < 0.00001. Studies of this variant with EMSA and luciferase assays showed a higher affinity for transcription factors and an increase of 2.5 times in LDLR transcriptional activity (T-allele vs C-allele). At multivariate analysis, this polymorphism with the lipoprotein(a) and age explained ≈ 10% of LDL-cholesterol variability. Conclusion Our results suggest that the T-allele at the g.3131 T > C SNP is associated with LDL-cholesterol levels, and explains part of the LDL-cholesterol variability. As a plausible

  10. Stimulators of the soluble guanylyl cyclase: promising functional insights from rare coding atherosclerosis-related GUCY1A3 variants.

    PubMed

    Wobst, Jana; von Ameln, Simon; Wolf, Bernhard; Wierer, Michael; Dang, Tan An; Sager, Hendrik B; Tennstedt, Stephanie; Hengstenberg, Christian; Koesling, Doris; Friebe, Andreas; Braun, Siegmund L; Erdmann, Jeanette; Schunkert, Heribert; Kessler, Thorsten

    2016-07-01

    Stimulators of the soluble guanylyl cyclase (sGC) are emerging therapeutic agents in cardiovascular diseases. Genetic alterations of the GUCY1A3 gene, which encodes the α1 subunit of the sGC, are associated with coronary artery disease. Studies investigating sGC stimulators in subjects with CAD and carrying risk-related variants in sGC are, however, lacking. Here, we functionally investigate the impact of coding GUCY1A3 variants on sGC activity and the therapeutic potential of sGC stimulators in vitro. In addition to a known loss-of-function variant, eight coding variants in GUCY1A3 were cloned and expressed in HEK 293 cells. Protein levels and dimerization capability with the β1 subunit were analysed by immunoblotting and co-immunoprecipitation, respectively. All α1 variants found in MI patients dimerized with the β1 subunit. Protein levels were reduced by 72 % in one variant (p < 0.01). Enzymatic activity was analysed using cGMP radioimmunoassay after stimulation with a nitric oxide (NO) donor. Five variants displayed decreased cGMP production upon NO stimulation (p < 0.001). The addition of the sGC stimulator BAY 41-2272 increased cGMP formation in all of these variants (p < 0.01). Except for the variant leading to decreased protein level, cGMP amounts reached the wildtype NO-induced level after addition of BAY 41-2272. In conclusion, rare coding variants in GUCY1A3 lead to reduced cGMP formation which can be rescued by a sGC stimulator in vitro. These results might therefore represent the starting point for discovery of novel treatment strategies for patients at risk with coding GUCY1A3 variants. PMID:27342234

  11. Strategies of Functional Foods Promote Sleep in Human Being

    PubMed Central

    Zeng, Yawen; Yang, Jiazhen; Du, Juan; Pu, Xiaoying; Yang, Xiaomen; Yang, Shuming; Yang, Tao

    2014-01-01

    Sleep is a vital segment of life, however, the mechanisms of diet promoting sleep are unclear and are the focus of research. Insomnia is a general sleep disorder and functional foods are known to play a key role in the prevention of insomnia. A number of studies have demonstrated that major insomnia risk factors in human being are less functional foods in dietary. There are higher functional components in functional foods promoting sleep, including tryptophan, GABA, calcium, potassium, melatonin, pyridoxine, L-ornithine and hexadecanoic acid; but wake-promoting neurochemical factors include serotonin, noradrenalin, acetylcholine, histamine, orexin and so on. The factors promoting sleep in human being are the functional foods include barley grass powder, whole grains, maca, panax, Lingzhi, asparagus powder, lettuce, cherry, kiwifruits, walnut, schisandra wine, and milk; Barley grass powder with higher GABA and calcium, as well as potassium is the most ideal functional food promoting sleep, however, the sleep duration for modern humans is associated with food structure of ancient humans. In this review, we put forward possible mechanisms of functional components in foods promoting sleep. Although there is clear relevance between sleep and diet, their molecular mechanisms need to be studied further. PMID:26005400

  12. Promoting Efficacy Research on Functional Analytic Psychotherapy

    ERIC Educational Resources Information Center

    Maitland, Daniel W. M.; Gaynor, Scott T.

    2012-01-01

    Functional Analytic Psychotherapy (FAP) is a form of therapy grounded in behavioral principles that utilizes therapist reactions to shape target behavior. Despite a growing literature base, there is a paucity of research to establish the efficacy of FAP. As a general approach to psychotherapy, and how the therapeutic relationship produces change,…

  13. Promoting Executive Function in the Classroom

    ERIC Educational Resources Information Center

    Meltzer, Lynn

    2010-01-01

    Accessible and practical, this book helps teachers incorporate executive function processes--such as planning, organizing, prioritizing, and self-checking--into the classroom curriculum. Chapters provide effective strategies for optimizing what K-12 students learn by improving how they learn. Noted authority Lynn Meltzer and her research…

  14. Functional Classification of BRCA2 DNA Variants by Splicing Assays in a Large Minigene with 9 Exons

    PubMed Central

    Acedo, Alberto; Hernández-Moro, Cristina; Curiel-García, Álvaro; Díez-Gómez, Beatriz; Velasco, Eladio A

    2015-01-01

    Numerous pathogenic DNA variants impair the splicing mechanism in human genetic diseases. Minigenes are optimal approaches to test variants under the splicing viewpoint without the need of patient samples. We aimed to design a robust minigene construct of the breast cancer gene BRCA2 in order to investigate the impact of variants on splicing. BRCA2 exons 19–27 (MGBR2_ex19–27) were cloned in the new vector pSAD. It produced a large transcript of the expected size (2,174 nucleotides) and exon structure (V1-ex19-27-V2). Splicing assays showed that 18 (17 splice-site and 1 silencer variants) out of 40 candidate DNA variants induced aberrant patterns. Twenty-four anomalous transcripts were accurately detected by fluorescent-RT-PCR that were generated by exon-skipping, alternative site usage, and intron-retention events. Fourteen variants induced major anomalies and were predicted to disrupt protein function so they could be classified as pathogenic. Furthermore, minigene mimicked previously reported patient RNA outcomes of seven variants supporting the reproducibility of minigene assays. Therefore, a relevant fraction of variants are involved in breast cancer through splicing alterations. MGBR2_ex19–27 is the largest reported BRCA2 minigene and constitutes a valuable tool for the functional and clinical classification of sequence variations. PMID:25382762

  15. Thirteen new patients with guanidinoacetate methyltransferase deficiency and functional characterization of nineteen novel missense variants in the GAMT gene.

    PubMed

    Mercimek-Mahmutoglu, Saadet; Ndika, Joseph; Kanhai, Warsha; de Villemeur, Thierry Billette; Cheillan, David; Christensen, Ernst; Dorison, Nathalie; Hannig, Vickie; Hendriks, Yvonne; Hofstede, Floris C; Lion-Francois, Laurence; Lund, Allan M; Mundy, Helen; Pitelet, Gaele; Raspall-Chaure, Miquel; Scott-Schwoerer, Jessica A; Szakszon, Katalin; Valayannopoulos, Vassili; Williams, Monique; Salomons, Gajja S

    2014-04-01

    Guanidinoacetate methyltransferase deficiency (GAMT-D) is an autosomal recessively inherited disorder of creatine biosynthesis. Creatine deficiency on cranial proton magnetic resonance spectroscopy, and elevated guanidinoacetate levels in body fluids are the biomarkers of GAMT-D. In 74 patients, 50 different mutations in the GAMT gene have been identified with missense variants being the most common. Clinical and biochemical features of the patients with missense variants were obtained from their physicians using a questionnaire. In 20 patients, 17 missense variants, 25% had a severe, 55% a moderate, and 20% a mild phenotype. The effect of these variants on GAMT enzyme activity was overexpressed using primary GAMT-D fibroblasts: 17 variants retained no significant activity and are therefore considered pathogenic. Two additional variants, c.22C>A (p.Pro8Thr) and c.79T>C (p.Tyr27His) (the latter detected in control cohorts) are in fact not pathogenic as these alleles restored GAMT enzyme activity, although both were predicted to be possibly damaging by in silico analysis. We report 13 new patients with GAMT-D, six novel mutations and functional analysis of 19 missense variants, all being included in our public LOVD database. Our functional assay is important for the confirmation of the pathogenicity of identified missense variants in the GAMT gene. PMID:24415674

  16. Rs6295 promoter variants of the serotonin type 1A receptor are differentially activated by c-Jun in vitro and correlate to transcript levels in human epileptic brain tissue.

    PubMed

    Pernhorst, Katharina; van Loo, Karen M J; von Lehe, Marec; Priebe, Lutz; Cichon, Sven; Herms, Stefan; Hoffmann, Per; Helmstaedter, Christoph; Sander, Thomas; Schoch, Susanne; Becker, Albert J

    2013-03-01

    Many brain disorders, including epilepsy, migraine and depression, manifest with episodic symptoms that may last for various time intervals. Transient alterations of neuronal function such as related to serotonin homeostasis generally underlie this phenomenon. Several nucleotide polymorphisms (SNPs) in gene promoters associated with these diseases have been described. For obvious reasons, their regulatory roles on gene expression particularly in human brain tissue remain largely enigmatic. The rs6295 G-/C-allelic variant is located in the promoter region of the human HTR1a gene, encoding the G-protein-coupled receptor for 5-hydroxytryptamine (5HT1AR). In addition to reported transcriptional repressor binding, our bioinformatic analyses predicted a reduced binding affinity of the transcription factor (TF) c-Jun for the G-allele. In vitro luciferase transfection assays revealed c-Jun to (a) activate the rs6295 C- significantly stronger than the G-allelic variant and (b) antagonize efficiently the repressive effect of Hes5 on the promoter. The G-allele of rs6295 is known to be associated with aspects of major depression and migraine. In order to address a potential role of rs6295 variants in human brain tissue, we have isolated DNA and mRNA from fresh frozen hippocampal tissue of pharmacoresistant temporal lobe epilepsy (TLE) patients (n=140) after epilepsy surgery for seizure control. We carried out SNP genotyping studies and mRNA analyses in order to determine HTR1a mRNA expression in human hippocampal samples stratified according to the rs6295 allelic variant. The mRNA expression of HTR1a was significantly more abundant in hippocampal mRNA of TLE patients homozygous for the rs6295 C-allele as compared to those with the GG-genotype. These data may point to a novel, i.e., rs6295 allelic variant and c-Jun dependent transcriptional 5HT1AR 'receptoropathy'. PMID:23333373

  17. Association Between a Functional Variant Downstream of TNFAIP3 and Systemic Lupus Erythematosus

    PubMed Central

    Adrianto, Indra; Wen, Feng; Templeton, Amanda; Wiley, Graham; King, Jarrod B.; Lessard, Christopher J.; Bates, Jared S.; Hu, Yanqing; Kelly, Jennifer A.; Kaufman, Kenneth M.; Guthridge, Joel M.; Alarcón-Riquelme, Marta E.; Anaya, Juan-Manuel; Bae, Sang-Cheol; Bang, So-Young; Boackle, Susan A.; Brown, Elizabeth E.; Petri, Michelle A.; Gallant, Caroline; Ramsey-Goldman, Rosalind; Reveille, John D.; Vila, Luis M.; Criswell, Lindsey A.; Edberg, Jeffrey C.; Freedman, Barry I.; Gregersen, Peter K.; Gilkeson, Gary S.; Jacob, Chaim O.; James, Judith A.; Kamen, Diane L.; Kimberly, Robert P.; Martin, Javier; Merrill, Joan T.; Niewold, Timothy B.; Park, So-Yeon; Pons-Estel, Bernardo A.; Scofield, R. Hal; Stevens, Anne M.; Tsao, Betty P.; Vyse, Timothy J.; Langefeld, Carl D.; Harley, John B.; Moser, Kathy L.; Webb, Carol F.; Humphrey, Mary Beth; Montgomery, Courtney Gray; Gaffney, Patrick M.

    2011-01-01

    Systemic Lupus Erythematosus (SLE, OMIM 152700) is an autoimmune disease characterized by self-reactive antibodies resulting in systemic inflammation and organ failure. TNFAIP3, encoding the ubiquitin-modifying enzyme A20, is an established susceptibility locus for SLE. By fine mapping and genomic resequencing in ethnically diverse populations we fully characterized the TNFAIP3 risk haplotype and isolated a novel TT>A polymorphic dinucleotide associated with SLE in subjects of European (P = 1.58 × 10−8; odds ratio (OR) = 1.70) and Korean (P = 8.33 × 10−10; OR = 2.54) ancestry. This variant, located in a region of high conservation and regulatory potential, bound a nuclear protein complex comprised of NF-κB subunits with reduced avidity. Furthermore, compared with the non-risk haplotype, the haplotype carrying this variant resulted in reduced TNFAIP3 mRNA and A20 protein expression. These results establish this TT>A variant as the most likely functional polymorphism responsible for the association between TNFAIP3 and SLE. PMID:21336280

  18. Cohesion promotes nucleolar structure and function.

    PubMed

    Harris, Bethany; Bose, Tania; Lee, Kenneth K; Wang, Fei; Lu, Shuai; Ross, Rhonda Trimble; Zhang, Ying; French, Sarah L; Beyer, Ann L; Slaughter, Brian D; Unruh, Jay R; Gerton, Jennifer L

    2014-02-01

    The cohesin complex contributes to ribosome function, although the molecular mechanisms involved are unclear. Compromised cohesin function is associated with a class of diseases known as cohesinopathies. One cohesinopathy, Roberts syndrome (RBS), occurs when a mutation reduces acetylation of the cohesin Smc3 subunit. Mutation of the cohesin acetyltransferase is associated with impaired rRNA production, ribosome biogenesis, and protein synthesis in yeast and human cells. Cohesin binding to the ribosomal DNA (rDNA) is evolutionarily conserved from bacteria to human cells. We report that the RBS mutation in yeast (eco1-W216G) exhibits a disorganized nucleolus and reduced looping at the rDNA. RNA polymerase I occupancy of the genes remains normal, suggesting that recruitment is not impaired. Impaired rRNA production in the RBS mutant coincides with slower rRNA cleavage. In addition to the RBS mutation, mutations in any subunit of the cohesin ring are associated with defects in ribosome biogenesis. Depletion or artificial destruction of cohesion in a single cell cycle is associated with loss of nucleolar integrity, demonstrating that the defects at the rDNA can be directly attributed to loss of cohesion. Our results strongly suggest that organization of the rDNA provided by cohesion is critical for formation and function of the nucleolus. PMID:24307683

  19. Investigation and Functional Characterization of Rare Genetic Variants in the Adipose Triglyceride Lipase in a Large Healthy Working Population

    PubMed Central

    Coassin, Stefan; Schweiger, Martina; Kloss-Brandstätter, Anita; Lamina, Claudia; Haun, Margot; Erhart, Gertraud; Paulweber, Bernhard; Rahman, Yusof; Olpin, Simon; Wolinski, Heimo; Cornaciu, Irina; Zechner, Rudolf; Zimmermann, Robert; Kronenberg, Florian

    2010-01-01

    Recent studies demonstrated a strong influence of rare genetic variants on several lipid-related traits. However, their impact on free fatty acid (FFA) plasma concentrations, as well as the role of rare variants in a general population, has not yet been thoroughly addressed. The adipose triglyceride lipase (ATGL) is encoded by the PNPLA2 gene and catalyzes the rate-limiting step of lipolysis. It represents a prominent candidate gene affecting FFA concentrations. We therefore screened the full genomic region of ATGL for mutations in 1,473 randomly selected individuals from the SAPHIR (Salzburg Atherosclerosis Prevention program in subjects at High Individual Risk) Study using a combined Ecotilling and sequencing approach and functionally investigated all detected protein variants by in-vitro studies. We observed 55 novel mostly rare genetic variants in this general population sample. Biochemical evaluation of all non-synonymous variants demonstrated the presence of several mutated but mostly still functional ATGL alleles with largely varying residual lipolytic activity. About one-quarter (3 out of 13) of the investigated variants presented a marked decrease or total loss of catalytic function. Genetic association studies using both continuous and dichotomous approaches showed a shift towards lower plasma FFA concentrations for rare variant carriers and an accumulation of variants in the lower 10%-quantile of the FFA distribution. However, the generally rather small effects suggest either only a secondary role of rare ATGL variants on the FFA levels in the SAPHIR population or a recessive action of ATGL variants. In contrast to these rather small effects, we describe here also the first patient with “neutral lipid storage disease with myopathy” (NLSDM) with a point mutation in the catalytic dyad, but otherwise intact protein. PMID:21170305

  20. Gastric cancer associated variant of DNA polymerase beta (Leu22Pro) promotes DNA replication associated double strand breaks

    PubMed Central

    Rozacky, Jenna; Nemec, Antoni A.; Sweasy, Joann B.; Kidane, Dawit

    2015-01-01

    DNA polymerase beta (Pol β) is a key enzymefor the protection against oxidative DNA lesions via itsrole in base excision repair (BER). Approximately 1/3 of tumors studied to date express Pol β variant proteins, and several tumors overexpress Pol β. Pol β possesses DNA polymerase and dRP lyase activities, both of which are known to be important for efficient BER. The dRP lyase activity resides within the 8kDa amino terminal domain of Pol β, is responsible for removal of the 5′ phosphate group (5′-dRP). The DNA polymerase subsequently fills the gaps. Previously, we demonstrated that the human gastric cancer-associated variant of Pol β (Leu22Pro (L22P)) lacks dRP lyase function in vitro. Here, we report that L22P-expressing cells harbor significantly increased replication associated DNA double strand breaks (DSBs) and defective maintenance of the nascent DNA strand (NDS) during replication stress. Moreover, L22P-expressing cells are sensitive to PARP1 inhibitors, which suggests trapped PARP1 binds to the 5′-dRP group and blocks replications forks, resulting in fork collapse and DSBs. Our data suggest that the normal function of the dRP lyase is critical to maintain replication fork integrity and prevent replication fork collapse to DSBs and cellular transformation. PMID:26090616

  1. Gastric cancer associated variant of DNA polymerase beta (Leu22Pro) promotes DNA replication associated double strand breaks.

    PubMed

    Rozacky, Jenna; Nemec, Antoni A; Sweasy, Joann B; Kidane, Dawit

    2015-09-15

    DNA polymerase beta (Pol β) is a key enzyme for the protection against oxidative DNA lesions via its role in base excision repair (BER). Approximately 1/3 of tumors studied to date express Pol β variant proteins, and several tumors overexpress Pol β. Pol β possesses DNA polymerase and dRP lyase activities, both of which are known to be important for efficient BER. The dRP lyase activity resides within the 8kDa amino terminal domain of Pol β, is responsible for removal of the 5' phosphate group (5'-dRP). The DNA polymerase subsequently fills the gaps. Previously, we demonstrated that the human gastric cancer-associated variant of Pol β (Leu22Pro (L22P)) lacks dRP lyase function in vitro. Here, we report that L22P-expressing cells harbor significantly increased replication associated DNA double strand breaks (DSBs) and defective maintenance of the nascent DNA strand (NDS) during replication stress. Moreover, L22P-expressing cells are sensitive to PARP1 inhibitors, which suggests trapped PARP1 binds to the 5'-dRP group and blocks replications forks, resulting in fork collapse and DSBs. Our data suggest that the normal function of the dRP lyase is critical to maintain replication fork integrity and prevent replication fork collapse to DSBs and cellular transformation. PMID:26090616

  2. CAUSEL: An epigenome and genome editing pipeline for establishing function of non-coding GWAS variants

    PubMed Central

    Spisak, Sandor; Lawrenson, Kate; Fu, Yanfang; Csabai, Istvan; Cottman, Rebecca T.; Haiman, Christopher; Han, Ying; Seo, Ji-Heui; Lenci, Romina; Li, Qiyuan; Tisza, Viktoria; Szallasi, Zoltan; Herbert, Zachery T.; Chabot, Matthew; Pomerantz, Mark; Solymosi, Norbert; Gayther, Simon; Joung, J. Keith; Freedman, Matthew L.

    2016-01-01

    The vast majority of disease-associated single nucleotide polymorphisms (SNPs) mapped by genome-wide association studies (GWAS) are located in the non-protein coding genome, but establishing the functional and mechanistic roles of these sequence variants has proven challenging. Here, we describe a general pipeline in which candidate functional SNPs are first evaluated by fine-mapping, epigenomic profiling, and epigenome editing and then interrogated for causal function by using genome editing to create isogenic cell lines. To validate this approach, we analyzed the 6q22.1 prostate cancer risk locus and identified rs339331 as the top scoring SNP. Epigenome editing confirmed that rs339331 possessed regulatory potential. Using transcription activator-like effector nuclease (TALEN)-mediated genome-editing, we created a panel of isogenic 22Rv1 prostate cancer cell lines representing all three genotypes (TT, TC, CC) at rs339331. Introduction of the “T” risk allele increased transcription of the RFX6 gene, increased HOXB13 binding at the rs339331 region, and increased deposition of the enhancer-associated H3K4me2 histone mark at the rs339331 region. The cell lines also differed in cellular morphology and adhesion, and pathway analysis of differentially expressed genes suggested an influence of androgens. In summary, we have developed and validated a widely accessible approach to establish functional causality for non-coding sequence variants identified by GWAS. PMID:26398868

  3. SLC6A3 coding variant Ala559Val found in two autism probands alters dopamine transporter function and trafficking.

    PubMed

    Bowton, E; Saunders, C; Reddy, I A; Campbell, N G; Hamilton, P J; Henry, L K; Coon, H; Sakrikar, D; Veenstra-VanderWeele, J M; Blakely, R D; Sutcliffe, J; Matthies, H J G; Erreger, K; Galli, A

    2014-01-01

    Emerging evidence associates dysfunction in the dopamine (DA) transporter (DAT) with the pathophysiology of autism spectrum disorder (ASD). The human DAT (hDAT; SLC6A3) rare variant with an Ala to Val substitution at amino acid 559 (hDAT A559V) was previously reported in individuals with bipolar disorder or attention-deficit hyperactivity disorder (ADHD). We have demonstrated that this variant is hyper-phosphorylated at the amino (N)-terminal serine (Ser) residues and promotes an anomalous DA efflux phenotype. Here, we report the novel identification of hDAT A559V in two unrelated ASD subjects and provide the first mechanistic description of its impaired trafficking phenotype. DAT surface expression is dynamically regulated by DAT substrates including the psychostimulant amphetamine (AMPH), which causes hDAT trafficking away from the plasma membrane. The integrity of DAT trafficking directly impacts DA transport capacity and therefore dopaminergic neurotransmission. Here, we show that hDAT A559V is resistant to AMPH-induced cell surface redistribution. This unique trafficking phenotype is conferred by altered protein kinase C β (PKCβ) activity. Cells expressing hDAT A559V exhibit constitutively elevated PKCβ activity, inhibition of which restores the AMPH-induced hDAT A559V membrane redistribution. Mechanistically, we link the inability of hDAT A559V to traffic in response to AMPH to the phosphorylation of the five most distal DAT N-terminal Ser. Mutation of these N-terminal Ser to Ala restores AMPH-induced trafficking. Furthermore, hDAT A559V has a diminished ability to transport AMPH, and therefore lacks AMPH-induced DA efflux. Pharmacological inhibition of PKCβ or Ser to Ala substitution in the hDAT A559V background restores AMPH-induced DA efflux while promoting intracellular AMPH accumulation. Although hDAT A559V is a rare variant, it has been found in multiple probands with neuropsychiatric disorders associated with imbalances in DA neurotransmission

  4. Identification of a functional variant in the KIF5A-CYP27B1-METTL1-FAM119B locus associated with multiple sclerosis

    PubMed Central

    Alcina, Antonio; Fedetz, Maria; Fernández, Óscar; Saiz, Albert; Izquierdo, Guillermo; Lucas, Miguel; Leyva, Laura; García-León, Juan-Antonio; Abad-Grau, María del Mar; Alloza, Iraide; Antigüedad, Alfredo; Garcia-Barcina, María J; Vandenbroeck, Koen; Varadé, Jezabel; de la Hera, Belén; Arroyo, Rafael; Comabella, Manuel; Montalban, Xavier; Petit-Marty, Natalia; Navarro, Arcadi; Otaegui, David; Olascoaga, Javier; Blanco, Yolanda; Urcelay, Elena; Matesanz, Fuencisla

    2013-01-01

    Background and aim Several studies have highlighted the association of the 12q13.3–12q14.1 region with coeliac disease, type 1 diabetes, rheumatoid arthritis and multiple sclerosis (MS); however, the causal variants underlying diseases are still unclear. The authors sought to identify the functional variant of this region associated with MS. Methods Tag-single nucleotide polymorphism (SNP) analysis of the associated region encoding 15 genes was performed in 2876 MS patients and 2910 healthy Caucasian controls together with expression regulation analyses. Results rs6581155, which tagged 18 variants within a region where 9 genes map, was sufficient to model the association. This SNP was in total linkage disequilibrium (LD) with other polymorphisms that associated with the expression levels of FAM119B, AVIL, TSFM, TSPAN31 and CYP27B1 genes in different expression quantitative trait loci studies. Functional annotations from Encyclopedia of DNA Elements (ENCODE) showed that six out of these rs6581155-tagged-SNPs were located in regions with regulatory potential and only one of them, rs10877013, exhibited allele-dependent (ratio A/G=9.5-fold) and orientation-dependent (forward/reverse=2.7-fold) enhancer activity as determined by luciferase reporter assays. This enhancer is located in a region where a long-range chromatin interaction among the promoters and promoter-enhancer of several genes has been described, possibly affecting their expression simultaneously. Conclusions This study determines a functional variant which alters the enhancer activity of a regulatory element in the locus affecting the expression of several genes and explains the association of the 12q13.3–12q14.1 region with MS. PMID:23160276

  5. Influence of loss of function MC1R variants in genetic susceptibility of familial melanoma in Spain.

    PubMed

    de Torre, Carlos; Garcia-Casado, Zaida; Martínez-Escribano, Jorge A; Botella-Estrada, Rafael; Bañuls, Jose; Oliver, Vicente; Mercader, Pedro; Azaña, Jose M; Frias, Javier; Nagore, Eduardo

    2010-08-01

    We explored the presence of germline alterations in CDK4 exon 2, CDKN2A and MC1R in a hospital-based study of 89 melanoma cases from 89 families with at least two members affected by cutaneous melanoma. A total of 30% of the melanoma kindreds studied were carriers of CDKN2A variants, and three of these variants were known predominant alleles that have been identified earlier in Mediterranean populations (p.G101W, p.V59G and c.358delG). We observed a higher frequency of nonsynonymous MC1R variants in these Spanish melanoma kindreds (72%) with respect to the general population (60%). We observed a higher frequency of nonsynonymous MC1R variants in this Spanish melanoma kindred (72%) respect to general population (60%). A new classification of MC1R variants based on their functional effects over melanocortin-1 receptor, including the dominant-negative effect of some of them in heterozygotes, suggested an association of loss of function MC1R variants and multiple primary melanoma cases from melanoma kindred (odds ratio: 6.07, 95% confidence interval: 1.35-27.20). This study proposes the relevance of loss of function MC1R variants in the risk of melanoma in multiple primary melanoma cases with family history from areas with low melanoma incidence rate. PMID:20539244

  6. Loss-of-function variants in endothelial lipase are a cause of elevated HDL cholesterol in humans

    PubMed Central

    Edmondson, Andrew C.; Brown, Robert J.; Kathiresan, Sekar; Cupples, L. Adrienne; Demissie, Serkalem; Manning, Alisa Knodle; Jensen, Majken K.; Rimm, Eric B.; Wang, Jian; Rodrigues, Amrith; Bamba, Vaneeta; Khetarpal, Sumeet A.; Wolfe, Megan L.; DerOhannessian, Stephanie; Li, Mingyao; Reilly, Muredach P.; Aberle, Jens; Evans, David; Hegele, Robert A.; Rader, Daniel J.

    2009-01-01

    Elevated plasma concentrations of HDL cholesterol (HDL-C) are associated with protection from atherosclerotic cardiovascular disease. Animal models indicate that decreased expression of endothelial lipase (LIPG) is inversely associated with HDL-C levels, and genome-wide association studies have identified LIPG variants as being associated with HDL-C levels in humans. We hypothesized that loss-of-function mutations in LIPG may result in elevated HDL-C and therefore performed deep resequencing of LIPG exons in cases with elevated HDL-C levels and controls with decreased HDL-C levels. We identified a significant excess of nonsynonymous LIPG variants unique to cases with elevated HDL-C. In vitro lipase activity assays demonstrated that these variants significantly decreased endothelial lipase activity. In addition, a meta-analysis across 5 cohorts demonstrated that the low-frequency Asn396Ser variant is significantly associated with increased HDL-C, while the common Thr111Ile variant is not. Functional analysis confirmed that the Asn396Ser variant has significantly decreased lipase activity both in vitro and in vivo, while the Thr111Ile variant has normal lipase activity. Our results establish that loss-of-function mutations in LIPG lead to increased HDL-C levels and support the idea that inhibition of endothelial lipase may be an effective mechanism to raise HDL-C. PMID:19287092

  7. Loss-of-function variants in HIVEP2 are a cause of intellectual disability.

    PubMed

    Srivastava, Siddharth; Engels, Hartmut; Schanze, Ina; Cremer, Kirsten; Wieland, Thomas; Menzel, Moritz; Schubach, Max; Biskup, Saskia; Kreiß, Martina; Endele, Sabine; Strom, Tim M; Wieczorek, Dagmar; Zenker, Martin; Gupta, Siddharth; Cohen, Julie; Zink, Alexander M; Naidu, SakkuBai

    2016-04-01

    Intellectual disability (ID) affects 2-3% of the population. In the past, many genetic causes of ID remained unidentified due to its vast heterogeneity. Recently, whole exome sequencing (WES) studies have shown that de novo variants underlie a significant portion of sporadic cases of ID. Applying WES to patients with ID or global developmental delay at different centers, we identified three individuals with distinct de novo variants in HIVEP2 (human immunodeficiency virus type I enhancer binding protein), which belongs to a family of zinc-finger-containing transcriptional proteins involved in growth and development. Two of the variants were nonsense changes, and one was a 1 bp deletion resulting in a premature stop codon that was reported previously without clinical detail. In silico prediction programs suggest loss-of-function in the mutated allele leading to haploinsufficiency as a putative mechanism in all three individuals. All three patients presented with moderate-to-severe ID, minimal structural brain anomalies, hypotonia, and mild dysmorphic features. Growth parameters were in the normal range except for borderline microcephaly at birth in one patient. Two of the patients exhibited behavioral anomalies including hyperactivity and aggression. Published functional data suggest a neurodevelopmental role for HIVEP2, and several of the genes regulated by HIVEP2 are implicated in brain development, for example, SSTR-2, c-Myc, and genes of the NF-κB pathway. In addition, HIVEP2-knockout mice exhibit several working memory deficits, increased anxiety, and hyperactivity. On the basis of the genotype-phenotype correlation and existing functional data, we propose HIVEP2 as a causative ID gene. PMID:26153216

  8. Complementation of the Function of Glycoprotein H of Human Herpesvirus 6 Variant A by Glycoprotein H of Variant B in the Virus Life Cycle

    PubMed Central

    Oyaizu, Hiroko; Tang, Huamin; Ota, Megumi; Takenaka, Nobuyuki; Ozono, Keiichi; Yamanishi, Koichi

    2012-01-01

    Human herpesvirus 6 (HHV-6) is a T-cell-tropic betaherpesvirus. HHV-6 can be classified into two variants, HHV-6 variant A (HHV-6A) and HHV-6B, based on genetic, antigenic, and cell tropisms, although the homology of their entire genomic sequences is nearly 90%. The HHV-6A glycoprotein complex gH/gL/gQ1/gQ2 is a viral ligand that binds to the cellular receptor human CD46. Because gH has 94.3% amino acid identity between the variants, here we examined whether gH from one variant could complement its loss in the other. Recently, we successfully reconstituted HHV-6A from its cloned genome in a bacterial artificial chromosome (BAC) (rHHV-6ABAC). Using this system, we constructed HHV-6ABAC DNA containing the HHV-6B gH (BgH) gene instead of the HHV-6A gH (AgH) gene in Escherichia coli. Recombinant HHV-6ABAC expressing BgH (rHHV-6ABAC-BgH) was successfully reconstituted. In addition, a monoclonal antibody that blocks HHV-6B but not HHV-6A infection neutralized rHHV-6ABAC-BgH but not rHHV-6ABAC. These results indicate that HHV-6B gH can complement the function of HHV-6A gH in the viral infectious cycle. PMID:22647694

  9. A Variant of GJD2, Encoding for Connexin 36, Alters the Function of Insulin Producing β-Cells

    PubMed Central

    Cigliola, Valentina; Populaire, Celine; Pierri, Ciro L.; Deutsch, Samuel; Haefliger, Jacques-Antoine; Fadista, João; Lyssenko, Valeriya; Groop, Leif; Rueedi, Rico; Thorel, Fabrizio; Herrera, Pedro Luis; Meda, Paolo

    2016-01-01

    Signalling through gap junctions contributes to control insulin secretion and, thus, blood glucose levels. Gap junctions of the insulin-producing β-cells are made of connexin 36 (Cx36), which is encoded by the GJD2 gene. Cx36-null mice feature alterations mimicking those observed in type 2 diabetes (T2D). GJD2 is also expressed in neurons, which share a number of common features with pancreatic β-cells. Given that a synonymous exonic single nucleotide polymorphism of human Cx36 (SNP rs3743123) associates with altered function of central neurons in a subset of epileptic patients, we investigated whether this SNP also caused alterations of β-cell function. Transfection of rs3743123 cDNA in connexin-lacking HeLa cells resulted in altered formation of gap junction plaques and cell coupling, as compared to those induced by wild type (WT) GJD2 cDNA. Transgenic mice expressing the very same cDNAs under an insulin promoter revealed that SNP rs3743123 expression consistently lead to a post-natal reduction of islet Cx36 levels and β-cell survival, resulting in hyperglycemia in selected lines. These changes were not observed in sex- and age-matched controls expressing WT hCx36. The variant GJD2 only marginally associated to heterogeneous populations of diabetic patients. The data document that a silent polymorphism of GJD2 is associated with altered β-cell function, presumably contributing to T2D pathogenesis. PMID:26959991

  10. HTRA1 promoter variant differentiates polypoidal choroidal vasculopathy from exudative age-related macular degeneration

    PubMed Central

    Ng, Tsz Kin; Liang, Xiao Ying; Lai, Timothy Y. Y.; Ma, Li; Tam, Pancy O. S.; Wang, Jian Xiong; Chen, Li Jia; Chen, Haoyu; Pang, Chi Pui

    2016-01-01

    Exudative age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) share similar abnormal choroidal vasculature, but responses to treatments are different. In this study, we sequenced the whole HTRA1 gene and its promoter by direct sequencing in a Hong Kong Chinese PCV cohort. We identified rs11200638, c.34delCinsTCCT, c.59C>T, rs1049331 and rs2293870 significantly associated with PCV. Notably, rs2672598 was significantly associated with exudative AMD (p = 1.31 × 10−4) than PCV (p = 0.11). Logistic regression indicated that rs2672598 (p = 2.27 × 10−3) remained significant after adjusting for rs11200638 in exudative AMD. Moreover, the rs11200638-rs2672598 joint genotype AA-CC conferred higher risk to exudative AMD (43.11 folds) than PCV (3.68 folds). Promoter analysis showed that rs2672598 C-allele showed higher luciferase expression than wildtype T-allele (p = 0.026), independent of rs11200638 genotype (p = 0.621). Coherently, vitreous humor HTRA1 expression with rs2672598 CC genotype was significantly higher than that with TT genotype by 2.56 folds (p = 0.02). Furthermore, rs2672598 C-allele was predicted to alter the transcription factor binding sites, but not rs11200638 A-allele. Our results revealed that HTRA1 rs2672598 is more significantly associated with exudative AMD than PCV in ARMS2/HTRA1 region, and it is responsible for elevated HTRA1 transcriptional activity and HTRA1 protein expression. PMID:27338780

  11. HTRA1 promoter variant differentiates polypoidal choroidal vasculopathy from exudative age-related macular degeneration.

    PubMed

    Ng, Tsz Kin; Liang, Xiao Ying; Lai, Timothy Y Y; Ma, Li; Tam, Pancy O S; Wang, Jian Xiong; Chen, Li Jia; Chen, Haoyu; Pang, Chi Pui

    2016-01-01

    Exudative age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) share similar abnormal choroidal vasculature, but responses to treatments are different. In this study, we sequenced the whole HTRA1 gene and its promoter by direct sequencing in a Hong Kong Chinese PCV cohort. We identified rs11200638, c.34delCinsTCCT, c.59C>T, rs1049331 and rs2293870 significantly associated with PCV. Notably, rs2672598 was significantly associated with exudative AMD (p = 1.31 × 10(-4)) than PCV (p = 0.11). Logistic regression indicated that rs2672598 (p = 2.27 × 10(-3)) remained significant after adjusting for rs11200638 in exudative AMD. Moreover, the rs11200638-rs2672598 joint genotype AA-CC conferred higher risk to exudative AMD (43.11 folds) than PCV (3.68 folds). Promoter analysis showed that rs2672598 C-allele showed higher luciferase expression than wildtype T-allele (p = 0.026), independent of rs11200638 genotype (p = 0.621). Coherently, vitreous humor HTRA1 expression with rs2672598 CC genotype was significantly higher than that with TT genotype by 2.56 folds (p = 0.02). Furthermore, rs2672598 C-allele was predicted to alter the transcription factor binding sites, but not rs11200638 A-allele. Our results revealed that HTRA1 rs2672598 is more significantly associated with exudative AMD than PCV in ARMS2/HTRA1 region, and it is responsible for elevated HTRA1 transcriptional activity and HTRA1 protein expression. PMID:27338780

  12. Enrich: software for analysis of protein function by enrichment and depletion of variants

    PubMed Central

    Fowler, Douglas M.; Araya, Carlos L.; Gerard, Wayne; Fields, Stanley

    2011-01-01

    Summary: Measuring the consequences of mutation in proteins is critical to understanding their function. These measurements are essential in such applications as protein engineering, drug development, protein design and genome sequence analysis. Recently, high-throughput sequencing has been coupled to assays of protein activity, enabling the analysis of large numbers of mutations in parallel. We present Enrich, a tool for analyzing such deep mutational scanning data. Enrich identifies all unique variants (mutants) of a protein in high-throughput sequencing datasets and can correct for sequencing errors using overlapping paired-end reads. Enrich uses the frequency of each variant before and after selection to calculate an enrichment ratio, which is used to estimate fitness. Enrich provides an interactive interface to guide users. It generates user-accessible output for downstream analyses as well as several visualizations of the effects of mutation on function, thereby allowing the user to rapidly quantify and comprehend sequence–function relationships. Availability and Implementation: Enrich is implemented in Python and is available under a FreeBSD license at http://depts.washington.edu/sfields/software/enrich/. Enrich includes detailed documentation as well as a small example dataset. Contact: dfowler@uw.edu; fields@uw.edu Supplementary Information: Supplementary data is available at Bioinformatics online. PMID:22006916

  13. Isoform and Splice-Variant Specific Functions of Dynamin-2 Revealed by Analysis of Conditional Knock-Out Cells

    PubMed Central

    Liu, Ya-Wen; Surka, Mark C.; Schroeter, Thomas; Lukiyanchuk, Vasyl

    2008-01-01

    Dynamin (Dyn) is a multifunctional GTPase implicated in several cellular events, including endocytosis, intracellular trafficking, cell signaling, and cytokinesis. The mammalian genome encodes three isoforms, Dyn1, Dyn2, and Dyn3, and several splice variants of each, leading to the suggestion that distinct isoforms and/or distinct splice variants might mediate distinct cellular functions. We generated a conditional Dyn2 KO cell line and performed knockout and reconstitution experiments to explore the isoform- and splice variant specific cellular functions of ubiquitously expressed Dyn2. We find that Dyn2 is required for clathrin-mediated endocytosis (CME), p75 export from the Golgi, and PDGF-stimulated macropinocytosis and cytokinesis, but not for other endocytic pathways. Surprisingly, CME and p75 exocytosis were efficiently rescued by reintroduction of Dyn2, but not Dyn1, suggesting that these two isoforms function differentially in vesicular trafficking in nonneuronal cells. Both isoforms rescued macropinocytosis and cytokinesis, suggesting that dynamin function in these processes might be mechanistically distinct from its role in CME. Although all four Dyn2 splice variants could equally restore CME, Dyn2ba and -bb were more effective at restoring p75 exocytosis. This splice variant specificity correlated with their differential targeting to the Golgi. These studies reveal isoform and splice-variant specific functions for Dyn2. PMID:18923138

  14. Isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells.

    PubMed

    Liu, Ya-Wen; Surka, Mark C; Schroeter, Thomas; Lukiyanchuk, Vasyl; Schmid, Sandra L

    2008-12-01

    Dynamin (Dyn) is a multifunctional GTPase implicated in several cellular events, including endocytosis, intracellular trafficking, cell signaling, and cytokinesis. The mammalian genome encodes three isoforms, Dyn1, Dyn2, and Dyn3, and several splice variants of each, leading to the suggestion that distinct isoforms and/or distinct splice variants might mediate distinct cellular functions. We generated a conditional Dyn2 KO cell line and performed knockout and reconstitution experiments to explore the isoform- and splice variant specific cellular functions of ubiquitously expressed Dyn2. We find that Dyn2 is required for clathrin-mediated endocytosis (CME), p75 export from the Golgi, and PDGF-stimulated macropinocytosis and cytokinesis, but not for other endocytic pathways. Surprisingly, CME and p75 exocytosis were efficiently rescued by reintroduction of Dyn2, but not Dyn1, suggesting that these two isoforms function differentially in vesicular trafficking in nonneuronal cells. Both isoforms rescued macropinocytosis and cytokinesis, suggesting that dynamin function in these processes might be mechanistically distinct from its role in CME. Although all four Dyn2 splice variants could equally restore CME, Dyn2ba and -bb were more effective at restoring p75 exocytosis. This splice variant specificity correlated with their differential targeting to the Golgi. These studies reveal isoform and splice-variant specific functions for Dyn2. PMID:18923138

  15. Postnatally-transmitted HIV-1 Envelope variants have similar neutralization-sensitivity and function to that of nontransmitted breast milk variants

    PubMed Central

    2013-01-01

    Background Breastfeeding is a leading cause of infant HIV-1 infection in the developing world, yet only a minority of infants exposed to HIV-1 via breastfeeding become infected. As a genetic bottleneck severely restricts the number of postnatally-transmitted variants, genetic or phenotypic properties of the virus Envelope (Env) could be important for the establishment of infant infection. We examined the efficiency of virologic functions required for initiation of infection in the gastrointestinal tract and the neutralization sensitivity of HIV-1 Env variants isolated from milk of three postnatally-transmitting mothers (n=13 viruses), five clinically-matched nontransmitting mothers (n=16 viruses), and seven postnatally-infected infants (n = 7 postnatally-transmitted/founder (T/F) viruses). Results There was no difference in the efficiency of epithelial cell interactions between Env virus variants from the breast milk of transmitting and nontransmitting mothers. Moreover, there was similar efficiency of DC-mediated trans-infection, CCR5-usage, target cell fusion, and infectivity between HIV-1 Env-pseudoviruses from nontransmitting mothers and postnatal T/F viruses. Milk Env-pseudoviruses were generally sensitive to neutralization by autologous maternal plasma and resistant to breast milk neutralization. Infant T/F Env-pseudoviruses were equally sensitive to neutralization by broadly-neutralizing monoclonal and polyclonal antibodies as compared to nontransmitted breast milk Env variants. Conclusion Postnatally-T/F Env variants do not appear to possess a superior ability to interact with and cross a mucosal barrier or an exceptional resistance to neutralization that define their capability to initiate infection across the infant gastrointestinal tract in the setting of preexisting maternal antibodies. PMID:23305422

  16. Isolation and Functional Characterization of Bidirectional Promoters in Rice

    PubMed Central

    Wang, Rui; Yan, Yan; Zhu, Menglin; Yang, Mei; Zhou, Fei; Chen, Hao; Lin, Yongjun

    2016-01-01

    Bidirectional promoters, which show great application potential in genetic improvement of plants, have aroused great research interest recently. However, most bidirectional promoters were cloned individually in the studies of single genes. Here, we initiatively combined RNA-seq data and cDNA microarray data to discover the potential bidirectional promoters in rice genome. Based on the expression level and correlation of each adjacent and oppositely transcribed gene pair, we selected four candidate gene pairs. Then, the intergenic region between each pair was isolated and cloned into a dual reporter vector pDX2181 for functional identification. GUS and GFP assays of the transgenic plants indicated that all the intergenic regions showed bidirectional expression activity in various tissues. Through 5′ and 3′ deletion analysis on one of the above bidirectional promoters, we identified the enhancing region which sharply increased its bidirectional expression efficiency and the essential regions respectively responsible for its 5′ and 3′ basic expression activity. The bidirectional arrangement of the four gene pairs in six gramineous plants was also analyzed, showing the conserved characteristics of the four bidirectional promoters identified in our study. In addition, two novel cis-sequences conserved in the four bidirectional promoters were discovered by bioinformatic identification. Our study proposes a feasible method for selecting, cloning, and functionally identifying bidirectional promoters as well as for discovering their bidirectional regulatory regions and conserved sequences in rice. PMID:27303432

  17. Cloning, expression, and functional characterization of the rat Pax6 5a orthologous splicing variant.

    PubMed

    Wei, Fei; Li, Min; Cheng, Sai-Yu; Wen, Liang; Liu, Ming-Hua; Shuai, Jie

    2014-08-15

    Pax6 functions as a pleiotropic regulator in eye development and neurogenesis. Its splice variant Pax6 5a has been cloned in many vertebrate species including human and mouse, but never in rat. This study focused on the cloning and characterization of the Pax6 5a orthologous splicing variant in rat. It was cloned from Sprague-Dawley rats 10 days post coitum (E10) by RT-PCR and was sequenced for comparison with Pax6 sequences in the GenBank by BLAST. The rat Pax6 5a was revealed to contain an additional 42 bp insertion at the paired domain. At the nucleotide level, the rat Pax6 5a coding sequence (1,311 bp) had a higher degree of homology to the mouse (96% identical) than to the human (93% identical) sequence. At the amino acid (aa) level, rat PAX6 5a shares 99.8% identity with the mouse sequence and 99.5% with the human sequence. The splice variant is preferentially expressed in the rat E10 embryonic headfolds and not in the trunk of neurula. Its effects on the proliferation of rat mesenchymal stem cells (rMSCs) were preliminarily evaluated by the MTT assay. Both pLEGFP-Pax6 5a-transfected cells and pLEGFP-Pax6-transfected cells exhibited a similar growth curve (P>0.05), suggesting that the Pax6 5a has a similar effect on the proliferation of rMSCs as Pax6. PMID:24952136

  18. Expression and function of a novel variant of estrogen receptor-α36 in murine airways.

    PubMed

    Jia, Shuping; Zhang, Xintian; He, David Z Z; Segal, Manav; Berro, Abdo; Gerson, Trevor; Wang, Zhaoyi; Casale, Thomas B

    2011-11-01

    Evidence suggests that estrogen signaling is involved in sex differences in the prevalence rates and control of asthma, but the expression patterns of estrogen receptor variants and estrogen function in the lung are not well established. We investigated the expression of major estrogen receptor variants occurring naturally and after the development of allergen-induced airway hyperreactivity in a murine model of allergic asthma, along with the role of estrogen signaling in small-airway ciliary motion and smooth muscle contraction. Female BALB/c mice were sensitized with ovalbumin, and estrogen receptor expression patterns were examined by immunofluorescence and Western blot analysis. Time-lapse video and photodiode-based displacement measurement systems were used to assess the effects of estrogen signaling on airway ciliary beat frequency and smooth muscle contraction. We found that a novel variant of estrogen receptor (ER)-α, ER-α36, is expressed in airway epithelial and smooth muscle cells. ER-α36 was predominately localized on the plasma membranes of airway cells. After sensitization to allergen, the expression levels of ER-α36 increased significantly (P < 0.01), whereas the expression of ER-β and ER-α66 did not significantly change. Estrogen treatment in vitro resulted in a rapid increase in airway cilia motion in a dose-dependent fashion, but did not exert any effect on airway smooth muscle contraction. We speculate that the up-regulation of estrogen receptor expression associated with allergen-induced airway hyperresponsiveness may constitute a protective mechanism to facilitate the clearance of mucus. The identification and localization of specific estrogen receptor subtypes in the lung could lead to newer therapeutic avenues aimed at addressing sex differences of asthma susceptibility. PMID:21642591

  19. Screening of GNAL variants in Brazilian patients with isolated dystonia reveals a novel mutation with partial loss of function.

    PubMed

    Dos Santos, Camila Oliveira; Masuho, Ikuo; da Silva-Júnior, Francisco Pereira; Barbosa, Egberto Reis; Silva, Sonia Maria Cesar Azevedo; Borges, Vanderci; Ferraz, Henrique Ballalai; Rocha, Maria Sheila Guimarães; Limongi, João Carlos Papaterra; Martemyanov, Kirill A; de Carvalho Aguiar, Patricia

    2016-04-01

    GNAL was identified as a cause of dystonia in patients from North America, Europe and Asia. In this study, we aimed to investigate the prevalence of GNAL variants in Brazilian patients with dystonia. Ninety-one patients with isolated idiopathic dystonia, negative for THAP1 and TOR1A mutations, were screened for GNAL variants by Sanger sequencing. Functional characterization of the Gαolf protein variant was performed using the bioluminescence resonance energy transfer assay. A novel heterozygous nonsynonymous variant (p. F133L) was identified in a patient with cervical and laryngeal dystonia since the third decade of life, with no family history. This variant was not identified in healthy Brazilian controls and was not described in 63,000 exomas of the ExAC database. The F133L mutant exhibited significantly elevated levels of basal BRET and severely diminished amplitude of response elicited by dopamine, that both indicate substantial functional impairment of Gαolf in transducing receptor signals, which could be involved in dystonia pathophysiology. GNAL mutations are not a common cause of dystonia in the Brazilian population and have a lower prevalence than THAP1 and TOR1A mutations. We present a novel variant that results in partial Gαolf loss of function. PMID:26810727

  20. BDNF-Val66Met variant and adolescent stress interact to promote susceptibility to anorexic behavior in mice.

    PubMed

    Madra, M; Zeltser, L M

    2016-01-01

    There is an urgent need to identify therapeutic targets for anorexia nervosa (AN) because current medications do not impact eating behaviors that drive AN's high mortality rate. A major obstacle to developing new treatments is the lack of animal models that recapitulate the pattern of disease onset typically observed in human populations. Here we describe a translational mouse model to study interactions between genetic, psychological and biological risk factors that promote anorexic behavior. We combined several factors that are consistently associated with increased risk of AN-adolescent females, genetic predisposition to anxiety imposed by the BDNF-Val66Met gene variant, social isolation stress and caloric restriction (CR). Approximately 40% of the mice with all of these risk factors will exhibit severe self-imposed dietary restriction, sometimes to the point of death. We systematically varied the risk factors outlined above to explore how they interact to influence anorexic behavior. We found that the Val66Met genotype markedly increases the likelihood and severity of abnormal feeding behavior triggered by CR, but only when CR is imposed in the peri-pubertal period. Incidence of anorexic behavior in our model is dependent on juvenile exposure to social stress and can be extinguished by adolescent handling, but is discordant from anxiety-like behavior. Thus, this study characterized gene × environment interactions during adolescence that could be the underlying driver of abnormal eating behavior in certain AN patients, and represents a promising system to identify possible targets for therapeutic intervention. PMID:27045846

  1. BDNF-Val66Met variant and adolescent stress interact to promote susceptibility to anorexic behavior in mice

    PubMed Central

    Madra, M; Zeltser, L M

    2016-01-01

    There is an urgent need to identify therapeutic targets for anorexia nervosa (AN) because current medications do not impact eating behaviors that drive AN's high mortality rate. A major obstacle to developing new treatments is the lack of animal models that recapitulate the pattern of disease onset typically observed in human populations. Here we describe a translational mouse model to study interactions between genetic, psychological and biological risk factors that promote anorexic behavior. We combined several factors that are consistently associated with increased risk of AN—adolescent females, genetic predisposition to anxiety imposed by the BDNF-Val66Met gene variant, social isolation stress and caloric restriction (CR). Approximately 40% of the mice with all of these risk factors will exhibit severe self-imposed dietary restriction, sometimes to the point of death. We systematically varied the risk factors outlined above to explore how they interact to influence anorexic behavior. We found that the Val66Met genotype markedly increases the likelihood and severity of abnormal feeding behavior triggered by CR, but only when CR is imposed in the peri-pubertal period. Incidence of anorexic behavior in our model is dependent on juvenile exposure to social stress and can be extinguished by adolescent handling, but is discordant from anxiety-like behavior. Thus, this study characterized gene × environment interactions during adolescence that could be the underlying driver of abnormal eating behavior in certain AN patients, and represents a promising system to identify possible targets for therapeutic intervention. PMID:27045846

  2. Color Enhancement in Endoscopic Images Using Adaptive Sigmoid Function and Space Variant Color Reproduction.

    PubMed

    Imtiaz, Mohammad S; Wahid, Khan A

    2015-01-01

    Modern endoscopes play an important role in diagnosing various gastrointestinal (GI) tract related diseases. The improved visual quality of endoscopic images can provide better diagnosis. This paper presents an efficient color image enhancement method for endoscopic images. It is achieved in two stages: image enhancement at gray level followed by space variant chrominance mapping color reproduction. Image enhancement is achieved by performing adaptive sigmoid function and uniform distribution of sigmoid pixels. Secondly, a space variant chrominance mapping color reproduction is used to generate new chrominance components. The proposed method is used on low contrast color white light images (WLI) to enhance and highlight the vascular and mucosa structures of the GI tract. The method is also used to colorize grayscale narrow band images (NBI) and video frames. The focus value and color enhancement factor show that the enhancement level in the processed image is greatly increased compared to the original endoscopic image. The overall contrast level of the processed image is higher than the original image. The color similarity test has proved that the proposed method does not add any additional color which is not present in the original image. The algorithm has low complexity with an execution speed faster than other related methods. PMID:26089969

  3. Serotonin transporter variant drives preventable gastrointestinal abnormalities in development and function

    PubMed Central

    Margolis, Kara Gross; Li, Zhishan; Stevanovic, Korey; Saurman, Virginia; Anderson, George M.; Snyder, Isaac; Blakely, Randy D.; Gershon, Michael D.

    2016-01-01

    Autism spectrum disorder (ASD) is an increasingly common behavioral condition that frequently presents with gastrointestinal (GI) disturbances. It is not clear, however, how gut dysfunction relates to core ASD features. Multiple, rare hyperfunctional coding variants of the serotonin (5-HT) transporter (SERT, encoded by SLC6A4) have been identified in ASD. Expression of the most common SERT variant (Ala56) in mice increases 5-HT clearance and causes ASD-like behaviors. Here, we demonstrated that Ala56-expressing mice display GI defects that resemble those seen in mice lacking neuronal 5-HT. These defects included enteric nervous system hypoplasia, slow GI transit, diminished peristaltic reflex activity, and proliferation of crypt epithelial cells. An opposite phenotype was seen in SERT-deficient mice and in progeny of WT dams given the SERT antagonist fluoxetine. The reciprocal phenotypes that resulted from increased or decreased SERT activity support the idea that 5-HT signaling regulates enteric neuronal development and can, when disturbed, cause long-lasting abnormalities of GI function. Administration of a 5-HT4 agonist to Ala56 mice during development prevented Ala56-associated GI perturbations, suggesting that excessive SERT activity leads to inadequate 5-HT4–mediated neurogenesis. We propose that deficient 5-HT signaling during development may contribute to GI and behavioral features of ASD. The consequences of therapies targeting SERT during pregnancy warrant further evaluation. PMID:27111230

  4. Serotonin transporter variant drives preventable gastrointestinal abnormalities in development and function.

    PubMed

    Margolis, Kara Gross; Li, Zhishan; Stevanovic, Korey; Saurman, Virginia; Israelyan, Narek; Anderson, George M; Snyder, Isaac; Veenstra-VanderWeele, Jeremy; Blakely, Randy D; Gershon, Michael D

    2016-06-01

    Autism spectrum disorder (ASD) is an increasingly common behavioral condition that frequently presents with gastrointestinal (GI) disturbances. It is not clear, however, how gut dysfunction relates to core ASD features. Multiple, rare hyperfunctional coding variants of the serotonin (5-HT) transporter (SERT, encoded by SLC6A4) have been identified in ASD. Expression of the most common SERT variant (Ala56) in mice increases 5-HT clearance and causes ASD-like behaviors. Here, we demonstrated that Ala56-expressing mice display GI defects that resemble those seen in mice lacking neuronal 5-HT. These defects included enteric nervous system hypoplasia, slow GI transit, diminished peristaltic reflex activity, and proliferation of crypt epithelial cells. An opposite phenotype was seen in SERT-deficient mice and in progeny of WT dams given the SERT antagonist fluoxetine. The reciprocal phenotypes that resulted from increased or decreased SERT activity support the idea that 5-HT signaling regulates enteric neuronal development and can, when disturbed, cause long-lasting abnormalities of GI function. Administration of a 5-HT4 agonist to Ala56 mice during development prevented Ala56-associated GI perturbations, suggesting that excessive SERT activity leads to inadequate 5-HT4-mediated neurogenesis. We propose that deficient 5-HT signaling during development may contribute to GI and behavioral features of ASD. The consequences of therapies targeting SERT during pregnancy warrant further evaluation. PMID:27111230

  5. Fine mapping of the celiac disease-associated LPP locus reveals a potential functional variant

    PubMed Central

    Almeida, Rodrigo; Ricaño-Ponce, Isis; Kumar, Vinod; Deelen, Patrick; Szperl, Agata; Trynka, Gosia; Gutierrez-Achury, Javier; Kanterakis, Alexandros; Westra, Harm-Jan; Franke, Lude; Swertz, Morris A.; Platteel, Mathieu; Bilbao, Jose Ramon; Barisani, Donatella; Greco, Luigi; Mearin, Luisa; Wolters, Victorien M.; Mulder, Chris; Mazzilli, Maria Cristina; Sood, Ajit; Cukrowska, Bozena; Núñez, Concepción; Pratesi, Riccardo; Withoff, Sebo; Wijmenga, Cisca

    2014-01-01

    Using the Immunochip for genotyping, we identified 39 non-human leukocyte antigen (non-HLA) loci associated to celiac disease (CeD), an immune-mediated disease with a worldwide frequency of ∼1%. The most significant non-HLA signal mapped to the intronic region of 70 kb in the LPP gene. Our aim was to fine map and identify possible functional variants in the LPP locus. We performed a meta-analysis in a cohort of 25 169 individuals from six different populations previously genotyped using Immunochip. Imputation using data from the Genome of the Netherlands and 1000 Genomes projects, followed by meta-analysis, confirmed the strong association signal on the LPP locus (rs2030519, P = 1.79 × 10−49), without any novel associations. The conditional analysis on this top SNP-indicated association to a single common haplotype. By performing haplotype analyses in each population separately, as well as in a combined group of the four populations that reach the significant threshold after correction (P < 0.008), we narrowed down the CeD-associated region from 70 to 2.8 kb (P = 1.35 × 10−44). By intersecting regulatory data from the ENCODE project, we found a functional SNP, rs4686484 (P = 3.12 × 10−49), that maps to several B-cell enhancer elements and a highly conserved region. This SNP was also predicted to change the binding motif of the transcription factors IRF4, IRF11, Nkx2.7 and Nkx2.9, suggesting its role in transcriptional regulation. We later found significantly low levels of LPP mRNA in CeD biopsies compared with controls, thus our results suggest that rs4686484 is the functional variant in this locus, while LPP expression is decreased in CeD. PMID:24334606

  6. Pseudomonas aeruginosa Rugose Small-Colony Variants Have Adaptations That Likely Promote Persistence in the Cystic Fibrosis Lung▿ †

    PubMed Central

    Starkey, Melissa; Hickman, Jason H.; Ma, Luyan; Zhang, Niu; De Long, Susan; Hinz, Aaron; Palacios, Sergio; Manoil, Colin; Kirisits, Mary Jo; Starner, Timothy D.; Wozniak, Daniel J.; Harwood, Caroline S.; Parsek, Matthew R.

    2009-01-01

    Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats, ranging from soil to immunocompromised people. The formation of surface-associated communities called biofilms is one factor thought to enhance colonization and persistence in these diverse environments. Another factor is the ability of P. aeruginosa to diversify genetically, generating phenotypically distinct subpopulations. One manifestation of diversification is the appearance of colony morphology variants on solid medium. Both laboratory biofilm growth and chronic cystic fibrosis (CF) airway infections produce rugose small-colony variants (RSCVs) characterized by wrinkled, small colonies and an elevated capacity to form biofilms. Previous reports vary on the characteristics attributable to RSCVs. Here we report a detailed comparison of clonally related wild-type and RSCV strains isolated from both CF sputum and laboratory biofilm cultures. The clinical RSCV had many characteristics in common with biofilm RSCVs. Transcriptional profiling and Biolog phenotypic analysis revealed that RSCVs display increased expression of the pel and psl polysaccharide gene clusters, decreased expression of motility functions, and a defect in growth on some amino acid and tricarboxylic acid cycle intermediates as sole carbon sources. RSCVs also elicited a reduced chemokine response from polarized airway epithelium cells compared to wild-type strains. A common feature of all RSCVs analyzed in this study is increased levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP). To assess the global transcriptional effects of elevated c-di-GMP levels, we engineered an RSCV strain that had elevated c-di-GMP levels but did not autoaggregate. Our results showed that about 50 genes are differentially expressed in response to elevated intracellular c-di-GMP levels. Among these genes are the pel and psl genes, which are upregulated, and flagellum and pilus genes, which are downregulated. RSCV

  7. Functional Evaluation of Nine Missense-Type Variants of the Human DNA Glycosylase Enzyme MUTYH in the Japanese Population.

    PubMed

    Shinmura, Kazuya; Kato, Hisami; Goto, Masanori; Yamada, Hidetaka; Tao, Hong; Nakamura, Satoki; Sugimura, Haruhiko

    2016-04-01

    Biallelic germline mutations of MUTYH, the gene encoding DNA glycosylase, cause MUTYH-associated polyposis (MAP), characterized by multiple colorectal adenomas and carcinoma(s). However, a considerable number of MUTYH variants are still functionally uncharacterized. Herein, we report the results of functional evaluation of nine missense-type MUTYH variant proteins in the Japanese population. The DNA glycosylase activity and ability to suppress mutations caused by 8-hydroxyguanine, an oxidized form of guanine, were examined for the nine variants of type 2 MUTYH, a nuclear form of the enzyme, by DNA cleavage activity assay and supF forward mutation assay, respectively. Both activities were severely defective in the p.N210S MUTYH type 2 variant corresponding to p.N238S in the reference MUTYH form and partially defective in p.R219G variant corresponding to p.R247G, but nearly fully retained in seven other variants examined. Our results suggest that p.N238S and p.R247G are likely to be pathogenic alleles for MAP. PMID:26694661

  8. A homozygous loss-of-function variant in MYH11 in a case with megacystis-microcolon-intestinal hypoperistalsis syndrome

    PubMed Central

    Gauthier, Julie; Ouled Amar Bencheikh, Bouchra; Hamdan, Fadi F; Harrison, Steven M; Baker, Linda A; Couture, Françoise; Thiffault, Isabelle; Ouazzani, Reda; Samuels, Mark E; Mitchell, Grant A; Rouleau, Guy A; Michaud, Jacques L; Soucy, Jean- François

    2015-01-01

    Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is characterized by marked dilatation of the bladder and microcolon and decreased intestinal peristalsis. Recent studies indicate that heterozygous variants in ACTG2, which codes for a smooth muscle actin, cause MMIHS. However, such variants do not explain MMIHS cases that show an autosomal recessive mode of inheritance. We performed exome sequencing in a newborn with MMIHS and prune belly phenotype whose parents are consanguineous and identified a homozygous variant (c.3598A>T: p.Lys1200Ter) in MYH11, which codes for the smooth muscle myosin heavy chain. Previous studies showed that loss of Myh11 function in mice causes a bladder and intestinal phenotype that is highly reminiscent of MMIHS. All together, these observations strongly suggest that loss-of-function variants in MYH11 cause MMIHS. The documentation of variants in ACTG2 and MYH11 thus points to the involvement of the contractile apparatus of the smooth muscle in MMIHS. Interestingly, dominant-negative variants in MYH11 have previously been shown to cause thoracic aortic aneurism and dilatation. Different mechanisms of MYH11 disruption may thus lead to distinct patterns of smooth muscle dysfunction. PMID:25407000

  9. A homozygous loss-of-function variant in MYH11 in a case with megacystis-microcolon-intestinal hypoperistalsis syndrome.

    PubMed

    Gauthier, Julie; Ouled Amar Bencheikh, Bouchra; Hamdan, Fadi F; Harrison, Steven M; Baker, Linda A; Couture, Françoise; Thiffault, Isabelle; Ouazzani, Reda; Samuels, Mark E; Mitchell, Grant A; Rouleau, Guy A; Michaud, Jacques L; Soucy, Jean-François

    2015-09-01

    Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is characterized by marked dilatation of the bladder and microcolon and decreased intestinal peristalsis. Recent studies indicate that heterozygous variants in ACTG2, which codes for a smooth muscle actin, cause MMIHS. However, such variants do not explain MMIHS cases that show an autosomal recessive mode of inheritance. We performed exome sequencing in a newborn with MMIHS and prune belly phenotype whose parents are consanguineous and identified a homozygous variant (c.3598A>T: p.Lys1200Ter) in MYH11, which codes for the smooth muscle myosin heavy chain. Previous studies showed that loss of Myh11 function in mice causes a bladder and intestinal phenotype that is highly reminiscent of MMIHS. All together, these observations strongly suggest that loss-of-function variants in MYH11 cause MMIHS. The documentation of variants in ACTG2 and MYH11 thus points to the involvement of the contractile apparatus of the smooth muscle in MMIHS. Interestingly, dominant-negative variants in MYH11 have previously been shown to cause thoracic aortic aneurism and dilatation. Different mechanisms of MYH11 disruption may thus lead to distinct patterns of smooth muscle dysfunction. PMID:25407000

  10. Bacterial XylRs and synthetic promoters function as genetically encoded xylose biosensors in Saccharomyces cerevisiae.

    PubMed

    Teo, Wei Suong; Chang, Matthew Wook

    2015-02-01

    Lignocellulosic biomass is a sustainable and abundant starting material for biofuel production. However, lignocellulosic hydrolysates contain not only glucose, but also other sugars including xylose which cannot be metabolized by the industrial workhorse Saccharomyces cerevisiae. Hence, engineering of xylose assimilating S. cerevisiae has been much studied, including strain optimization strategies. In this work, we constructed genetically encoded xylose biosensors that can control protein expression upon detection of xylose sugars. These were constructed with the constitutive expression of heterologous XylR repressors, which function as protein sensors, and cloning of synthetic promoters with XylR operator sites. Three XylR variants and the corresponding synthetic promoters were used: XylR from Tetragenococcus halophile, Clostridium difficile, and Lactobacillus pentosus. To optimize the biosensor, two promoters with different strengths were used to express the XylR proteins. The ability of XylR to repress yEGFP expression from the synthetic promoters was demonstrated. Furthermore, xylose sugars added exogenously to the cells were shown to regulate gene expression. We envision that the xylose biosensors can be used as a tool to engineer and optimize yeast that efficiently utilizes xylose as carbon source for growth and biofuel production. PMID:24975936

  11. Functional Properties of Rare Missense Variants of Human CDH13 Found in Adult Attention Deficit/Hyperactivity Disorder (ADHD) Patients

    PubMed Central

    Mavroconstanti, Thegna; Johansson, Stefan; Winge, Ingeborg; Knappskog, Per M.; Haavik, Jan

    2013-01-01

    The CDH13 gene codes for T-cadherin, a GPI-anchored protein with cell adhesion properties that is highly expressed in the brain and cardiovascular system. Previous studies have suggested that CDH13 may be a promising candidate gene for Attention Deficit/Hyperactivity Disorder (ADHD). The aims of this study were to identify, functionally characterize, and estimate the frequency of coding CDH13 variants in adult ADHD patients and controls. We performed sequencing of the CDH13 gene in 169 Norwegian adult ADHD patients and 63 controls and genotyping of the identified variants in 641 patients and 668 controls. Native and green fluorescent protein tagged wild type and variant CDH13 proteins were expressed and studied in CHO and HEK293 cells, respectively. Sequencing identified seven rare missense CDH13 variants, one of which was novel. By genotyping, we found a cumulative frequency of these rare variants of 2.9% in controls and 3.2% in ADHD patients, implying that much larger samples are needed to obtain adequate power to study the genetic association between ADHD and rare CDH13 variants. Protein expression and localization studies in CHO cells and HEK293 cells showed that the wild type and mutant proteins were processed according to the canonical processing of GPI-anchored proteins. Although some of the mutations were predicted to severely affect protein secondary structure and stability, no significant differences were observed between the expression levels and distribution of the wild type and mutant proteins in either HEK293 or CHO cells. This is the first study where the frequency of coding CDH13 variants in patients and controls is reported and also where the functional properties of these variants are examined. Further investigations are needed to conclude whether CDH13 is involved in the pathogenesis of ADHD or other conditions. PMID:23936508

  12. Functional properties of rare missense variants of human CDH13 found in adult attention deficit/hyperactivity disorder (ADHD) patients.

    PubMed

    Mavroconstanti, Thegna; Johansson, Stefan; Winge, Ingeborg; Knappskog, Per M; Haavik, Jan

    2013-01-01

    The CDH13 gene codes for T-cadherin, a GPI-anchored protein with cell adhesion properties that is highly expressed in the brain and cardiovascular system. Previous studies have suggested that CDH13 may be a promising candidate gene for Attention Deficit/Hyperactivity Disorder (ADHD). The aims of this study were to identify, functionally characterize, and estimate the frequency of coding CDH13 variants in adult ADHD patients and controls. We performed sequencing of the CDH13 gene in 169 Norwegian adult ADHD patients and 63 controls and genotyping of the identified variants in 641 patients and 668 controls. Native and green fluorescent protein tagged wild type and variant CDH13 proteins were expressed and studied in CHO and HEK293 cells, respectively. Sequencing identified seven rare missense CDH13 variants, one of which was novel. By genotyping, we found a cumulative frequency of these rare variants of 2.9% in controls and 3.2% in ADHD patients, implying that much larger samples are needed to obtain adequate power to study the genetic association between ADHD and rare CDH13 variants. Protein expression and localization studies in CHO cells and HEK293 cells showed that the wild type and mutant proteins were processed according to the canonical processing of GPI-anchored proteins. Although some of the mutations were predicted to severely affect protein secondary structure and stability, no significant differences were observed between the expression levels and distribution of the wild type and mutant proteins in either HEK293 or CHO cells. This is the first study where the frequency of coding CDH13 variants in patients and controls is reported and also where the functional properties of these variants are examined. Further investigations are needed to conclude whether CDH13 is involved in the pathogenesis of ADHD or other conditions. PMID:23936508

  13. dbWGFP: a database and web server of human whole-genome single nucleotide variants and their functional predictions.

    PubMed

    Wu, Jiaxin; Wu, Mengmeng; Li, Lianshuo; Liu, Zhuo; Zeng, Wanwen; Jiang, Rui

    2016-01-01

    The recent advancement of the next generation sequencing technology has enabled the fast and low-cost detection of all genetic variants spreading across the entire human genome, making the application of whole-genome sequencing a tendency in the study of disease-causing genetic variants. Nevertheless, there still lacks a repository that collects predictions of functionally damaging effects of human genetic variants, though it has been well recognized that such predictions play a central role in the analysis of whole-genome sequencing data. To fill this gap, we developed a database named dbWGFP (a database and web server of human whole-genome single nucleotide variants and their functional predictions) that contains functional predictions and annotations of nearly 8.58 billion possible human whole-genome single nucleotide variants. Specifically, this database integrates 48 functional predictions calculated by 17 popular computational methods and 44 valuable annotations obtained from various data sources. Standalone software, user-friendly query services and free downloads of this database are available at http://bioinfo.au.tsinghua.edu.cn/dbwgfp. dbWGFP provides a valuable resource for the analysis of whole-genome sequencing, exome sequencing and SNP array data, thereby complementing existing data sources and computational resources in deciphering genetic bases of human inherited diseases. PMID:26989155

  14. dbWGFP: a database and web server of human whole-genome single nucleotide variants and their functional predictions

    PubMed Central

    Wu, Jiaxin; Wu, Mengmeng; Li, Lianshuo; Liu, Zhuo; Zeng, Wanwen; Jiang, Rui

    2016-01-01

    The recent advancement of the next generation sequencing technology has enabled the fast and low-cost detection of all genetic variants spreading across the entire human genome, making the application of whole-genome sequencing a tendency in the study of disease-causing genetic variants. Nevertheless, there still lacks a repository that collects predictions of functionally damaging effects of human genetic variants, though it has been well recognized that such predictions play a central role in the analysis of whole-genome sequencing data. To fill this gap, we developed a database named dbWGFP (a database and web server of human whole-genome single nucleotide variants and their functional predictions) that contains functional predictions and annotations of nearly 8.58 billion possible human whole-genome single nucleotide variants. Specifically, this database integrates 48 functional predictions calculated by 17 popular computational methods and 44 valuable annotations obtained from various data sources. Standalone software, user-friendly query services and free downloads of this database are available at http://bioinfo.au.tsinghua.edu.cn/dbwgfp. dbWGFP provides a valuable resource for the analysis of whole-genome sequencing, exome sequencing and SNP array data, thereby complementing existing data sources and computational resources in deciphering genetic bases of human inherited diseases. PMID:26989155

  15. An Artificial Reaction Promoter Modulates Mitochondrial Functions via Chemically Promoting Protein Acetylation.

    PubMed

    Shindo, Yutaka; Komatsu, Hirokazu; Hotta, Kohji; Ariga, Katsuhiko; Oka, Kotaro

    2016-01-01

    Acetylation, which modulates protein function, is an important process in intracellular signalling. In mitochondria, protein acetylation regulates a number of enzymatic activities and, therefore, modulates mitochondrial functions. Our previous report showed that tributylphosphine (PBu3), an artificial reaction promoter that promotes acetylransfer reactions in vitro, also promotes the reaction between acetyl-CoA and an exogenously introduced fluorescent probe in mitochondria. In this study, we demonstrate that PBu3 induces the acetylation of mitochondrial proteins and a decrease in acetyl-CoA concentration in PBu3-treated HeLa cells. This indicates that PBu3 can promote the acetyltransfer reaction between acetyl-CoA and mitochondrial proteins in living cells. PBu3-induced acetylation gradually reduced mitochondrial ATP concentrations in HeLa cells without changing the cytoplasmic ATP concentration, suggesting that PBu3 mainly affects mitochondrial functions. In addition, pyruvate, which is converted into acetyl-CoA in mitochondria and transiently increases ATP concentrations in the absence of PBu3, elicited a further decrease in mitochondrial ATP concentrations in the presence of PBu3. Moreover, the application and removal of PBu3 reversibly alternated mitochondrial fragmentation and elongation. These results indicate that PBu3 enhances acetyltransfer reactions in mitochondria and modulates mitochondrial functions in living cells. PMID:27374857

  16. An Artificial Reaction Promoter Modulates Mitochondrial Functions via Chemically Promoting Protein Acetylation

    PubMed Central

    Shindo, Yutaka; Komatsu, Hirokazu; Hotta, Kohji; Ariga, Katsuhiko; Oka, Kotaro

    2016-01-01

    Acetylation, which modulates protein function, is an important process in intracellular signalling. In mitochondria, protein acetylation regulates a number of enzymatic activities and, therefore, modulates mitochondrial functions. Our previous report showed that tributylphosphine (PBu3), an artificial reaction promoter that promotes acetylransfer reactions in vitro, also promotes the reaction between acetyl-CoA and an exogenously introduced fluorescent probe in mitochondria. In this study, we demonstrate that PBu3 induces the acetylation of mitochondrial proteins and a decrease in acetyl-CoA concentration in PBu3-treated HeLa cells. This indicates that PBu3 can promote the acetyltransfer reaction between acetyl-CoA and mitochondrial proteins in living cells. PBu3-induced acetylation gradually reduced mitochondrial ATP concentrations in HeLa cells without changing the cytoplasmic ATP concentration, suggesting that PBu3 mainly affects mitochondrial functions. In addition, pyruvate, which is converted into acetyl-CoA in mitochondria and transiently increases ATP concentrations in the absence of PBu3, elicited a further decrease in mitochondrial ATP concentrations in the presence of PBu3. Moreover, the application and removal of PBu3 reversibly alternated mitochondrial fragmentation and elongation. These results indicate that PBu3 enhances acetyltransfer reactions in mitochondria and modulates mitochondrial functions in living cells. PMID:27374857

  17. Meiotic Interactors of a Mitotic Gene TAO3 Revealed by Functional Analysis of its Rare Variant.

    PubMed

    Gupta, Saumya; Radhakrishnan, Aparna; Nitin, Rachana; Raharja-Liu, Pandu; Lin, Gen; Steinmetz, Lars M; Gagneur, Julien; Sinha, Himanshu

    2016-01-01

    Studying the molecular consequences of rare genetic variants has the potential to identify novel and hitherto uncharacterized pathways causally contributing to phenotypic variation. Here, we characterize the functional consequences of a rare coding variant of TAO3, previously reported to contribute significantly to sporulation efficiency variation in Saccharomyces cerevisiae During mitosis, the common TAO3 allele interacts with CBK1-a conserved NDR kinase. Both TAO3 and CBK1 are components of the RAM signaling network that regulates cell separation and polarization during mitosis. We demonstrate that the role of the rare allele TAO3(4477C) in meiosis is distinct from its role in mitosis by being independent of ACE2-a RAM network target gene. By quantitatively measuring cell morphological dynamics, and expressing the TAO3(4477C) allele conditionally during sporulation, we show that TAO3 has an early role in meiosis. This early role of TAO3 coincides with entry of cells into meiotic division. Time-resolved transcriptome analyses during early sporulation identified regulators of carbon and lipid metabolic pathways as candidate mediators. We show experimentally that, during sporulation, the TAO3(4477C) allele interacts genetically with ERT1 and PIP2, regulators of the tricarboxylic acid cycle and gluconeogenesis metabolic pathways, respectively. We thus uncover a meiotic functional role for TAO3, and identify ERT1 and PIP2 as novel regulators of sporulation efficiency. Our results demonstrate that studying the causal effects of genetic variation on the underlying molecular network has the potential to provide a more extensive understanding of the pathways driving a complex trait. PMID:27317780

  18. A Functional Variant of the Dimethylarginine Dimethylaminohydrolase-2 Gene Is Associated with Insulin Sensitivity

    PubMed Central

    Andreozzi, Francesco; Presta, Ivan; Mannino, Gaia Chiara; Scarpelli, Daniela; Di Silvestre, Sara; Di Pietro, Natalia; Succurro, Elena; Sciacqua, Angela; Pandolfi, Assunta; Consoli, Agostino; Hribal, Marta Letizia; Perticone, Francesco; Sesti, Giorgio

    2012-01-01

    Background Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of endothelial nitric oxide synthase, which was associated with insulin resistance. Dimethylarginine dimethylaminohydrolase (DDAH) is the major determinant of plasma ADMA. Examining data from the DIAGRAM+ (Diabetes Genetics Replication And Meta-analysis), we identified a variant (rs9267551) in the DDAH2 gene nominally associated with type 2 diabetes (P = 3×10−5). Methodology/Principal Findings initially, we assessed the functional impact of rs9267551 in human endothelial cells (HUVECs), observing that the G allele had a lower transcriptional activity resulting in reduced expression of DDAH2 and decreased NO production in primary HUVECs naturally carrying it. We then proceeded to investigate whether this variant is associated with insulin sensitivity in vivo. To this end, two cohorts of nondiabetic subjects of European ancestry were studied. In sample 1 (n = 958) insulin sensitivity was determined by the insulin sensitivity index (ISI), while in sample 2 (n = 527) it was measured with a euglycemic-hyperinsulinemic clamp. In sample 1, carriers of the GG genotype had lower ISI than carriers of the C allele (67±33 vs.79±44; P = 0.003 after adjusting for age, gender, and BMI). ADMA levels were higher in subjects carrying the GG genotype than in carriers of the C allele (0.68±0.14 vs. 0.57±0.14 µmol/l; P = 0.04). In sample 2, glucose disposal was lower in GG carriers as compared with C carriers (9.3±4.1 vs. 11.0±4.2 mg×Kg−1 free fat mass×min−1; P = 0.009). Conclusions/Significance A functional polymorphism of the DDAH2 gene may confer increased risk for type 2 diabetes by affecting insulin sensitivity throughout increased ADMA levels. PMID:22558392

  19. Complexity of murine cardiomyocyte miRNA biogenesis, sequence variant expression and function.

    PubMed

    Humphreys, David T; Hynes, Carly J; Patel, Hardip R; Wei, Grace H; Cannon, Leah; Fatkin, Diane; Suter, Catherine M; Clancy, Jennifer L; Preiss, Thomas

    2012-01-01

    microRNAs (miRNAs) are critical to heart development and disease. Emerging research indicates that regulated precursor processing can give rise to an unexpected diversity of miRNA variants. We subjected small RNA from murine HL-1 cardiomyocyte cells to next generation sequencing to investigate the relevance of such diversity to cardiac biology. ∼40 million tags were mapped to known miRNA hairpin sequences as deposited in miRBase version 16, calling 403 generic miRNAs as appreciably expressed. Hairpin arm bias broadly agreed with miRBase annotation, although 44 miR* were unexpectedly abundant (>20% of tags); conversely, 33 -5p/-3p annotated hairpins were asymmetrically expressed. Overall, variability was infrequent at the 5' start but common at the 3' end of miRNAs (5.2% and 52.3% of tags, respectively). Nevertheless, 105 miRNAs showed marked 5' isomiR expression (>20% of tags). Among these was miR-133a, a miRNA with important cardiac functions, and we demonstrated differential mRNA targeting by two of its prevalent 5' isomiRs. Analyses of miRNA termini and base-pairing patterns around Drosha and Dicer cleavage regions confirmed the known bias towards uridine at the 5' most position of miRNAs, as well as supporting the thermodynamic asymmetry rule for miRNA strand selection and a role for local structural distortions in fine tuning miRNA processing. We further recorded appreciable expression of 5 novel miR*, 38 extreme variants and 8 antisense miRNAs. Analysis of genome-mapped tags revealed 147 novel candidate miRNAs. In summary, we revealed pronounced sequence diversity among cardiomyocyte miRNAs, knowledge of which will underpin future research into the mechanisms involved in miRNA biogenesis and, importantly, cardiac function, disease and therapy. PMID:22319597

  20. Characterization of the antigenic and functional domains of a Mycoplasma synoviae variant vlhA gene.

    PubMed

    Khiari, Awatef Béjaoui; Mardassi, Boutheina Ben Abdelmoumen

    2012-05-01

    The Mycoplasma synoviae haemagglutinin gene, vlhA, encodes two major immunodominant and surface-exposed membrane proteins, MSPB and MSPA. Both products are antigenically variable but only MSPA mediates binding to erythrocytes. Previously we have shown that M. synoviae type strain WVU 1853 could express a variant vlhA gene, referred to as MS2/28.1, with a considerably shorter and divergent MSPA region. A finding that prompted detailed characterization of its antigenic and functional properties. Here we mutagenized each of the six opal codons of the variant MS2/28.1 vlhA member into tryptophan, thus allowing its expression in Escherichia coli as well as its cleavage products, MSPB and MSPA. In addition, we expressed 5 contiguous regions of MS2/28.1 extending from the last part of MSPB to the C-terminus of MSPA. Colony immunostaining with region-specific antisera mapped antigenic variation to the N-terminal half of MS2/28.1 MSPA. No haemagglutinating activity was observed for MSPB, but consistent haemadsorption inhibition was mapped to the region extending from amino acid 325 to 344. Inhibition of both haemagglutination and haemadsorption activities were obtained with sera directed against the C-terminal region of MSPA, with the highest titers (1/320 and 1/160, respectively) being recorded for its last 60 residues. Importantly, antibodies to this region also yielded the highest metabolic inhibition titer of 1/1280. Overall, aside from mapping the functional domains of a M. synoviae highly divergent haemagglutinin gene, this study shows that the C-terminal half of its MSPA region induced the highest titers of antibodies inhibiting haemagglutination, haemadsorption, and metabolism. PMID:22176762

  1. Meiotic Interactors of a Mitotic Gene TAO3 Revealed by Functional Analysis of its Rare Variant

    PubMed Central

    Gupta, Saumya; Radhakrishnan, Aparna; Nitin, Rachana; Raharja-Liu, Pandu; Lin, Gen; Steinmetz, Lars M.; Gagneur, Julien; Sinha, Himanshu

    2016-01-01

    Studying the molecular consequences of rare genetic variants has the potential to identify novel and hitherto uncharacterized pathways causally contributing to phenotypic variation. Here, we characterize the functional consequences of a rare coding variant of TAO3, previously reported to contribute significantly to sporulation efficiency variation in Saccharomyces cerevisiae. During mitosis, the common TAO3 allele interacts with CBK1—a conserved NDR kinase. Both TAO3 and CBK1 are components of the RAM signaling network that regulates cell separation and polarization during mitosis. We demonstrate that the role of the rare allele TAO3(4477C) in meiosis is distinct from its role in mitosis by being independent of ACE2—a RAM network target gene. By quantitatively measuring cell morphological dynamics, and expressing the TAO3(4477C) allele conditionally during sporulation, we show that TAO3 has an early role in meiosis. This early role of TAO3 coincides with entry of cells into meiotic division. Time-resolved transcriptome analyses during early sporulation identified regulators of carbon and lipid metabolic pathways as candidate mediators. We show experimentally that, during sporulation, the TAO3(4477C) allele interacts genetically with ERT1 and PIP2, regulators of the tricarboxylic acid cycle and gluconeogenesis metabolic pathways, respectively. We thus uncover a meiotic functional role for TAO3, and identify ERT1 and PIP2 as novel regulators of sporulation efficiency. Our results demonstrate that studying the causal effects of genetic variation on the underlying molecular network has the potential to provide a more extensive understanding of the pathways driving a complex trait. PMID:27317780

  2. Vitamin D-responsive SGPP2 variants associated with lung cell expression and lung function

    PubMed Central

    2013-01-01

    Background Vitamin D is associated with lung health in epidemiologic studies, but mechanisms mediating observed associations are poorly understood. This study explores mechanisms for an effect of vitamin D in lung through an in vivo gene expression study, an expression quantitative trait loci (eQTL) analysis in lung tissue, and a population-based cohort study of sequence variants. Methods Microarray analysis investigated the association of gene expression in small airway epithelial cells with serum 25(OH)D in adult non-smokers. Sequence variants in candidate genes identified by the microarray were investigated in a lung tissue eQTL database, and also in relation to cross-sectional pulmonary function in the Health, Aging, and Body Composition (Health ABC) study, stratified by race, with replication in the Framingham Heart Study (FHS). Results 13 candidate genes had significant differences in expression by serum 25(OH)D (nominal p < 0.05), and a genome-wide significant eQTL association was detected for SGPP2. In Health ABC, SGPP2 SNPs were associated with FEV1 in both European- and African-Americans, and the gene-level association was replicated in European-American FHS participants. SNPs in 5 additional candidate genes (DAPK1, FSTL1, KAL1, KCNS3, and RSAD2) were associated with FEV1 in Health ABC participants. Conclusions SGPP2, a sphingosine-1-phosphate phosphatase, is a novel vitamin D-responsive gene associated with lung function. The identified associations will need to be followed up in further studies. PMID:24274704

  3. Functional Validation of Rare Human Genetic Variants Involved in Homologous Recombination Using Saccharomyces cerevisiae

    PubMed Central

    Lee, Min-Soo; Yu, Mi; Kim, Kyoung-Yeon; Park, Geun-Hee; Kwack, KyuBum; Kim, Keun P.

    2015-01-01

    Systems for the repair of DNA double-strand breaks (DSBs) are necessary to maintain genome integrity and normal functionality of cells in all organisms. Homologous recombination (HR) plays an important role in repairing accidental and programmed DSBs in mitotic and meiotic cells, respectively. Failure to repair these DSBs causes genome instability and can induce tumorigenesis. Rad51 and Rad52 are two key proteins in homologous pairing and strand exchange during DSB-induced HR; both are highly conserved in eukaryotes. In this study, we analyzed pathogenic single nucleotide polymorphisms (SNPs) in human RAD51 and RAD52 using the Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant from Tolerant (SIFT) algorithms and observed the effect of mutations in highly conserved domains of RAD51 and RAD52 on DNA damage repair in a Saccharomyces cerevisiae-based system. We identified a number of rad51 and rad52 alleles that exhibited severe DNA repair defects. The functionally inactive SNPs were located near ATPase active site of Rad51 and the DNA binding domain of Rad52. The rad51-F317I, rad52-R52W, and rad52-G107C mutations conferred hypersensitivity to methyl methane sulfonate (MMS)-induced DNA damage and were defective in HR-mediated DSB repair. Our study provides a new approach for detecting functional and loss-of-function genetic polymorphisms and for identifying causal variants in human DNA repair genes that contribute to the initiation or progression of cancer. PMID:25938495

  4. Weaver Syndrome‐Associated EZH2 Protein Variants Show Impaired Histone Methyltransferase Function In Vitro

    PubMed Central

    Yap, Damian B.; Lewis, M.E. Suzanne; Chijiwa, Chieko; Ramos‐Arroyo, Maria A.; Tkachenko, Natália; Milano, Valentina; Fradin, Mélanie; McKinnon, Margaret L.; Townsend, Katelin N.; Xu, Jieqing; Van Allen, M.I.; Ross, Colin J.D.; Dobyns, William B.; Weaver, David D.; Gibson, William T.

    2016-01-01

    ABSTRACT Weaver syndrome (WS) is a rare congenital disorder characterized by generalized overgrowth, macrocephaly, specific facial features, accelerated bone age, intellectual disability, and susceptibility to cancers. De novo mutations in the enhancer of zeste homolog 2 (EZH2) have been shown to cause WS. EZH2 is a histone methyltransferase that acts as the catalytic agent of the polycomb‐repressive complex 2 (PRC2) to maintain gene repression via methylation of lysine 27 on histone H3 (H3K27). Functional studies investigating histone methyltransferase activity of mutant EZH2 from various cancers have been reported, whereas WS‐associated mutations remain poorly characterized. To investigate the role of EZH2 in WS, we performed functional studies using artificially assembled PRC2 complexes containing mutagenized human EZH2 that reflected the codon changes predicted from patients with WS. We found that WS‐associated amino acid alterations reduce the histone methyltransferase function of EZH2 in this in vitro assay. Our results support the hypothesis that WS is caused by constitutional mutations in EZH2 that alter the histone methyltransferase function of PRC2. However, histone methyltransferase activities of different EZH2 variants do not appear to correlate directly with the phenotypic variability between WS patients and individuals with a common c.553G>C (p.Asp185His) polymorphism in EZH2. PMID:26694085

  5. Anti-candidal activity of genetically engineered histatin variants with multiple functional domains.

    PubMed

    Oppenheim, Frank G; Helmerhorst, Eva J; Lendenmann, Urs; Offner, Gwynneth D

    2012-01-01

    The human bodily defense system includes a wide variety of innate antimicrobial proteins. Histatins are small molecular weight proteins produced by the human salivary glands that exhibit antifungal and antibacterial activities. While evolutionarily old salivary proteins such as mucins and proline-rich proteins contain large regions of tandem repeats, relatively young proteins like histatins do not contain such repeated domains. Anticipating that domain duplications have a functional advantage, we genetically engineered variants of histatin 3 with one, two, three, or four copies of the functional domain by PCR and splice overlap. The resulting proteins, designated reHst3 1-mer, reHist3 2-mer, reHis3 3-mer and reHist3 4-mer, exhibited molecular weights of 4,062, 5,919, 7,777, and 9,634 Da, respectively. The biological activities of these constructs were evaluated in fungicidal assays toward Candida albicans blastoconidia and germinated cells. The antifungal activities per mole of protein increased concomitantly with the number of functional domains present. This increase, however, was higher than could be anticipated from the molar concentration of functional domains present in the constructs. The demonstrated increase in antifungal activity may provide an evolutionary explanation why such domain multiplication is a frequent event in human salivary proteins. PMID:23251551

  6. Pain modality- and sex-specific effects of COMT genetic functional variants

    PubMed Central

    Belfer, Inna; Segall, Samantha K.; Lariviere, William R.; Smith, Shad B.; Dai, Feng; Slade, Gary G.; Rashid, Naim U.; Mogil, Jeffrey S.; Campbell, Claudia; Edwards, Robert; Liu, Qian; Bair, Eric; Maixner, William; Diatchenko, Luda

    2013-01-01

    The enzyme catechol-O-methyltransferase (COMT) metabolizes catecholamine neurotransmitters involved in a number of physiological functions including pain perception. Both human and mouse COMT genes possess functional polymorphisms contributing to inter-individual variability in pain phenotypes such as sensitivity to noxious stimuli, severity of clinical pain and response to pain treatment. In this study, we found that the effects of Comt functional variation in mice are modality-specific. Spontaneous inflammatory nociception and thermal nociception behaviors were correlated the most with the presence of the B2 SINE transposon insertion residing in the 3’UTR mRNA region. Similarly, in humans, COMT functional haplotypes were associated with thermal pain perception and with capsaicin-induced pain. Furthermore, COMT genetic variations contributed to pain behaviors in mice and pain ratings in humans in a sex-specific manner. The ancestral Comt variant, without a B2 SINE insertion, was more strongly associated with sensitivity to capsaicin in female versus male mice. In humans, the haplotype coding for low COMT activity increased capsaicin-induced pain perception in women, but not men. These findings reemphasize the fundamental contribution of COMT to pain processes, and provide a fine-grained resolution of this contribution at the genetic level that can be used to guide future studies in the area of pain genetics. PMID:23701723

  7. Weaver Syndrome-Associated EZH2 Protein Variants Show Impaired Histone Methyltransferase Function In Vitro.

    PubMed

    Cohen, Ana S A; Yap, Damian B; Lewis, M E Suzanne; Chijiwa, Chieko; Ramos-Arroyo, Maria A; Tkachenko, Natália; Milano, Valentina; Fradin, Mélanie; McKinnon, Margaret L; Townsend, Katelin N; Xu, Jieqing; Van Allen, M I; Ross, Colin J D; Dobyns, William B; Weaver, David D; Gibson, William T

    2016-03-01

    Weaver syndrome (WS) is a rare congenital disorder characterized by generalized overgrowth, macrocephaly, specific facial features, accelerated bone age, intellectual disability, and susceptibility to cancers. De novo mutations in the enhancer of zeste homolog 2 (EZH2) have been shown to cause WS. EZH2 is a histone methyltransferase that acts as the catalytic agent of the polycomb-repressive complex 2 (PRC2) to maintain gene repression via methylation of lysine 27 on histone H3 (H3K27). Functional studies investigating histone methyltransferase activity of mutant EZH2 from various cancers have been reported, whereas WS-associated mutations remain poorly characterized. To investigate the role of EZH2 in WS, we performed functional studies using artificially assembled PRC2 complexes containing mutagenized human EZH2 that reflected the codon changes predicted from patients with WS. We found that WS-associated amino acid alterations reduce the histone methyltransferase function of EZH2 in this in vitro assay. Our results support the hypothesis that WS is caused by constitutional mutations in EZH2 that alter the histone methyltransferase function of PRC2. However, histone methyltransferase activities of different EZH2 variants do not appear to correlate directly with the phenotypic variability between WS patients and individuals with a common c.553G>C (p.Asp185His) polymorphism in EZH2. PMID:26694085

  8. Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

    PubMed Central

    Aprile-Garcia, Fernando; Metzger, Michael W.; Paez-Pereda, Marcelo; Stadler, Herbert; Acuña, Matías; Liberman, Ana C.; Senin, Sergio A.; Gerez, Juan; Hoijman, Esteban; Refojo, Damian; Mitkovski, Mišo; Panhuysen, Markus; Stühmer, Walter; Holsboer, Florian; Deussing, Jan M.; Arzt, Eduardo

    2016-01-01

    The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations. PMID:26986975

  9. Promoters of Escherichia coli versus Promoter Islands: Function and Structure Comparison

    PubMed Central

    Panyukov, Valeriy V.; Ozoline, Olga N.

    2013-01-01

    Expression of bacterial genes takes place under the control of RNA polymerase with exchangeable σ-subunits and multiple transcription factors. A typical promoter region contains one or several overlapping promoters. In the latter case promoters have the same or different σ-specificity and are often subjected to different regulatory stimuli. Genes, transcribed from multiple promoters, have on average higher expression levels. However, recently in the genome of Escherichia coli we found 78 regions with an extremely large number of potential transcription start points (promoter islands, PIs). It was shown that all PIs interact with RNA polymerase in vivo and are able to form transcriptionally competent open complexes both in vitro and in vivo but their transcriptional activity measured by oligonucleotide microarrays was very low, if any. Here we confirmed transcriptional defectiveness of PIs by analyzing the 5′-end specific RNA-seq data, but showed their ability to produce short oligos (9–14 bases). This combination of functional properties indicated a deliberate suppression of transcriptional activity within PIs. According to our data this suppression may be due to a specific conformation of the DNA double helix, which provides an ideal platform for interaction with both RNA polymerase and the histone-like nucleoid protein H-NS. The genomic DNA of E.coli contains therefore several dozen sites optimized by evolution for staying in a heterochromatin-like state. Since almost all promoter islands are associated with horizontally acquired genes, we offer them as specific components of bacterial evolution involved in acquisition of foreign genetic material by turning off the expression of toxic or useless aliens or by providing optimal promoter for beneficial genes. The putative molecular mechanism underlying the appearance of promoter islands within recipient genomes is discussed. PMID:23717391

  10. Common Variants in Promoter of ADTRP Associate with Early-Onset Coronary Artery Disease in a Southern Han Chinese Population

    PubMed Central

    Huang, Lei; Wu, Qiu-Ping; Tang, Shuang-Bo; Luo, Bin; Liu, Shui-Ping; Liu, Xiao-Shan; Li, Zhao-Hui; Quan, Li; Li, Yue; Shi, He; Lv, Guo-Li; Zhao, Jian; Cheng, Jian-Ding; Liu, Chao

    2015-01-01

    The first genome-wide association study for coronary artery disease (CAD) in the Han Chinese population, we reported recently, had identified rs6903956 in gene ADTRP on chromosome 6p24.1 as a novel susceptibility locus for CAD. The risk allele of rs6903956 was associated with decreased mRNA expression of ADTRP. To further study the correlation of ADTRP expression and CAD, in this study we evaluated the associations of eight common variants in the expression-regulating regions of ADTRP with CAD in the Southern Han Chinese population. Rs169790 in 3’UTR, rs2076189 in 5’UTR, four SNPs (rs2076188, rs7753407, rs11966356 and rs1018383) in promoter, and two SNPs (rs3734273, rs80355771) in the last intron of ADTRP were genotyped in 1716 CAD patients and 1572 controls. The correlations between these loci and total or early-onset CAD were investigated. None of these loci was discovered to associate with total CAD (P > 0.05). However, with early-onset CAD, significant both allelic and genotypic associations of rs7753407, rs11966356 and rs1018383 were identified, after adjustment for risk factors of age, gender, hypertension, diabetes, lipid profiles and smoking (adjusted P < 0.05). A haplotype AGCG (constructed by rs2076188, rs7753407, rs11966356 and rs1018383) was identified to protect subjects from early-onset CAD (OR = 0.332, 95% CI = 0.105–0.879, adjusted P = 0.010). Real-time quantitative reverse transcription polymerase chain reaction assay showed that the risk alleles of the associated loci were significantly associated with decreased expression of ADTRP mRNA. Moreover, the average level of ADTRP mRNA expression in early-onset CAD cases was significantly lower than that in controls. Our results provide new evidence supporting the association of ADTRP with the pathogenesis of early-onset CAD. PMID:26375920

  11. Population-Specific Risk of Type 2 Diabetes Conferred by HNF4A P2 Promoter Variants

    PubMed Central

    Barroso, Inês; Luan, Jian’an; Wheeler, Eleanor; Whittaker, Pamela; Wasson, Jon; Zeggini, Eleftheria; Weedon, Michael N.; Hunt, Sarah; Venkatesh, Ranganath; Frayling, Timothy M.; Delgado, Marcos; Neuman, Rosalind J.; Zhao, Jinghua; Sherva, Richard; Glaser, Benjamin; Walker, Mark; Hitman, Graham; McCarthy, Mark I.; Hattersley, Andrew T.; Permutt, M. Alan; Wareham, Nicholas J.; Deloukas, Panagiotis

    2008-01-01

    OBJECTIVE—Single nucleotide polymorphisms (SNPs) in the P2 promoter region of HNF4A were originally shown to be associated with predisposition for type 2 diabetes in Finnish, Ashkenazi, and, more recently, Scandinavian populations, but they generated conflicting results in additional populations. We aimed to investigate whether data from a large-scale mapping approach would replicate this association in novel Ashkenazi samples and in U.K. populations and whether these data would allow us to refine the association signal. RESEARCH DESIGN AND METHODS—Using a dense linkage disequilibrium map of 20q, we selected SNPs from a 10-Mb interval centered on HNF4A. In a staged approach, we first typed 4,608 SNPs in case-control populations from four U.K. populations and an Ashkenazi population (n = 2,516). In phase 2, a subset of 763 SNPs was genotyped in 2,513 additional samples from the same populations. RESULTS—Combined analysis of both phases demonstrated association between HNF4A P2 SNPs (rs1884613 and rs2144908) and type 2 diabetes in the Ashkenazim (n = 991; P < 1.6 × 10−6). Importantly, these associations are significant in a subset of Ashkenazi samples (n = 531) not previously tested for association with P2 SNPs (odds ratio [OR] ∼1.7; P < 0.002), thus providing replication within the Ashkenazim. In the U.K. populations, this association was not significant (n = 4,022; P > 0.5), and the estimate for the OR was much smaller (OR 1.04; [95%CI 0.91–1.19]). CONCLUSIONS—These data indicate that the risk conferred by HNF4A P2 is significantly different between U.K. and Ashkenazi populations (P < 0.00007), suggesting that the underlying causal variant remains unidentified. Interactions with other genetic or environmental factors may also contribute to this difference in risk between populations. PMID:18728231

  12. Association of adiponectin promoter variants with traits and clusters of metabolic syndrome in Arabs: family-based study.

    PubMed

    Zadjali, F; Al-Yahyaee, S; Hassan, M O; Albarwani, S; Bayoumi, R A

    2013-09-25

    Plasma levels of adiponectin are decreased in type 2 diabetes, obesity and hypertension. Our aim was to use a family-based analysis to identify the genetic variants of the adiponectin (ADIPOQ) gene that are associated with obesity, insulin resistance, dyslipidemia and hypertension, among Arabs. We screened 328 Arabs in one large extended family for single nucleotide polymorphisms (SNPs) in the promoter region of the ADIPOQ gene. Two common SNPs were detected: rs17300539 and rs266729. Evidences of association between traits related to the metabolic syndrome and the SNPs were studied by implementing quantitative genetic association analysis. Results showed that SNP rs266729 was significantly associated with body weight (p-value=0.001), waist circumference (p-value=0.037), BMI (p-value=0.015) and percentage of total body fat (p-value=0.003). Up to 4.1% of heritability of obesity traits was explained by the rs266729 locus. Further cross-sectional analysis showed that carriers of the G allele had significantly higher values of waist circumference, BMI and percentage of total body fat (p-values 0.014, 0.004 and 0.032, respectively). No association was detected between SNP rs266729 and other clusters of metabolic syndrome or their traits except for HOMA-IR and fasting plasma insulin levels, p-values 0.035 and 0.004, respectively. In contrast, both measured genotype and cross-sectional analysis failed to detect an association between the SNP rs17300539 with traits and clusters of metabolic syndrome. In conclusion, we showed family-based evidence of association of SNP rs266729 at ADIPOQ gene with traits defining obesity in Arab population. This is important for future prediction and prevention of obesity in population where obesity is in an increasing trend. PMID:23845780

  13. Common variants in the CRP promoter are associated with a high C-reactive protein level in Kawasaki disease.

    PubMed

    Kim, Jae-Jung; Yun, Sin Weon; Yu, Jeong Jin; Yoon, Kyung Lim; Lee, Kyung-Yil; Kil, Hong-Ryang; Kim, Gi Beom; Han, Myung Ki; Song, Min Seob; Lee, Hyoung Doo; Byeon, Jung Hye; Sohn, Saejung; Hong, Young Mi; Jang, Gi Young; Lee, Jong-Keuk

    2015-02-01

    Kawasaki disease (KD) is an acute self-limiting form of vasculitis that afflicts infants and children and manifests as fever and signs of mucocutaneous inflammation. Children with KD show various laboratory inflammatory abnormalities, such as elevations in their white blood cell (WBC) count, C-reactive protein (CRP) level, and erythrocyte sedimentation rate (ESR). We here performed a genome-wide association study (GWAS) of 178 KD patients to identify the genetic loci that influence 10 important KD laboratory markers: WBC count, neutrophil count, platelet count, CRP, ESR, hemoglobin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, and total protein. A total of 165 loci passed our arbitrary stage 1 threshold for replication (p < 1 × 10(-5)). Of these, only 2 SNPs (rs12068753 and rs4786091) demonstrated a significant association with the CRP level in replication study of 473 KD patients (p < 0.05). The SNP located at the CRP locus (rs12068753) demonstrated the most significant association with CRP in KD patients (beta = 4.73 and p = 1.20 × 10(-6) according to the stage 1 GWAS; beta = 3.65 and p = 1.35 × 10(-8) according to the replication study; beta = 3.97 and p = 1.11 × 10(-13) according to combined analysis) and explained 8.1% of the phenotypic variation observed. However, this SNP did not demonstrate any significant association with CRP in the general population (beta = 0.37 and p = 0.1732) and only explained 0.1% of the phenotypic variation in this instance. Furthermore, rs12068753 did not affect the development of coronary artery lesions or intravenous immunoglobulin resistance in KD patients. These results indicate that common variants in the CRP promoter can play an important role in the CRP levels in KD. PMID:25266886

  14. MIR retroposon exonization promotes evolutionary variability and generates species-specific expression of IGF-1 splice variants.

    PubMed

    Annibalini, Giosuè; Bielli, Pamela; De Santi, Mauro; Agostini, Deborah; Guescini, Michele; Sisti, Davide; Contarelli, Serena; Brandi, Giorgio; Villarini, Anna; Stocchi, Vilberto; Sette, Claudio; Barbieri, Elena

    2016-05-01

    Insulin-like growth factor (IGF) -1 is a pleiotropic hormone exerting mitogenic and anti-apoptotic effects. Inclusion or exclusion of exon 5 into the IGF-1 mRNA gives rise to three transcripts, IGF-1Ea, IGF-1Eb and IGF-1Ec, which yield three different C-terminal extensions called Ea, Eb and Ec peptides. The biological significance of the IGF-1 splice variants and how the E-peptides affect the actions of mature IGF-1 are largely unknown. In this study we investigated the origin and conservation of the IGF-1 E-peptides and we compared the pattern of expression of the IGF-1 isoforms in vivo, in nine mammalian species, and in vitro using human and mouse IGF-1 minigenes. Our analysis showed that only IGF-1Ea is conserved among all vertebrates, whereas IGF-1Eb and IGF-1Ec are an evolutionary novelty originated from the exonization of a mammalian interspersed repetitive-b (MIR-b) element. Both IGF-1Eb and IGF-1Ec mRNAs were constitutively expressed in all mammalian species analyzed but their expression ratio varies greatly among species. Using IGF-1 minigenes we demonstrated that divergence in cis-acting regulatory elements between human and mouse conferred species-specific features to the exon 5 region. Finally, the protein-coding sequences of exon 5 showed low rate of synonymous mutations and contain disorder-promoting amino acids, suggesting a regulatory role for these domains. In conclusion, exonization of a MIR-b element in the IGF-1 gene determined gain of exon 5 during mammalian evolution. Alternative splicing of this novel exon added new regulatory elements at the mRNA and protein level potentially able to regulate the mature IGF-1 across tissues and species. PMID:27048986

  15. Rare loss-of-function variants in SETD1A are associated with schizophrenia and developmental disorders.

    PubMed

    Singh, Tarjinder; Kurki, Mitja I; Curtis, David; Purcell, Shaun M; Crooks, Lucy; McRae, Jeremy; Suvisaari, Jaana; Chheda, Himanshu; Blackwood, Douglas; Breen, Gerome; Pietiläinen, Olli; Gerety, Sebastian S; Ayub, Muhammad; Blyth, Moira; Cole, Trevor; Collier, David; Coomber, Eve L; Craddock, Nick; Daly, Mark J; Danesh, John; DiForti, Marta; Foster, Alison; Freimer, Nelson B; Geschwind, Daniel; Johnstone, Mandy; Joss, Shelagh; Kirov, Georg; Körkkö, Jarmo; Kuismin, Outi; Holmans, Peter; Hultman, Christina M; Iyegbe, Conrad; Lönnqvist, Jouko; Männikkö, Minna; McCarroll, Steve A; McGuffin, Peter; McIntosh, Andrew M; McQuillin, Andrew; Moilanen, Jukka S; Moore, Carmel; Murray, Robin M; Newbury-Ecob, Ruth; Ouwehand, Willem; Paunio, Tiina; Prigmore, Elena; Rees, Elliott; Roberts, David; Sambrook, Jennifer; Sklar, Pamela; Clair, David St; Veijola, Juha; Walters, James T R; Williams, Hywel; Sullivan, Patrick F; Hurles, Matthew E; O'Donovan, Michael C; Palotie, Aarno; Owen, Michael J; Barrett, Jeffrey C

    2016-04-01

    By analyzing the whole-exome sequences of 4,264 schizophrenia cases, 9,343 controls and 1,077 trios, we identified a genome-wide significant association between rare loss-of-function (LoF) variants in SETD1A and risk for schizophrenia (P = 3.3 × 10(-9)). We found only two heterozygous LoF variants in 45,376 exomes from individuals without a neuropsychiatric diagnosis, indicating that SETD1A is substantially depleted of LoF variants in the general population. Seven of the ten individuals with schizophrenia carrying SETD1A LoF variants also had learning difficulties. We further identified four SETD1A LoF carriers among 4,281 children with severe developmental disorders and two more carriers in an independent sample of 5,720 Finnish exomes, both with notable neuropsychiatric phenotypes. Together, our observations indicate that LoF variants in SETD1A cause a range of neurodevelopmental disorders, including schizophrenia. Combining these data with previous common variant evidence, we suggest that epigenetic dysregulation, specifically in the histone H3K4 methylation pathway, is an important mechanism in the pathogenesis of schizophrenia. PMID:26974950

  16. Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor

    PubMed Central

    Herraiz, Cecilia; Olivares, Conchi; Castejón-Griñán, Maria; Abrisqueta, Marta; Jiménez-Cervantes, Celia; García-Borrón, José Carlos

    2015-01-01

    The melanocortin 1 receptor gene (MC1R) expressed in melanocytes is a major determinant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melanocyte stimulating hormone (αMSH). Human MC1R has an inefficient poly(A) site allowing intergenic splicing with its downstream neighbour Tubulin-β-III (TUBB3). Intergenic splicing produces two MC1R isoforms, designated Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. It has been reported that exposure to ultraviolet radiation (UVR) might promote an isoform switch from canonical MC1R (MC1R-001) to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and human melanoma cells, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affinity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than wild-type, but ERK activation upon binding of αMSH was unimpaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters. PMID:26657157

  17. A Single Nucleotide Polymorphism in the Il17ra Promoter Is Associated with Functional Severity of Ankylosing Spondylitis

    PubMed Central

    Vidal-Castiñeira, Jose Ramón; López-Vázquez, Antonio; Diaz-Peña, Roberto; Diaz-Bulnes, Paula; Martinez-Camblor, Pablo; Coto, Eliecer; Coto-Segura, Pablo; Bruges-Armas, Jacome; Pinto, Jose Antonio; Blanco, Francisco Jose; Sánchez, Alejandra; Mulero, Juan; Queiro, Ruben; Lopez-Larrea, Carlos

    2016-01-01

    The aim of this study was to identify new genetic variants associated with the severity of ankylosing spondylitis (AS). We sequenced the exome of eight patients diagnosed with AS, selected on the basis of the severity of their clinical parameters. We identified 27 variants in exons and regulatory regions. The contribution of candidate variants found to AS severity was validated by genotyping two Spanish cohorts consisting of 180 cases/300 controls and 419 cases/656 controls. Relationships of SNPs and clinical variables with the Bath Ankylosing Spondylitis Disease Activity and Functional Indices BASDAI and BASFI were analyzed. BASFI was standardized by adjusting for the duration of the disease since the appearance of the first symptoms. Refining the analysis of SNPs in the two cohorts, we found that the rs4819554 minor allele G in the promoter of the IL17RA gene was associated with AS (p<0.005). This variant was also associated with the BASFI score. Classifying AS patients by the severity of their functional status with respect to BASFI/disease duration of the 60th, 65th, 70th and 75th percentiles, we found the association increased from p60 to p75 (cohort 1: p<0.05 to p<0.01; cohort 2: p<0.01 to p<0.005). Our findings indicate a genetic role for the IL17/ILRA axis in the development of severe forms of AS. PMID:27415816

  18. A Single Nucleotide Polymorphism in the Il17ra Promoter Is Associated with Functional Severity of Ankylosing Spondylitis.

    PubMed

    Vidal-Castiñeira, Jose Ramón; López-Vázquez, Antonio; Diaz-Peña, Roberto; Diaz-Bulnes, Paula; Martinez-Camblor, Pablo; Coto, Eliecer; Coto-Segura, Pablo; Bruges-Armas, Jacome; Pinto, Jose Antonio; Blanco, Francisco Jose; Sánchez, Alejandra; Mulero, Juan; Queiro, Ruben; Lopez-Larrea, Carlos

    2016-01-01

    The aim of this study was to identify new genetic variants associated with the severity of ankylosing spondylitis (AS). We sequenced the exome of eight patients diagnosed with AS, selected on the basis of the severity of their clinical parameters. We identified 27 variants in exons and regulatory regions. The contribution of candidate variants found to AS severity was validated by genotyping two Spanish cohorts consisting of 180 cases/300 controls and 419 cases/656 controls. Relationships of SNPs and clinical variables with the Bath Ankylosing Spondylitis Disease Activity and Functional Indices BASDAI and BASFI were analyzed. BASFI was standardized by adjusting for the duration of the disease since the appearance of the first symptoms. Refining the analysis of SNPs in the two cohorts, we found that the rs4819554 minor allele G in the promoter of the IL17RA gene was associated with AS (p<0.005). This variant was also associated with the BASFI score. Classifying AS patients by the severity of their functional status with respect to BASFI/disease duration of the 60th, 65th, 70th and 75th percentiles, we found the association increased from p60 to p75 (cohort 1: p<0.05 to p<0.01; cohort 2: p<0.01 to p<0.005). Our findings indicate a genetic role for the IL17/ILRA axis in the development of severe forms of AS. PMID:27415816

  19. A loss-of-function variant of the antiviral molecule MAVS is associated with a subset of systemic lupus patients

    PubMed Central

    Pothlichet, Julien; Niewold, Timothy B; Vitour, Damien; Solhonne, Brigitte; Crow, Mary K; Si-Tahar, Mustapha

    2011-01-01

    Dysregulation of the antiviral immune response may contribute to autoimmune diseases. Here, we hypothesized that altered expression or function of MAVS, a key molecule downstream of the viral sensors RIG-I and MDA-5, may impair antiviral cell signalling and thereby influence the risk for systemic lupus erythematosus (SLE), the prototype autoimmune disease. We used molecular techniques to screen non-synonymous single nucleotide polymorphisms (SNPs) in the MAVS gene for functional significance in human cell lines and identified one critical loss-of-function variant (C79F, rs11905552). This SNP substantially reduced expression of type I interferon (IFN) and other proinflammatory mediators and was found almost exclusively in the African-American population. Importantly, in African-American SLE patients, the C79F allele was associated with low type I IFN production and absence of anti-RNA-binding protein autoantibodies. These serologic associations were not related to a distinct, functionally neutral, MAVS SNP Q198K. Hence, this is the first demonstration that an uncommon genetic variant in the MAVS gene has a functional impact upon the anti-viral IFN pathway in vivo in humans and is associated with a novel sub-phenotype in SLE. This study demonstrates the utility of functional data in selecting rare variants for genetic association studies, allowing for fewer comparisons requiring statistical correction and for alternate lines of evidence implicating the particular variant in disease. PMID:21268286

  20. Resting state functional connectivity differences between behavioral variant frontotemporal dementia and Alzheimer's disease

    PubMed Central

    Hafkemeijer, Anne; Möller, Christiane; Dopper, Elise G. P.; Jiskoot, Lize C.; Schouten, Tijn M.; van Swieten, John C.; van der Flier, Wiesje M.; Vrenken, Hugo; Pijnenburg, Yolande A. L.; Barkhof, Frederik; Scheltens, Philip; van der Grond, Jeroen; Rombouts, Serge A. R. B.

    2015-01-01

    Introduction: Alzheimer's disease (AD) and behavioral variant frontotemporal dementia (bvFTD) are the most common types of early-onset dementia. Early differentiation between both types of dementia may be challenging due to heterogeneity and overlap of symptoms. Here, we apply resting state functional magnetic resonance imaging (fMRI) to study functional brain connectivity differences between AD and bvFTD. Methods: We used resting state fMRI data of 31 AD patients, 25 bvFTD patients, and 29 controls from two centers specialized in dementia. We studied functional connectivity throughout the entire brain, applying two different analysis techniques, studying network-to-region and region-to-region connectivity. A general linear model approach was used to study group differences, while controlling for physiological noise, age, gender, study center, and regional gray matter volume. Results: Given gray matter differences, we observed decreased network-to-region connectivity in bvFTD between (a) lateral visual cortical network and lateral occipital and cuneal cortex, and (b) auditory system network and angular gyrus. In AD, we found decreased network-to-region connectivity between the dorsal visual stream network and lateral occipital and parietal opercular cortex. Region-to-region connectivity was decreased in bvFTD between superior temporal gyrus and cuneal, supracalcarine, intracalcarine cortex, and lingual gyrus. Conclusion: We showed that the pathophysiology of functional brain connectivity is different between AD and bvFTD. Our findings support the hypothesis that resting state fMRI shows disease-specific functional connectivity differences and is useful to elucidate the pathophysiology of AD and bvFTD. However, the group differences in functional connectivity are less abundant than has been shown in previous studies. PMID:26441584

  1. Variants of the yeast MAPK Mpk1 are fully functional independently of activation loop phosphorylation.

    PubMed

    Goshen-Lago, Tal; Goldberg-Carp, Anat; Melamed, Dganit; Darlyuk-Saadon, Ilona; Bai, Chen; Ahn, Natalie G; Admon, Arie; Engelberg, David

    2016-09-01

    MAP kinases of the ERK family are conserved from yeast to humans. Their catalytic activity is dependent on dual phosphorylation of their activation loop's TEY motif, catalyzed by MAPK kinases (MEKs). Here we studied variants of Mpk1, a yeast orthologue of Erk, which is essential for cell wall integrity. Cells lacking MPK1, or the genes encoding the relevant MEKs, MKK1 and MKK2, do not proliferate under cell wall stress, imposed, for example, by caffeine. Mutants of Mpk1, Mpk1(Y268C) and Mpk1(Y268A), function independently of Mkk1 and Mkk2. We show that these variants are phosphorylated at their activation loop in mkk1∆mkk2∆ and mkk1∆mkk2∆pbs2∆ste7∆ cells, suggesting that they autophosphorylate. However, strikingly, when Y268C/A mutations were combined with the kinase-dead mutation, K54R, or mutations at the TEY motif, T190A+Y192F, the resulting proteins still allowed mkk1∆mkk2∆ cells to proliferate under caffeine stress. Mutating the equivalent residue, Tyr-280/Tyr-261, in Erk1/Erk2 significantly impaired Erk1/2's catalytic activity. This study describes the first case in which a MAPK, Erk/Mpk1, imposes a phenotype via a mechanism that is independent of TEY phosphorylation and an unusual case in which an equivalent mutation in a highly conserved domain of yeast and mammalian Erks causes an opposite effect. PMID:27413009

  2. Identification and functional characterization of four transient receptor potential ankyrin 1 variants in Apolygus lucorum (Meyer-Dür).

    PubMed

    Fu, T; Hull, J J; Yang, T; Wang, G

    2016-08-01

    As signal integrators that respond to various physical and chemical stimuli, transient receptor potential (TRP) channels fulfil critical functional roles in the sensory systems of both vertebrate and invertebrate organisms. Here, four variants of TRP ankyrin 1 (TRPA1) were identified and cloned from the green plant bug, Apolygus lucorum. Spatiotemporal expression profiling across development and in different adult tissues revealed that the highest relative-transcript levels occurred in first-instar nymphs and antennae, respectively. In Xenopus laevis-based functional assays, Apo. lucorum TRPA1-A (AlucTRPA1-A), AlucTRPA1-B and AlucTRPA1-C were activated by increasing the temperature from 20 to 40 °C with no significant desensitization observed after repeated temperature stimuli. The activation temperature of AlucTRPA1-A and AlucTRPA1-B was < 25 °C, whereas the activation temperature of AlucTRPA1-C was between 25 and 30 °C. Amongst the variants, only AlucTRPA1-A and AlucTRPA1-C were directly activated by high concentrations of allyl isothiocyanate, cinnamaldehyde and citronellal. Taken together, these results suggest that AlucTRPA1 variants may function in vivo as both thermal and chemical sensors, with the four variants potentially mediating different physiological functions. This study not only enriches our understanding of TRPA1 function in Hemiptera (Miridae), but also offers a foundation for developing new pest control strategies. PMID:27038267

  3. Clinical polyomavirus BK variants with agnogene deletion are non-functional but rescued by trans-complementation

    SciTech Connect

    Myhre, Marit Renee; Olsen, Gunn-Hege; Gosert, Rainer; Hirsch, Hans H.; Rinaldo, Christine Hanssen

    2010-03-01

    High-level replication of polyomavirus BK (BKV) in kidney transplant recipients is associated with the emergence of BKV variants with rearranged (rr) non-coding control region (NCCR) increasing viral early gene expression and cytopathology. Cloning and sequencing revealed the presence of a BKV quasispecies which included non-functional variants when assayed in a recombinant virus assay. Here we report that the rr-NCCR of BKV variants RH-3 and RH-12, both bearing a NCCR deletion including the 5' end of the agnoprotein coding sequence, mediated early and late viral reporter gene expression in kidney cells. However, in a recombinant virus they failed to produce infectious progeny despite large T-antigen and VP1 expression and the formation of nuclear virus-like particles. Infectious progeny was generated when the agnogene was reconstructed in cis or agnoprotein provided in trans from a co-existing BKV rr-NCCR variant. We conclude that complementation can rescue non-functional BKV variants in vitro and possibly in vivo.

  4. Modifying lipid rafts promotes regeneration and functional recovery.

    PubMed

    Tassew, Nardos G; Mothe, Andrea J; Shabanzadeh, Alireza P; Banerjee, Paromita; Koeberle, Paulo D; Bremner, Rod; Tator, Charles H; Monnier, Philippe P

    2014-08-21

    Ideal strategies to ameliorate CNS damage should promote both neuronal survival and axon regeneration. The receptor Neogenin promotes neuronal apoptosis. Its ligand prevents death, but the resulting repulsive guidance molecule a (RGMa)-Neogenin interaction also inhibits axonal growth, countering any prosurvival benefits. Here, we explore strategies to inhibit Neogenin, thus simultaneously enhancing survival and regeneration. We show that bone morphogenetic protein (BMP) and RGMa-dependent recruitment of Neogenin into lipid rafts requires an interaction between RGMa and Neogenin subdomains. RGMa or Neogenin peptides that prevent this interaction, BMP inhibition by Noggin, or reduction of membrane cholesterol all block Neogenin raft localization, promote axon outgrowth, and prevent neuronal apoptosis. Blocking Neogenin raft association influences axonal pathfinding, enhances survival in the developing CNS, and promotes survival and regeneration in the injured adult optic nerve and spinal cord. Moreover, lowering cholesterol disrupts rafts and restores locomotor function after spinal cord injury. These data reveal a unified strategy to promote both survival and regeneration in the CNS. PMID:25127134

  5. Functional implications of a psychiatric risk variant within CACNA1C in induced human neurons

    PubMed Central

    Yoshimizu, Takao; Pan, Jen Q.; Mungenast, Alison E.; Madison, Jon M.; Su, Susan; Ketterman, Josh; Ongur, Dost; McPhie, Donna; Cohen, Bruce; Perlis, Roy; Tsai, Li-Huei

    2014-01-01

    Psychiatric disorders have clear heritable risk. Several large-scale genome-wide association studies have revealed a strong association between susceptibility for psychiatric disorders, including bipolar disease, schizophrenia, and major depression, and a haplotype located in an intronic region of the L-type voltage gated calcium channel (VGCC) subunit gene CACNA1C (peak associated SNP rs1006737), making it one of the most replicable and consistent associations in psychiatric genetics. In the current study, we used induced human neurons to reveal a functional phenotype associated with this psychiatric risk variant. We generated induced human neurons, or iN cells, from more than 20 individuals harboring homozygous risk genotypes, heterozygous, or homozygous non-risk genotypes at the rs1006737 locus. Using these iNs, we performed electrophysiology and quantitative PCR experiments that demonstrated increased L-type VGCC current density as well as increased mRNA expression of CACNA1C in induced neurons homozygous for the risk genotype, compared to non-risk genotypes. These studies demonstrate that the risk genotype at rs1006737 is associated with significant functional alterations in human induced neurons, and may direct future efforts at developing novel therapeutics for the treatment of psychiatric disease. PMID:25403839

  6. Functional implications of a psychiatric risk variant within CACNA1C in induced human neurons.

    PubMed

    Yoshimizu, T; Pan, J Q; Mungenast, A E; Madison, J M; Su, S; Ketterman, J; Ongur, D; McPhie, D; Cohen, B; Perlis, R; Tsai, L-H

    2015-02-01

    Psychiatric disorders have clear heritable risk. Several large-scale genome-wide association studies have revealed a strong association between susceptibility for psychiatric disorders, including bipolar disease, schizophrenia and major depression, and a haplotype located in an intronic region of the L-type voltage-gated calcium channel (VGCC) subunit gene CACNA1C (peak associated SNP rs1006737), making it one of the most replicable and consistent associations in psychiatric genetics. In the current study, we used induced human neurons to reveal a functional phenotype associated with this psychiatric risk variant. We generated induced human neurons, or iN cells, from more than 20 individuals harboring homozygous risk genotypes, heterozygous or homozygous non-risk genotypes at the rs1006737 locus. Using these iNs, we performed electrophysiology and quantitative PCR experiments that demonstrated increased L-type VGCC current density as well as increased mRNA expression of CACNA1C in iNs homozygous for the risk genotype, compared with non-risk genotypes. These studies demonstrate that the risk genotype at rs1006737 is associated with significant functional alterations in human iNs, and may direct future efforts at developing novel therapeutics for the treatment of psychiatric disease. PMID:25403839

  7. Characterization of functional methylomes by next-generation capture sequencing identifies novel disease-associated variants

    PubMed Central

    Allum, Fiona; Shao, Xiaojian; Guénard, Frédéric; Simon, Marie-Michelle; Busche, Stephan; Caron, Maxime; Lambourne, John; Lessard, Julie; Tandre, Karolina; Hedman, Åsa K.; Kwan, Tony; Ge, Bing; Rönnblom, Lars; McCarthy, Mark I.; Deloukas, Panos; Richmond, Todd; Burgess, Daniel; Spector, Timothy D.; Tchernof, André; Marceau, Simon; Lathrop, Mark; Vohl, Marie-Claude; Pastinen, Tomi; Grundberg, Elin; Ahmadi, Kourosh R.; Ainali, Chrysanthi; Barrett, Amy; Bataille, Veronique; Bell, Jordana T.; Buil, Alfonso; Dermitzakis, Emmanouil T.; Dimas, Antigone S.; Durbin, Richard; Glass, Daniel; Hassanali, Neelam; Ingle, Catherine; Knowles, David; Krestyaninova, Maria; Lindgren, Cecilia M.; Lowe, Christopher E.; Meduri, Eshwar; di Meglio, Paola; Min, Josine L.; Montgomery, Stephen B.; Nestle, Frank O.; Nica, Alexandra C.; Nisbet, James; O'Rahilly, Stephen; Parts, Leopold; Potter, Simon; Sandling, Johanna; Sekowska, Magdalena; Shin, So-Youn; Small, Kerrin S.; Soranzo, Nicole; Surdulescu, Gabriela; Travers, Mary E.; Tsaprouni, Loukia; Tsoka, Sophia; Wilk, Alicja; Yang, Tsun-Po; Zondervan, Krina T.

    2015-01-01

    Most genome-wide methylation studies (EWAS) of multifactorial disease traits use targeted arrays or enrichment methodologies preferentially covering CpG-dense regions, to characterize sufficiently large samples. To overcome this limitation, we present here a new customizable, cost-effective approach, methylC-capture sequencing (MCC-Seq), for sequencing functional methylomes, while simultaneously providing genetic variation information. To illustrate MCC-Seq, we use whole-genome bisulfite sequencing on adipose tissue (AT) samples and public databases to design AT-specific panels. We establish its efficiency for high-density interrogation of methylome variability by systematic comparisons with other approaches and demonstrate its applicability by identifying novel methylation variation within enhancers strongly correlated to plasma triglyceride and HDL-cholesterol, including at CD36. Our more comprehensive AT panel assesses tissue methylation and genotypes in parallel at ∼4 and ∼3 M sites, respectively. Our study demonstrates that MCC-Seq provides comparable accuracy to alternative approaches but enables more efficient cataloguing of functional and disease-relevant epigenetic and genetic variants for large-scale EWAS. PMID:26021296

  8. Novel application of luciferase assay for the in vitro functional assessment of KAL1 variants in three females with septo-optic dysplasia (SOD)

    PubMed Central

    McCabe, Mark J.; Hu, Youli; Gregory, Louise C.; Gaston-Massuet, Carles; Alatzoglou, Kyriaki S.; Saldanha, José W.; Gualtieri, Angelica; Thankamony, Ajay; Hughes, Ieuan; Townshend, Sharron; Martinez-Barbera, Juan-Pedro; Bouloux, Pierre-Marc; Dattani, Mehul T.

    2015-01-01

    KAL1 is implicated in 5% of Kallmann syndrome cases, a disorder which genotypically overlaps with septo-optic dysplasia (SOD). To date, a reporter-based assay to assess the functional consequences of KAL1 mutations is lacking. We aimed to develop a luciferase assay for novel application to functional assessment of rare KAL1 mutations detected in a screen of 422 patients with SOD. Quantitative analysis was performed using L6-myoblasts stably expressing FGFR1, transfected with a luciferase-reporter vector containing elements of the FGF-responsive osteocalcin promoter. The two variants assayed [p.K185N, p.P291T], were detected in three females with SOD (presenting with optic nerve hypoplasia, midline and pituitary defects). Our novel assay revealed significant decreases in transcriptional activity [p.K185N: 21% (p < 0.01); p.P291T: 40% (p < 0.001)]. Our luciferase-reporter assay, developed for assessment of KAL1 mutations, determined that two variants in females with hypopituitarism/SOD are loss-of-function; demonstrating that this assay is suitable for quantitative assessment of mutations in this gene. PMID:26375424

  9. Deleterious Rare Variants Reveal Risk for Loss of GABAA Receptor Function in Patients with Genetic Epilepsy and in the General Population.

    PubMed

    Hernandez, Ciria C; Klassen, Tara L; Jackson, Laurel G; Gurba, Katharine; Hu, Ningning; Noebels, Jeffrey L; Macdonald, Robert L

    2016-01-01

    Genetic epilepsies (GEs) account for approximately 50% of all seizure disorders, and familial forms include mutations in single GABAA receptor subunit genes (GABRs). In 144 sporadic GE cases (GECs), exome sequencing of 237 ion channel genes identified 520 GABR variants. Among these variants, 33 rare variants in 11 GABR genes were present in 24 GECs. To assess functional risk of variants in GECs, we selected 8 variants found in GABRA, 3 in GABRB, and 3 in GABRG and compared them to 18 variants found in the general population for GABRA1 (n = 9), GABRB3 (n = 7), and GABRG2 (n = 2). To identify deleterious variants and gain insight into structure-function relationships, we studied the gating properties, surface expression and structural perturbations of the 32 variants. Significant reduction of GABAA receptor function was strongly associated with variants scored as deleterious and mapped within the N-terminal and transmembrane domains. In addition, 12 out of 17 variants mapped along the β+/α- GABA binding interface, were associated with reduction in channel gating and were predicted to cause structural rearrangements of the receptor by in silico simulations. Missense or nonsense mutations of GABRA1, GABRB3 and GABRG2 primarily impair subunit biogenesis. In contrast, GABR variants affected receptor function by impairing gating, suggesting that different mechanisms are operating in GABR epilepsy susceptibility variants and disease-causing mutations. The functional impact of single GABR variants found in individuals with sporadic GEs warrants the use of molecular diagnosis and will ultimately improve the treatment of genetic epilepsies by using a personalized approach. PMID:27622563

  10. Disentangling the Effects of Colocalizing Genomic Annotations to Functionally Prioritize Non-coding Variants within Complex-Trait Loci

    PubMed Central

    Trynka, Gosia; Westra, Harm-Jan; Slowikowski, Kamil; Hu, Xinli; Xu, Han; Stranger, Barbara E.; Klein, Robert J.; Han, Buhm; Raychaudhuri, Soumya

    2015-01-01

    Identifying genomic annotations that differentiate causal from trait-associated variants is essential to fine mapping disease loci. Although many studies have identified non-coding functional annotations that overlap disease-associated variants, these annotations often colocalize, complicating the ability to use these annotations for fine mapping causal variation. We developed a statistical approach (Genomic Annotation Shifter [GoShifter]) to assess whether enriched annotations are able to prioritize causal variation. GoShifter defines the null distribution of an annotation overlapping an allele by locally shifting annotations; this approach is less sensitive to biases arising from local genomic structure than commonly used enrichment methods that depend on SNP matching. Local shifting also allows GoShifter to identify independent causal effects from colocalizing annotations. Using GoShifter, we confirmed that variants in expression quantitative trail loci drive gene-expression changes though DNase-I hypersensitive sites (DHSs) near transcription start sites and independently through 3′ UTR regulation. We also showed that (1) 15%–36% of trait-associated loci map to DHSs independently of other annotations; (2) loci associated with breast cancer and rheumatoid arthritis harbor potentially causal variants near the summits of histone marks rather than full peak bodies; (3) variants associated with height are highly enriched in embryonic stem cell DHSs; and (4) we can effectively prioritize causal variation at specific loci. PMID:26140449

  11. Functional analysis and transcriptional regulation of porcine six transmembrane epithelial antigen of prostate 4 (STEAP4) gene and its novel variant in hepatocytes.

    PubMed

    Wang, S B; Lei, T; Zhou, L L; Zheng, H L; Zeng, C P; Liu, N; Yang, Z Q; Chen, X D

    2013-03-01

    Six-transmembrane epithelial antigen of prostate 4 (STEAP4) plays a critical role in modulating inflammatory response and protecting metabolic function. However, the role of STEAP4 in hepatocytes is not well understood, and the mechanism of STEAP4 action remains elusive. Here, we report the molecular characterization of porcine STEAP4 and its novel splice variant (STEAP4v), then the metabolic and anti-inflammatory roles of porcine STEAP4 and STEAP4v were investigated in HepG2 liver cells. The results revealed that overexpression of STEAP4, but not STEAP4v, suppresses triglyceride (TG) content and ameliorates the up-regulation of the transcription of the genes necessary for de novo lipogenesis and gluconeogenesis elicited by FFAs treatment. In RAW264.7 macrophage cells, transient transfection of STEAP4v, to a greater extent than STEAP4, repressed the transcription of TNFα and IL-6. In HepG2 cells with LPS treatment, the endogenous mRNA and protein levels of STEAP4 and STEAP4v were up-regulated, which was accompanied by a concurrent increase in C/EBPβ mRNA and protein levels. Thirdly, the functional regulation of STEAP4 was explored, which revealed that the porcine STEAP4 promoter activity was significantly up-regulated by C/EBPβ. The progressive deletions and mutations demonstrated that the C/EBPβ binding motif situated at -73/-59 bp is an essential component required for promoter activity of STEAP4 gene. Chromatin immunoprecipitation (ChIP) assays determined that C/EBPβ can directly interact with the steap4 promoter DNA. In conclusion, our data demonstrated that C/EBPβ directly regulates the roles of STEAP4 and its novel variant in attenuating lipogenesis, gluconeogenesis or/and inflammation elicited by FFAs or LPS in hepatocytes. PMID:23262293

  12. Associations of functional alanine-glyoxylate aminotransferase 2 gene variants with atrial fibrillation and ischemic stroke.

    PubMed

    Seppälä, Ilkka; Kleber, Marcus E; Bevan, Steve; Lyytikäinen, Leo-Pekka; Oksala, Niku; Hernesniemi, Jussi A; Mäkelä, Kari-Matti; Rothwell, Peter M; Sudlow, Cathie; Dichgans, Martin; Mononen, Nina; Vlachopoulou, Efthymia; Sinisalo, Juha; Delgado, Graciela E; Laaksonen, Reijo; Koskinen, Tuomas; Scharnagl, Hubert; Kähönen, Mika; Markus, Hugh S; März, Winfried; Lehtimäki, Terho

    2016-01-01

    Asymmetric and symmetric dimethylarginines (ADMA and SDMA) impair nitric oxide bioavailability and have been implicated in the pathogenesis of atrial fibrillation (AF). Alanine-glyoxylate aminotransferase 2 (AGXT2) is the only enzyme capable of metabolizing both of the dimethylarginines. We hypothesized that two functional AGXT2 missense variants (rs37369, V140I; rs16899974, V498L) are associated with AF and its cardioembolic complications. Association analyses were conducted using 1,834 individulas with AF and 7,159 unaffected individuals from two coronary angiography cohorts and a cohort comprising patients undergoing clinical exercise testing. In coronary angiography patients without structural heart disease, the minor A allele of rs16899974 was associated with any AF (OR = 2.07, 95% CI 1.59-2.68), and with paroxysmal AF (OR = 1.98, 95% CI 1.44-2.74) and chronic AF (OR = 2.03, 95% CI 1.35-3.06) separately. We could not replicate the association with AF in the other two cohorts. However, the A allele of rs16899974 was nominally associated with ischemic stroke risk in the meta-analysis of WTCCC2 ischemic stroke cohorts (3,548 cases, 5,972 controls) and with earlier onset of first-ever ischemic stroke (360 cases) in the cohort of clinical exercise test patients. In conclusion, AGXT2 variations may be involved in the pathogenesis of AF and its age-related thromboembolic complications. PMID:26984639

  13. Compensating for velocity truncation during subaperture polishing by controllable and time-variant tool influence functions.

    PubMed

    Dong, Zhichao; Cheng, Haobo; Tam, Hon-Yuen

    2015-02-10

    The velocity-varying regime used in deterministic subaperture polishing employs a time-invariant tool influence function (TIF) to figure localized surface errors by varying the transverse velocities of polishing tools. Desired transverse velocities have to be truncated if they exceed the maximal velocity of computer numerical control (CNC) machines, which induces excessive material removal and reduces figuring efficiency (FE). A time-variant (TV) TIF regime is presented, in which a TIF serves as a variable to compensate for excessive material removal when the transverse velocities are truncated. Compared with other methods, the TV-TIF regime exhibits better performance in terms of convergence rate, FE, and versatility; its operability can also be strengthened by a TIF library. Comparative experiments were conducted on a magnetorheological finishing machine to validate the effectiveness of the TV-TIF regime. Without a TV-TIF, the tool made an unwished dent (depth of 76 nm) at the center because of the velocity truncation problem. Through compensation with a TV-TIF, the dent was completely removed by the second figuring process, and a TV-TIF improved the FE from 0.029 to 0.066  mm(3)/h. PMID:25968037

  14. Generation of Functional Fluorescent BK Channels by Random Insertion of GFP Variants

    PubMed Central

    Giraldez, Teresa; Hughes, Thomas E.; Sigworth, Fred J.

    2005-01-01

    The yellow and cyan variants of green fluorescent protein (GFP) constitute an excellent pair for fluorescence resonance energy transfer (FRET) and can be used to study conformational rearrangements of proteins. Our aim was to develop a library of fluorescent large conductance voltage- and Ca2+-gated channels (BK or slo channels) for future use in FRET studies. We report the results of a random insertion of YFP and CFP into multiple sites of the α subunit of the hslo channel using a Tn5 transposon-based technique. 55 unique fluorescent fusion proteins were obtained and tested for cell surface expression and channel function. 19 constructs are expressed at the plasma membrane and show voltage and Ca2+-dependent currents. In 16 of them the voltage and Ca2+ dependence is very similar to the wild-type channel. Two insertions in the Ca2+ bowl and one in the RCK2 domain showed a strong shift in the G-V curve. The remaining 36 constructs were retained intracellularly; a solubility assay suggests that these proteins are not forming intracellular aggregates. The “success rate” of 19 out of 55 hslo insertion constructs compares very favorably with other studies of random GFP fusions. PMID:16260837

  15. The catecholamine biosynthetic enzyme dopamine β-hydroxylase (DBH): first genome-wide search positions trait-determining variants acting additively in the proximal promoter

    PubMed Central

    Mustapic, Maja; Maihofer, Adam X.; Mahata, Manjula; Chen, Yuqing; Baker, Dewleen G.; O'Connor, Daniel T.; Nievergelt, Caroline M.

    2014-01-01

    Dopamine beta-hydroxylase (DBH) is the biosynthetic enzyme catalyzing formation of norepinephrine. Changes in DBH expression or activity have been implicated in the pathogenesis of cardiovascular and neuropsychiatric disorders. Genetic determination of DBH enzymatic activity and its secretion are only incompletely understood. We began with a genome-wide association search for loci contributing to DBH activity in human plasma. Initially, in a population sample of European ancestry, we identified the proximal DBH promoter as a region harboring three common trait-determining variants (top hit rs1611115, P = 7.2 × 10−51). We confirmed their effects on transcription and showed that the three variants each acted additively on gene expression. Results were replicated in a population sample of Native American descent (top hit rs1611115, P = 4.1 × 10−15). Jointly, DBH variants accounted for 57% of DBH trait variation. We further identified a genome-wide significant SNP at the LOC338797 locus on chromosome 12 as trans-quantitative trait locus (QTL) (rs4255618, P = 4.62 × 10−8). Conditional analyses on DBH identified a third genomic region contributing to DBH variation: a likely cis-QTL adjacent to DBH in SARDH (rs7040170, P = 1.31 × 10−14) on chromosome 9q. We conclude that three common SNPs in the DBH promoter act additively to control phenotypic variation in DBH levels, and that two additional novel loci (SARDH and LOC338797) may also contribute to the expression of this catecholamine biosynthetic trait. Identification of DBH variants with strong effects makes it possible to take advantage of Mendelian randomization approaches to test causal effects of this intermediate trait on disease. PMID:24986918

  16. Identification and in vivo functional characterization of novel compound heterozygous BMP1 variants in osteogenesis imperfecta.

    PubMed

    Cho, Sung Yoon; Asharani, P V; Kim, Ok-Hwa; Iida, Aritoshi; Miyake, Noriko; Matsumoto, Naomichi; Nishimura, Gen; Ki, Chang-Seok; Hong, Geehay; Kim, Su Jin; Sohn, Young Bae; Park, Sung Won; Lee, Jieun; Kwun, Younghee; Carney, Thomas J; Huh, Rimm; Ikegawa, Shiro; Jin, Dong-Kyu

    2015-02-01

    Osteogenesis imperfecta (OI) comprises a heterogeneous group of disorders that are characterized by susceptibility to bone fractures, and range in severity from a subtle increase in fracture frequency to death in the perinatal period. Most patients have defects in type I collagen biosynthesis with autosomal-dominant inheritance, but many autosomal-recessive genes have been reported. We applied whole-exome sequencing to identify mutations in a Korean OI patient who had an umbilical hernia, frequent fractures, a markedly short stature, delayed motor development, scoliosis, and dislocation of the radial head, with a bowed radius and ulna. We identified two novel variants in the BMP1 gene: c.808A>G and c.1297G>T. The former variant caused a missense change p.(Met270Val) and the latter variant caused the skipping of exon 10. The hypofunctional nature of the two variants was demonstrated in a zebrafish assay. PMID:25402547

  17. The rare DAT coding variant Val559 perturbs DA neuron function, changes behavior, and alters in vivo responses to psychostimulants

    PubMed Central

    Mergy, Marc A.; Gowrishankar, Raajaram; Gresch, Paul J.; Gantz, Stephanie C.; Williams, John; Davis, Gwynne L.; Wheeler, C. Austin; Stanwood, Gregg D.; Hahn, Maureen K.; Blakely, Randy D.

    2014-01-01

    Despite the critical role of the presynaptic dopamine (DA) transporter (DAT, SLC6A3) in DA clearance and psychostimulant responses, evidence that DAT dysfunction supports risk for mental illness is indirect. Recently, we identified a rare, nonsynonymous Slc6a3 variant that produces the DAT substitution Ala559Val in two male siblings who share a diagnosis of attention-deficit hyperactivity disorder (ADHD), with other studies identifying the variant in subjects with bipolar disorder (BPD) and autism spectrum disorder (ASD). Previously, using transfected cell studies, we observed that although DAT Val559 displays normal total and surface DAT protein levels, and normal DA recognition and uptake, the variant transporter exhibits anomalous DA efflux (ADE) and lacks capacity for amphetamine (AMPH)-stimulated DA release. To pursue the significance of these findings in vivo, we engineered DAT Val559 knock-in mice, and here we demonstrate in this model the presence of elevated extracellular DA levels, altered somatodendritic and presynaptic D2 DA receptor (D2R) function, a blunted ability of DA terminals to support depolarization and AMPH-evoked DA release, and disruptions in basal and psychostimulant-evoked locomotor behavior. Together, our studies demonstrate an in vivo functional impact of the DAT Val559 variant, providing support for the ability of DAT dysfunction to impact risk for mental illness. PMID:25331903

  18. A Germline Variant in the PANX1 Gene Has Reduced Channel Function and Is Associated with Multisystem Dysfunction.

    PubMed

    Shao, Qing; Lindstrom, Kristin; Shi, Ruoyang; Kelly, John; Schroeder, Audrey; Juusola, Jane; Levine, Kara L; Esseltine, Jessica L; Penuela, Silvia; Jackson, Michael F; Laird, Dale W

    2016-06-10

    Pannexin1 (PANX1) is probably best understood as an ATP release channel involved in paracrine signaling. Given its ubiquitous expression, PANX1 pathogenic variants would be expected to lead to disorders involving multiple organ systems. Using whole exome sequencing, we discovered the first patient with a homozygous PANX1 variant (c.650G→A) resulting in an arginine to histidine substitution at position 217 (p.Arg217His). The 17-year-old female has intellectual disability, sensorineural hearing loss requiring bilateral cochlear implants, skeletal defects, including kyphoscoliosis, and primary ovarian failure. Her consanguineous parents are each heterozygous for this variant but are not affected by the multiorgan syndromes noted in the proband. Expression of the p.Arg217His mutant in HeLa, N2A, HEK293T, and Ad293 cells revealed normal PANX1 glycosylation and cell surface trafficking. Dye uptake, ATP release, and electrophysiological measurements revealed p.Arg217His to be a loss-of-function variant. Co-expression of the mutant with wild-type PANX1 suggested the mutant was not dominant-negative to PANX1 channel function. Collectively, we demonstrate a PANX1 missense change associated with human disease in the first report of a "PANX1-related disorder." PMID:27129271

  19. The rare DAT coding variant Val559 perturbs DA neuron function, changes behavior, and alters in vivo responses to psychostimulants.

    PubMed

    Mergy, Marc A; Gowrishankar, Raajaram; Gresch, Paul J; Gantz, Stephanie C; Williams, John; Davis, Gwynne L; Wheeler, C Austin; Stanwood, Gregg D; Hahn, Maureen K; Blakely, Randy D

    2014-11-01

    Despite the critical role of the presynaptic dopamine (DA) transporter (DAT, SLC6A3) in DA clearance and psychostimulant responses, evidence that DAT dysfunction supports risk for mental illness is indirect. Recently, we identified a rare, nonsynonymous Slc6a3 variant that produces the DAT substitution Ala559Val in two male siblings who share a diagnosis of attention-deficit hyperactivity disorder (ADHD), with other studies identifying the variant in subjects with bipolar disorder (BPD) and autism spectrum disorder (ASD). Previously, using transfected cell studies, we observed that although DAT Val559 displays normal total and surface DAT protein levels, and normal DA recognition and uptake, the variant transporter exhibits anomalous DA efflux (ADE) and lacks capacity for amphetamine (AMPH)-stimulated DA release. To pursue the significance of these findings in vivo, we engineered DAT Val559 knock-in mice, and here we demonstrate in this model the presence of elevated extracellular DA levels, altered somatodendritic and presynaptic D2 DA receptor (D2R) function, a blunted ability of DA terminals to support depolarization and AMPH-evoked DA release, and disruptions in basal and psychostimulant-evoked locomotor behavior. Together, our studies demonstrate an in vivo functional impact of the DAT Val559 variant, providing support for the ability of DAT dysfunction to impact risk for mental illness. PMID:25331903

  20. Impact of protective IL-2 allelic variants on CD4+ Foxp3+ regulatory T cell function in situ and resistance to autoimmune diabetes in NOD mice.

    PubMed

    Sgouroudis, Evridiki; Albanese, Alexandre; Piccirillo, Ciriaco A

    2008-11-01

    Type I diabetes (T1D) susceptibility is inherited through multiple insulin-dependent diabetes (Idd) genes. NOD.B6 Idd3 congenic mice, introgressed with an Idd3 allele from T1D-resistant C57BL/6 mice (Idd3(B6)), show a marked resistance to T1D compared with control NOD mice. The protective function of the Idd3 locus is confined to the Il2 gene, whose expression is critical for naturally occurring CD4(+)Foxp3(+) regulatory T (nT(reg)) cell development and function. In this study, we asked whether Idd3(B6) protective alleles in the NOD mouse model confer T1D resistance by promoting the cellular frequency, function, or homeostasis of nT(reg) cells in vivo. We show that resistance to T1D in NOD.B6 Idd3 congenic mice correlates with increased levels of IL-2 mRNA and protein production in Ag-activated diabetogenic CD4(+) T cells. We also observe that protective IL2 allelic variants (Idd3(B6) resistance allele) also favor the expansion and suppressive functions of CD4(+)Foxp3(+) nT(reg) cells in vitro, as well as restrain the proliferation, IL-17 production, and pathogenicity of diabetogenic CD4(+) T cells in vivo more efficiently than control do nT(reg) cells. Lastly, the resistance to T1D in Idd3 congenic mice does not correlate with an augmented systemic frequency of CD4(+)Foxp3(+) nT(reg) cells but more so with the ability of protective IL2 allelic variants to promote the expansion of CD4(+)Foxp3(+) nT(reg) cells directly in the target organ undergoing autoimmune attack. Thus, protective, IL2 allelic variants impinge the development of organ-specific autoimmunity by bolstering the IL-2 producing capacity of self-reactive CD4(+) T cells and, in turn, favor the function and homeostasis of CD4(+)Foxp3(+) nT(reg) cells in vivo. PMID:18941219

  1. Plant growth-promoting rhizobacteria and root system functioning

    PubMed Central

    Vacheron, Jordan; Desbrosses, Guilhem; Bouffaud, Marie-Lara; Touraine, Bruno; Moënne-Loccoz, Yvan; Muller, Daniel; Legendre, Laurent; Wisniewski-Dyé, Florence; Prigent-Combaret, Claire

    2013-01-01

    The rhizosphere supports the development and activity of a huge and diversified microbial community, including microorganisms capable to promote plant growth. Among the latter, plant growth-promoting rhizobacteria (PGPR) colonize roots of monocots and dicots, and enhance plant growth by direct and indirect mechanisms. Modification of root system architecture by PGPR implicates the production of phytohormones and other signals that lead, mostly, to enhanced lateral root branching and development of root hairs. PGPR also modify root functioning, improve plant nutrition and influence the physiology of the whole plant. Recent results provided first clues as to how PGPR signals could trigger these plant responses. Whether local and/or systemic, the plant molecular pathways involved remain often unknown. From an ecological point of view, it emerged that PGPR form coherent functional groups, whose rhizosphere ecology is influenced by a myriad of abiotic and biotic factors in natural and agricultural soils, and these factors can in turn modulate PGPR effects on roots. In this paper, we address novel knowledge and gaps on PGPR modes of action and signals, and highlight recent progress on the links between plant morphological and physiological effects induced by PGPR. We also show the importance of taking into account the size, diversity, and gene expression patterns of PGPR assemblages in the rhizosphere to better understand their impact on plant growth and functioning. Integrating mechanistic and ecological knowledge on PGPR populations in soil will be a prerequisite to develop novel management strategies for sustainable agriculture. PMID:24062756

  2. Association of variants in genes involved in pancreatic β-cell development and function with type 2 diabetes in North Indians.

    PubMed

    Chavali, Sreenivas; Mahajan, Anubha; Tabassum, Rubina; Dwivedi, Om Prakash; Chauhan, Ganesh; Ghosh, Saurabh; Tandon, Nikhil; Bharadwaj, Dwaipayan

    2011-10-01

    Variants in genes involved in pancreatic β-cell development and function are known to cause monogenic forms of type 2 diabetes and are also associated with complex form. In this study, we studied the genetic association of polymorphisms in such important genes with type 2 diabetes in the high-risk Indians. We genotyped 91 polymorphisms in 19 genes (ABCC8, HNF1A, HNF1B, HNF4A, INS, INSM1, ISL1, KCNJ11, MAFA, MNX1, NEUROD1, NEUROG3, NKX2.2, NKX6.1, PAX4, PAX6, PDX1, USF1 and WFS1) in 2025 unrelated North Indians of Indo-European ethnicity comprising of 1019 diabetic and 1006 non-diabetic subjects. HNF4A promoter P2 polymorphisms rs1884613 and rs2144908, which are in high linkage disequilibrium, showed significant association with type 2 diabetes (odds ratio (OR)=1.37 (95% confidence interval (CI) 1.19-1.57), P=9.4 × 10(-6) for rs1884613 and OR=1.37 (95%CI 1.20-1.57), P=6.0 × 10(-6) for rs2144908), as previously shown in other populations. We observed body mass index-dependent association of these variants with type 2 diabetes in normal-weight/lean subjects. Variants in USF1, ABCC8, ISL1 and KCNJ11 showed nominal association, while haplotypes in these genes were significantly associated. rs3812704 upstream of NEUROG3 significantly increased risk for type 2 diabetes in normal-weight/lean subjects (OR=1.68 (95%CI 1.25-2.24), P=4.9 × 10(-4)). Thus, pancreatic β-cell development and function genes contribute to susceptibility to type 2 diabetes in North Indians. PMID:21814221

  3. Functional characterization and molecular mechanism exploration of three granulin epithelin precursor splice variants in biomineralization of the pearl oyster Pinctada fucata.

    PubMed

    Zhao, Mi; He, Maoxian; Huang, Xiande; Wang, Qi; Shi, Yu

    2016-02-01

    The granulin/epithelin precursor (GEP) encodes a glycoprotein precursor which exhibits pleiotropic tissue growth factor activity with multiple functions. Here, GEP was isolated and its role in the shell biomineralization process of the pearl oyster Pinctada fucata was investigated. Three forms of GEP mRNA were isolated from the pearl oyster (designated PfGEP-1, PfGEP-2 and PfGEP-3). Genomic DNA flanking the splicing region of the PfGEP variants was sequenced and it was found that PfGEP-2 splices out Exon 4, whereas PfGEP-3 splices out Exon 3 compared to PfGEP-1. PfGEP-1 (1505 amino acids) consists of 18 granulin domains, whereas PfGEP-2 (1459 amino acids) and PfGEP-3 (1471 amino acids) consist of 17.5 granulin domains, respectively. Analyses of PfGEP-1 and PfGEP-3 mRNA showed differential patterns in the tissues and developmental stages. Western blotting results showed that the three splice variants can translate to proteins in HEK293T cells. A knockdown experiment using PfGEP dsRNA showed decreased PfGEP-1/PfGEP-3 and PfMSX mRNA, and irregular crystallization of the nacreous layer using scanning electron microscopy. In luciferase assays, co-transfection of PfGEP-1 could activate as well as repress luciferase expression of the reporter plasmid driven by the PfMSX promoter, whereas PfGEP-3 stimulated the expression, elucidating the molecular mechanisms involved in the correlation between PfGEP and PfMSX. These results suggested that GEP variants might function differently during the biomineralization process, which provides new knowledge on the mechanism regulating nacre formation. PMID:26388260

  4. Impaired coactivator activity of the Gly{sub 482} variant of peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) on mitochondrial transcription factor A (Tfam) promoter

    SciTech Connect

    Choi, Yon-Sik; Hong, Jung-Man; Lim, Sunny; Ko, Kyung Soo; Pak, Youngmi Kim . E-mail: ymkimpak@amc.seoul.kr

    2006-06-09

    Mitochondrial dysfunction may cause diabetes or insulin resistance. Peroxisome proliferation-activated receptor-{gamma} (PPAR-{gamma}) coactivator-1 {alpha} (PGC-1{alpha}) increases mitochondrial transcription factor A (Tfam) resulting in mitochondrial DNA content increase. An association between a single nucleotide polymorphism (SNP), G1444A(Gly482Ser), of PGC-1{alpha} coding region and insulin resistance has been reported in some ethnic groups. In this study, we investigated whether a change of glycine to serine at codon 482 of PGC-1{alpha} affected the Tfam promoter activity. The cDNA of PGC-1{alpha} variant bearing either glycine or serine at 482 codon was transfected into Chang human hepatocyte cells. The PGC-1{alpha} protein bearing glycine had impaired coactivator activity on Tfam promoter-mediated luciferase. We analyzed the PGC-1{alpha} genotype G1444A and mitochondrial DNA (mtDNA) copy number from 229 Korean leukocyte genomic DNAs. Subjects with Gly/Gly had a 20% lower amount of peripheral blood mtDNA than did subjects with Gly/Ser and Ser/Ser (p < 0.05). No correlation was observed between diabetic parameters and PGC-1{alpha} genotypes in Koreans. These results suggest that PGC-1{alpha} variants with Gly/Gly at 482nd amino acid may impair the Tfam transcription, a regulatory function of mitochondrial biogenesis, resulting in dysfunctional mtDNA replication.

  5. Promoter variants in interleukin-6 and tumor necrosis factor alpha and risk of coronary artery disease in a population from Western India

    PubMed Central

    Bhanushali, Aparna A; Das, B R

    2013-01-01

    INTRODUCTION: A central component of the atherosclerotic process is inflammation. Single nucleotide polymorphisms (SNPs) present in the promoter region of various cytokines can lead to altered levels of the transcript and a state of low-grade inflammation exacerbating the risk of coronary artery disease (CAD). The present work tries to understand the role of permissive promoter variants in the interleukin-6 gene (IL-6-174G/C) and the tumor necrosis factor alpha (TNFα-308G/A) in the causation of CAD and also dyslipidemia. MATERIALS AND METHODS: Genotyping was conducted on 100 cases of CAD and 150 controls by the allele termination assay SNaPshot. Biochemical parameters were determined by routine enzymatic endpoint methods. The results were analyzed by appropriate statistical methods. RESULTS: No differences in the minor allele frequency IL-6-174G/C SNP were seen between cases and controls (0.13 vs. 0.12). The differences in the allele frequency of TNFα-308A between cases (6%) and controls (2%) have led to an odds ratio, 3.370; 95% confidence interval, 1.039-11.543; P=0.033 in the univariate analysis. In the final logistic regression analysis, however none of the variants were associated with an increased risk of CAD. CONCLUSIONS: In summary, no association of the permissive promoter variants in the IL-6 gene and the TNFα gene were seen with an increased CAD risk. These and other studies highlight the importance of doing population specific studies. PMID:24497708

  6. Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis

    PubMed Central

    Lewis, Michele D.; Park, Hyun Woo; Brand, Randall E.; Gelrud, Andres; Anderson, Michelle A.; Banks, Peter A.; Conwell, Darwin; Lawrence, Christopher; Romagnuolo, Joseph; Baillie, John; Alkaade, Samer; Cote, Gregory; Gardner, Timothy B.; Amann, Stephen T.; Slivka, Adam; Sandhu, Bimaljit; Aloe, Amy; Kienholz, Michelle L.; Yadav, Dhiraj; Barmada, M. Michael; Bahar, Ivet; Lee, Min Goo; Whitcomb, David C.

    2014-01-01

    CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR

  7. Examining quality function deployment in safety promotion in Sweden.

    PubMed

    Kullberg, Agneta; Nordqvist, Cecilia; Lindqvist, Kent; Timpka, Toomas

    2014-09-01

    The first-hand needs and demands of laypersons are not always considered when safety promotion programmes are being developed. We compared focal areas for interventions identified from residents' statements of safety needs with focal areas for interventions identified by local government professionals in a Swedish urban community certified by the international Safe Community movement supported by the World Health Organization. Quantitative and qualitative data on self-expressed safety needs from 787 housing residents were transformed into an intervention design, using the quality function deployment (QFD) technique and compared with the safety intervention programme developed by professionals at the municipality administrative office. The outcome of the comparison was investigated with regard to implications for the Safe Community movement. The QFD analysis identified the initiation and maintenance of social integrative processes in housing areas as the most highly prioritized interventions among the residents, but failed to highlight the safety needs of several vulnerable groups (the elderly, infants and persons with disabilities). The intervention programme designed by the public health professionals did not address the social integrative processes, but it did highlight the vulnerable groups. This study indicates that the QFD technique is suitable for providing residential safety promotion efforts with a quality orientation from the layperson's perspective. Views of public health professionals have to be included to ascertain that the needs of socially deprived residents are adequately taken into account. QFD can augment the methodological toolbox for safety promotion programmes, including interventions in residential areas. PMID:23322486

  8. In-vitro characterization of novel and functional regulatory SNPs in the promoter region of IL2 and IL2R alpha in a Gabonese population

    PubMed Central

    2012-01-01

    Background The selection pressure imposed by the parasite has a functional consequence on the immune genes, leading to altered immune function in which regulatory T cells (Tregs) induced by parasites during infectious challenges modulate or thwart T effector cell mechanism. Methods We identified and investigated regulatory polymorphisms in the immune gene IL2 and its receptor IL2R alpha (also known as CD25) in Gabonese individuals exposed to plentiful parasitic infections. Results We identified two reported variants each for IL2 and its receptor IL2R alpha gene loci. Also identified were two novel variants, -83 /-84 CT deletions (ss410961576) for IL2 and -409C/T (ss410961577) for IL2R alpha. We further validated all identified promoter variants for their allelic gene expression using transient transfection assays. Three promoter variants of the IL2 locus revealed no significant expression of the reporter gene. The identified novel variant (ss410961577C/T) of the IL2R alpha revealed a significant higher expression of the reporter gene in comparison to the major allele (P<0.05). In addition, the rs12722616C/T variant of the IL2R alpha locus altered the transcription factor binding site TBP (TATA box binding protein) and C/EBP beta (CCAAT/enhancer binding protein beta) that are believed to regulate the Treg function. Conclusions The identification and validation of such regulatory polymorphisms in the immune genes may provide a basis for future studies on parasite susceptibility in a population where T cell functions are compromised. PMID:23217119

  9. A role for coding functional variants in HNF4A in Type 2 Diabetes susceptibility

    PubMed Central

    Jafar-Mohammadi, B; Groves, C J; Gjesing, A P; Herrera, B M; Winckler, W; Stringham, H M; Morris, A P; Lauritzen, T; Doney, A S F; Morris, A D; Weedon, M N; Swift, A J; Kuusisto, J; Laakso, M; Altshuler, D; Hattersley, A T; Collins, F S; Boehnke, M; Hansen, T; Pedersen, O; Palmer, C N A; Frayling, T M; Gloyn, A L; McCarthy, M I

    2011-01-01

    Aims/hypothesis Rare mutations in the gene (HNF4A) encoding the transcription factor HNF-4A account for ~5% of cases of maturity-onset diabetes of the young (MODY) and more frequent variants in this gene may be involved in multifactorial forms of diabetes. Two low frequency, non-synonymous variants in HNF4A (V255M, minor allele frequency [MAF] ~0.1%, T130I, MAF ~3.0%), known to influence downstream HNF-4A target gene expression, are of interest but previous type 2 diabetes association reports were inconclusive. We aimed to evaluate the contribution of these variants to type 2 diabetes susceptibility through large-scale association analysis. Methods We genotyped both variants in at least 5745 cases and 14756 population controls from the UK and Denmark. We also undertook an expanded association-analysis including previously reported and novel genotype data obtained in Danish, Finnish, Canadian and Swedish samples. A meta-analysis incorporating all published association studies of the T130I variant was subsequently carried out in a maximum sample size of 14279 cases and 26835 controls. Results We found no association between V255M and type 2 diabetes in either the initial (p=0.28) or expanded analysis (p=0.44). However, T130I demonstrated a modest association with type 2 diabetes in the UK and Danish samples (additive per allele OR 1.17 [1.08-1.28]; p=1.5×10−4), which was strengthened in the meta-analysis (OR 1.20 [1.10-1.30]; p=2.1×10−5). Conclusions/interpretation Our data are consistent with T130I as a low frequency variant influencing type 2 diabetes risk, but are not conclusive when judged against stringent standards for genome-wide significance. This study exemplifies the difficulties encountered in association testing of low frequency variants. PMID:20878384

  10. Functional FEN1 genetic variants and haplotypes are associated with glioma risk.

    PubMed

    Chen, Yi-Dong; Zhang, Xiaojiao; Qiu, Xiao-Guang; Li, Jinpeng; Yuan, Qipeng; Jiang, Tao; Yang, Ming

    2013-01-01

    As a tumor suppressor, FEN1 plays an essential role in keeping genomic instability and preventing tumorigenesis. There are two functional genetic variants (-69G>A and 4150G>T) in the FEN1 gene, which have been associated with DNA damage levels in coke-oven workers as well as risks of lung cancer, hepatocellular carcinoma, esophageal cancer, gastric cancer and colorectal cancer in general populations. However, it is still unknown how these polymorphisms and their haplotypes are associated with glioma risk. Therefore, we investigated the role of these polymorphisms in glioma development using a case-control design in a Chinese population. The impact of the haplotypes constructed by these two polymorphisms on glioma risk was also examined. It was observed that the FEN1-69GG or 4150GG genotype were significantly associated to increased glioma risk compared with the -69AA or 4150TT genotype [Odds ratios (OR) = 1.87, 95 % confidence interval (CI) = 1.23-2.85, P = 0.003; or OR = 1.87, 95 % CI = 1.23-2.84, P = 0.003). The associations were more pronounced among female subjects (For -69AG or GG genotype: OR = 2.35, 95 % CI = 1.22-4.52; for 4150TG or GG genotype: OR = 2.33, 95 % CI = 1.21-4.48) and patients with grade 1 or 2 disease (For -69AG or GG genotype: OR = 2.21, 95 % CI = 1.20-4.05; for 4150TG or GG genotype: OR = 2.45, 95 % CI = 1.31-4.58). Additionally, the G(-69)G(4150) haplotype was also significantly associated with increased glioma risk compared with the A(-69)T(4150) haplotype. Our results suggest that FEN1 polymorphisms and haplotypes are associated with glioma risk. PMID:23184144