Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection

PubMed Central

KC, R.; Srivastava, A.; Wilkowski, J. M.; Richter, C. E.; Shavit, J. A.; Burke, D. T.; Bielas, S. L.

2016-01-01

CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies. PMID:27557703

Single-tube, non-isotopic, multiplex PCR/OLA assay and sequence-coded separation for simultaneous screening of 31 cystic fibrosis mutations

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

Brinson, E.C.; Adriano, T.; Bloch, W.

1994-09-01

We have developed a rapid, single-tube, non-isotopic assay that screens a patient sample for the presence of 31 cystic fibrosis (CF) mutations. This assay can identify these mutations in a single reaction tube and a single electrophoresis run. Sample preparation is a simple, boil-and-go procedure, completed in less than an hour. The assay is composed of a 15-plex PCR, followed by a 61-plex oligonucleotide ligation assay (OLA), and incorporates a novel detection scheme, Sequence Coded Separation. Initially, the multiplex PCR amplifies 15 relevant segments of the CFTR gene, simultaneously. These PCR amplicons serve as templates for the multiplex OLA, whichmore » detects the normal or mutant allele at all loci, simultaneously. Each polymorphic site is interrogated by three oligonucleotide probes, a common probe and two allele-specific probes. Each common probe is tagged with a fluorescent dye, and the competing normal and mutant allelic probes incorporate different, non-nucleotide, mobility modifiers. These modifiers are composed of hexaethylene oxide (HEO) units, incorporated as HEO phosphoramidite monomers during automated DNA synthesis. The OLA is based on both probe hybridization and the ability of DNA ligase to discriminate single base mismatches at the junction between paired probes. Each single tube assay is electrophoresed in a single gel lane of a 4-color fluorescent DNA sequencer (Applied Biosystems, Model 373A). Each of the ligation products is identified by its unique combination of electrophoretic mobility and one of three colors. The fourth color is reserved for the in-lane size standard, used by GENESCAN{sup TM} software (Applied Biosystems) to size the OLA electrophoresis products. The Genotyper{sub TM} software (Applied Biosystems) decodes these Sequence-Coded-Separation data to create a patient summary report for all loci tested.« less

Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ

PubMed Central

Mignardi, Marco; Mezger, Anja; Qian, Xiaoyan; La Fleur, Linnea; Botling, Johan; Larsson, Chatarina; Nilsson, Mats

2015-01-01

In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ. PMID:26240388

Analyses of Genotypes and Phenotypes of Ten Chinese Patients with Wolf-Hirschhorn Syndrome by Multiplex Ligation-dependent Probe Amplification and Array Comparative Genomic Hybridization

PubMed Central

Yang, Wen-Xu; Pan, Hong; Li, Lin; Wu, Hai-Rong; Wang, Song-Tao; Bao, Xin-Hua; Jiang, Yu-Wu; Qi, Yu

2016-01-01

Background: Wolf-Hirschhorn syndrome (WHS) is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the features of Chinese WHS patients. This study aimed to characterize the clinical and molecular cytogenetic features of Chinese WHS patients using the combination of multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (array CGH). Methods: Clinical information was collected from ten patients with WHS. Genomic DNA was extracted from the peripheral blood of the patients. The deletions were analyzed by MLPA and array CGH. Results: All patients exhibited the core clinical symptoms of WHS, including severe growth delay, a Greek warrior helmet facial appearance, differing degrees of intellectual disability, and epilepsy or electroencephalogram anomalies. The 4p deletions ranged from 2.62 Mb to 17.25 Mb in size and included LETM1, WHSC1, and FGFR3. Conclusions: The combined use of MLPA and array CGH is an effective and specific means to diagnose WHS and allows for the precise identification of the breakpoints and sizes of deletions. The deletion of genes in the WHS candidate region is closely correlated with the core WHS phenotype. PMID:26960370

Feasibility and Outcomes of Multiplex Ligation-Dependent Probe Amplification on Buccal Smears as a Screening Method for Microdeletions and Duplications among 300 Adults with an Intellectual Disability of Unknown Aetiology

ERIC Educational Resources Information Center

Peppink, D.; Douma-Kloppenburg, D. D.; de Rooij-Askes, E. S. P.; van Zoest, I. M.; Evenhuis, H. M.; Gille, J. J. P.; van Hagen, J. M.

2008-01-01

Background: Determining the aetiology of intellectual disability (ID) enables anticipation of specific comorbidity and can thus be beneficial. Blood sampling, however, is considered stressful for people with ID. Our aim was to evaluate the feasibility of a non-invasive screening technique of nine microdeletions/duplications among adults with ID of…

[Multiplex Ligation - dependent Probe Amplification (MLPA) as a screening test in children with developmental defects and intellectual disability of unknown etiology].

PubMed

Laczmańska, Izabela; Jakubiak, Aleksandra; Slęzak, Ryszard; Pesz, Karolina; Stembalska, Agnieszka; Laczmański, Lukasz; Sąsiadek, Maria M; Smigiel, Robert

2011-01-01

Developmental delay and intellectual disability are significant medical and social problems which concern 1-3% of population. The etiology remains unknown in over half of the cases. To evaluate the efficiency of MLPA (Multiplex Ligation-dependent Probe Amplification) as a screening test in diagnosis of patients with developmental delay and/or intellectual disability. 313 MLPA tests were performed in 256 patients with developmental delay and/ or intellectual disability with unknown etiology. MLPA test was made after exclusion of genetic disorders possible to diagnose by dysmorphological examination or using specifi c genetic tests. Positive results were confirmed by FISH analysis with appropriate probes. Chromosomal microaberrations were identifi ed in 15 patients (4,8%): deletions of 1p36 in 4 cases, in one case deletion of 22q11.21, 22q13.33, SNRPN1, 4ptel, 6qtel, 7q11.23, 16ptel, 18qtel as well as one ca se of deletion 3ptel/duplication 15qtel; deletion 18qtel/duplication Xqtel, and also duplication 7q11.23. Detail clinical analysis was performed in patients with diagnosed microaberrations in MLPA test. The molecular MLPA test, screening for chromosomal microaberration syndromes, should be performed in each patient with developmental delay and/or intellectual disability of unknown etiology and normal cytogenetic analysis, even if congenital defects and positive familial history do not exist.

Can we rely on the multiplex ligation-dependent probe amplification method (MLPA) for prenatal diagnosis?

PubMed Central

Omrani, Mir Davood; Azizi, Faezeh; Rajabibazl, Masoumeh; Safavi Naini, Niloufar; Omrani, Sara; Abbasi, Arezo Mona; Saleh Gargari, Soraya

2014-01-01

Background: The major aneuploidies that are diagnosed prenatally involve the autosomal chromosomes 13, 18, and 21, as well as sex chromosomes, X and Y. Because multiplex ligation-dependent probe amplification (MLPA) is rapid and non-invasive, it has replaced traditional culture methods for the screening and diagnosis of common aneuploidies in some countries. Objective: To evaluate the sensitivity and specificity of MLPA in a cross-sectional descriptive study for the detection of chromosomal aneuploidies in comparison to other methods. Materials and Methods: Genomic DNA was extracted from the peripheral blood samples of 10 normal controls and the amniotic fluid of 55 patients. Aneuploidies screening of chromosomes 13, 18, 21, X and Y were carried out using specific MLPA probe mixes (P095-A2). For comparison purposes, samples were also tested by Quantitative Fluorescent-PCR (QF-PCR) and routine chromosomal culture method. Results: Using this specific MLPA technique and data-analyzing software (Genemarker v1.85), one case was diagnosed with 45, X (e.g. Monosomy X or Turner’s Syndrome), and the remaining 54 cases revealed normal karyotypes. These results were concordant with routine chromosomal culture and QF-PCR findings. Conclusion: The experiment demonstrates that MLPA can provide a rapid and accurate clinical method for prenatal identification of common chromosomal aneuploidies with 100% sensitivity and 100% specificity. PMID:24976821

[Molecular diagnosis of spinal muscular atrophy by multiplex ligation-dependent probe amplification].

PubMed

Zeng, Jian; Ke, Long-feng; Deng, Xiao-jun; Cai, Mei-ying; Tu, Xiang-dong; Lan, Feng-hua

2008-12-16

To investigate the effect of multiplex ligation-dependent probe amplification (MLPA) in molecular diagnosis of spinal muscular atrophy (SMA). Peripheral blood samples were collected from 13 SMA patients, 31 parents of SMA patients, 50 healthy individuals without family history of SMA, and 10 specimens of amniotic fluid from these families were collected too. Genomic DNA was analyzed by MLPA, conventional PCR-RFLP, and allele-specific PCR. In complete agreement with the results of conventional PCR-RFLP and allele-specific PCR, MLPA analysis showed that all of the 13 patients had homozygous deletion of the survival of motor neuron 1 (SMN1) gene, and there was significant difference between the SMA severity (type I to type III) and SMN2 copy number (P < 0.05). Of the 31 parents 29 (93.5%) had 1 copy of SMN1, 2 (6.5%) had 2 copies of SMN1. Of the 50 healthy individuals, 1 (2.0%) had 1 copy of SMN1, 48 (96.0%) had 2 copies of SMN1, and 1 (2.0%) had 3 copies. The SMN1 copy number of the parents was significantly higher than that of the healthy individuals (P < 0.01). Two of the 10 fetuses had homozygous deletion of SMN1. The MLPA technique has proved to be an accurate and reliable tool for the molecular diagnosis of SMA, both in patients and in healthy carriers.

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

PubMed

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

2015-01-01

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

[Detection of large deletions in X linked Alport syndrome using competitive multiplex fluorescence polymerase chain reaction].

PubMed

Wang, F; Zhang, Y Q; Ding, J; Yu, L X

2017-10-18

To evaluate the ability of multiplex competitive fluorescence polymerase chain reaction in detection of large deletion and duplication genotypes of X-linked Alport syndrome. Clinical diagnosis of X-linked Alport syndrome was based on either abnormal staining of type IV collagen α5 chain in the epidermal basement membrane alone or with abnormal staining of type IV collagen α5 chain in the glomerular basement membrane and Bowman's capsule/ultrastructural changes in the glomerular basement membrane typical of Alport syndrome. A total of 20 unrelated Chinese patients (13 males and 7 females) clinically diagnosed as X-linked Alport syndrome were included in the study. Their genotypes were unknown. Control subjects included a male patient with other renal disease and two patients who had large deletions in COL4A5 gene detected by multiplex ligation-dependent probe amplification. Genomic DNA was isolated from peripheral blood leukocytes in all the participants. Multiplex competitive fluorescence polymerase chain reaction was used to coamplify 53 exons of COL4A5 gene and four reference genes in a single reaction. When a deletion removed exon 1 of COL4A5 gene was identified, the same method was used to coamplify the first 4 exons of COL4A5 and COL4A6 genes, a promoter shared by COL4A5 and COL4A6 genes, and three reference genes in a single reaction. Any copy number loss suggested by this method was verified by electrophoresis of corresponding polymerase chain reaction amplified products or DNA sequencing to exclude possible DNA variations in the primer regions. Genotypes of two positive controls identified by multiplex competitive fluorescence polymerase chain reaction were consistent with those detected by multiplex ligation-dependent probe amplification. Deletions were identified in 6 of the 20 patients, including two large deletions removing the 5' part of both COL4A5 and COL4A6 genes with the breakpoint located in the second intron of COL4A6, two large deletions removing more than 30 exons of COL4A5 gene, one large deletion removing at least 1 exon of COL4A5 gene, and one small deletion involving 13 bps. No duplication was found. Our results show that multiplex competitive fluorescence polymerase chain reaction is a good alternative to classical techniques for large deletion genotyping in X-linked Alport syndrome.

The -(α)(5.2) Deletion Detected in a Uruguayan Family: First Case Report in the Americas.

PubMed

Soler, Ana María; Schelotto, Magdalena; de Oliveira Mota, Natalia; Dorta Ferreira, Roberta; Sonati, Maria de Fatima; da Luz, Julio Abayubá

2016-08-01

In Uruguay, α-thalassemia (α-thal) mutations were introduced predominantly by Mediterranean European immigrant populations and by slave trade of African populations. A patient with anemia with hypochromia and microcytosis, refractory to iron treatment and with normal hemoglobin (Hb) electrophoresis was analyzed for α-thal mutations by multiplex gap-polymerase chain reaction (gap-PCR), automated sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses. Agarose gel electrophoresis of the multiplex gap-PCR showed a band of unexpected size (approximately 700 bp) in the samples from the proband and mother. Automated sequencing of the amplified fragment showed the presence of the -(α)(5.2) deletion (NG_000006.1: g.32867_38062del5196) [an α-thal-1 deletion of 5196 nucleotides (nts)]. The MLPA analysis of the proband's sample also showed the presence of the -(α)(5.2) deletion in heterozygous state. We report here the presence of the -(α)(5.2) deletion, for the first time in the Americas, in a Uruguayan family with Italian ancestry, detected with a previously described multiplex gap-PCR.

A valveless rotary microfluidic device for multiplex point mutation identification based on ligation-rolling circle amplification.

PubMed

Heo, Hyun Young; Chung, Soyi; Kim, Yong Tae; Kim, Do Hyun; Seo, Tae Seok

2016-04-15

Genetic variations such as single nucleotide polymorphism (SNP) and point mutations are important biomarkers to monitor disease prognosis and diagnosis. In this study, we developed a novel rotary microfluidic device which can perform multiplex SNP typing on the mutation sites of TP53 genes. The microdevice consists of three glass layers: a channel wafer, a Ti/Pt electrode-patterned resistance temperature detector (RTD) wafer, and a rotary plate in which twelve reaction chambers were fabricated. A series of sample injection, ligation-rolling circle amplification (L-RCA) reaction, and fluorescence detection of the resultant amplicons could be executed by rotating the top rotary plate, identifying five mutation points related with cancer prognosis. The use of the rotary plate eliminates the necessity of microvalves and micropumps to control the microfluidic flow in the channel, simplifying the chip design and chip operation for multiplex SNP detection. The proposed microdevice provides an advanced genetic analysis platform in terms of multiplexity, simplicity, and portability in the fields of biomedical diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

Methylation-Specific Multiplex Ligation-Dependent Probe Amplification and Identification of Deletion Genetic Subtypes in Prader-Willi Syndrome

PubMed Central

Henkhaus, Rebecca S.; Kim, Soo-Jeong; Kimonis, Virginia E.; Gold, June-Anne; Dykens, Elisabeth M.; Driscoll, Daniel J.

2012-01-01

Purpose: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are complex neurodevelopmental disorders caused by loss of expression of imprinted genes from the 15q11-q13 region depending on the parent of origin. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) kits from MRC-Holland (Amsterdam, The Netherlands) were used to detect PWS and AS deletion subtypes. We report our experience with two versions of the MS-MLPA-PWS/AS kit (original A1 and newer B1) in determining methylation status and deletion subtypes in individuals with PWS. Methods: MS-MLPA analysis was performed on DNA isolated from a large cohort of PWS subjects with the MS-MLPA-PWS/AS-A1 and -B1 probe sets. Results: Both MS-MLPA kits will identify deletions in the 15q11-q13 region but the original MS-MLPA-A1 kit has a higher density of probes at the telomeric end of the 15q11-q13 region, which is more useful for identifying individuals with atypical deletions. The newer B1 kit contains more probes in the imprinting center (IC) and adjoining small noncoding RNAs useful in identifying small microdeletions. Conclusion: The A1 kit identified the typical deletions and smaller atypical deletions, whereas the B1 kit was more informative for identifying microdeletions including the IC and SNORD116 regions. Both kits should be made available for accurate characterization of PWS/AS deletion subtypes as well as evaluating for IC and SNORD116 microdeletions. PMID:21977908

Prevalence of MLH1 constitutional epimutations as a cause of Lynch syndrome in unselected versus selected consecutive series of patients with colorectal cancer.

PubMed

Castillejo, Adela; Hernández-Illán, Eva; Rodriguez-Soler, María; Pérez-Carbonell, Lucía; Egoavil, Cecilia; Barberá, Victor M; Castillejo, María-Isabel; Guarinos, Carla; Martínez-de-Dueñas, Eduardo; Juan, María-Jose; Sánchez-Heras, Ana-Beatriz; García-Casado, Zaida; Ruiz-Ponte, Clara; Brea-Fernández, Alejandro; Juárez, Miriam; Bujanda, Luis; Clofent, Juan; Llor, Xavier; Andreu, Montserrat; Castells, Antoni; Carracedo, Angel; Alenda, Cristina; Payá, Artemio; Jover, Rodrigo; Soto, José-Luis

2015-07-01

The prevalence of MLH1 constitutional epimutations in the general population is unknown. We sought to analyse the prevalence of MLH1 constitutional epimutations in unselected and selected series of patients with colorectal cancer (CRC). Patients with diagnoses of CRC (n=2123) were included in the unselected group. For comparison, a group of 847 selected patients with CRC who fulfilled the revised Bethesda guidelines (rBG) were also included. Somatic and constitutional MLH1 methylation was assayed via methylation-specific multiplex ligation-dependent probe amplification of cases lacking MLH1 expression. Germline alterations in mismatch-repair (MMR) genes were assessed via Sanger sequencing and methylation-specific multiplex ligation-dependent probe amplification. Loss of MLH1 expression occurred in 5.5% of the unselected series and 12.5% of the selected series (p<0.0001). No constitutional epimutations in MLH1 were detected in the unselected population (0/62); five cases from the selected series were positive for MLH1 epimutations (15.6%, 5/32; p=0.004). Our results suggest a negligible prevalence of MLH1 constitutional epimutations in unselected cases of CRC. Therefore, MLH1 constitutional epimutation analysis should be conducted only for patients who fulfil the rBG and who lack MLH1 expression with methylated MLH1. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Expanding the mutational spectrum in Johanson-Blizzard syndrome: identification of whole exon deletions and duplications in the UBR1 gene by multiplex ligation-dependent probe amplification analysis.

PubMed

Sukalo, Maja; Schäflein, Eva; Schanze, Ina; Everman, David B; Rezaei, Nima; Argente, Jesús; Lorda-Sanchez, Isabel; Deshpande, Charu; Takahashi, Tsutomu; Kleger, Alexander; Zenker, Martin

2017-11-01

Johanson-Blizzard syndrome (JBS, MIM #243800) is a very rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency, nasal wing hypoplasia, hypodontia, and other abnormalities. JBS is caused by mutations of the UBR1 gene (MIM *605981), encoding a ubiquitin ligase of the N-end rule pathway. Molecular findings in a total of 65 unrelated patients with a clinical diagnosis of JBS who were previously screened for UBR1 mutations by Sanger sequencing were reviewed and cases lacking a disease-causing UBR1 mutation on either one or both alleles were included in this study. In order to discover mutations that are not detectable by Sanger sequencing, we designed a probe set for multiplex ligation-dependent probe amplification (MLPA) analysis of the UBR1 gene and analyzed the copy number status of all 47 UBR1 exons. Our previous studies using Sanger sequencing could detect mutations in 93.1% of 130 disease-associated UBR1 alleles. Six patients with a highly suggestive clinical diagnosis of JBS and unsolved genotype were included in this study. MLPA analysis detected six alleles harboring exon deletions/duplications, thereby raising the mutation detection rate in the entire cohort to 97.7% (127/130 alleles). We conclude that single or multi-exon deletions or duplications account for a substantial proportion of JBS-associated UBR1 mutations. © 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

Aneuploidy identification in pre-B acute lymphoblastic leukemia patients at diagnosis by Multiplex Ligation-dependent Probe Amplification (MLPA).

PubMed

Vázquez-Reyes, A; Bobadilla-Morales, L; Barba-Barba, C; Macías-Salcedo, G; Serafín-Saucedo, G; Velázquez-Rivera, M E; Almodóvar-Cuevas, M C; Márquez-Mora, A; Pimentel-Gutiérrez, H J; Ortega-de-la-Torre, C; Cruz-Osorio, R M; Nava-Gervasio, S; Rivera-Vargas, J; Sánchez-Zubieta, F; Corona-Rivera, J R; Corona-Rivera, A

2017-08-01

Three-quarters of the patients with acute lymphoblastic leukemia (ALL), show numerical or structural chromosomal alterations, which are important factors in leukemogenesis. The use of Multiplex Ligation-dependent Probes Amplification (MLPA) has been mainly limited for searching copy number alterations of genes, suggesting that MLPA could detect numerical alterations in cancer. However, the use of MLPA in pediatrics to analyze subtelomeric sequences for aneuploidy detection has not been considered in previous studies. The aim of this study was to identify aneuploidy for the first time using MLPA and correlate the results with karyotype and DNA-index (DI), from preB ALL patients. Forty-two bone marrow samples were analyzed by cytogenetics and flow cytometry to determine the DI. The chromosomal gains and/or losses were detected by the SALSA MLPA P036 Subtelomere Mix 1 probemix ® . The chromosomal number matched in 36 out of 42 samples between MLPA and karyotype (R 2 =0.7829, p=3.7×10 -10 ), 18/42 between MLPA and DI (R 2 =0.1556, p=0.023), and 20/42 between karyotype and DI (R 2 =0.1509, p=0.015). MLPA results correlated with karyotype and DI. The use of MLPA led us to identify a gained marker chromosome. Our results indicate that MLPA could be a useful and fast alternative tool for aneuploidy identification in pediatric leukemia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Improved multiplex ligation-dependent probe amplification analysis identifies a deleterious PMS2 allele generated by recombination with crossover between PMS2 and PMS2CL.

PubMed

Wernstedt, Annekatrin; Valtorta, Emanuele; Armelao, Franco; Togni, Roberto; Girlando, Salvatore; Baudis, Michael; Heinimann, Karl; Messiaen, Ludwine; Staehli, Noemie; Zschocke, Johannes; Marra, Giancarlo; Wimmer, Katharina

2012-09-01

Heterozygous PMS2 germline mutations are associated with Lynch syndrome. Up to one third of these mutations are genomic deletions. Their detection is complicated by a pseudogene (PMS2CL), which--owing to extensive interparalog sequence exchange--closely resembles PMS2 downstream of exon 12. A recently redesigned multiplex ligation-dependent probe amplification (MLPA) assay identifies PMS2 copy number alterations with improved reliability when used with reference DNAs containing equal numbers of PMS2- and PMS2CL-specific sequences. We selected eight such reference samples--all publicly available--and used them with this assay to study 13 patients with PMS2-defective colorectal tumors. Three presented deleterious alterations: an Alu-mediated exon deletion; a 125-kb deletion encompassing PMS2 and four additional genes (two with tumor-suppressing functions); and a novel deleterious hybrid PMS2 allele produced by recombination with crossover between PMS2 and PMS2CL, with the breakpoint in intron 10 (the most 5' breakpoint of its kind reported thus far). We discuss mechanisms that might generate this allele in different chromosomal configurations (and their diagnostic implications) and describe an allele-specific PCR assay that facilitates its detection. Our data indicate that the redesigned PMS2 MLPA assay is a valid first-line option. In our series, it identified roughly a quarter of all PMS2 mutations. Copyright © 2012 Wiley Periodicals, Inc.

Improved Multiplex Ligation-Dependent Probe Amplification Analysis Identifies a Deleterious PMS2 Allele Generated by Recombination with Crossover Between PMS2 and PMS2CL

PubMed Central

Wernstedt, Annekatrin; Valtorta, Emanuele; Armelao, Franco; Togni, Roberto; Girlando, Salvatore; Baudis, Michael; Heinimann, Karl; Messiaen, Ludwine; Staehli, Noemie; Zschocke, Johannes; Marra, Giancarlo; Wimmer, Katharina

2012-01-01

Heterozygous PMS2 germline mutations are associated with Lynch syndrome. Up to one third of these mutations are genomic deletions. Their detection is complicated by a pseudogene (PMS2CL), which – owing to extensive interparalog sequence exchange – closely resembles PMS2 downstream of exon 12. A recently redesigned multiplex ligation-dependent probe amplification (MLPA) assay identifies PMS2 copy number alterations with improved reliability when used with reference DNAs containing equal numbers of PMS2- and PMS2CL-specific sequences. We selected eight such reference samples – all publicly available – and used them with this assay to study 13 patients with PMS2-defective colorectal tumors. Three presented deleterious alterations: an Alu-mediated exon deletion; a 125-kb deletion encompassing PMS2 and four additional genes (two with tumor-suppressing functions); and a novel deleterious hybrid PMS2 allele produced by recombination with crossover between PMS2 and PMS2CL, with the breakpoint in intron 10 (the most 5′ breakpoint of its kind reported thus far). We discuss mechanisms that might generate this allele in different chromosomal configurations (and their diagnostic implications) and describe an allele-specific PCR assay that facilitates its detection. Our data indicate that the redesigned PMS2 MLPA assay is a valid first-line option. In our series, it identified roughly a quarter of all PMS2 mutations. © 2012 Wiley Periodicals, Inc. PMID:22585707

A Comprehensive Strategy for Accurate Mutation Detection of the Highly Homologous PMS2.

PubMed

Li, Jianli; Dai, Hongzheng; Feng, Yanming; Tang, Jia; Chen, Stella; Tian, Xia; Gorman, Elizabeth; Schmitt, Eric S; Hansen, Terah A A; Wang, Jing; Plon, Sharon E; Zhang, Victor Wei; Wong, Lee-Jun C

2015-09-01

Germline mutations in the DNA mismatch repair gene PMS2 underlie the cancer susceptibility syndrome, Lynch syndrome. However, accurate molecular testing of PMS2 is complicated by a large number of highly homologous sequences. To establish a comprehensive approach for mutation detection of PMS2, we have designed a strategy combining targeted capture next-generation sequencing (NGS), multiplex ligation-dependent probe amplification, and long-range PCR followed by NGS to simultaneously detect point mutations and copy number changes of PMS2. Exonic deletions (E2 to E9, E5 to E9, E8, E10, E14, and E1 to E15), duplications (E11 to E12), and a nonsense mutation, p.S22*, were identified. Traditional multiplex ligation-dependent probe amplification and Sanger sequencing approaches cannot differentiate the origin of the exonic deletions in the 3' region when PMS2 and PMS2CL share identical sequences as a result of gene conversion. Our approach allows unambiguous identification of mutations in the active gene with a straightforward long-range-PCR/NGS method. Breakpoint analysis of multiple samples revealed that recurrent exon 14 deletions are mediated by homologous Alu sequences. Our comprehensive approach provides a reliable tool for accurate molecular analysis of genes containing multiple copies of highly homologous sequences and should improve PMS2 molecular analysis for patients with Lynch syndrome. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Log-PCR: a new tool for immediate and cost-effective diagnosis of up to 85% of dystrophin gene mutations.

PubMed

Trimarco, Amelia; Torella, Annalaura; Piluso, Giulio; Maria Ventriglia, Vega; Politano, Luisa; Nigro, Vincenzo

2008-06-01

Duchenne (DMD) and Becker (BMD) muscular dystrophies are caused by mutations in the dystrophin gene. Despite the progress in the technologies of mutation detection, the disease of one third of patients escapes molecular definition because the labor and expense involved has precluded analyzing the entire gene. Novel techniques with higher detection rates, such as multiplex ligation-dependent probe amplification and multiplex amplifiable probe hybridization, have been introduced. We approached the challenge of multiplexing by modifying the PCR chemistry. We set up a rapid protocol that analyzes all dystrophin exons and flanking introns (57.5 kb). We grouped exons according to their effect on the reading frame and ran 2 PCR reactions for DMD mutations and 2 reactions for BMD mutations under the same conditions. The PCR products are evenly spaced logarithmically on the gel (Log-PCR) in an order that reproduces their chromosomal locations. This strategy enables both simultaneous mapping of all the mutation borders and distinguishing between DMD and BMD. As a proof of principle, we reexamined samples from 506 patients who had received a DMD or BMD diagnosis. We observed gross rearrangements in 428 of the patients (84.6%; 74.5% deletions and 10.1% duplications). We also recognized a much broader spectrum of mutations and identified 14.6% additional cases. This study is the first exhaustive investigation of this subject and has made possible the development of a cost-effective test for diagnosing a larger proportion of cases. The benefit of this approach may allow more focused efforts for discovering small or deep-intronic mutations among the few remaining undiagnosed cases. The same protocol can be extended to set up Log-PCRs for other high-throughput applications.

Duchenne Muscular Dystrophy and Becker Muscular Dystrophy Confirmed by Multiplex Ligation-Dependent Probe Amplification: Genotype-Phenotype Correlation in a Large Cohort.

PubMed

Vengalil, Seena; Preethish-Kumar, Veeramani; Polavarapu, Kiran; Mahadevappa, Manjunath; Sekar, Deepha; Purushottam, Meera; Thomas, Priya Treesa; Nashi, Saraswathi; Nalini, Atchayaram

2017-01-01

Studies of cases of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) confirmed by multiplex ligation-dependent probe amplification (MLPA) have determined the clinical characteristics, genotype, and relations between the reading frame and phenotype for different countries. This is the first such study from India. A retrospective genotype-phenotype analysis of 317 MLPA-confirmed patients with DMD or BMD who visited the neuromuscular clinic of a quaternary referral center in southern India. The 317 patients comprised 279 cases of DMD (88%), 32 of BMD (10.1%), and 6 of intermediate phenotype (1.9%). Deletions accounted for 91.8% of cases, with duplications causing the remaining 8.2%. There were 254 cases of DMD (91%) with deletions and 25 (9%) due to duplications, and 31 cases (96.8%) of BMD with deletions and 1 (3.2%) due to duplication. All six cases of intermediate type were due to deletions. The most-common mutation was a single-exon deletion. Deletions of six or fewer exons constituted 68.8% of cases. The deletion of exon 50 was the most common. The reading-frame rule held in 90% of DMD and 94% of BMD cases. A tendency toward a lower IQ and earlier wheelchair dependence was observed with distal exon deletions, though a significant correlation was not found. The reading-frame rule held in 90% to 94% of children, which is consistent with reports from other parts of the world. However, testing by MLPA is a limitation, and advanced sequencing methods including analysis of the structure of mutant dystrophin is needed for more-accurate assessments of the genotype-phenotype correlation.

Optimized MOL-PCR for Characterization of Microbial Pathogens.

PubMed

Wuyts, Véronique; Roosens, Nancy H C; Bertrand, Sophie; Marchal, Kathleen; De Keersmaecker, Sigrid C J

2016-01-06

Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL-PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL-PCR assay for subtyping of Salmonella Typhimurium. Copyright © 2016 John Wiley & Sons, Inc.

Analysis of genomic alterations in neuroblastoma by multiplex ligation-dependent probe amplification and array comparative genomic hybridization: a comparison of results.

PubMed

Combaret, Valérie; Iacono, Isabelle; Bréjon, Stéphanie; Schleiermacher, Gudrun; Pierron, Gäelle; Couturier, Jérôme; Bergeron, Christophe; Blay, Jean-Yves

2012-12-01

In cases of neuroblastoma, recurring genetic alterations--losses of the 1p, 3p, 4p, and 11q and/or gains of 1q, 2p, and 17q chromosome arms--are currently used to define the therapeutic strategy in therapeutic protocols for low- and intermediate-risk patients. Different genome-wide analysis techniques, such as array comparative genomic hybridization (aCGH) or multiplex ligation-dependent probe amplification (MLPA), have been suggested for detecting chromosome segmental abnormalities. In this study, we compared the results of the two technologies in the analyses of the DNA of tumor samples from 91 neuroblastoma patients. Similar results were obtained with the two techniques for 75 samples (82%). In five cases (5.5%), the MLPA results were not interpretable. Discrepancies between the aCGH and MLPA results were observed in 11 cases (12%). Among the discrepancies, a 18q21.2-qter gain and 16p11.2 and 11q14.1-q14.3 losses were detected only by aCGH. The MLPA results showed that the 7p, 7q, and 14q chromosome arms were affected in six cases, while in two cases, 2p and 17q gains were observed; these results were confirmed by neither aCGH nor fluorescence in situ hybridization (FISH) analysis. Because of the higher sensitivity and specificity of genome-wide information, reasonable cost, and shorter time of aCGH analysis, we recommend the aCGH procedure for the analysis of genomic alterations in neuroblastoma. Copyright © 2012 Elsevier Inc. All rights reserved.

Genetic heterogeneity in uveal melanoma assessed by multiplex ligation-dependent probe amplification.

PubMed

Dopierala, Justyna; Damato, Bertil E; Lake, Sarah L; Taktak, Azzam F G; Coupland, Sarah E

2010-10-01

To determine intratumor genetic heterogeneity in uveal melanoma (UM) by multiplex ligation-dependent probe amplification (MLPA) in formalin-fixed, paraffin-embedded (FFPE) tumor tissues. DNA was extracted from whole tumor sections and from two to nine different areas microdissected from 32 FFPE UMs. Thirty-one loci on chromosomes 1, 3, 6, and 8 were tested with MLPA for copy number changes. The tumor was considered heterogeneous at a locus if (1) the difference in dosage quotients (DQs) of any two areas was 0.2 or more, and (2) the DQs of the areas belonged to different ranges. Comparison of MLPA data obtained from microdissected areas of the UMs showed heterogeneity in 1 to 26 examined loci in 24 (75%) tumors, with only 25% of the tumors being homogeneous. Intratumor heterogeneity of 3p12.2, 6p21.2, and 8q11.23 was most common, occurring in >30% of the UMs. Gains of chromosome 3 were observed in four UMs, with three of these tumors showing the highest degree of heterogeneity. Copy number variation was associated with differences in tumor cell type, but not with differences in tumor pigmentation or reactive inflammation. UMs with genetic heterogeneity across multiple sample sites showed equivocal MLPA results when the whole tumor section was examined. These results suggest that different clones dilute MLPA results. Heterogeneity of chromosomal abnormalities of chromosomes 1, 3, 6, and 8 is present in most UMs. This heterogeneity causes equivocal MLPA results. One random tumor sample may not be representative of the whole tumor and, therefore, may be insufficient for prognostic testing.

Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants.

PubMed

Haghshenas, Maryam; Akbari, Mohammad Taghi; Karizi, Shohreh Zare; Deilamani, Faravareh Khordadpoor; Nafissi, Shahriar; Salehi, Zivar

2016-06-01

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50-79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.

Extending the phenotype of monosomy 1p36 syndrome and mapping of a critical region for obesity and hyperphagia.

PubMed

D'Angelo, Carla S; Kohl, Ilana; Varela, Monica Castro; de Castro, Cláudia I E; Kim, Chong A; Bertola, Débora R; Lourenço, Charles M; Koiffmann, Célia P

2010-01-01

Rearrangements of 1p36 are the most frequently detected abnormalities in diagnostic testing for chromosomal cryptic imbalances and include variably sized simple terminal deletions, derivative chromosomes, interstitial deletions, and complex rearrangements. These rearrangements result in the specific pattern of malformation and neurodevelopmental disabilities that characterizes monosomy 1p36 syndrome. Thus far, no individual gene within this region has been conclusively determined to be causative of any component of the phenotype. Nor is it known if the rearrangements convey phenotypes via a haploinsufficiency mechanism or through a position effect. We have used multiplex ligation-dependent probe amplification to screen for deletions of 1p36 in a group of 154 hyperphagic and overweight/obese, PWS negative individuals, and in a separate group of 83 patients initially sent to investigate a variety of other conditions. The strategy allowed the identification and delineation of rearrangements in nine subjects with a wide spectrum of clinical presentations. Our work reinforces the association of monosomy 1p36 and obesity and hyperphagia, and further suggests that these features may be associated with non-classical manifestations of this disorder in addition to a submicroscopic deletion of approximately 2-3 Mb in size. Multiplex ligation probe amplification using the monosomy 1p36 syndrome-specific kit coupled to the subtelomeric kit is an effective approach to identify and delineate rearrangements at 1p36.

ProteinSeq: High-Performance Proteomic Analyses by Proximity Ligation and Next Generation Sequencing

PubMed Central

Vänelid, Johan; Siegbahn, Agneta; Ericsson, Olle; Fredriksson, Simon; Bäcklin, Christofer; Gut, Marta; Heath, Simon; Gut, Ivo Glynne; Wallentin, Lars; Gustafsson, Mats G.; Kamali-Moghaddam, Masood; Landegren, Ulf

2011-01-01

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 µl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use. PMID:21980495

Dosage analysis of cancer predisposition genes by multiplex ligation-dependent probe amplification

PubMed Central

Bunyan, D J; Eccles, D M; Sillibourne, J; Wilkins, E; Thomas, N Simon; Shea-Simonds, J; Duncan, P J; Curtis, C E; Robinson, D O; Harvey, J F; Cross, N C P

2004-01-01

Multiplex ligation-dependent probe amplification (MLPA) is a recently described method for detecting gross deletions or duplications of DNA sequences, aberrations which are commonly overlooked by standard diagnostic analysis. To determine the incidence of copy number variants in cancer predisposition genes from families in the Wessex region, we have analysed the hMLH1 and hMSH2 genes in patients with hereditary nonpolyposis colorectal cancer (HNPCC), BRCA1 and BRCA2 in families with hereditary breast/ovarian cancer (BRCA) and APC in patients with familial adenomatous polyposis coli (FAP). Hereditary nonpolyposis colorectal cancer (n=162) and FAP (n=74) probands were fully screened for small mutations, and cases for which no causative abnormality were found (HNPCC, n=122; FAP, n=24) were screened by MLPA. Complete or partial gene deletions were identified in seven cases for hMSH2 (5.7% of mutation-negative HNPCC; 4.3% of all HNPCC), no cases for hMLH1 and six cases for APC (25% of mutation negative FAP; 8% of all FAP). For BRCA1 and BRCA2, a partial mutation screen was performed and 136 mutation-negative cases were selected for MLPA. Five deletions and one duplication were found for BRCA1 (4.4% of mutation-negative BRCA cases) and one deletion for BRCA2 (0.7% of mutation-negative BRCA cases). Cost analysis indicates it is marginally more cost effective to perform MLPA prior to point mutation screening, but the main advantage gained by prescreening is a greatly reduced reporting time for the patients who are positive. These data demonstrate that dosage analysis is an essential component of genetic screening for cancer predisposition genes. PMID:15475941

Subtelomeric multiplex ligation-dependent probe amplification as a supplement for rapid prenatal detection of fetal chromosomal aberrations.

PubMed

Chen, Xiangnan; Li, Huanzheng; Mao, Yijian; Xu, Xueqin; Lv, Jiaojiao; Zhou, Lili; Lin, Xiaoling; Tang, Shaohua

2014-01-01

Pregnant women with high-risk indications are highly suspected of fetal chromosomal aberrations. To determine whether Multiplex Ligation-dependent Probe Amplification (MLPA) using subtelomeric probe mixes (P036-E2 and P070-B2) is a reliable method for rapid detection of fetal chromosomal aberrations. The subtelomeric MLPA probe mixes were used to evaluate 50 blood samples from healthy individuals. 168 amniocytes and 182 umbilical cord blood samples from high-risk fetuses were analyzed using the same subtelomeric MLPA probe sets. Karyotyping was also performed in all cases of high-risk pregnancies, and single nucleotide polymorphism array analysis was used to confirm submicroscopic and ambiguous results from MLPA/karyotyping. Subtelomeric MLPA analysis of normal samples showed normal result in all cases by use of P036-E2 probe mix, while P070-B2 probe mix gave normal results for all but one case. In one normal control case P070-B2 produced a duplicated signal of probe for 13q34. In the high-risk group, totally 44 chromosomal abnormalities were found by karyotyping and MLPA, including 23 aneuploidies and 21 rearrangements or mosaics. MLPA detected all 23 aneuploidies, 12 rearrangements and 1 mosaic. Importantly, MLPA revealed 4 chromosomal translocations, 2 small supernumerary marker chromosomes (sSMCs), and 3 subtelomeric imbalances that were not well characterized or not detectable by karyotyping. However, MLPA showed negetive results for the remaining 8 rearrangements or mosaics, including 3 low mosaic aneuploidies, 1 inherited sSMC, and 4 paracentric inversions. Results suggest that combined use of subtelomeric MLPA and karyotyping may be an alternative method for using karyotype analyses alone in rapid detection of aneuploidies, rearrangements, and sSMCs.

Validation of the multiplex ligation-dependent probe amplification assay and its application on the distribution study of the major alleles of 17 blood group systems in Chinese donors from Guangzhou.

PubMed

Ji, Yanli; Wen, Jizhi; Veldhuisen, Barbera; Haer-Wigman, Lonneke; Wang, Zhen; Lodén-van Straaten, Martin; Wei, Ling; Luo, Guangping; Fu, Yongshui; van der Schoot, C Ellen

2017-02-01

Genotyping platforms for common red blood cell (RBC) antigens have been successfully applied in Caucasian and black populations but not in Chinese populations. In this study, a genotyping assay based on multiplex ligation-dependent probe amplification (MLPA) technology was applied in a Chinese population to validate the MLPA probes. Subsequently, the comprehensive distribution of 17 blood group systems also was obtained. DNA samples from 200 Chinese donors were extracted and genotyped using the blood-MLPA assay. To confirm the MLPA results, a second independent genotyping assay (ID Core+) was conducted in 40 donors, and serological typing of 14 blood-group antigens was performed in 91 donors. In donors who had abnormal copy numbers of an allele (DI and GYPB) determined by MLPA, additional experiments were performed (polymerase chain reaction, sequencing, and flow cytometry analysis). The genotyping results obtained using the blood-MLPA and ID Core+ assays were consistent. Serological data were consistent with the genotyping results except for one donor who had a Lu(a-b-) phenotype. Of the 17 blood group systems, the distribution of the MNS, Duffy, Kidd, Diego, Yt, and Dombrock systems was polymorphic. The Mur and St a antigens of the MNS system were distributed with a frequency of 9% (18 of 200) and 2% (4 of 200), respectively. One donor with chimerism and one who carried a novel DI*02(A845V) allele, which predicts the depression of Di b antigen expression, were identified. The blood-MLPA assay could easily identify the common blood-group alleles and correctly predicted phenotype in the Chinese population. The Mur and St a antigens were distributed with high frequency in a Southern Chinese Han population. © 2016 AABB.

Molecular Analysis-Based Genetic Characterization of a Cohort of Patients with Duchenne and Becker Muscular Dystrophy in Eastern China.

PubMed

Zhao, Hui-Hui; Sun, Xue-Ping; Shi, Ming-Chao; Yi, Yong-Xiang; Cheng, Hong; Wang, Xing-Xia; Xu, Qing-Cheng; Ma, Hong-Ming; Wu, Hao-Quan; Jin, Qing-Wen; Niu, Qi

2018-04-05

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-linked recessive neuromuscular disorders caused by mutations in dystrophin gene. Multiplex polymerase chain reaction (multiplex PCR) and multiplex ligation-dependent probe amplification (MLPA) are the most common methods for detecting dystrophin gene mutations. This study aimed to contrast the two methods and discern the genetic characterization of patients with DMD/BMD in Eastern China. We collected 121 probands, 64 mothers of probands, and 15 fetuses in our study. The dystrophin gene was detected by multiplex PCR primarily in 28 probands, and MLPA was used in multiplex PCR-negative cases subsequently. The dystrophin gene of the remaining 93 probands and 62 female potential carriers was tested by MLPA directly. In fetuses, multiplex PCR and MLPA were performed on 4 fetuses and 10 fetuses, respectively. In addition, sequencing was also performed in 4 probands with negative MLPA. We found that 61.98% of the subjects had genetic mutations including deletions (50.41%) and duplications (11.57%). There were 43.75% of mothers as carriers of the mutation. In 15 fetuses, 2 out of 7 male fetuses were found to be unhealthy and 2 out of 8 female fetuses were found to be carriers. Exons 3-26 and 45-52 have the maximum frequency in mutation regions. In the frequency of exons individually, exon 47 and exon 50 were the most common in deleted regions and exons 5, 6, and 7 were found most frequently in duplicated regions. MLPA has better productivity and sensitivity than multiplex PCR. Prenatal diagnosis should be applied in DMD high-risk fetuses to reduce the disease incidence. Furthermore, it is the responsibility of physicians to inform female carriers the importance of prenatal diagnosis.

Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

PubMed

Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

2015-02-01

In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on paraffin-embedded material to test for other diagnostically, prognostically, or therapeutically relevant genomic mutations in lipomatous tumors.

DNA hypermethylation profiles in squamous cell carcinoma of the vulva.

PubMed

Stephen, Josena K; Chen, Kang Mei; Raitanen, Misa; Grénman, Seija; Worsham, Maria J

2009-01-01

Gene silencing through promoter hypermethylation is a growing concept in the development of human cancers. In this study, we examined the contribution of aberrant methylation of promoter regions in methylation-prone tumor suppressors to the pathogenesis of vulvar cancer. Thirteen cell lines from 12 patients with squamous cell carcinoma of the vulva were evaluated for aberrant methylation status and gene copy number alterations, concomitantly, using the methylation-specific multiplex ligation-dependent probe amplification assay. Of the 22 tumor suppressor genes examined, aberrant methylation was observed for 9 genes: tumor protein p73 (TP73), fragile histidine triad (FHIT), von Hippel-Lindau (VHL), adenomatosis polyposis coli (APC), estrogen receptor 1 (ESR1), cyclin-dependent kinase inhibitor 2B (CDKN2B), death-associated protein kinase 1 (DAPK1), glutathione S-transferase pi (GSTP1), and immunoglobin superfamily, member 4 (IGSF4). The most frequently methylated genes included TP73 in 9 of 13 cell lines, and IGSF4, DAPK1, and FHIT in 3 of 13 cell lines. Methylation-specific polymerase chain reaction was performed for TP73 and FHIT to confirm aberrant methylation by methylation-specific multiplex ligation-dependent probe amplification. In the context of gene copy number and methylation status, both copies of the TP73 gene were hypermethylated. Loss or decreased mRNA expression of TP73 and IGSF4 by reverse transcription polymerase chain reaction confirmed aberrant methylation. Frequent genetic alterations of loss and gain of gene copy number included gain of GSTP1 and multiple endocrine neoplasia type 1 (MEN1), and loss of malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) and IGSF4 in over 50% of the squamous cell carcinoma of the vulva cell lines. These findings underscore the contribution of both genetic and epigenetic events to the underlying pathogenesis of squamous cell carcinoma of the vulva.

Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction

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

Day, D.J.; Barany, F.; Speiser, P.W.

Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia, an inherited inability to synthesize cortisol that occurs in 1 in 10,000-15,000 births. Affected females are born with ambiguous genitalia, a condition that can be ameliorated by administering dexamethasone to the mother for most of gestation. Prenatal diagnosis is required for accurate treatment of affected females as well as for genetic counseling purposes. Approximately 95% of mutations causing this disorder result from recombinations between the gene encoding the 21-hydroxylase enzyme (CYP21) and a linked, highly homologous pseudogene (CYP21P). Approximately 20% of these mutations are gene deletions, and themore » remainder are gene conversions that transfer any of nine deleterious mutations from the CYP21P pseudogene to CYP21. We describe a methodology for genetic diagnosis of 21-hydroxylase deficiency that utilizes gene-specific PCR amplification in conjunction with thermostable DNA ligase to discriminate single nucleotide variations in a multiplexed ligation detection assay. The assay has been designed to be used with either fluorescent or radioactive detection of ligation products by electrophoresis on denaturing acrylamide gels and is readily adaptable for use in other disease systems. 30 refs., 5 figs.« less

Modular Ligation Extension of Guide RNA Operons (LEGO) for Multiplexed dCas9 Regulation of Metabolic Pathways in Saccharomyces cerevisiae.

PubMed

Deaner, Matthew; Holzman, Allison; Alper, Hal S

2018-04-16

Metabolic engineering typically utilizes a suboptimal step-wise gene target optimization approach to parse a highly connected and regulated cellular metabolism. While the endonuclease-null CRISPR/Cas system has enabled gene expression perturbations without genetic modification, it has been mostly limited to small sets of gene targets in eukaryotes due to inefficient methods to assemble and express large sgRNA operons. In this work, we develop a TEF1p-tRNA expression system and demonstrate that the use of tRNAs as splicing elements flanking sgRNAs provides higher efficiency than both Pol III and ribozyme-based expression across a variety of single sgRNA and multiplexed contexts. Next, we devise and validate a scheme to allow modular construction of tRNA-sgRNA (TST) operons using an iterative Type IIs digestion/ligation extension approach, termed CRISPR-Ligation Extension of sgRNA Operons (LEGO). This approach enables facile construction of large TST operons. We demonstrate this utility by constructing a metabolic rewiring prototype for 2,3-butanediol production in 2 distinct yeast strain backgrounds. These results demonstrate that our approach can act as a surrogate for traditional genetic modification on a much shorter design-cycle timescale. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modulation-frequency encoded multi-color fluorescent DNA analysis in an optofluidic chip.

PubMed

Dongre, Chaitanya; van Weerd, Jasper; Besselink, Geert A J; Vazquez, Rebeca Martinez; Osellame, Roberto; Cerullo, Giulio; van Weeghel, Rob; van den Vlekkert, Hans H; Hoekstra, Hugo J W M; Pollnau, Markus

2011-02-21

We introduce a principle of parallel optical processing to an optofluidic lab-on-a-chip. During electrophoretic separation, the ultra-low limit of detection achieved with our set-up allows us to record fluorescence from covalently end-labeled DNA molecules. Different sets of exclusively color-labeled DNA fragments-otherwise rendered indistinguishable by spatio-temporal coincidence-are traced back to their origin by modulation-frequency-encoded multi-wavelength laser excitation, fluorescence detection with a single ultrasensitive, albeit color-blind photomultiplier, and Fourier analysis decoding. As a proof of principle, fragments obtained by multiplex ligation-dependent probe amplification from independent human genomic segments, associated with genetic predispositions to breast cancer and anemia, are simultaneously analyzed.

Association of an α-globin gene cluster duplication and heterozygous β-thalassemia in a patient with a severe thalassemia syndrome.

PubMed

Jiang, Hua; Liu, Sha; Zhang, Yong-Ling; Wan, Jun-Hui; Li, Ru; Li, Dong-Zhi

2015-01-01

We describe a new case of a β-thalassemia (β-thal) heterozygote with the mutation IVS-II-654 (C>T) presenting with a transfusion-dependent phenotype. Multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (CGH) analyses of the α-globin gene cluster revealed a full duplication of the α-globin genes including the upstream regulatory element. The duplicated allele and the normal allele in trans resulted in a total of six active α-globin genes. The severe clinical phenotype seemed to be related to the considerable excess of the α- and β-globin deficit caused by the presence of the β-thal. α-Globin cluster duplication should be considered in patients heterozygous for β-thal who show a more severe phenotype than β-thal trait.

Influence of prophylactic probiotics and selective decontamination on bacterial translocation in patients undergoing pancreatic surgery: a randomized controlled trial.

PubMed

Diepenhorst, Gwendolyn M P; van Ruler, Oddeke; Besselink, Marc G H; van Santvoort, Hjalmar C; Wijnandts, Paul R; Renooij, Willem; Gouma, Dirk J; Gooszen, Hein G; Boermeester, Marja A

2011-01-01

Bacterial translocation (BT) is suspected to play a major role in the development of infections in surgical patients. However, the clinical association between intestinal barrier dysfunction, BT, and septic morbidity has remained unconfirmed. The objective of this study was to study BT in patients undergoing major abdominal surgery and the effects of probiotics, selective decontamination of the digestive tract (SDD), and standard treatment on intestinal barrier function. In a randomized controlled setting, 30 consecutive patients planned for elective pylorus-preserving pancreaticoduodenectomy (PPPD) were allocated to receive perioperatively probiotics, SDD, or standard treatment. To assess intestinal barrier function, intestinal fatty acid-binding protein (mucosal damage) and polyethylene glycol recovery (intestinal permeability) in urine were measured perioperatively. BT was assessed by real-time polymerase chain reaction and multiplex ligation-dependent probe amplification (MLPA) in mesenteric lymph nodes (MLNs) harvested early (baseline control) and at the end of surgery ("end-of-surgery" MLNs, after 3h in PPPD patients). Polymerase chain reaction detected bacterial DNA in 18 of 27 end-of-surgery MLNs and in 13 of 23 control MLNs (P = 0.378). Probiotics and SDD had no significant effect on the number of positive MLNs or the change in bacterial DNA during operation. Multiplex ligation-dependent probe amplification analysis showed significantly increased expression of only 4 of 30 inflammatory mediator-related genes in end-of-surgery compared with early sampled MLN (P < 0.05). Polyethylene glycol recovery was unaffected by operation, probiotics and SDD as compared with standard treatment. Intestinal fatty acid-binding protein levels were increased shortly postoperatively only in patients treated with SDD (P = 0.02). Probiotics and SDD did not influence BT, intestinal permeability, or inflammatory mediator expression. Bacterial translocation after abdominal surgery may be part of normal antigen-sampling processes of the gut.

Prevalence and spectrum of large deletions or duplications in the major long QT syndrome-susceptibility genes and implications for long QT syndrome genetic testing.

PubMed

Tester, David J; Benton, Amber J; Train, Laura; Deal, Barbara; Baudhuin, Linnea M; Ackerman, Michael J

2010-10-15

Long QT syndrome (LQTS) is a cardiac channelopathy associated with syncope, seizures, and sudden death. Approximately 75% of LQTS is due to mutations in genes encoding for 3 cardiac ion channel α-subunits (LQT1 to LQT3). However, traditional mutational analyses have limited detection capabilities for atypical mutations such as large gene rearrangements. We set out to determine the prevalence and spectrum of large deletions/duplications in the major LQTS-susceptibility genes in unrelated patients who were mutation negative after point mutation analysis of LQT1- to LQT12-susceptibility genes. Forty-two unrelated, clinically strong LQTS patients were analyzed using multiplex ligation-dependent probe amplification, a quantitative fluorescent technique for detecting multiple exon deletions and duplications. The SALSA multiplex ligation-dependent probe amplification LQTS kit from MRC-Holland was used to analyze the 3 major LQTS-associated genes, KCNQ1, KCNH2, and SCN5A, and the 2 minor genes, KCNE1 and KCNE2. Overall, 2 gene rearrangements were found in 2 of 42 unrelated patients (4.8%, confidence interval 1.7 to 11). A deletion of KCNQ1 exon 3 was identified in a 10-year-old Caucasian boy with a corrected QT duration of 660 ms, a personal history of exercise-induced syncope, and a family history of syncope. A deletion of KCNQ1 exon 7 was identified in a 17-year-old Caucasian girl with a corrected QT duration of 480 ms, a personal history of exercise-induced syncope, and a family history of sudden cardiac death. In conclusion, because nearly 5% of patients with genetically elusive LQTS had large genomic rearrangements involving the canonical LQTS-susceptibility genes, reflex genetic testing to investigate genomic rearrangements may be of clinical value. Copyright © 2010 Elsevier Inc. All rights reserved.

Germline mutations in PMS2 and MLH1 in individuals with solitary loss of PMS2 expression in colorectal carcinomas from the Colon Cancer Family Registry Cohort.

PubMed

Rosty, Christophe; Clendenning, Mark; Walsh, Michael D; Eriksen, Stine V; Southey, Melissa C; Winship, Ingrid M; Macrae, Finlay A; Boussioutas, Alex; Poplawski, Nicola K; Parry, Susan; Arnold, Julie; Young, Joanne P; Casey, Graham; Haile, Robert W; Gallinger, Steven; Le Marchand, Loïc; Newcomb, Polly A; Potter, John D; DeRycke, Melissa; Lindor, Noralane M; Thibodeau, Stephen N; Baron, John A; Win, Aung Ko; Hopper, John L; Jenkins, Mark A; Buchanan, Daniel D

2016-02-19

Immunohistochemistry for DNA mismatch repair proteins is used to screen for Lynch syndrome in individuals with colorectal carcinoma (CRC). Although solitary loss of PMS2 expression is indicative of carrying a germline mutation in PMS2, previous studies reported MLH1 mutation in some cases. We determined the prevalence of MLH1 germline mutations in a large cohort of individuals with a CRC demonstrating solitary loss of PMS2 expression. This cohort study included 88 individuals affected with a PMS2-deficient CRC from the Colon Cancer Family Registry Cohort. Germline PMS2 mutation analysis (long-range PCR and multiplex ligation-dependent probe amplification) was followed by MLH1 mutation testing (Sanger sequencing and multiplex ligation-dependent probe amplification). Of the 66 individuals with complete mutation screening, we identified a pathogenic PMS2 mutation in 49 (74%), a pathogenic MLH1 mutation in 8 (12%) and a MLH1 variant of uncertain clinical significance predicted to be damaging by in silico analysis in 3 (4%); 6 (9%) carried variants likely to have no clinical significance. Missense point mutations accounted for most alterations (83%; 9/11) in MLH1. The MLH1 c.113A> G p.Asn38Ser mutation was found in 2 related individuals. One individual who carried the MLH1 intronic mutation c.677+3A>G p.Gln197Argfs*8 leading to the skipping of exon 8, developed 2 tumours, both of which retained MLH1 expression. A substantial proportion of CRCs with solitary loss of PMS2 expression are associated with a deleterious MLH1 germline mutation supporting the screening for MLH1 in individuals with tumours of this immunophenotype, when no PMS2 mutation has been identified. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Germline mutations in PMS2 and MLH1 in individuals with solitary loss of PMS2 expression in colorectal carcinomas from the Colon Cancer Family Registry Cohort

PubMed Central

Rosty, Christophe; Clendenning, Mark; Walsh, Michael D; Eriksen, Stine V; Southey, Melissa C; Winship, Ingrid M; Macrae, Finlay A; Boussioutas, Alex; Parry, Susan; Arnold, Julie; Young, Joanne P; Casey, Graham; Haile, Robert W; Gallinger, Steven; Le Marchand, Loïc; Newcomb, Polly A; Potter, John D; DeRycke, Melissa; Lindor, Noralane M; Thibodeau, Stephen N; Baron, John A; Win, Aung Ko; Hopper, John L; Jenkins, Mark A; Buchanan, Daniel D

2016-01-01

Objectives Immunohistochemistry for DNA mismatch repair proteins is used to screen for Lynch syndrome in individuals with colorectal carcinoma (CRC). Although solitary loss of PMS2 expression is indicative of carrying a germline mutation in PMS2, previous studies reported MLH1 mutation in some cases. We determined the prevalence of MLH1 germline mutations in a large cohort of individuals with a CRC demonstrating solitary loss of PMS2 expression. Design This cohort study included 88 individuals affected with a PMS2-deficient CRC from the Colon Cancer Family Registry Cohort. Germline PMS2 mutation analysis (long-range PCR and multiplex ligation-dependent probe amplification) was followed by MLH1 mutation testing (Sanger sequencing and multiplex ligation-dependent probe amplification). Results Of the 66 individuals with complete mutation screening, we identified a pathogenic PMS2 mutation in 49 (74%), a pathogenic MLH1 mutation in 8 (12%) and a MLH1 variant of uncertain clinical significance predicted to be damaging by in silico analysis in 3 (4%); 6 (9%) carried variants likely to have no clinical significance. Missense point mutations accounted for most alterations (83%; 9/11) in MLH1. The MLH1 c.113A> G p.Asn38Ser mutation was found in 2 related individuals. One individual who carried the MLH1 intronic mutation c.677+3A>G p.Gln197Argfs*8 leading to the skipping of exon 8, developed 2 tumours, both of which retained MLH1 expression. Conclusions A substantial proportion of CRCs with solitary loss of PMS2 expression are associated with a deleterious MLH1 germline mutation supporting the screening for MLH1 in individuals with tumours of this immunophenotype, when no PMS2 mutation has been identified. PMID:26895986

Determination of absolute expression profiles using multiplexed miRNA analysis

PubMed Central

Song, Jee Hoon; Cheng, Yulan; Saeui, Christopher T.; Cheung, Douglas G.; Croce, Carlo M.; Yarema, Kevin J.; Meltzer, Stephen J.; Liu, Kelvin J.; Wang, Tza-Huei

2017-01-01

Accurate measurement of miRNA expression is critical to understanding their role in gene expression as well as their application as disease biomarkers. Correct identification of changes in miRNA expression rests on reliable normalization to account for biological and technological variance between samples. Ligo-miR is a multiplex assay designed to rapidly measure absolute miRNA copy numbers, thus reducing dependence on biological controls. It uses a simple 2-step ligation process to generate length coded products that can be quantified using a variety of DNA sizing methods. We demonstrate Ligo-miR’s ability to quantify miRNA expression down to 20 copies per cell sensitivity, accurately discriminate between closely related miRNA, and reliably measure differential changes as small as 1.2-fold. Then, benchmarking studies were performed to show the high correlation between Ligo-miR, microarray, and TaqMan qRT-PCR. Finally, Ligo-miR was used to determine copy number profiles in a number of breast, esophageal, and pancreatic cell lines and to demonstrate the utility of copy number analysis for providing layered insight into expression profile changes. PMID:28704432

A novel approach for copy number variation analysis by combining multiplex PCR with matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

PubMed

Gao, Yonghui; Chen, Xiaoli; Wang, Jianhua; Shangguan, Shaofang; Dai, Yaohua; Zhang, Ting; Liu, Junling

2013-06-20

With the increasing interest in copy number variation as it pertains to human genomic variation, common phenotypes, and disease susceptibility, there is a pressing need for methods to accurately identify copy number. In this study, we developed a simple approach that combines multiplex PCR with matrix-assisted laser desorption ionization time-of-flight mass spectrometry for submicroscopic copy number variation detection. Two pairs of primers were used to simultaneously amplify query and endogenous control regions in the same reaction. Using a base extension reaction, the two amplicons were then distinguished and quantified in a mass spectrometry map. The peak ratio between the test region and the endogenous control region was manually calculated. The relative copy number could be determined by comparing the peak ratio between the test and control samples. This method generated a copy number measurement comparable to those produced by two other commonly used methods - multiplex ligation-dependent probe amplification and quantitative real-time PCR. Furthermore, it can discriminate a wide range of copy numbers. With a typical 384-format SpectroCHIP, at least six loci on 384 samples can be analyzed simultaneously in a hexaplex assay, making this assay adaptable for high throughput, and potentially applicable for large-scale association studies. Copyright © 2013 Elsevier B.V. All rights reserved.

L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs)

PubMed Central

Qi, Xiaoquan; Bakht, Saleha; Devos, Katrien M.; Gale, Mike D.; Osbourn, Anne

2001-01-01

A flexible, non-gel-based single nucleotide polymorphism (SNP) detection method is described. The method adopts thermostable ligation for allele discrimination and rolling circle amplification (RCA) for signal enhancement. Clear allelic discrimination was achieved after staining of the final reaction mixtures with Cybr-Gold and visualisation by UV illumination. The use of a compatible buffer system for all enzymes allows the reaction to be initiated and detected in the same tube or microplate well, so that the experiment can be scaled up easily for high-throughput detection. Only a small amount of DNA (i.e. 50 ng) is required per assay, and use of carefully designed short padlock probes coupled with generic primers and probes make the SNP detection cost effective. Biallelic assay by hybridisation of the RCA products with fluorescence dye-labelled probes is demonstrated, indicating that ligation-RCA (L-RCA) has potential for multiplexed assays. PMID:11713336

Optimised padlock probe ligation and microarray detection of multiple (non-authorised) GMOs in a single reaction

PubMed Central

Prins, Theo W; van Dijk, Jeroen P; Beenen, Henriek G; Van Hoef, AM Angeline; Voorhuijzen, Marleen M; Schoen, Cor D; Aarts, Henk JM; Kok, Esther J

2008-01-01

Background To maintain EU GMO regulations, producers of new GM crop varieties need to supply an event-specific method for the new variety. As a result methods are nowadays available for EU-authorised genetically modified organisms (GMOs), but only to a limited extent for EU-non-authorised GMOs (NAGs). In the last decade the diversity of genetically modified (GM) ingredients in food and feed has increased significantly. As a result of this increase GMO laboratories currently need to apply many different methods to establish to potential presence of NAGs in raw materials and complex derived products. Results In this paper we present an innovative method for detecting (approved) GMOs as well as the potential presence of NAGs in complex DNA samples containing different crop species. An optimised protocol has been developed for padlock probe ligation in combination with microarray detection (PPLMD) that can easily be scaled up. Linear padlock probes targeted against GMO-events, -elements and -species have been developed that can hybridise to their genomic target DNA and are visualised using microarray hybridisation. In a tenplex PPLMD experiment, different genomic targets in Roundup-Ready soya, MON1445 cotton and Bt176 maize were detected down to at least 1%. In single experiments, the targets were detected down to 0.1%, i.e. comparable to standard qPCR. Conclusion Compared to currently available methods this is a significant step forward towards multiplex detection in complex raw materials and derived products. It is shown that the PPLMD approach is suitable for large-scale detection of GMOs in real-life samples and provides the possibility to detect and/or identify NAGs that would otherwise remain undetected. PMID:19055784

Optimised padlock probe ligation and microarray detection of multiple (non-authorised) GMOs in a single reaction.

PubMed

Prins, Theo W; van Dijk, Jeroen P; Beenen, Henriek G; Van Hoef, Am Angeline; Voorhuijzen, Marleen M; Schoen, Cor D; Aarts, Henk J M; Kok, Esther J

2008-12-04

To maintain EU GMO regulations, producers of new GM crop varieties need to supply an event-specific method for the new variety. As a result methods are nowadays available for EU-authorised genetically modified organisms (GMOs), but only to a limited extent for EU-non-authorised GMOs (NAGs). In the last decade the diversity of genetically modified (GM) ingredients in food and feed has increased significantly. As a result of this increase GMO laboratories currently need to apply many different methods to establish to potential presence of NAGs in raw materials and complex derived products. In this paper we present an innovative method for detecting (approved) GMOs as well as the potential presence of NAGs in complex DNA samples containing different crop species. An optimised protocol has been developed for padlock probe ligation in combination with microarray detection (PPLMD) that can easily be scaled up. Linear padlock probes targeted against GMO-events, -elements and -species have been developed that can hybridise to their genomic target DNA and are visualised using microarray hybridisation.In a tenplex PPLMD experiment, different genomic targets in Roundup-Ready soya, MON1445 cotton and Bt176 maize were detected down to at least 1%. In single experiments, the targets were detected down to 0.1%, i.e. comparable to standard qPCR. Compared to currently available methods this is a significant step forward towards multiplex detection in complex raw materials and derived products. It is shown that the PPLMD approach is suitable for large-scale detection of GMOs in real-life samples and provides the possibility to detect and/or identify NAGs that would otherwise remain undetected.

Sensitivity and specificity: twin goals of proteomics assays. Can they be combined?

PubMed

Wilson, Robert

2013-04-01

A major ambition of proteomics is the provision of assays that can diagnose disease and monitor therapies. These assays are required to be sensitive and specific for individual proteins, and in most cases to quantify more than one protein in the same sample. The two main technologies currently used for proteomics assays are based on mass spectrometry and panels of affinity molecules such as antibodies. In the first part of this review the most sensitive existing assays based on these technologies are described and compared with the gold standard of ELISA. Analytical sensitivity is defined and related to the limit of detection, and analytical specificity is defined and shown to depend on molecular proofreading steps, similar to those applied in living systems whenever there is a need for high fidelity. It is shown that at present neither mass spectrometry nor panels of affinity molecules offer the necessary combination of sensitivity and specificity required for multiplexed assays. In the second part of this review the growing numbers of assays that use additional proofreading steps to combine sensitivity with specificity are described. These include assays based on proximity ligation and slow off-rate modified aptamers. Finally the review considers what improvements might be possible in the near future, and concludes that further development of proteomics assays incorporating advanced proofreading steps are most likely to provide the necessary combination of sensitivity and specificity, without incurring high development costs.

Midterm Clinical Outcomes after Modified High Ligation and Segmental Stripping of Incompetent Small Saphenous Veins

PubMed Central

Hong, Ki Pyo

2015-01-01

Background The aim of this study was to evaluate the midterm clinical outcomes after modified high ligation and segmental stripping of small saphenous vein (SSV) varicosities. Methods Between January 2010 and March 2013, 62 patients (69 legs) with isolated primary small saphenous varicose veins were enrolled in this study. The outcomes measured were reflux in the remaining distal SSV, the recurrence of varicose veins, the improvement of preoperative symptoms, and the rate of postoperative complications. Results No major complications occurred. No instances of the recurrence of varicose veins at previous stripping sites were noted. Three legs (4.3%) showed reflux in the remaining distal small saphenous veins. The preoperative symptoms were found to have improved in 96.4% of the cases. Conclusion In the absence of flush ligation of the saphenopopliteal junction, modified high ligation and segmental stripping of small saphenous vein varicosities with preoperative duplex marking is an effective treatment method for reducing postoperative complications and the recurrence of SSV incompetence. PMID:26665106

Universal multiplex PCR and CE for quantification of SMN1/SMN2 genes in spinal muscular atrophy.

PubMed

Wang, Chun-Chi; Chang, Jan-Gowth; Jong, Yuh-Jyh; Wu, Shou-Mei

2009-04-01

We established a universal multiplex PCR and CE to calculate the copy number of survival motor neuron (SMN1 and SMN2) genes for clinical screening of spinal muscular atrophy (SMA). In this study, one universal fluorescent primer was designed and applied for multiplex PCR of SMN1, SMN2 and two internal standards (CYBB and KRIT1). These amplicons were separated by conformation sensitive CE. Mixture of hydroxyethyl cellulose and hydroxypropyl cellulose were used in this CE system. Our method provided the potential to separate two 390-bp PCR products that differ in a single nucleotide. Differentiation and quantification of SMN1 and SMN2 are essential for clinical screening of SMA patients and carriers. The DNA samples included 22 SMA patients, 45 parents of SMA patients (obligatory carriers) and 217 controls. For evaluating accuracy, those 284 samples were blind-analyzed by this method and denaturing high pressure liquid chromatography (DHPLC). Eight of the total samples showed different results. Among them, two samples were diagnosed as having only SMN2 gene by DHPLC, however, they contained both SMN1 and SMN2 by our method. They were further confirmed by DNA sequencing. Our method showed good agreement with the DNA sequencing. The multiplex ligation-dependent probe amplification (MLPA) was used for confirming the other five samples, and showed the same results with our CE method. For only one sample, our CE showed different results with MLPA and DNA sequencing. One out of 284 samples (0.35%) belonged to mismatching. Our method provided a better accurate method and convenient method for clinical genotyping of SMA disease.

Sensitive and specific miRNA detection method using SplintR Ligase

PubMed Central

Jin, Jingmin; Vaud, Sophie; Zhelkovsky, Alexander M.; Posfai, Janos; McReynolds, Larry A.

2016-01-01

We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR® Ligase). This two-step method involves ligation of adjacent DNA oligonucleotides hybridized to a miRNA followed by real-time quantitative PCR (qPCR). SplintR Ligase is 100X faster than either T4 DNA Ligase or T4 RNA Ligase 2 for RNA splinted DNA ligation. Only a 4–6 bp overlap between a DNA probe and miRNA was required for efficient ligation by SplintR Ligase. This property allows more flexibility in designing miRNA-specific ligation probes than methods that use reverse transcriptase for cDNA synthesis of miRNA. The qPCR SplintR ligation assay is sensitive; it can detect a few thousand molecules of miR-122. For miR-122 detection the SplintR qPCR assay, using a FAM labeled double quenched DNA probe, was at least 40× more sensitive than the TaqMan assay. The SplintR method, when coupled with NextGen sequencing, allowed multiplex detection of miRNAs from brain, kidney, testis and liver. The SplintR qPCR assay is specific; individual let-7 miRNAs that differ by one nucleotide are detected. The rapid kinetics and ability to ligate DNA probes hybridized to RNA with short complementary sequences makes SplintR Ligase a useful enzyme for miRNA detection. PMID:27154271

The clinical phenotype of Lynch syndrome due to germ-line PMS2 mutations.

PubMed

Senter, Leigha; Clendenning, Mark; Sotamaa, Kaisa; Hampel, Heather; Green, Jane; Potter, John D; Lindblom, Annika; Lagerstedt, Kristina; Thibodeau, Stephen N; Lindor, Noralane M; Young, Joanne; Winship, Ingrid; Dowty, James G; White, Darren M; Hopper, John L; Baglietto, Laura; Jenkins, Mark A; de la Chapelle, Albert

2008-08-01

Although the clinical phenotype of Lynch syndrome (also known as hereditary nonpolyposis colorectal cancer) has been well described, little is known about disease in PMS2 mutation carriers. Now that mutation detection methods can discern mutations in PMS2 from mutations in its pseudogenes, more mutation carriers have been identified. Information about the clinical significance of PMS2 mutations is crucial for appropriate counseling. Here, we report the clinical characteristics of a large series of PMS2 mutation carriers. We performed PMS2 mutation analysis using long-range polymerase chain reaction and multiplex ligation-dependent probe amplification for 99 probands diagnosed with Lynch syndrome-associated tumors showing isolated loss of PMS2 by immunohistochemistry. Penetrance was calculated using a modified segregation analysis adjusting for ascertainment. Germ-line PMS2 mutations were detected in 62% of probands (n = 55 monoallelic; 6 biallelic). Among families with monoallelic PMS2 mutations, 65.5% met revised Bethesda guidelines. Compared with the general population, in mutation carriers, the incidence of colorectal cancer was 5.2-fold higher, and the incidence of endometrial cancer was 7.5-fold higher. In North America, this translates to a cumulative cancer risk to age 70 years of 15%-20% for colorectal cancer, 15% for endometrial cancer, and 25%-32% for any Lynch syndrome-associated cancer. No elevated risk for non-Lynch syndrome-associated cancers was observed. PMS2 mutations contribute significantly to Lynch syndrome, but the penetrance for monoallelic mutation carriers appears to be lower than that for the other mismatch repair genes. Modified counseling and cancer surveillance guidelines for PMS2 mutation carriers are proposed.

"Atypical" Pleomorphic Lipomatous Tumor: A Clinicopathologic, Immunohistochemical and Molecular Study of 21 Cases, Emphasizing its Relationship to Atypical Spindle Cell Lipomatous Tumor and Suggesting a Morphologic Spectrum (Atypical Spindle Cell/Pleomorphic Lipomatous Tumor).

PubMed

Creytens, David; Mentzel, Thomas; Ferdinande, Liesbeth; Lecoutere, Evelyne; van Gorp, Joost; Atanesyan, Lilit; de Groot, Karel; Savola, Suvi; Van Roy, Nadine; Van Dorpe, Jo; Flucke, Uta

2017-11-01

The classification of the until recently poorly explored group of atypical adipocytic neoplasms with spindle cell features, for which recently the term atypical spindle cell lipomatous tumor (ASLT) has been proposed, remains challenging. Recent studies have proposed ASLT as a unique entity with (in at least a significant subset of cases) a specific genetic background, namely deletions/losses of 13q14, including RB1 and its flanking genes RCBTB2, DLEU1, and ITM2B. Similar genetic aberrations have been reported in pleomorphic liposarcomas (PLSs). This prompted us to investigate a series of 21 low-grade adipocytic neoplasms with a pleomorphic lipoma-like appearance, but with atypical morphologic features (including atypical spindle cells, pleomorphic [multinucleated] cells, pleomorphic lipoblasts and poor circumscription), for which we propose the term "atypical" pleomorphic lipomatous tumor (APLT). Five cases of PLS were also included in this study. We used multiplex ligation-dependent probe amplification to evaluate genetic changes of 13q14. In addition, array-based comparative genomic hybridization was performed on 4 APLTs and all PLSs. Multiplex ligation-dependent probe amplification showed consistent loss of RB1 and its flanking gene RCBTB2 in all cases of APLT. This genetic alteration was also present in all PLSs, suggesting genetic overlap, in addition to morphologic overlap, with APLTs. However, array-based comparative genomic hybridization demonstrated more complex genetic alterations with more losses and gains in PLSs compared with APLTs. APLTs arose in the subcutis (67%) more frequently than in the deep (subfascial) soft tissues (33%). With a median follow-up of 42 months, recurrences were documented in 2 of 12 APLTs for which a long follow-up was available. Herein, we also demonstrate that APLTs share obvious overlapping morphologic, immunohistochemical, genetic and clinical characteristics with the recently defined ASLT, suggesting that they are related lesions that form a spectrum (atypical spindle cell/pleomorphic lipomatous tumor).

Clinical utility of multiplex ligation-dependent probe amplification technique in identification of aetiology of unexplained mental retardation: a study in 203 Indian patients.

PubMed

Boggula, Vijay R; Shukla, Anju; Danda, Sumita; Hariharan, Sankar V; Nampoothiri, Sheela; Kumar, Rashmi; Phadke, Shubha R

2014-01-01

Developmental delay (DD)/mental retardation also described as intellectual disability (ID), is seen in 1-3 per cent of general population. Diagnosis continues to be a challenge at clinical level. With the advancement of new molecular cytogenetic techniques such as cytogenetic microarray (CMA), multiplex ligation-dependent probe amplification (MLPA) techniques, many microdeletion/microduplication syndromes with DD/ID are now delineated. MLPA technique can probe 40-50 genomic regions in a single reaction and is being used for evaluation of cases with DD/ID. In this study we evaluated the clinical utility of MLPA techniques with different probe sets to identify the aetiology of unexplained mental retardation in patients with ID/DD. A total of 203 randomly selected DD/ID cases with/without malformations were studied. MLPA probe sets for subtelomeric regions (P070/P036) and common microdeletions/microduplications (P245-A2) and X-chromosome (P106) were used. Positive cases with MLPA technique were confirmed using either fluorescence in situ hybridization (FISH) or follow up confirmatory MLPA probe sets. The overall detection rate was found to be 9.3 per cent (19 out of 203). The detection rates were 6.9 and 7.4 per cent for common microdeletion/microduplication and subtelomeric probe sets, respectively. No abnormality was detected with probe set for X-linked ID. The subtelomeric abnormalities detected included deletions of 1p36.33, 4p, 5p, 9p, 9q, 13q telomeric regions and duplication of 9pter. The deletions/duplications detected in non telomeric regions include regions for Prader Willi/Angelman regions, Williams syndrome, Smith Magenis syndrome and Velocardiofacial syndrome. Our results show that the use of P245-A2 and P070/P036-E1 probes gives good diagnostic yield. Though MLPA cannot probe the whole genome like cytogenetic microarray, due to its ease and relative low cost it is an important technique for evaluation of cases with DD/ID.

Highly multiplexed simultaneous detection of RNAs and proteins in single cells.

PubMed

Frei, Andreas P; Bava, Felice-Alessio; Zunder, Eli R; Hsieh, Elena W Y; Chen, Shih-Yu; Nolan, Garry P; Gherardini, Pier Federico

2016-03-01

To enable the detection of expression signatures specific to individual cells, we developed PLAYR (proximity ligation assay for RNA), a method for highly multiplexed transcript quantification by flow and mass cytometry that is compatible with standard antibody staining. When used with mass cytometry, PLAYR allowed for the simultaneous quantification of more than 40 different mRNAs and proteins. In primary cells, we quantified multiple transcripts, with the identity and functional state of each analyzed cell defined on the basis of the expression of a separate set of transcripts or proteins. By expanding high-throughput deep phenotyping of cells beyond protein epitopes to include RNA expression, PLAYR opens a new avenue for the characterization of cellular metabolism.

Clinical and molecular characterization of Chilean patients with Léri-Weill dyschondrosteosis.

PubMed

Rodríguez, Fernando Adrián; Unanue, Nancy; Hernandez, María Isabel; Basaure, Javiera; Heath, Karen Elise; Cassorla, Fernando

2013-01-01

Léri-Weill dyschondrosteosis (LWD) is a mesomelic dysplasia with disproportionate short stature associated with short stature homeobox-containing gene (SHOX) haploinsufficiency. The objective of this study was to improve the diagnosis of patients with suspected LWD through molecular analysis. Twelve patients from 11 families with a clinical diagnosis of LWD were analyzed with multiplex ligation-dependent probe amplification to detect deletions and duplications of SHOX and its enhancer regions. High resolution melting and sequencing was employed to screen for mutations in SHOX coding exons. The molecular-based screening strategy applied in these patients allowed detection of five SHOX deletions and two previously unreported SHOX missense mutations. Molecular studies confirmed the clinical diagnosis of LWD in seven out of 12 patients, which provided support for therapeutic decisions and improved genetic counseling in their families.

CYP1B1 copy number variation is not a major contributor to primary congenital glaucoma.

PubMed

Souzeau, Emmanuelle; Hayes, Melanie; Ruddle, Jonathan B; Elder, James E; Staffieri, Sandra E; Kearns, Lisa S; Mackey, David A; Zhou, Tiger; Ridge, Bronwyn; Burdon, Kathryn P; Dubowsky, Andrew; Craig, Jamie E

2015-01-01

To evaluate the prevalence and the diagnostic utility of testing for CYP1B1 copy number variation (CNV) in primary congenital glaucoma (PCG) cases unexplained by CYP1B1 point mutations in The Australian and New Zealand Registry of Advanced Glaucoma. In total, 50 PCG cases either heterozygous for disease-causing variants or with no CYP1B1 sequence variants were included in the study. CYP1B1 CNV was analyzed by Multiplex Ligation-dependent Probe Amplification (MLPA). No deletions or duplications were found in any of the cases. This is the first study to report on CYP1B1 CNV in PCG cases. Our findings show that this mechanism is not a major contributor to the phenotype and is of limited diagnostic utility.

Population-based rare variant detection via pooled exome or custom hybridization capture with or without individual indexing.

PubMed

Ramos, Enrique; Levinson, Benjamin T; Chasnoff, Sara; Hughes, Andrew; Young, Andrew L; Thornton, Katherine; Li, Allie; Vallania, Francesco L M; Province, Michael; Druley, Todd E

2012-12-06

Rare genetic variation in the human population is a major source of pathophysiological variability and has been implicated in a host of complex phenotypes and diseases. Finding disease-related genes harboring disparate functional rare variants requires sequencing of many individuals across many genomic regions and comparing against unaffected cohorts. However, despite persistent declines in sequencing costs, population-based rare variant detection across large genomic target regions remains cost prohibitive for most investigators. In addition, DNA samples are often precious and hybridization methods typically require large amounts of input DNA. Pooled sample DNA sequencing is a cost and time-efficient strategy for surveying populations of individuals for rare variants. We set out to 1) create a scalable, multiplexing method for custom capture with or without individual DNA indexing that was amenable to low amounts of input DNA and 2) expand the functionality of the SPLINTER algorithm for calling substitutions, insertions and deletions across either candidate genes or the entire exome by integrating the variant calling algorithm with the dynamic programming aligner, Novoalign. We report methodology for pooled hybridization capture with pre-enrichment, indexed multiplexing of up to 48 individuals or non-indexed pooled sequencing of up to 92 individuals with as little as 70 ng of DNA per person. Modified solid phase reversible immobilization bead purification strategies enable no sample transfers from sonication in 96-well plates through adapter ligation, resulting in 50% less library preparation reagent consumption. Custom Y-shaped adapters containing novel 7 base pair index sequences with a Hamming distance of ≥2 were directly ligated onto fragmented source DNA eliminating the need for PCR to incorporate indexes, and was followed by a custom blocking strategy using a single oligonucleotide regardless of index sequence. These results were obtained aligning raw reads against the entire genome using Novoalign followed by variant calling of non-indexed pools using SPLINTER or SAMtools for indexed samples. With these pipelines, we find sensitivity and specificity of 99.4% and 99.7% for pooled exome sequencing. Sensitivity, and to a lesser degree specificity, proved to be a function of coverage. For rare variants (≤2% minor allele frequency), we achieved sensitivity and specificity of ≥94.9% and ≥99.99% for custom capture of 2.5 Mb in multiplexed libraries of 22-48 individuals with only ≥5-fold coverage/chromosome, but these parameters improved to ≥98.7 and 100% with 20-fold coverage/chromosome. This highly scalable methodology enables accurate rare variant detection, with or without individual DNA sample indexing, while reducing the amount of required source DNA and total costs through less hybridization reagent consumption, multi-sample sonication in a standard PCR plate, multiplexed pre-enrichment pooling with a single hybridization and lesser sequencing coverage required to obtain high sensitivity.

Modified Functional Superficial Parotidectomy With Ligation of the Major Branch of the Parotid Duct Extending to the Superficial Lobe.

PubMed

Chang, Jung Woo; Leem, Soo Seong; Choi, Hwan Jun; Lee, Jang Hyun

2017-05-01

A functional superficial parotidectomy can maintain salivary function by preserving the Stensen duct. However, this technique still brings the possibility of salivary leakage, because major branches of the parotid duct from the resected site do not get ligated. To reduce this complication, this study introduces a modified technique with major branch ligation. From December 2008 to February 2015, 14 patients who underwent superficial parotidectomy were divided into 2 groups. Group A was treated with the modified functional superficial parotidectomy involving the major branch between the superficial lobe and parotid duct. Group B was treated with the conventional superficial parotidectomy without involving the major branch of the parotid duct. The clinical complications, period of Hemovac usage, and surgical duration were noted in each group. Two of 8 patients in group A had a major branch from Stensen duct that was ligated, and there was no evidence of salivary leakage or sialocele in any of the patients of group A, whereas group B contained 2 cases of salivary leakage, one of which became sialocele. Group A had a significantly longer Hemovac maintenance period than group B (P < 0.05), and the duration of surgery was also significantly different between the 2 groups (P < 0.05). Because a solitary major branch of the main parotid duct occasionally extends toward the superficial lobe, our modified technique-functional superficial parotidectomy with ligation of the major branch toward the superficial lobe-is a useful option for treatment of a benign parotid mass in such cases.

Mapping RNA Structure In Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq).

PubMed

Watters, Kyle E; Lucks, Julius B

2016-01-01

Mapping RNA structure with selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry has proven to be a versatile method for characterizing RNA structure in a variety of contexts. SHAPE reagents covalently modify RNAs in a structure-dependent manner to create adducts at the 2'-OH group of the ribose backbone at nucleotides that are structurally flexible. The positions of these adducts are detected using reverse transcriptase (RT) primer extension, which stops one nucleotide before the modification, to create a pool of cDNAs whose lengths reflect the location of SHAPE modification. Quantification of the cDNA pools is used to estimate the "reactivity" of each nucleotide in an RNA molecule to the SHAPE reagent. High reactivities indicate nucleotides that are structurally flexible, while low reactivities indicate nucleotides that are inflexible. These SHAPE reactivities can then be used to infer RNA structures by restraining RNA structure prediction algorithms. Here, we provide a state-of-the-art protocol describing how to perform in vitro RNA structure probing with SHAPE chemistry using next-generation sequencing to quantify cDNA pools and estimate reactivities (SHAPE-Seq). The use of next-generation sequencing allows for higher throughput, more consistent data analysis, and multiplexing capabilities. The technique described herein, SHAPE-Seq v2.0, uses a universal reverse transcription priming site that is ligated to the RNA after SHAPE modification. The introduced priming site allows for the structural analysis of an RNA independent of its sequence.

CNV analysis in 169 patients with bladder exstrophy-epispadias complex.

PubMed

von Lowtzow, Catharina; Hofmann, Andrea; Zhang, Rong; Marsch, Florian; Ebert, Anne-Karoline; Rösch, Wolfgang; Stein, Raimund; Boemers, Thomas M; Hirsch, Karin; Marcelis, Carlo; Feitz, Wouter F J; Brusco, Alfredo; Migone, Nicola; Di Grazia, Massimo; Moebus, Susanne; Nöthen, Markus M; Reutter, Heiko; Ludwig, Michael; Draaken, Markus

2016-04-30

The bladder exstrophy-epispadias complex (BEEC) represents the severe end of the congenital uro-rectal malformation spectrum. Initial studies have implicated rare copy number variations (CNVs), including recurrent duplications of chromosomal region 22q11.21, in BEEC etiology. To detect further CNVs, array analysis was performed in 169 BEEC patients. Prior to inclusion, 22q11.21 duplications were excluded using multiplex ligation-dependent probe amplification. Following the application of stringent filter criteria, seven rare CNVs were identified: n = 4, not present in 1307 in-house controls; n = 3, frequency of <0.002 in controls. These CNVs ranged from 1 to 6.08 Mb in size. To identify smaller CNVs, relaxed filter criteria used in the detection of previously reported BEEC associated chromosomal regions were applied. This resulted in the identification of six additional rare CNVs: n = 4, not present in 1307 in-house controls; n = 2, frequency <0.0008 in controls. These CNVs ranged from 0.03-0.08 Mb in size. For 10 of these 13 CNVs, confirmation and segregation analyses were performed (5 of maternal origin; 5 of paternal origin). Interestingly, one female with classic bladder extrophy carried a 1.18 Mb duplication of 22q11.1, a chromosomal region that is associated with cat eye syndrome. A number of rare CNVs were identified in BEEC patients, and these represent candidates for further evaluation. Rare inherited CNVs may constitute modifiers of, or contributors to, multifactorial BEEC phenotypes.

Identification of rare and novel deletions that cause (δβ)0-thalassaemia and hereditary persistence of foetal haemoglobin in Indian population.

PubMed

Mayuranathan, Thiyagaraj; Rayabaram, Janakiram; Das, Reena; Arora, Neeraj; Edison, Eunice S; Chandy, Mammen; Srivastava, Alok; Velayudhan, Shaji R

2014-06-01

Hereditary persistence of foetal haemoglobin (HPFH) and (δβ)(0) -thalassaemia are conditions caused by large deletions that involve δ- and β-globin genes in the β-globin cluster, and they are characterized by increased haemoglobin (HbF) levels in adults. Significant phenotypic diversity is observed between the different mutations that cause these conditions. Molecular characterization of these deletions is important for accurate molecular diagnosis, and they will also provide the information on the cis-acting genetic regulatory elements present in the β-globin cluster. We performed gap-PCR, multiplex ligation-dependent probe amplification (MLPA), quantitative fluorescent multiplex PCR (QF-MPCR) and DNA sequencing to detect and characterize the deletions in the β-globin cluster. We characterized six different deletions resulting in (δβ)(0) -thalassaemia or HPFH in 51 unrelated families. With the help of multiple genetic tools, we performed comprehensive genetic analysis of HPFH and (δβ)(0) -thalassaemia in Indian population and could define the molecular basis of these conditions in this population. We also identified two novel HPFH mutations, 49.98 kb (HPFH-9) and 86.7 kb (HPFH-10) deletions, in this population. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Single quantum dot analysis enables multiplexed point mutation detection by gap ligase chain reaction.

PubMed

Song, Yunke; Zhang, Yi; Wang, Tza-Huei

2013-04-08

Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and a tedious assay processes. In this report, an assay technology is proposed which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single-molecule coincidence detection, and the superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single-molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cerebellum volume in high-risk offspring from multiplex alcohol dependence families: Association with allelic variation in GABRA2 and BDNF

PubMed Central

Hill, Shirley Y.; Wang, Shuhui; Carter, Howard; Tessner, Kevin; Holmes, Brian; McDermott, Michael; Zezza, Nicholas; Stiffler, Scott

2012-01-01

Offspring from families with multiple cases of alcohol dependence have a greater likelihood of developing alcohol dependence (AD) and related substance use disorders. Greater susceptibility for developing these disorders may be related to structural differences in brain circuits that influence the salience of rewards or modify the efficiency of information processing and AD susceptibility. We examined the cerebellum of 71 adolescent/young adult high-risk (HR) offspring from families with multiple cases of alcohol dependence (multiplex families), and 60 low-risk (LR) controls with no family history of alcohol or drug dependence who were matched for age, gender, socioeconomic status and IQ, with attention given to possible effects of personal use of substances and maternal use during pregnancy. Magnetic resonance images were acquired on a General Electric 1.5-Tesla scanner and manually traced (BRAINS2) blind to clinical information. GABRA2 and BDNF variation were tested for their association with cerebellar volumes. High-risk offspring from multiplex AD families showed greater total volume of the cerebellum and total gray matter (GM), in comparison with LR controls. An interaction between allelic variation in GABRA2 and BDNF genes was associated with GM volumes, suggesting that inherited variation in these genes may promote early developmental differences in neuronal proliferation of the cerebellum. PMID:22047728

Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms.

PubMed

Cottenet, Geoffrey; Blancpain, Carine; Sonnard, Véronique; Chuah, Poh Fong

2013-08-01

Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers' perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.

A novel Alu-mediated microdeletion at 11p13 removes WT1 in a patient with cryptorchidism and azoospermia.

PubMed

Seabra, Catarina M; Quental, Sofia; Neto, Ana Paula; Carvalho, Filipa; Gonçalves, João; Oliveira, João Paulo; Fernandes, Susana; Sousa, Mário; Barros, Alberto; Amorim, António; Lopes, Alexandra M

2014-09-01

This article describes a patient with cryptorchidism and nonobstructive azoospermia presenting a novel microdeletion of approximately 1 Mb at 11p13. It was confirmed by multiplex ligation-dependent probe amplification that this heterozygous deletion spanned nine genes (WT1, EIF3M, CCDC73, PRRG4, QSER1, DEPDC7, TCP11L1, CSTF3 and HIPK3) and positioned the breakpoints within highly homologous repetitive elements. As far as is known, this is the smallest deletion as-yet described encompassing the WT1 gene and was detected only once in a total of 32 Portuguese patients with isolated uni- or bilateral cryptorchidism. These findings suggest that molecular analysis in patients with genitourinary features suggestive of WT1 impairment, namely cryptorchidism and renal abnormalities, may reveal cryptic genetic defects. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

A large deletion in the succinate dehydrogenase B gene (SDHB) in a Japanese patient with abdominal paraganglioma and concomitant metastasis.

PubMed

Kodama, Hitomi; Iihara, Masatoshi; Nissato, Sumiko; Isobe, Kazumasa; Kawakami, Yasushi; Okamoto, Takahiro; Takekoshi, Kazuhiro

2010-01-01

Recently, mutations in nuclear genes encoding two mitochondrial complex II subunit proteins, Succinate dehydrogenase D (SDHD) and SDHB, have been found to be associated with the development of familial pheochromocytomas and paragangliomas (hereditary pheochromocytoma/paraganglioma syndrome: HPPS). Growing evidence suggests that the mutation of SDHB is highly associated with abdominal paraganglioma and the following distant metastasis (malignant paraganglioma). In the present study, we used multiplex ligation dependent probe amplification (MLPA) analysis to identify a large heterozygous SDHB gene deletion encompassing sequences corresponding to the promoter region, in addition to exon 1 and exon 2 malignant paraganglioma patient in whom previously characterized SDHB mutations were undetectable. This is the first Japanese case report of malignant paraganglioma, with a large SDHB deletions. Our present findings strongly support the notion that large deletions in the SDHB gene should be considered in patients lacking characterized SDHB mutations.

DMD mutation spectrum analysis in 613 Chinese patients with dystrophinopathy.

PubMed

Guo, Ruolan; Zhu, Guosheng; Zhu, Huimin; Ma, Ruiyu; Peng, Ying; Liang, Desheng; Wu, Lingqian

2015-08-01

Dystrophinopathy is a group of inherited diseases caused by mutations in the DMD gene. Within the dystrophinopathy spectrum, Duchenne and Becker muscular dystrophies are common X-linked recessive disorders that mainly feature striated muscle necrosis. We combined multiplex ligation-dependent probe amplification with Sanger sequencing to detect large deletions/duplications and point mutations in the DMD gene in 613 Chinese patients. A total of 571 (93.1%) patients were diagnosed, including 428 (69.8%) with large deletions/duplications and 143 (23.3%) with point mutations. Deletion/duplication breakpoints gathered mostly in introns 44-55. Reading frame rules could explain 88.6% of deletion mutations. We identified seventy novel point mutations that had not been previously reported. Spectrum expansion and genotype-phenotype analysis of DMD mutations on such a large sample size in Han Chinese population would provide new insights into the pathogenic mechanism underlying dystrophinopathies.

Characterization of 4 New Mutations in the CYBB Gene in 10 Iranian Families With X-linked Chronic Granulomatous Disease.

PubMed

Teimourian, Shahram; Sazgara, Faezeh; de Boer, Martin; van Leeuwen, Karin; Roos, Dirk; Lashkary, Sharzad; Chavoshzadeh, Zahra; Nabavi, Mohammad; Bemanian, Mohammad Hassan; Isaian, Anna

2018-04-26

Chronic granulomatous disease (CGD) is an inherited disease of the innate immune system that results from defects in 1 of the 5 subunits of nicotinamide adenine dinucleotide phosphate oxidase complex and leads to life-threatening infections with granuloma formation. During 3 years of study, we recognized 10 male patients with X-linked CGD from a tertiary referral center for immune deficiencies in Iran. The CGD patients were diagnosed according to clinical features and biochemical tests, including nitroblue tetrazolium and dihydrorhodamine-1, 2, 3 tests, performed on patients and their mothers. In all patients, Western blot analysis showed a gp91 phenotype. Mutation screening by single strand conformation polymorphism and multiplex ligation-dependent probe amplification analysis of the CYBB gene encoding gp91, followed by sequencing, showed 9 different mutations, 4 of them novel as far as we know.

[Application of droplet digital PCR technology for genetic testing and prenatal diagnosis of spinal muscular atrophy].

PubMed

Zou, Yang; Xu, Peiwen; Li, Jie; Huang, Sexin; Gao, Ming; Kang, Ranran; Gao, Xuan; Gao, Yuan

2016-10-01

To explore the clinical application of droplet digital PCR (ddPCR) for genetic testing and prenatal diagnosis of spinal muscular atrophy (SMA) with deletion of SMN1 gene exon 7. A total of 138 clinical samples, including 121 peripheral blood, 13 amniotic fluid, 2 umbilical cord blood and 2 chorionic villi from 56 SMA families, were tested by both ddPCR and multiplex ligation-dependent probe amplification (MLPA). Results of the two approaches were analyzed with commercial software QuantaSoft (ddPCR) and Coffalyser (MLPA), respectively. Among the 138 cases, 25 had two copies, 84 had one copy, and 29 had null copy of exon 7 of the SMN1 gene. The results of ddPCR and MLPA were completely consistent. As a rapid, precise and economically efficient method, ddPCR will provide a new choice for genetic testing of SMA.

A case report: Becker muscular dystrophy presenting with epilepsy and dysgnosia induced by duplication mutation of Dystrophin gene.

PubMed

Miao, Jing; Feng, Jia-Chun; Zhu, Dan; Yu, Xue-Fan

2016-12-12

Becker muscular dystrophy (BMD), a genetic disorder of X-linked recessive inheritance, typically presents with gradually progressive muscle weakness. The condition is caused by mutations of Dystrophin gene located at Xp21.2. Epilepsy is an infrequent manifestation of BMD, while cases of BMD with dysgnosia are extremely rare. We describe a 9-year-old boy with BMD, who presented with epilepsy and dysgnosia. Serum creatine kinase level was markedly elevated (3665 U/L). Wechsler intelligence tests showed a low intelligence quotient (IQ = 65). Electromyogram showed slight myogenic changes and skeletal muscle biopsy revealed muscular dystrophy. Immunohistochemical staining showed partial positivity of sarcolemma for dystrophin-N. Multiplex ligation-dependent probe amplification revealed a duplication mutation in exons 37-44 in the Dystrophin gene. The present case report helps to better understand the clinical and genetic features of BMD.

High-Throughput Amplicon-Based Copy Number Detection of 11 Genes in Formalin-Fixed Paraffin-Embedded Ovarian Tumour Samples by MLPA-Seq

PubMed Central

Kondrashova, Olga; Love, Clare J.; Lunke, Sebastian; Hsu, Arthur L.; Waring, Paul M.; Taylor, Graham R.

2015-01-01

Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity. PMID:26569395

Compositional Bias in Naïve and Chemically-modified Phage-Displayed Libraries uncovered by Paired-end Deep Sequencing.

PubMed

He, Bifang; Tjhung, Katrina F; Bennett, Nicholas J; Chou, Ying; Rau, Andrea; Huang, Jian; Derda, Ratmir

2018-01-19

Understanding the composition of a genetically-encoded (GE) library is instrumental to the success of ligand discovery. In this manuscript, we investigate the bias in GE-libraries of linear, macrocyclic and chemically post-translationally modified (cPTM) tetrapeptides displayed on the M13KE platform, which are produced via trinucleotide cassette synthesis (19 codons) and NNK-randomized codon. Differential enrichment of synthetic DNA {S}, ligated vector {L} (extension and ligation of synthetic DNA into the vector), naïve libraries {N} (transformation of the ligated vector into the bacteria followed by expression of the library for 4.5 hours to yield a "naïve" library), and libraries chemically modified by aldehyde ligation and cysteine macrocyclization {M} characterized by paired-end deep sequencing, detected a significant drop in diversity in {L} → {N}, but only a minor compositional difference in {S} → {L} and {N} → {M}. Libraries expressed at the N-terminus of phage protein pIII censored positively charged amino acids Arg and Lys; libraries expressed between pIII domains N1 and N2 overcame Arg/Lys-censorship but introduced new bias towards Gly and Ser. Interrogation of biases arising from cPTM by aldehyde ligation and cysteine macrocyclization unveiled censorship of sequences with Ser/Phe. Analogous analysis can be used to explore library diversity in new display platforms and optimize cPTM of these libraries.

[Investigation of RNA viral genome amplification by multiple displacement amplification technique].

PubMed

Pang, Zheng; Li, Jian-Dong; Li, Chuan; Liang, Mi-Fang; Li, De-Xin

2013-06-01

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.

Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins

NASA Astrophysics Data System (ADS)

Bondalapati, Somasekhar; Jbara, Muhammad; Brik, Ashraf

2016-05-01

Methods to prepare proteins that include a specific modification at a desired position are essential for understanding their cellular functions and physical properties in living systems. Chemical protein synthesis, which relies on the chemoselective ligation of unprotected peptides, enables the preparation of modified proteins that are not easily fabricated by other methods. In contrast to recombinant approaches, chemical synthesis can be used to prepare protein analogues such as D-proteins, which are useful in protein structure determination and the discovery of novel therapeutics. Post-translationally modifying proteins is another example where chemical protein synthesis proved itself as a powerful approach for preparing samples with high homogeneity and in workable quantities. In this Review, we discuss the basic principles of the field, focusing on novel chemoselective peptide ligation approaches such as native chemical ligation and the recent advances based on this method with a proven record of success in the synthesis of highly important protein targets.

Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

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

Lovrinovic, Marina; Niemeyer, Christof M.

2005-09-30

We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter weremore » ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.« less

Multiplex quantification of 16S rDNA of predominant bacteria group within human fecal samples by polymerase chain reaction--ligase detection reaction (PCR-LDR).

PubMed

Li, Kai; Chen, Bei; Zhou, Yuxun; Huang, Rui; Liang, Yinming; Wang, Qinxi; Xiao, Zhenxian; Xiao, Junhua

2009-03-01

A new method, based on ligase detection reaction (LDR), was developed for quantitative detection of multiplex PCR amplicons of 16S rRNA genes present in complex mixtures (specifically feces). LDR has been widely used in single nucleotide polymorphism (SNP) assay but never applied for quantification of multiplex PCR products. This method employs one pair of DNA probes, one of which is labeled with fluorescence for signal capture, complementary to the target sequence. For multiple target sequence analysis, probes were modified with different lengths of polyT at the 5' end and 3' end. Using a DNA sequencer, these ligated probes were separated and identified by size and dye color. Then, relative abundance of target DNA were normalized and quantified based on the fluorescence intensities and exterior size standards. 16S rRNA gene of three preponderant bacteria groups in human feces: Clostridium coccoides, Bacteroides and related genera, and Clostridium leptum group, were amplified and cloned into plasmid DNA so as to make standard curves. After PCR-LDR analysis, a strong linear relationship was found between the florescence intensity and the diluted plasmid DNA concentrations. Furthermore, based on this method, 100 human fecal samples were quantified for the relative abundance of the three bacterial groups. Relative abundance of C. coccoides was significantly higher in elderly people in comparison with young adults, without gender differences. Relative abundance of Bacteroides and related genera and C. leptum group were significantly higher in young and middle aged than in the elderly. Regarding the whole set of sample, C. coccoides showed the highest relative abundance, followed by decreasing groups Bacteroides and related genera, and C. leptum. These results imply that PCR-LDR can be feasible and flexible applied to large scale epidemiological studies.

A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens.

PubMed

Rundell, Mark S; Pingle, Maneesh; Das, Sanchita; Hussain, Aashiq; Ocheretina, Oksana; Charles, Macarthur; Larone, Davise H; Spitzer, Eric D; Golightly, Linnie; Barany, Francis

2014-06-01

Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/ligation detection reaction (LDR) assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay were assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91% to 100% (median 100%) depending on the species. For the majority of organisms, the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens. Copyright © 2014 Elsevier Inc. All rights reserved.

Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

PubMed

Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

2011-01-01

In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

Photophysics of self-assembled zinc porphyrin-bidentate diamine ligand complexes.

PubMed

Danger, Brook R; Bedient, Krysta; Maiti, Manisankar; Burgess, Ian J; Steer, Ronald P

2010-10-21

The effects of complexation--by bidentate nitrogen-containing ligands such as pyrazine and 4,4'-bipyridine commonly used for porphyrin self-assembly--on the photophysics of the model metalloporphyrin, ZnTPP, are reported. Ligation to form the 5-coordinate species introduces an intramolecular charge transfer (ITC) state that, depending on the oxidation and reduction potentials of the electron donor and acceptor, can become involved in the excited state relaxation processes. For ZnTPP, ligation with pyridine has little effect on excited state relaxation following either Q-band or Soret band excitation. However, coordination of ZnTPP with pyrazine and bipyridine causes the S(2) (Soret) state of the ligated species to decay almost exclusively via an S(2)-ICT-S(1) pathway, while affecting the S(1) decay route only slightly. In these 5-coordinate species the S(2)-ICT-S(1) decay route is ultrafast and nearly quantitative. Literature redox data for other bidentate ligands such as DABCO and multidentate ligands commonly used for pophyrin assembly suggest that the ITC states introduced by them could also modify the excited state relaxation dynamics of a wide variety of multiporphyrin arrays.

A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.

PubMed

Ma, Xingliang; Zhang, Qunyu; Zhu, Qinlong; Liu, Wei; Chen, Yan; Qiu, Rong; Wang, Bin; Yang, Zhongfang; Li, Heying; Lin, Yuru; Xie, Yongyao; Shen, Rongxin; Chen, Shuifu; Wang, Zhi; Chen, Yuanling; Guo, Jingxin; Chen, Letian; Zhao, Xiucai; Dong, Zhicheng; Liu, Yao-Guang

2015-08-01

CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

CRISPR/Cas9-Based Multiplex Genome Editing in Monocot and Dicot Plants.

PubMed

Ma, Xingliang; Liu, Yao-Guang

2016-07-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome targeting system has been applied to a variety of organisms, including plants. Compared to other genome-targeting technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR/Cas9 system is easier to use and has much higher editing efficiency. In addition, multiple "single guide RNAs" (sgRNAs) with different target sequences can be designed to direct the Cas9 protein to multiple genomic sites for simultaneous multiplex editing. Here, we present a procedure for highly efficient multiplex genome targeting in monocot and dicot plants using a versatile and robust CRISPR/Cas9 vector system, emphasizing the construction of binary constructs with multiple sgRNA expression cassettes in one round of cloning using Golden Gate ligation. We also describe the genotyping of targeted mutations in transgenic plants by direct Sanger sequencing followed by decoding of superimposed sequencing chromatograms containing biallelic or heterozygous mutations using the Web-based tool DSDecode. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Multiplexed methylation profiles of tumor suppressor genes and clinical outcome in oligodendroglial tumors.

PubMed

Kuo, Lu-Ting; Lu, Hsueh-Yi; Lee, Chien-Chang; Tsai, Jui-Chang; Lai, Hong-Shiee; Tseng, Ham-Min; Kuo, Meng-Fai; Tu, Yong-Kwang

2016-08-01

Aberrant methylation has been associated with transcriptional inactivation of tumor-related genes in a wide spectrum of human neoplasms. The influence of DNA methylation in oligodendroglial tumors is not fully understood. Genomic DNA was isolated from 61 oligodendroglial tumors for analysis of methylation using methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). We correlated methylation status with clinicopathological findings and outcome. The genes found to be most frequently methylated in oligodendroglial tumors were RASSF1A (80.3%), CASP8 (70.5%), and CDKN2A (52.5%). Kaplan-Meier survival curve analysis demonstrated longer duration of progression-free survival in patients with 19q loss, aged less than 38 years, and with a proliferative index of less than 5%. Methylation of the ESR1 promoter is significantly associated with shorter duration of overall survival and progression-free survival, and that methylation of IGSF4 and RASSF1A is significantly associated with shorter duration of progression-free survival. However, none of the methylation status of ESR1, IGSF4, and RASSF1A was of prognostic value for survival in a multivariate Cox model. A number of novel and interesting epigenetic alterations were identified in this study. The findings highlight the importance of methylation profiles in oligodendroglial tumors and their possible involvement in tumorigenesis. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Assembly of a biocompatible triazole-linked gene by one-pot click-DNA ligation

NASA Astrophysics Data System (ADS)

Kukwikila, Mikiembo; Gale, Nittaya; El-Sagheer, Afaf H.; Brown, Tom; Tavassoli, Ali

2017-11-01

The chemical synthesis of oligonucleotides and their enzyme-mediated assembly into genes and genomes has significantly advanced multiple scientific disciplines. However, these approaches are not without their shortcomings; enzymatic amplification and ligation of oligonucleotides into genes and genomes makes automation challenging, and site-specific incorporation of epigenetic information and/or modified bases into large constructs is not feasible. Here we present a fully chemical one-pot method for the assembly of oligonucleotides into a gene by click-DNA ligation. We synthesize the 335 base-pair gene that encodes the green fluorescent protein iLOV from ten functionalized oligonucleotides that contain 5ʹ-azide and 3ʹ-alkyne units. The resulting click-linked iLOV gene contains eight triazoles at the sites of chemical ligation, and yet is fully biocompatible; it is replicated by DNA polymerases in vitro and encodes a functional iLOV protein in Escherichia coli. We demonstrate the power and potential of our one-pot gene-assembly method by preparing an epigenetically modified variant of the iLOV gene.

The Ufm1 Cascade

PubMed Central

Daniel, Jens; Liebau, Eva

2014-01-01

The ubiquitin-fold modifier 1 (Ufm1) is a posttranslational modifier that belongs to the ubiquitin-like protein (UBL) family. Ufm1 is present in nearly all eukaryotic organisms, with the exception of fungi. It resembles ubiquitin in its ability to be ligated to other proteins, as well as in the mechanism of ligation. While the Ufm1 cascade has been implicated in endoplasmic reticulum functions and cell cycle control, its biological role still remains poorly understood. In this short review, we summarize the current state of Ufm1 research and its potential role in human diseases, like diabetes, ischemic heart disease and cancer. PMID:24921187

Friction behavior of self-ligating and conventional brackets with different ligature systems.

PubMed

Szczupakowski, Alexandra; Reimann, Susanne; Dirk, Cornelius; Keilig, Ludger; Weber, Anna; Jäger, Andreas; Bourauel, Christoph

2016-07-01

Self-ligating brackets are widely believed to offer better clinical efficiency and, in particular, less friction. Thus, the goal of this in vitro investigation was to assess the friction behavior of different bracket/archwire/ligature combinations during simulated canine retraction. An important aspect of this work was to determine whether conventional bracket systems behave differently in passive or active self-ligating brackets used with a Slide™ ligature, an elastic ligature, or a steel ligature. Three conventional (Contour, Class One; Discovery(®), Dentaurum; Mystique MB, GAC) and six self-ligating (Carriere SL, Class One; Clarity™ SL, 3M Unitek; Damon3, Ormco; In-Ovation(®) C, GAC; Speed Appliance, Speed System™; QuicKlear(®), Forestadent(®)) bracket systems were analyzed. All brackets featured a 0.022″ slot (0.56 mm). Each conventional system was tested with a steel ligature (0.25 mm; Remanium(®), Dentaurum), an elastic ligature (1.3 mm in diameter; Dentalastics, Dentaurum), and a modified elastic ligature (Slide™; Leone(®)). Each combination was used with four archwires, including rectangular stainless steel (0.46 × 0.64 mm, 0.018 × 0.025″, Dentaurum), rectangular nickel-titanium with Teflon coating (0.46 × 0.64 mm, 0.018 × 0.025″, Forestadent(®)), round coaxial nickel-titanium (0.46 mm, 0.018″, Speed), and half-round/half-square (D-profile) stainless steel (0.46 mm, 0.018″, Speed). In the orthodontic measurement and simulation system (OMSS), retraction of a canine was simulated on a Frasaco model replicated in resin. Based on the force systems, the respective friction values were determined. For each combination of materials, five brackets of the same type were tested and five single measurements performed. Friction values were found to vary distinctly with the different combinations, modifiers being the ligature systems and the archwire types. Any significant friction differences between the steel-ligated, Slide™-ligated, and self-ligated brackets were sporadic. All three systems were associated with average friction values of 40 %. Active self-ligating brackets and elastic-ligated conventional brackets, by contrast, generally differed significantly from the three above-mentioned bracket systems and showed distinctly higher friction values averaging 59 and 67 %, respectively. While passive self-ligating bracket systems have frequently been touted as advantageous in the literature, they should not be regarded as the only favorable system. Steel-ligated and Slide™-ligated conventional bracket systems are capable of offering similar friction performance.

Covalent Chemical Ligation Strategy for Mono- and Polyclonal Immunoglobulins at Their Nucleotide Binding Sites.

PubMed

Lac, Diana; Feng, Chun; Bhardwaj, Gaurav; Le, Huong; Tran, Jimmy; Xing, Li; Fung, Gabriel; Liu, Ruiwu; Cheng, Holland; Lam, Kit S

2016-01-20

Nonspecific ligation methods have been traditionally used to chemically modify immunoglobulins. Site-specific ligation of compounds (toxins or ligands) to antibodies has become increasingly important in the fields of therapeutic antibody-drug conjugates and bispecific antibodies. In this present study, we took advantage of the reported nucleotide-binding pocket (NBP) in the Fab arms of immunoglobulins by developing indole-based, 5-fluoro-2,4-dinitrobenzene-derivatized OBOC peptide libraries for the identification of affinity elements that can be used as site-specific derivatization agents against both mono- and polyclonal antibodies. Ligation can occur at any one of the few lysine residues located at the NBP. Immunoconjugates resulting from such affinity elements can be used as therapeutics against cancer or infectious agents.

Identification of a novel large intragenic deletion in a family with Fanconi anemia: first molecular report from India and review of literature.

PubMed

Shukla, Pallavi; Rao, Anita; Ghosh, Kanjaksha; Vundinti, Babu Rao

2013-04-15

We report here an Indian case with Fanconi anemia (FA) presented with fever, pallor, short stature, hyperpigmentation and upper limb anomaly. Chromosome breakage analysis together with FANCD2 Western blot monoubiquitination assay confirmed the diagnosis as FA. Multiplex ligation-dependent probe amplification (MLPA) revealed a novel homozygous large intragenic deletion (exons 8-27 del) in the FANCA gene in the proband. His sib and parents were also analyzed and found to be heterozygous for the same mutation. We also reviewed the literature of FANCA large intragenic deletions found in FA patients from different countries and the mechanism involved in the formation of these deletions. To the best of our knowledge, this is the first molecular report from India on FA. The finding expands the mutation spectrum of the FANCA gene. Identification of the mutation confirms the diagnosis of FA at DNA level and helps in providing proper genetic counseling to the family. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification of de novo mutations of Duchénnè/Becker muscular dystrophies in southern Spain.

PubMed

Garcia, Susana; de Haro, Tomás; Zafra-Ceres, Mercedes; Poyatos, Antonio; Gomez-Capilla, Jose A; Gomez-Llorente, Carolina

2014-01-01

Duchénnè/Becker muscular dystrophies (DMD/BMD) are X-linked diseases, which are caused by a de novo gene mutation in one-third of affected males. The study objectives were to determine the incidence of DMD/BMD in Andalusia (Spain) and to establish the percentage of affected males in whom a de novo gene mutation was responsible. Multiplex ligation-dependent probe amplification (MLPA) technology was applied to determine the incidence of DMD/BMD in 84 males with suspicion of the disease and 106 female relatives. Dystrophin gene exon deletion (89.5%) or duplication (10.5%) was detected in 38 of the 84 males by MLPA technology; de novo mutations account for 4 (16.7%) of the 24 mother-son pairs studied. MLPA technology is adequate for the molecular diagnosis of DMD/BMD and establishes whether the mother carries the molecular alteration responsible for the disease, a highly relevant issue for genetic counseling.

Novel germline SDHD deletion associated with an unusual sympathetic head and neck paraganglioma.

PubMed

Cadiñanos, Juan; Llorente, José L; de la Rosa, Jorge; Villameytide, José A; Illán, Rafael; Durán, Noelia S; Murias, Eduardo; Cabanillas, Rubén

2011-08-01

Paragangliomas (PGLs) are rare tumors arising either from sympathetic or parasympathetic-associated chromaffin tissue. PGLs can occur either sporadically or as part of a hereditary syndrome. Sympathetic head and neck PGLs are extremely rare tumors and only a few cases have been reported to date. We report the pedigree of a patient with a head and neck PGL arising from the right sympathetic trunk. SDHD mutation analysis was performed using standard sequencing, multiplex ligation-dependent probe amplification, chromosome 11-specific comparative genome hybridization, and long-range/short-range polymerase chain reaction (PCR) approaches. A previously unreported chromosome 11q deletion encompassing 5 annotated genes (SDHD, DLAT, PIH1D2, C11Orf57, and TIMM8B) was detected in the proband. PGL families considered "mutation-negative" may be attributable to large gene deletions not detectable by standard sequencing methods. Therefore, deletion analysis should be offered to families or individuals at risk for hereditary PGLs. Copyright © 2010 Wiley Periodicals, Inc.

The First Report of a 290-bp Deletion in β-Globin Gene in the South of Iran

PubMed Central

Hamid, Mohammad; Nejad, Ladan Dawoody; Shariati, Gholamreza; Galehdari, Hamid; Saberi, Alihossein; Mohammadi-Anaei, Marziye

2017-01-01

Background: β-thalassemia is one of the most widespread diseases in the world, including Iran. In this study, we reported, for the first time, a 290-bp β-globin gene deletion in the south of Iran. Methods: Four individuals from three unrelated families with Arabic ethnic background were studied in Khuzestan Province. Red blood cell indices and hemoglobin analysis were carried out according to the standard methods. Genomic DNA was obtained from peripheral blood cells by salting out procedures. β-globin gene amplification, multiplex ligation-dependent probe amplification (MLPA), and DNA sequencing were performed. Results: The PCR followed by sequencing and MLPA test of the β-globin gene confirmed the presence of a 290-bp deletion in the heterozygous form, along with -88C>A mutation. All the individuals had elevated hemoglobin A2 and normal fetal hemoglobin levels. Conclusions: This mutation causes β0-thalassemia and can be highly useful for prenatal diagnosis in compound heterozygous condition with different β-globin gene mutations. PMID:26948378

Cryptic intragenic deletion of the SHOX gene in a family with Léri-Weill dyschondrosteosis detected by Multiplex Ligation-Dependent Probe Amplification (MLPA).

PubMed

Funari, Mariana F A; Jorge, Alexander A L; Pinto, Emilia M; Arnhold, Ivo J P; Mendonca, Berenice B; Nishi, Mirian Y

2008-11-01

LWD is associated to SHOX haploinsufficiency, in most cases, due to gene deletion. Generally FISH and microsatellite analysis are used to identify SHOX deletion. MLPA is a new method of detecting gene copy variation, allowing simultaneous analysis of several regions. Here we describe the presence of a SHOX intragenic deletion in a family with LWD, analyzed through different methodologies. Genomic DNA of 11 subjects from one family were studied by microsatellite analysis, direct sequencing and MLPA. FISH was performed in two affected individuals. Microsatellite analysis showed that all affected members shared the same haplotype suggesting the involvement of SHOX. MLPA detected an intragenic deletion involving exons IV-VIa, which was not detected by FISH and microsatellite analysis. In conclusion, the MLPA technique was proved to be the best solution on detecting this small deletion, it has the advantage of being less laborious also allowing the analysis of several regions simultaneously.

Precise Sequential DNA Ligation on A Solid Substrate: Solid-Based Rapid Sequential Ligation of Multiple DNA Molecules

PubMed Central

Takita, Eiji; Kohda, Katsunori; Tomatsu, Hajime; Hanano, Shigeru; Moriya, Kanami; Hosouchi, Tsutomu; Sakurai, Nozomu; Suzuki, Hideyuki; Shinmyo, Atsuhiko; Shibata, Daisuke

2013-01-01

Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation. PMID:23897972

The minimal amount of starting DNA for Agilent’s hybrid capture-based targeted massively parallel sequencing

PubMed Central

Chung, Jongsuk; Son, Dae-Soon; Jeon, Hyo-Jeong; Kim, Kyoung-Mee; Park, Gahee; Ryu, Gyu Ha; Park, Woong-Yang; Park, Donghyun

2016-01-01

Targeted capture massively parallel sequencing is increasingly being used in clinical settings, and as costs continue to decline, use of this technology may become routine in health care. However, a limited amount of tissue has often been a challenge in meeting quality requirements. To offer a practical guideline for the minimum amount of input DNA for targeted sequencing, we optimized and evaluated the performance of targeted sequencing depending on the input DNA amount. First, using various amounts of input DNA, we compared commercially available library construction kits and selected Agilent’s SureSelect-XT and KAPA Biosystems’ Hyper Prep kits as the kits most compatible with targeted deep sequencing using Agilent’s SureSelect custom capture. Then, we optimized the adapter ligation conditions of the Hyper Prep kit to improve library construction efficiency and adapted multiplexed hybrid selection to reduce the cost of sequencing. In this study, we systematically evaluated the performance of the optimized protocol depending on the amount of input DNA, ranging from 6.25 to 200 ng, suggesting the minimal input DNA amounts based on coverage depths required for specific applications. PMID:27220682

APC promoter 1B deletion in seven American families with familial adenomatous polyposis.

PubMed

Snow, A K; Tuohy, T M F; Sargent, N R; Smith, L J; Burt, R W; Neklason, D W

2015-10-01

Familial adenomatous polyposis (FAP) is a colorectal cancer predisposition syndrome caused by mutations in the adenomatous polyposis coli (APC) gene. Clinical genetic testing fails to identify disease causing mutations in up to 20% of clinically apparent FAP cases. Following the inclusion of multiplex ligation-dependent probe amplification (MLPA) probes specific for APC promoter 1B, seven probands were identified with a deletion of promoter 1B. Using haplotype analysis spanning the APC locus, the seven families appear to be identical by descent from a common founder. The clinical phenotype of 19 mutation carriers is classical FAP with colectomy at an average age of 24. The majority of cases had a large number of duodenal and gastric polyps. Measurements of allele-specific expression of APC mRNA using TaqMan assay confirmed that relative expression in the allele containing the promoter 1B deletion was reduced 42-98%, depending on tissue type. This study confirms the importance of APC promoter deletions as a cause of FAP and identifies a founder mutation in FAP patients from the United States. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Anatomy of an engineered NAD-binding site.

PubMed Central

Mittl, P. R.; Berry, A.; Scrutton, N. S.; Perham, R. N.; Schulz, G. E.

1994-01-01

The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2'-phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38-43). In order to analyze the structural changes involved, we have determined 4 high-resolution crystal structures, i.e., the structures of the wild-type enzyme (1.86 A resolution, R-factor of 16.8%), of the wild-type enzyme ligated with NADP (2.0 A, 20.8%), of the NAD-dependent mutant (1.74 A, 16.8%), and of the NAD-dependent mutant ligated with NAD (2.2 A, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NAD-binding site assumes a less rigid structure than the NADP site of the wild-type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 A from the sites of the mutations, this observation reminds us that mutations may have important long-range consequences that are difficult to anticipate. PMID:7833810

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

PubMed

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

2008-10-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1-25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

PubMed Central

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Žel, Jana; Gruden, Kristina

2008-01-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification. PMID:18710880

Aromatic thiol-mediated cleavage of N-O bonds enables chemical ubiquitylation of folded proteins

NASA Astrophysics Data System (ADS)

Weller, Caroline E.; Dhall, Abhinav; Ding, Feizhi; Linares, Edlaine; Whedon, Samuel D.; Senger, Nicholas A.; Tyson, Elizabeth L.; Bagert, John D.; Li, Xiaosong; Augusto, Ohara; Chatterjee, Champak

2016-09-01

Access to protein substrates homogenously modified by ubiquitin (Ub) is critical for biophysical and biochemical investigations aimed at deconvoluting the myriad biological roles for Ub. Current chemical strategies for protein ubiquitylation, however, employ temporary ligation auxiliaries that are removed under harsh denaturing conditions and have limited applicability. We report an unprecedented aromatic thiol-mediated N-O bond cleavage and its application towards native chemical ubiquitylation with the ligation auxiliary 2-aminooxyethanethiol. Our interrogation of the reaction mechanism suggests a disulfide radical anion as the active species capable of cleaving the N-O bond. The successful semisynthesis of full-length histone H2B modified by the small ubiquitin-like modifier-3 (SUMO-3) protein further demonstrates the generalizability and compatibility of our strategy with folded proteins.

BeeDoctor, a Versatile MLPA-Based Diagnostic Tool for Screening Bee Viruses

PubMed Central

De Smet, Lina; Ravoet, Jorgen; de Miranda, Joachim R.; Wenseleers, Tom; Mueller, Matthias Y.; Moritz, Robin F. A.; de Graaf, Dirk C.

2012-01-01

The long-term decline of managed honeybee hives in the world has drawn significant attention to the scientific community and bee-keeping industry. A high pathogen load is believed to play a crucial role in this phenomenon, with the bee viruses being key players. Most of the currently characterized honeybee viruses (around twenty) are positive stranded RNA viruses. Techniques based on RNA signatures are widely used to determine the viral load in honeybee colonies. High throughput screening for viral loads necessitates the development of a multiplex polymerase chain reaction approach in which different viruses can be targeted simultaneously. A new multiparameter assay, called “BeeDoctor”, was developed based on multiplex-ligation probe dependent amplification (MLPA) technology. This assay detects 10 honeybee viruses in one reaction. “BeeDoctor” is also able to screen selectively for either the positive strand of the targeted RNA bee viruses or the negative strand, which is indicative for active viral replication. Due to its sensitivity and specificity, the MLPA assay is a useful tool for rapid diagnosis, pathogen characterization, and epidemiology of viruses in honeybee populations. “BeeDoctor” was used for screening 363 samples from apiaries located throughout Flanders; the northern half of Belgium. Using the “BeeDoctor”, virus infections were detected in almost eighty percent of the colonies, with deformed wing virus by far the most frequently detected virus and multiple virus infections were found in 26 percent of the colonies. PMID:23144717

BeeDoctor, a versatile MLPA-based diagnostic tool for screening bee viruses.

PubMed

De Smet, Lina; Ravoet, Jorgen; de Miranda, Joachim R; Wenseleers, Tom; Mueller, Matthias Y; Moritz, Robin F A; de Graaf, Dirk C

2012-01-01

The long-term decline of managed honeybee hives in the world has drawn significant attention to the scientific community and bee-keeping industry. A high pathogen load is believed to play a crucial role in this phenomenon, with the bee viruses being key players. Most of the currently characterized honeybee viruses (around twenty) are positive stranded RNA viruses. Techniques based on RNA signatures are widely used to determine the viral load in honeybee colonies. High throughput screening for viral loads necessitates the development of a multiplex polymerase chain reaction approach in which different viruses can be targeted simultaneously. A new multiparameter assay, called "BeeDoctor", was developed based on multiplex-ligation probe dependent amplification (MLPA) technology. This assay detects 10 honeybee viruses in one reaction. "BeeDoctor" is also able to screen selectively for either the positive strand of the targeted RNA bee viruses or the negative strand, which is indicative for active viral replication. Due to its sensitivity and specificity, the MLPA assay is a useful tool for rapid diagnosis, pathogen characterization, and epidemiology of viruses in honeybee populations. "BeeDoctor" was used for screening 363 samples from apiaries located throughout Flanders; the northern half of Belgium. Using the "BeeDoctor", virus infections were detected in almost eighty percent of the colonies, with deformed wing virus by far the most frequently detected virus and multiple virus infections were found in 26 percent of the colonies.

Pseudohypoparathyroidism type Ib associated with novel duplications in the GNAS locus.

PubMed

Perez-Nanclares, Gustavo; Velayos, Teresa; Vela, Amaya; Muñoz-Torres, Manuel; Castaño, Luis

2015-01-01

Pseudohypoparathyroidism type 1b (PHP-Ib) is characterized by renal resistance to PTH (and, sometimes, a mild resistance to TSH) and absence of any features of Albright's hereditary osteodystrophy. Patients with PHP-Ib suffer of defects in the methylation pattern of the complex GNAS locus. PHP-Ib can be either sporadic or inherited in an autosomal dominant pattern. Whereas familial PHP-Ib is well characterized at the molecular level, the genetic cause of sporadic PHP-Ib cases remains elusive, although some molecular mechanisms have been associated with this subtype. The aim of the study was to investigate the molecular and imprinting defects in the GNAS locus in two unrelated patients with PHP-Ib. We have analyzed the GNAS locus by direct sequencing, Methylation-Specific Multiplex Ligation-dependent Probe Amplification, microsatellites, Quantitative Multiplex PCR of Short Fluorescent fragments and array-Comparative Genomic Hybridization studies in order to characterize two unrelated families with clinical features of PHP-Ib. We identified two duplications in the GNAS region in two patients with PHP-Ib: one of them, comprising ∼ 320 kb, occurred 'de novo' in the patient, whereas the other one, of ∼ 179 kb in length, was inherited from the maternal allele. In both cases, no other known genetic cause was observed. In this article, we describe the to-our-knowledge biggest duplications reported so far in the GNAS region. Both are associated to PHP-Ib, one of them occurring 'de novo' and the other one being maternally inherited.

Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification.

PubMed

Ebai, Tonge; Souza de Oliveira, Felipe Marques; Löf, Liza; Wik, Lotta; Schweiger, Caroline; Larsson, Anders; Keilholtz, Ulrich; Haybaeck, Johannes; Landegren, Ulf; Kamali-Moghaddam, Masood

2017-09-01

Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals. Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader. We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor 15 (GDF-15) by PLARCA and conventional sandwich ELISA or immuno-RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared to ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA. PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories. © 2017 American Association for Clinical Chemistry.

Synthesis of histone proteins by CPE ligation using a recombinant peptide as the C-terminal building block.

PubMed

Kawakami, Toru; Yoshikawa, Ryo; Fujiyoshi, Yuki; Mishima, Yuichi; Hojo, Hironobu; Tajima, Shoji; Suetake, Isao

2015-11-01

The post-translational modification of histones plays an important role in gene expression. We report herein on a method for synthesizing such modified histones by ligating chemically prepared N-terminal peptides and C-terminal recombinant peptide building blocks. Based on their chemical synthesis, core histones can be categorized as two types; histones H2A, H2B and H4 which contain no Cys residues, and histone H3 which contains a Cys residue(s) in the C-terminal region. A combination of native chemical ligation and desulphurization can be simply used to prepare histones without Cys residues. For the synthesis of histone H3, the endogenous Cys residue(s) must be selectively protected, while keeping the N-terminal Cys residue of the C-terminal building block that is introduced for purposes of chemical ligation unprotected. To this end, a phenacyl group was successfully utilized to protect endogenous Cys residue(s), and the recombinant peptide was ligated with a peptide containing a Cys-Pro ester (CPE) sequence as a thioester precursor. Using this approach it was possible to prepare all of the core histones H2A, H2B, H3 and H4 with any modifications. The resulting proteins could then be used to prepare a core histone library of proteins that have been post-translationally modified. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Simultaneous Detection of Genetically Modified Organisms in a Mixture by Multiplex PCR-Chip Capillary Electrophoresis.

PubMed

Patwardhan, Supriya; Dasari, Srikanth; Bhagavatula, Krishna; Mueller, Steffen; Deepak, Saligrama Adavigowda; Ghosh, Sudip; Basak, Sanjay

2015-01-01

An efficient PCR-based method to trace genetically modified food and feed products is in demand due to regulatory requirements and contaminant issues in India. However, post-PCR detection with conventional methods has limited sensitivity in amplicon separation that is crucial in multiplexing. The study aimed to develop a sensitive post-PCR detection method by using PCR-chip capillary electrophoresis (PCR-CCE) to detect and identify specific genetically modified organisms in their genomic DNA mixture by targeting event-specific nucleotide sequences. Using the PCR-CCE approach, novel multiplex methods were developed to detect MON531 cotton, EH 92-527-1 potato, Bt176 maize, GT73 canola, or GA21 maize simultaneously when their genomic DNAs in mixtures were amplified using their primer mixture. The repeatability RSD (RSDr) of the peak migration time was 0.06 and 3.88% for the MON531 and Bt176, respectively. The RSD (RSDR) of the Cry1Ac peak ranged from 0.12 to 0.40% in multiplex methods. The method was sensitive in resolving amplicon of size difference up to 4 bp. The PCR-CCE method is suitable to detect multiple genetically modified events in a composite DNA sample by tagging their event specific sequences.

Epigenetic events underlie the pathogenesis of sinonasal papillomas.

PubMed

Stephen, Josena K; Vaught, Lori E; Chen, Kang M; Sethi, Seema; Shah, Veena; Benninger, Michael S; Gardner, Glendon M; Schweitzer, Vanessa G; Khan, Mumtaz; Worsham, Maria J

2007-10-01

Benign inverted papillomas have been reported as monoclonal but lacking common genetic alterations identified in squamous cell carcinoma of the head and neck. Epigenetic changes alter the heritable state of gene expression and chromatin organization without change in DNA sequence. We investigated whether epigenetic events of aberrant promoter hypermethylation in genes known to be involved in squamous head and neck cancer underlie the pathogenesis of sinonasal papillomas. Ten formalin-fixed paraffin DNA samples from three inverted papilloma cases, two exophytic (everted) papilloma cases, and two cases with inverted and exophytic components were studied. DNA was obtained from microdissected areas of normal and papilloma areas and examined using a panel of 41 gene probes, designed to interrogate 35 unique genes for aberrant methylation status (22 genes) using the methylation-specific multiplex-ligation-specific polymerase assay. Methylation-specific PCR was employed to confirm aberrant methylation detected by the methylation-specific multiplex-ligation-specific polymerase assay. All seven cases indicated at least one epigenetic event of aberrant promoter hypermethylation. The CDKN2B gene was a consistent target of aberrant methylation in six of seven cases. Methylation-specific PCR confirmed hypermethylation of CDKN2B. Recurrent biopsies from two inverted papilloma cases had common epigenetic events. Promoter hypermethylation of CDKN2B was a consistent epigenetic event. Common epigenetic alterations in recurrent biopsies underscore a monoclonal origin for these lesions. Epigenetic events contribute to the underlying pathogenesis of benign inverted and exophytic papillomas. As a consistent target of aberrant promoter hypermethylation, CDKN2B may serve as an important epigenetic biomarker for gene reactivation studies.

Methylation profile analysis of DNA repair genes in hepatocellular carcinoma with MS-MLPA.

PubMed

Ozer, Ozge; Bilezikci, Banu; Aktas, Sema; Sahin, Feride I

2013-12-01

Hepatocellular carcinoma (HCC) is one of the rare tumors with well-defined risk factors. The multifactorial etiology of HCC can be explained by its complex molecular pathogenesis. In the current study, the methylation status of 7 genes involved in DNA repair mechanisms, namely MLH1, PMS2, MSH6, MSH2, MGMT, MSH3, and MLH3, was investigated in tumor samples from HCC patients, using the methylation-specific-multiplex ligated probe amplification method and the results were correlated with available clinical findings. The most common etiological factor in these cases was the presence of hepatitis B alone (47.2%). Among the 56 cases that were studied, promoter methylation was detected in at least one of the genes in 27 (48.2%) cases, only in 1 gene in 13 (23.2%) cases, and in >1 gene in 14 (25%) cases. Of the 7 genes investigated, methylation was most frequently observed in MSH3, in 14 (25%) cases. Methylation of at least 1 gene was significantly more frequent in patients with single tumors than multifocal tumors. There were significant differences regarding hepatitis B status, Child Class, tumor number, grade, and TNM stage in cases where PMS2 methylation was detected. Our results suggest that methylation of genes involved in mismatch repair may be responsible in the pathogenesis of HCC, and evaluating changes in multiple genes in these pathways simultaneously would be more informative. Despite being a robust and relatively inexpensive method, the methylation-specific-multiplex ligated probe amplification assay could be more extensively applied with improvements in the currently intricate data analysis component.

Enhancing the efficiency of sortase-mediated ligations through nickel-peptide complex formation.

PubMed

David Row, R; Roark, Travis J; Philip, Marina C; Perkins, Lorena L; Antos, John M

2015-08-14

A modified sortase A recognition motif containing a masked Ni(2+)-binding peptide was employed to boost the efficiency of sortase-catalyzed ligation reactions. Deactivation of the Ni(2+)-binding peptide using a Ni(2+) additive improved reaction performance at low to equimolar ratios of the glycine amine nucleophile and sortase substrate. The success of this approach was demonstrated with both peptide and protein substrates.

Enzyme-free and isothermal detection of microRNA based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction signal amplification.

PubMed

Oishi, Motoi

2015-05-01

An enzyme-free and isothermal microRNA (miRNA) detection method has been developed based on click-chemical ligation-assisted hybridization coupled with hybridization chain reaction (HCR) on magnetic beads (MBs). The click-chemical ligation between an azide-modified probe DNA and a dibenzocyclooctyne-modified probe DNA occurred through the hybridization of target miRNA (miR-141). HCR on MBs was performed by the addition of DNA hairpin monomers (H1 and H2). After magnetic separation and denaturation/rehybridization of HCR products ([H1/H2] n ), the resulting HCR products were analyzed by the fluorescence emitted from an intercalative dye, allowing amplification of the fluorescent signal. The proposed assay had a limit of detection of 0.55 fmol, which was 230-fold more sensitive than that of the HCR on the MBs coupled with a conventional sandwich hybridization assay (without click-chemical ligation) (limit of detection 127 fmol). Additionally, the proposed assay could discriminate between miR-141 and other miR-200 family members. In contrast to quantitative reverse transcription polymerase chain reaction techniques using enzymes and thermal cycling, this is an enzyme-free assay that can be conducted under isothermal conditions and can specifically detect miR-141 in fetal bovine serum.

Modified Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy for Hepatoblastoma in a Small Infant: How Far Can We Push the Envelope?

PubMed

Hong, Johnny C; Kim, Joohyun; Browning, Meghen; Wagner, Amy; Lerret, Stacee; Segura, Annette D; Zimmerman, Michael A

2017-08-01

: For patients with hepatoblastoma, a timely and complete resection of the tumor is critical to the patient's tumor recurrence-free survival. Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS), a 2-stage hepatectomy procedure, has revolutionized the surgical management of large hepatic tumors with insufficient future liver remnant (FLR) at presentation. Although existing data support the utility of ALPPS in adults with primary and metastatic hepatobiliary malignancy, the literature in children is scarce. To our knowledge, this is the first report showing clinical applicability and safety of the modified ALPPS procedure in a small infant (54 days old) with hepatoblastoma who presented with insufficient FLR. Our report suggests the modified ALPPS could potentially expand the surgical treatment alternative for small infants with large hepatoblastoma.

Detection of hepatitis C virus subtypes 6a, 6n, 6w and mixed infections using a modified multiplex real-time polymerase chain reaction protocol.

PubMed

Lee, Yuan-Ming; Chen, Yen-Ju; Lee, Cheng-Ming; Kuo, Lou-Hui; Wong, Wing-Wai; Chen, Yi-Ming Arthur

2011-12-01

In the past few years, many new subtypes in hepatitis C virus (HCV) genotype 6 have been identified. The aim of this study was to modify the multiplex real-time polymerase chain reaction (RT-PCR) protocol and use it to determine the HCV subtypes of a group of Taiwanese injection drug users (IDUs). We used 76 serum specimens collected in northern Taiwan in 2008. Multiplex RT-PCR was used for HCV subtyping among those serum samples having anti-HCV antibodies. Twenty cases were randomly selected for comparison with subtyping results from Inno-LiPa II tests and phylogenetic tree analysis using NS5B sequences. Multiplex RT-PCR assays showed that 60.5% (46/76) of IDUs had single HCV infection. Three out of 76 (3.9%) had double HCV infection (1b/6a, 2a/2b and 2b/6a). Besides this, 27.6% (21/76) had no HCV signal. One IDU had subtype 6n and two had subtype 6w infection. Inno-LiPa II tests misclassified all 6n and 6w cases as 1b subtype. Our modified multiplex RT-PCR protocol can be used to support molecular epidemiological studies and laboratory diagnoses of different HCV subtypes including genotype 6. Copyright © 2011. Published by Elsevier B.V.

Multiplex Droplet Digital PCR Protocols for Quantification of GM Maize Events.

PubMed

Dobnik, David; Spilsberg, Bjørn; Bogožalec Košir, Alexandra; Štebih, Dejan; Morisset, Dany; Holst-Jensen, Arne; Žel, Jana

2018-01-01

The standard-curve based simplex quantitative polymerase chain reaction (qPCR) has been the gold standard for DNA target quantification for more than a decade. The large and growing number of individual analyses needed to test for genetically modified organisms (GMOs) is reducing the cost-effectiveness of qPCR. Droplet digital PCR (ddPCR) enables absolute quantification without standard curves, avoids the amplification efficiency bias observed with qPCR, allows more accurate estimations at low target copy numbers and, in combination with multiplexing, significantly improves cost efficiency. Here we describe two protocols for multiplex quantification of GM maize events: (1) nondiscriminating, with multiplex quantification of targets as a group (12 GM maize lines) and (2) discriminating, with multiplex quantification of individual targets (events). The first enables the quantification of twelve European Union authorized GM maize events as a group with only two assays, but does not permit determination of the individual events present. The second protocol enables the quantification of four individual targets (three GM events and one endogene) in a single reaction. Both protocols can be modified for quantification of any other DNA target.

A LDR-PCR approach for multiplex polymorphisms genotyping of severely degraded DNA with fragment sizes <100 bp.

PubMed

Zhang, Zhen; Wang, Bao-Jie; Guan, Hong-Yu; Pang, Hao; Xuan, Jin-Feng

2009-11-01

Reducing amplicon sizes has become a major strategy for analyzing degraded DNA typical of forensic samples. However, amplicon sizes in current mini-short tandem repeat-polymerase chain reaction (PCR) and mini-sequencing assays are still not suitable for analysis of severely degraded DNA. In this study, we present a multiplex typing method that couples ligase detection reaction with PCR that can be used to identify single nucleotide polymorphisms and small-scale insertion/deletions in a sample of severely fragmented DNA. This method adopts thermostable ligation for allele discrimination and subsequent PCR for signal enhancement. In this study, four polymorphic loci were used to assess the ability of this technique to discriminate alleles in an artificially degraded sample of DNA with fragment sizes <100 bp. Our results showed clear allelic discrimination of single or multiple loci, suggesting that this method might aid in the analysis of extremely degraded samples in which allelic drop out of larger fragments is observed.

Nucleic acid sequence detection using multiplexed oligonucleotide PCR

DOEpatents

Nolan, John P [Santa Fe, NM; White, P Scott [Los Alamos, NM

2006-12-26

Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

Mode of genetic inheritance modifies the association of head circumference and autism-related symptoms: a cross-sectional study.

PubMed

Davis, Jonathan M; Keeney, Jonathon G; Sikela, James M; Hepburn, Susan

2013-01-01

Frequently individuals with autism spectrum disorder (ASD) have been noted with a larger head circumference (HC) than their typical developing peers. Biologic hypotheses suggest that an overly rapid brain growth leads to the core symptoms of ASD by impairing connectivity. Literature is divided however where deleterious, protective and null associations of HC with ASD symptoms in individuals with ASD have been found. Individuals (n = 1,416) from the Autism Genetic Resource Exchange with ASD were examined for associations of HC with ASD like symptoms. Mixed models controlling for sex, age, race/ethnicity, simplex/multiplex status and accounting for correlations between siblings were used. Interactions by simplex/multiplex were explored. Adjustments for height in a sub-population with available data were explored as well. A Significant interaction term (p = 0.03) suggested that the effect of HC was dependent on whether the individual was simplex or multiplex. In simplex individuals at mean age (8.9 years) 1 cm increase in head circumference was associated with a 24% increase in the odds of a high social diagnostic score from the Autism Diagnostic Interview-Revised (odds ratio  = 1.24, p = 0.01). There was no association in multiplex individuals. Additionally, individuals classified with a non-verbal IQ <70 were 90% simplex and had a significantly increased head circumference (0.7 cm p = 0.03) relative to a mid-range non-verbal IQ group. Interestingly, children classified with a >110 non-verbal IQ also had an increased HC (0.4 cm p = 0.04), relative to a mid-range non-verbal IQ group, and were 90% multiplex. HC effects do not appear to be confounded by height, however, larger samples with height information are needed. The potential link between brain growth and autism like symptoms is complex and could depend on specific etiologies. Further investigations accounting for a likely mode of inheritance will help identify an ASD subtype related to HC.

Universal strategies for the DNA-encoding of libraries of small molecules using the chemical ligation of oligonucleotide tags

PubMed Central

Litovchick, Alexander; Clark, Matthew A; Keefe, Anthony D

2014-01-01

The affinity-mediated selection of large libraries of DNA-encoded small molecules is increasingly being used to initiate drug discovery programs. We present universal methods for the encoding of such libraries using the chemical ligation of oligonucleotides. These methods may be used to record the chemical history of individual library members during combinatorial synthesis processes. We demonstrate three different chemical ligation methods as examples of information recording processes (writing) for such libraries and two different cDNA-generation methods as examples of information retrieval processes (reading) from such libraries. The example writing methods include uncatalyzed and Cu(I)-catalyzed alkyne-azide cycloadditions and a novel photochemical thymidine-psoralen cycloaddition. The first reading method “relay primer-dependent bypass” utilizes a relay primer that hybridizes across a chemical ligation junction embedded in a fixed-sequence and is extended at its 3′-terminus prior to ligation to adjacent oligonucleotides. The second reading method “repeat-dependent bypass” utilizes chemical ligation junctions that are flanked by repeated sequences. The upstream repeat is copied prior to a rearrangement event during which the 3′-terminus of the cDNA hybridizes to the downstream repeat and polymerization continues. In principle these reading methods may be used with any ligation chemistry and offer universal strategies for the encoding (writing) and interpretation (reading) of DNA-encoded chemical libraries. PMID:25483841

A Multiplex Enzymatic Machinery for Cellular Protein S-nitrosylation.

PubMed

Seth, Divya; Hess, Douglas T; Hausladen, Alfred; Wang, Liwen; Wang, Ya-Juan; Stamler, Jonathan S

2018-02-01

S-nitrosylation, the oxidative modification of Cys residues by nitric oxide (NO) to form S-nitrosothiols (SNOs), modifies all main classes of proteins and provides a fundamental redox-based cellular signaling mechanism. However, in contrast to other post-translational protein modifications, S-nitrosylation is generally considered to be non-enzymatic, involving multiple chemical routes. We report here that endogenous protein S-nitrosylation in the model organism E. coli depends principally upon the enzymatic activity of the hybrid cluster protein Hcp, employing NO produced by nitrate reductase. Anaerobiosis on nitrate induces both Hcp and nitrate reductase, thereby resulting in the S-nitrosylation-dependent assembly of a large interactome including enzymes that generate NO (NO synthase), synthesize SNO-proteins (SNO synthase), and propagate SNO-based signaling (trans-nitrosylases) to regulate cell motility and metabolism. Thus, protein S-nitrosylation by NO in E. coli is essentially enzymatic, and the potential generality of the multiplex enzymatic mechanism that we describe may support a re-conceptualization of NO-based cellular signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparison of Hi-C results using in-solution versus in-nucleus ligation.

PubMed

Nagano, Takashi; Várnai, Csilla; Schoenfelder, Stefan; Javierre, Biola-Maria; Wingett, Steven W; Fraser, Peter

2015-08-26

Chromosome conformation capture and various derivative methods such as 4C, 5C and Hi-C have emerged as standard tools to analyze the three-dimensional organization of the genome in the nucleus. These methods employ ligation of diluted cross-linked chromatin complexes, intended to favor proximity-dependent, intra-complex ligation. During development of single-cell Hi-C, we devised an alternative Hi-C protocol with ligation in preserved nuclei rather than in solution. Here we directly compare Hi-C methods employing in-nucleus ligation with the standard in-solution ligation. We show in-nucleus ligation results in consistently lower levels of inter-chromosomal contacts. Through chromatin mixing experiments we show that a significantly large fraction of inter-chromosomal contacts are the result of spurious ligation events formed during in-solution ligation. In-nucleus ligation significantly reduces this source of experimental noise, and results in improved reproducibility between replicates. We also find that in-nucleus ligation eliminates restriction fragment length bias found with in-solution ligation. These improvements result in greater reproducibility of long-range intra-chromosomal and inter-chromosomal contacts, as well as enhanced detection of structural features such as topologically associated domain boundaries. We conclude that in-nucleus ligation captures chromatin interactions more consistently over a wider range of distances, and significantly reduces both experimental noise and bias. In-nucleus ligation creates higher quality Hi-C libraries while simplifying the experimental procedure. We suggest that the entire range of 3C applications are likely to show similar benefits from in-nucleus ligation.

Multiplex quantification of 12 European Union authorized genetically modified maize lines with droplet digital polymerase chain reaction.

PubMed

Dobnik, David; Spilsberg, Bjørn; Bogožalec Košir, Alexandra; Holst-Jensen, Arne; Žel, Jana

2015-08-18

Presence of genetically modified organisms (GMO) in food and feed products is regulated in many countries. The European Union (EU) has implemented a threshold for labeling of products containing more than 0.9% of authorized GMOs per ingredient. As the number of GMOs has increased over time, standard-curve based simplex quantitative polymerase chain reaction (qPCR) analyses are no longer sufficiently cost-effective, despite widespread use of initial PCR based screenings. Newly developed GMO detection methods, also multiplex methods, are mostly focused on screening and detection but not quantification. On the basis of droplet digital PCR (ddPCR) technology, multiplex assays for quantification of all 12 EU authorized GM maize lines (per April first 2015) were developed. Because of high sequence similarity of some of the 12 GM targets, two separate multiplex assays were needed. In both assays (4-plex and 10-plex), the transgenes were labeled with one fluorescence reporter and the endogene with another (GMO concentration = transgene/endogene ratio). It was shown that both multiplex assays produce specific results and that performance parameters such as limit of quantification, repeatability, and trueness comply with international recommendations for GMO quantification methods. Moreover, for samples containing GMOs, the throughput and cost-effectiveness is significantly improved compared to qPCR. Thus, it was concluded that the multiplex ddPCR assays could be applied for routine quantification of 12 EU authorized GM maize lines. In case of new authorizations, the events can easily be added to the existing multiplex assays. The presented principle of quantitative multiplexing can be applied to any other domain.

Viral prevalence increases with regional colony abundance in honey bee drones (Apis mellifera L).

PubMed

Forfert, Nadège; Natsopoulou, Myrsini E; Paxton, Robert J; Moritz, Robin F A

2016-10-01

Transmission among colonies is a central feature for the epidemiology of honey bee pathogens. High colony abundance may promote transmission among colonies independently of apiary layout, making colony abundance a potentially important parameter determining pathogen prevalence in populations of honey bees. To test this idea, we sampled male honey bees (drones) from seven distinct drone congregation areas (DCA), and used their genotypes to estimate colony abundance at each site. A multiplex ligation dependent probe amplification assay (MLPA) was used to assess the prevalence of ten viruses, using five common viral targets, in individual drones. There was a significant positive association between colony abundance and number of viral infections. This result highlights the potential importance of high colony abundance for pathogen prevalence, possibly because high population density facilitates pathogen transmission. Pathogen prevalence in drones collected from DCAs may be a useful means of estimating the disease status of a population of honey bees during the mating season, especially for localities with a large number of wild or feral colonies. Copyright © 2016 Elsevier B.V. All rights reserved.

Heterogeneity in Phenotype of Usher-Congenital Hyperinsulinism Syndrome

PubMed Central

Al Mutair, Angham N.; Brusgaard, Klaus; Bin-Abbas, Bassam; Hussain, Khalid; Felimban, Naila; Al Shaikh, Adnan; Christesen, Henrik T.

2013-01-01

OBJECTIVE To evaluate the phenotype of 15 children with congenital hyperinsulinism (CHI) and profound hearing loss, known as Homozygous 11p15-p14 Deletion syndrome (MIM #606528). RESEARCH DESIGN AND METHODS Prospective clinical follow-up and genetic analysis by direct sequencing, multiplex ligation-dependent probe amplification, and microsatellite markers. RESULTS Genetic testing identified the previous described homozygous deletion in 11p15, USH1C:c.(90+592)_ABCC8:c.(2694–528)del. Fourteen patients had severe CHI demanding near-total pancreatectomy. In one patient with mild, transient neonatal hypoglycemia and nonautoimmune diabetes at age 11 years, no additional mutations were found in HNF1A, HNF4A, GCK, INS, and INSR. Retinitis pigmentosa was found in two patients aged 9 and 13 years. No patients had enteropathy or renal tubular defects. Neuromotor development ranged from normal to severe delay with epilepsy. CONCLUSIONS The phenotype of Homozygous 11p15-p14 Deletion syndrome, or Usher-CHI syndrome, includes any severity of neonatal-onset CHI and severe, sensorineural hearing loss. Retinitis pigmentosa and nonautoimmune diabetes may occur in adolescence. PMID:23150283

Mutation spectrum of Chinese patients with Bartter syndrome.

PubMed

Han, Yue; Lin, Yi; Sun, Qing; Wang, Shujuan; Gao, Yanxia; Shao, Leping

2017-11-24

Bartter syndrome (BS) has been rarely reported in Chinese population except for a few case reports. This investigation was aimed to analyze the mutations of the causal genes in sixteen Chinese patients with BS, and review their followup and treatment. Identify mutations by the next generation sequencing and the multiplex ligation-dependent probe amplification (MLPA). Clinical characteristics and biochemical findings at the first presentation as well as follow-up were reviewed. 15 different CLCNKB gene mutations were identified in fourteen patients with BS, including 11 novel ones. A novel missense mutation and a novel small deletion were found from SLC12A1 gene. A novel gross deletion was found in CLCNKA gene. A recurrent missense mutation was identified from BSND gene. We found that the whole gene deletion mutation of CLCNKB gene was the most frequent mutation (32%), and the rate of gross deletion was up to 50 percent in this group of Chinese patients. The present study has found 19 mutations, including 14 novel ones, which would enrich the human gene mutation database (HGMD) and provide valuable references to the genetic counseling and diagnosis of the Chinese population.

IKZF1 deletion is an independent predictor of outcome in pediatric acute lymphoblastic leukemia treated according to the ALL-BFM 2000 protocol.

PubMed

Dörge, Petra; Meissner, Barbara; Zimmermann, Martin; Möricke, Anja; Schrauder, André; Bouquin, Jean-Pierre; Schewe, Denis; Harbott, Jochen; Teigler-Schlegel, Andrea; Ratei, Richard; Ludwig, Wolf-Dieter; Koehler, Rolf; Bartram, Claus R; Schrappe, Martin; Stanulla, Martin; Cario, Gunnar

2013-03-01

IKZF1 gene deletions have been associated with a poor outcome in pediatric precursor B-cell acute lymphoblastic leukemia. To assess the prognostic relevance of IKZF1 deletions for patients treated on Berlin-Frankfurt-Münster Study Group trial ALL-BFM 2000, we screened 694 diagnostic acute lymphoblastic leukemia samples by Multiplex Ligation-dependent Probe Amplification. Patients whose leukemic cells bore IKZF1 deletions had a lower 5-year event-free survival (0.69±0.05 vs. 0.85±0.01; P<0.0001) compared to those without, mainly due to a higher cumulative incidence of relapses (0.21±0.04 vs. 0.10±0.01; P=0.001). Although IKZF1 deletions were significantly associated with the P2RY8-CRLF2 rearrangement, their prognostic value was found to be independent from this association. Thus, IKZF1 deletion is an independent predictor of treatment outcome and a strong candidate marker for integration in future treatment stratification strategies on ALL-BFM protocols. Clinicaltrials.gov identifier: NCT00430118.

11p15 duplication and 13q34 deletion with Beckwith-Wiedemann syndrome and factor VII deficiency.

PubMed

Jurkiewicz, Dorota; Kugaudo, Monika; Tańska, Anna; Wawrzkiewicz-Witkowska, Angelika; Tomaszewska, Agnieszka; Kucharczyk, Marzena; Cieślikowska, Agata; Ciara, Elżbieta; Krajewska-Walasek, Małgorzata

2015-06-01

Here we report a patient with 11p15.4p15.5 duplication and 13q34 deletion presenting with Beckwith-Wiedemann syndrome (BWS) and moderate deficiency of factor VII (FVII). The duplication was initially diagnosed on methylation-sensitive multiplex ligation-dependent probe amplification. Array comparative genome hybridization confirmed its presence and indicated a 13q34 distal deletion. The patient's clinical symptoms, including developmental delay and facial dysmorphism, were typical of BWS with paternal 11p15 trisomy. Partial 13q monosomy in this patient is associated with moderate deficiency of FVII and may also overlap with a few symptoms of paternal 11p15 trisomy such as developmental delay and some facial features. To our knowledge this is the first report of 11p15.4p15.5 duplication associated with deletion of 13q34 and FVII deficiency. Moreover, this report emphasizes the importance of detailed clinical as well as molecular examinations in patients with BWS features and developmental delay. © 2015 Japan Pediatric Society.

MTTE: an innovative strategy for the evaluation of targeted/exome enrichment efficiency

PubMed Central

Klonowska, Katarzyna; Handschuh, Luiza; Swiercz, Aleksandra; Figlerowicz, Marek; Kozlowski, Piotr

2016-01-01

Although currently available strategies for the preparation of exome-enriched libraries are well established, a final validation of the libraries in terms of exome enrichment efficiency prior to the sequencing step is of considerable importance. Here, we present a strategy for the evaluation of exome enrichment, i.e., the Multipoint Test for Targeted-enrichment Efficiency (MTTE), PCR-based approach utilizing multiplex ligation-dependent probe amplification with capillary electrophoresis separation. We used MTTE for the analysis of subsequent steps of the Illumina TruSeq Exome Enrichment procedure. The calculated values of enrichment-associated parameters (i.e., relative enrichment, relative clearance, overall clearance, and fold enrichment) and the comparison of MTTE results with the actual enrichment revealed the high reliability of our assay. Additionally, the MTTE assay enabled the determination of the sequence-associated features that may confer bias in the enrichment of different targets. Importantly, the MTTE is low cost method that can be easily adapted to the region of interest important for a particular project. Thus, the MTTE strategy is attractive for post-capture validation in a variety of targeted/exome enrichment NGS projects. PMID:27572310

MTTE: an innovative strategy for the evaluation of targeted/exome enrichment efficiency.

PubMed

Klonowska, Katarzyna; Handschuh, Luiza; Swiercz, Aleksandra; Figlerowicz, Marek; Kozlowski, Piotr

2016-10-11

Although currently available strategies for the preparation of exome-enriched libraries are well established, a final validation of the libraries in terms of exome enrichment efficiency prior to the sequencing step is of considerable importance. Here, we present a strategy for the evaluation of exome enrichment, i.e., the Multipoint Test for Targeted-enrichment Efficiency (MTTE), PCR-based approach utilizing multiplex ligation-dependent probe amplification with capillary electrophoresis separation. We used MTTE for the analysis of subsequent steps of the Illumina TruSeq Exome Enrichment procedure. The calculated values of enrichment-associated parameters (i.e., relative enrichment, relative clearance, overall clearance, and fold enrichment) and the comparison of MTTE results with the actual enrichment revealed the high reliability of our assay. Additionally, the MTTE assay enabled the determination of the sequence-associated features that may confer bias in the enrichment of different targets. Importantly, the MTTE is low cost method that can be easily adapted to the region of interest important for a particular project. Thus, the MTTE strategy is attractive for post-capture validation in a variety of targeted/exome enrichment NGS projects.

Siblings with opposite chromosome constitutions, dup(2q)/del(7q) and del(2q)/dup(7q).

PubMed

Shim, Sung Han; Shim, Jae Sun; Min, Kyunghoon; Lee, Hee Song; Park, Ji Eun; Park, Sang Hee; Hwang, Euna; Kim, Minyoung

2014-01-15

Chromosome 7q36 microdeletion syndrome is a rare genomic disorder characterized by underdevelopment of the brain, microcephaly, anomalies of the sex organs, and language problems. Developmental delay, intellectual disability, autistic spectrum disorders, BDMR syndrome, and unusual facial morphology are the key features of the chromosome 2q37 microdeletion syndrome. A genetic screening for two brothers with global developmental delay using high-resolution chromosomal analysis and subtelomeric multiplex ligation-dependent probe amplification revealed subtelomeric rearrangements on the same sites of 2q37.2 and 7q35, with reversed deletion and duplication. Both of them showed dysmorphic facial features, severe disability of physical and intellectual development, and abnormal genitalia with differential abnormalities in their phenotypes. The family did not have abnormal genetic phenotypes. According to the genetic analysis of their parents, adjacent-1 segregation from their mother's was suggested as a mechanism of their gene mutation. By comparing the phenotypes of our patients with previous reports on similar patients, we tried to obtain the information of related genes and their chromosomal locations. © 2013.

Heterogeneity in phenotype of usher-congenital hyperinsulinism syndrome: hearing loss, retinitis pigmentosa, and hyperinsulinemic hypoglycemia ranging from severe to mild with conversion to diabetes.

PubMed

Al Mutair, Angham N; Brusgaard, Klaus; Bin-Abbas, Bassam; Hussain, Khalid; Felimban, Naila; Al Shaikh, Adnan; Christesen, Henrik T

2013-03-01

To evaluate the phenotype of 15 children with congenital hyperinsulinism (CHI) and profound hearing loss, known as Homozygous 11p15-p14 Deletion syndrome (MIM #606528). Prospective clinical follow-up and genetic analysis by direct sequencing, multiplex ligation-dependent probe amplification, and microsatellite markers. Genetic testing identified the previous described homozygous deletion in 11p15, USH1C:c.(90+592)_ABCC8:c.(2694-528)del. Fourteen patients had severe CHI demanding near-total pancreatectomy. In one patient with mild, transient neonatal hypoglycemia and nonautoimmune diabetes at age 11 years, no additional mutations were found in HNF1A, HNF4A, GCK, INS, and INSR. Retinitis pigmentosa was found in two patients aged 9 and 13 years. No patients had enteropathy or renal tubular defects. Neuromotor development ranged from normal to severe delay with epilepsy. The phenotype of Homozygous 11p15-p14 Deletion syndrome, or Usher-CHI syndrome, includes any severity of neonatal-onset CHI and severe, sensorineural hearing loss. Retinitis pigmentosa and nonautoimmune diabetes may occur in adolescence.

SHOX gene and conserved noncoding element deletions/duplications in Colombian patients with idiopathic short stature.

PubMed

Sandoval, Gloria Tatiana Vinasco; Jaimes, Giovanna Carola; Barrios, Mauricio Coll; Cespedes, Camila; Velasco, Harvy Mauricio

2014-03-01

SHOX gene mutations or haploinsufficiency cause a wide range of phenotypes such as Leri Weill dyschondrosteosis (LWD), Turner syndrome, and disproportionate short stature (DSS). However, this gene has also been found to be mutated in cases of idiopathic short stature (ISS) with a 3-15% frequency. In this study, the multiplex ligation-dependent probe amplification (MLPA) technique was employed to determine the frequency of SHOX gene mutations and their conserved noncoding elements (CNE) in Colombian patients with ISS. Patients were referred from different centers around the county. From a sample of 62 patients, 8.1% deletions and insertions in the intragenic regions and in the CNE were found. This result is similar to others published in other countries. Moreover, an isolated case of CNE 9 duplication and a new intron 6b deletion in another patient, associated with ISS, are described. This is one of the first studies of a Latin American population in which deletions/duplications of the SHOX gene and its CNE are examined in patients with ISS.

SHOX gene and conserved noncoding element deletions/duplications in Colombian patients with idiopathic short stature

PubMed Central

Sandoval, Gloria Tatiana Vinasco; Jaimes, Giovanna Carola; Barrios, Mauricio Coll; Cespedes, Camila; Velasco, Harvy Mauricio

2014-01-01

SHOX gene mutations or haploinsufficiency cause a wide range of phenotypes such as Leri Weill dyschondrosteosis (LWD), Turner syndrome, and disproportionate short stature (DSS). However, this gene has also been found to be mutated in cases of idiopathic short stature (ISS) with a 3–15% frequency. In this study, the multiplex ligation-dependent probe amplification (MLPA) technique was employed to determine the frequency of SHOX gene mutations and their conserved noncoding elements (CNE) in Colombian patients with ISS. Patients were referred from different centers around the county. From a sample of 62 patients, 8.1% deletions and insertions in the intragenic regions and in the CNE were found. This result is similar to others published in other countries. Moreover, an isolated case of CNE 9 duplication and a new intron 6b deletion in another patient, associated with ISS, are described. This is one of the first studies of a Latin American population in which deletions/duplications of the SHOX gene and its CNE are examined in patients with ISS. PMID:24689071

Identification of the first multi-exonic WDR72 deletion in isolated amelogenesis imperfecta, and generation of a WDR72-specific copy number screening tool.

PubMed

Hentschel, Julia; Tatun, Dana; Parkhomchuk, Dmitri; Kurth, Ingo; Schimmel, Bettina; Heinrich-Weltzien, Roswitha; Bertzbach, Sabine; Peters, Hartmut; Beetz, Christian

2016-09-15

Amelogenesis imperfecta (AI) is a clinically and genetically heterogeneous disorder of tooth development which is due to aberrant deposition or composition of enamel. Both syndromic and isolated forms exist; they may be inherited in an X-linked, autosomal recessive, or autosomal dominant manner. WDR72 is one of ten currently known genes for recessive isolated AI; nine WDR72 mutations affecting single nucleotides have been described to date. Based on whole exome sequencing in a large consanguineous AI pedigree, we obtained evidence for presence of a multi-exonic WDR72 deletion. A home-made multiplex ligation-dependent probe amplification assay was used to confirm the aberration, to narrow its extent, and to identify heterozygous carriers. Our study extends the mutational spectrum for WDR72 to include large deletions, and supports a relevance of the previously proposed loss-of-function mechanism. It also introduces an easy-to-use and highly sensitive tool for detecting WDR72 copy number alterations. Copyright © 2016. Published by Elsevier B.V.

Characterisation of ATM mutations in Slavic Ataxia telangiectasia patients.

PubMed

Soukupova, Jana; Pohlreich, Petr; Seemanova, Eva

2011-09-01

Ataxia telangiectasia (AT) is a genomic instability syndrome characterised, among others, by progressive cerebellar degeneration, oculocutaneous telangiectases, immunodeficiency, elevated serum alpha-phetoprotein level, chromosomal breakage, hypersensitivity to ionising radiation and increased cancer risk. This autosomal recessive disorder is caused by mutations in the ataxia telangiectasia mutated (ATM) gene coding for serine/threonine protein kinase with a crucial role in response to DNA double-strand breaks. We characterised genotype and phenotype of 12 Slavic AT patients from 11 families. Mutation analysis included sequencing of the entire coding sequence, adjacent intron regions, 3'UTR and 5'UTR of the ATM gene and multiplex ligation-dependent probe amplification (MLPA) for the detection of large deletions/duplications at the ATM locus. The high incidence of new and individual mutations demonstrates a marked mutational heterogeneity of AT in the Czech Republic. Our data indicate that sequence analysis of the entire coding region of ATM is sufficient for a high detection rate of mutations in ATM and that MLPA analysis for the detection of deletions/duplications seems to be redundant in the Slavic population.

Normal exon copy number of the GLI2 and GLI3 genes in patients with esophageal atresia.

PubMed

Bednarczyk, D; Smigiel, R; Patkowski, D; Laczmanska, I; Lebioda, A; Laczmanski, L; Sasiadek, M M

2013-01-01

Esophageal atresia (EA) is a congenital developmental defect of the alimentary tract concerning the interruption of the esophagus with or without connection to the trachea. The incidence of EA is 1 in 3000-3500 of live-born infants, and occurs in both isolated and syndromic (in combination with abnormalities in other organ systems) forms. The molecular mechanisms underlying the development of EA are poorly understood. Knockout studies in mice indicate that genes like Sonic hedgehog, Gli2, and Gli3 play a role in the etiology of EA. These facts led us to hypothesize that Sonic hedgehog-GLI gene rearrangements are associated with EA in humans. To test this hypothesis, we screened patients with isolated and syndromic EA for GLI2 and/or GLI3 microrearrangements using methods to estimate the copy number (Multiplex Ligation-dependent Probe Amplification, real-time polymerase chain reaction). To our best knowledge this is the first study assessing copy number of GLI2 and GLI3 genes in patients with EA. © 2013 Wiley Periodicals, Inc. and the International Society for Diseases of the Esophagus.

Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 22 associated with cat eye syndrome.

PubMed

Chen, Chih-Ping; Ko, Tsang-Ming; Chen, Yi-Yung; Su, Jun-Wei; Wang, Wayseen

2013-09-15

We present prenatal diagnosis of mosaicism for a small supernumerary marker chromosome (sSMC) derived from chromosome 22 associated with cat eye syndrome (CES) using cultured amniocytes in a pregnancy with fetal microcephaly, intrauterine growth restriction, left renal hypoplasia, total anomalous pulmonary venous return with dominant right heart and right ear deformity. The sSMC was bisatellited and dicentric, and was characterized by multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (aCGH). The SALSA MLPA P250-B1 DiGeorge Probemix showed duplication of gene dosage in the CES region. aCGH showed a 1.26-Mb duplication at 22q11.1-q11.21 encompassing CECR1-CECR7. The sSMC was likely inv dup(22) (q11.21). Prenatal diagnosis of an sSMC(22) at amniocentesis should alert CES. MLPA, aCGH and fetal ultrasound are useful for rapid diagnosis of CES in case of prenatally detected sSMC(22). Copyright © 2013 Elsevier B.V. All rights reserved.

EvOligo: A Novel Software to Design and Group Libraries of Oligonucleotides Applicable for Nucleic Acid-Based Experiments.

PubMed

Milewski, Marek C; Kamel, Karol; Kurzynska-Kokorniak, Anna; Chmielewski, Marcin K; Figlerowicz, Marek

2017-10-01

Experimental methods based on DNA and RNA hybridization, such as multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification, or microarray analysis, require the use of mixtures of multiple oligonucleotides (primers or probes) in a single test tube. To provide an optimal reaction environment, minimal self- and cross-hybridization must be achieved among these oligonucleotides. To address this problem, we developed EvOligo, which is a software package that provides the means to design and group DNA and RNA molecules with defined lengths. EvOligo combines two modules. The first module performs oligonucleotide design, and the second module performs oligonucleotide grouping. The software applies a nearest-neighbor model of nucleic acid interactions coupled with a parallel evolutionary algorithm to construct individual oligonucleotides, and to group the molecules that are characterized by the weakest possible cross-interactions. To provide optimal solutions, the evolutionary algorithm sorts oligonucleotides into sets, preserves preselected parts of the oligonucleotides, and shapes their remaining parts. In addition, the oligonucleotide sets can be designed and grouped based on their melting temperatures. For the user's convenience, EvOligo is provided with a user-friendly graphical interface. EvOligo was used to design individual oligonucleotides, oligonucleotide pairs, and groups of oligonucleotide pairs that are characterized by the following parameters: (1) weaker cross-interactions between the non-complementary oligonucleotides and (2) more uniform ranges of the oligonucleotide pair melting temperatures than other available software products. In addition, in contrast to other grouping algorithms, EvOligo offers time-efficient sorting of paired and unpaired oligonucleotides based on various parameters defined by the user.

Cost-Effectiveness Analysis of Diagnosis of Duchenne/Becker Muscular Dystrophy in Colombia.

PubMed

Atehortúa, Sara C; Lugo, Luz H; Ceballos, Mateo; Orozco, Esteban; Castro, Paula A; Arango, Juan C; Mateus, Heidi E

2018-03-09

To determine the cost-effectiveness ratio of different courses of action for the diagnosis of Duchenne or Becker muscular dystrophy in Colombia. The cost-effectiveness analysis was performed from the Colombian health system perspective. Decision trees were constructed, and different courses of action were compared considering the following tests: immunohistochemistry (IHC), Western blot (WB), multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification (MLPA), and the complete sequencing of the dystrophin gene. The time horizon matched the duration of sample extraction and analysis. Transition probabilities were obtained from a systematic review. Costs were constructed with a type-case methodology using the consensus of experts and the valuation of resources from consulting laboratories and the 2001 Social Security Institute cost manual. Deterministic sensitivity and scenario analyses were performed with one or more unavailable alternatives. Costs were converted from Colombian pesos to US dollars using the 2014 exchange rate. In the base case, WB was the dominant strategy, with a cost of US $419.07 and a sensitivity of 100%. This approach remains the dominant strategy down to a 98.2% sensitivity and while costs do not exceed US $837.38. If WB was not available, IHC had the best cost-effectiveness ratio, followed by MLPA and sequencing. WB is a cost-effective alternative for the diagnosis of patients suspected of having Duchenne or Becker muscular dystrophy in the Colombian health system. The IHC test is rated as the second-best detection method. If these tests are not available, MLPA followed by sequencing would be the most cost-effective alternative. Copyright © 2018. Published by Elsevier Inc.

Efficient Bioconjugation of Protein Capture Agents to Biosensor Surfaces Using Aniline-Catalyzed Hydrazone Ligation

PubMed Central

Byeon, Ji-Yeon; Limpoco, F. T.; Bailey, Ryan C.

2010-01-01

Aniline-catalyzed hydrazone ligation between surface immobilized hydrazines and aldehyde-modified antibodies is shown to be an efficient method for attaching protein capture agents to model oxide-coated biosensor substrates. Silicon photonic microring resonators are used to directly evaluate the efficiency of this surface bioconjugate reaction at various pHs and in the presence or absence of aniline as a nucleophilic catalyst. It is found that aniline significantly increases the net antibody loading for surfaces functionalized over a pH range from 4.5 to 7.4, allowing derivatization of substrates with reduced incubation time and sample consumption. This increase in antibody loading directly results in more sensitive antigen detection when functionalized microrings are employed in a label-free immunoassay. Furthermore, these experiments also reveal an interesting pH dependent non-covalent binding trend that plays an important role in dictating the amount of antibody attached onto the substrate, highlighting the competing contributions of the bioconjugate reaction rate and the dynamic interactions that control opportunities for a solution-phase biomolecule to react with a substrate-bound reagent. PMID:20809595

Optimizing the molecular diagnosis of CDKL5 gene-related epileptic encephalopathy in boys.

PubMed

Mei, Davide; Darra, Francesca; Barba, Carmen; Marini, Carla; Fontana, Elena; Chiti, Laura; Parrini, Elena; Dalla Bernardina, Bernardo; Guerrini, Renzo

2014-11-01

Mutations involving the cyclin-dependent kinase-like 5 (CDKL5) gene cause an early onset epileptic encephalopathy (EE) with severe neurologic impairment and a skewed 12:1 female-to-male ratio. To date, 18 mutations have been described in boys. We analyzed our cohort of boys with early onset EE to assess the diagnostic yield of our molecular approach. We studied 74 boys who presented early onset severe seizures, including infantile spasms and developmental delay, in the setting of EE, using Sanger sequencing, next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA). We identified alterations involving CDKL5 in four boys (5.4%) using NGS in one and MLPA in three. Three of four mutations were indicative of somatic mosaicism. CDKL5 gene mutations accounted for 5.4% of boys with early onset EE. Somatic mosaic mutations might be even more represented than germline mutations, probably because their less deleterious effect enhances viability of the male embryo. The molecular approach used for CDKL5 screening remarkably influences the diagnostic yield in boys. Diagnosis is optimized by Sanger sequencing combined with array-based methods or MLPA; alternatively, NGS targeted resequencing designed to also detect copy number alterations, may be performed. Wiley Periodicals, Inc. © 2014 International League Against Epilepsy.

The effect of ligation on the load deflection characteristics of nickel titanium orthodontic wire.

PubMed

Kasuya, Shugo; Nagasaka, Satoshi; Hanyuda, Ai; Ishimura, Sadao; Hirashita, Ayao

2007-12-01

This study examined the effect of ligation on the load-deflection characteristics of nickel-titanium (NiTi) orthodontic wire. A modified three-point bending system was used for bending the NiTi round wire, which was inserted and ligated in the slots of three brackets, one of which was bonded to each of the three bender rods. Three different ligation methods, stainless steel ligature (SSL), slot lid (SL), and elastomeric ligature (EL), were employed, as well as a control with neither bracket nor ligation (NBL). The tests were repeated five times under each condition. Comparisons were made of load-deflection curve, load at maximum deflection of 2,000 microm, and load at a deflection of 1,500 microm during unloading. Analysis of Variance (ANOVA) and Dunnett's test were conducted to determine method difference (alpha = 0.05). The interaction between deflection and ligation was tested, using repeated-measures ANOVA (alpha = 0.05). The load values of the ligation groups were two to three times greater than the NBL group at a deflection of 1,500 microm during unloading: 4.37 N for EL, 3.90 N for SSL, 3.02 N for SL, and 1.49 N for NBL (P < 0.01). For the EL, a plateau region disappeared in the unloading curve. SL showed the smallest load. The ligation of the bracket wire may make NiTi wire exhibit a significantly heavier load than that traditionally expected. NiTi wire exhibited the majority of its true superelasticity with SL, whereas EL may act as a restraint on its superelasticity.

Multiplex polymerase chain reaction-capillary gel electrophoresis: a promising tool for GMO screening--assay for simultaneous detection of five genetically modified cotton events and species.

PubMed

Nadal, Anna; Esteve, Teresa; Pla, Maria

2009-01-01

A multiplex polymerase chain reaction assay coupled to capillary gel electrophoresis for amplicon identification by size and color (multiplex PCR-CGE-SC) was developed for simultaneous detection of cotton species and 5 events of genetically modified (GM) cotton. Validated real-time-PCR reactions targeting Bollgard, Bollgard II, Roundup Ready, 3006-210-23, and 281-24-236 junction sequences, and the cotton reference gene acp1 were adapted to detect more than half of the European Union-approved individual or stacked GM cotton events in one reaction. The assay was fully specific (<1.7% of false classification rate), with limit of detection values of 0.1% for each event, which were also achieved with simulated mixtures at different relative percentages of targets. The assay was further combined with a second multiplex PCR-CGE-SC assay to allow simultaneous detection of 6 cotton and 5 maize targets (two endogenous genes and 9 GM events) in two multiplex PCRs and a single CGE, making the approach more economic. Besides allowing simultaneous detection of many targets with adequate specificity and sensitivity, the multiplex PCR-CGE-SC approach has high throughput and automation capabilities, while keeping a very simple protocol, e.g., amplification and labeling in one step. Thus, it is an easy and inexpensive tool for initial screening, to be complemented with quantitative assays if necessary.

Layer-switching cost and optimality in information spreading on multiplex networks

PubMed Central

Min, Byungjoon; Gwak, Sang-Hwan; Lee, Nanoom; Goh, K. -I.

2016-01-01

We study a model of information spreading on multiplex networks, in which agents interact through multiple interaction channels (layers), say online vs. offline communication layers, subject to layer-switching cost for transmissions across different interaction layers. The model is characterized by the layer-wise path-dependent transmissibility over a contact, that is dynamically determined dependently on both incoming and outgoing transmission layers. We formulate an analytical framework to deal with such path-dependent transmissibility and demonstrate the nontrivial interplay between the multiplexity and spreading dynamics, including optimality. It is shown that the epidemic threshold and prevalence respond to the layer-switching cost non-monotonically and that the optimal conditions can change in abrupt non-analytic ways, depending also on the densities of network layers and the type of seed infections. Our results elucidate the essential role of multiplexity that its explicit consideration should be crucial for realistic modeling and prediction of spreading phenomena on multiplex social networks in an era of ever-diversifying social interaction layers. PMID:26887527

Multiplexed ChIP-Seq Using Direct Nucleosome Barcoding: A Tool for High-Throughput Chromatin Analysis.

PubMed

Chabbert, Christophe D; Adjalley, Sophie H; Steinmetz, Lars M; Pelechano, Vicent

2018-01-01

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) or microarray hybridization (ChIP-on-chip) are standard methods for the study of transcription factor binding sites and histone chemical modifications. However, these approaches only allow profiling of a single factor or protein modification at a time.In this chapter, we present Bar-ChIP, a higher throughput version of ChIP-Seq that relies on the direct ligation of molecular barcodes to chromatin fragments. Bar-ChIP enables the concurrent profiling of multiple DNA-protein interactions and is therefore amenable to experimental scale-up, without the need for any robotic instrumentation.

Urokinase-type plasminogen activator receptor (uPAR) ligation induces a raft-localized integrin signaling switch that mediates the hypermotile phenotype of fibrotic fibroblasts.

PubMed

Grove, Lisa M; Southern, Brian D; Jin, Tong H; White, Kimberly E; Paruchuri, Sailaja; Harel, Efrat; Wei, Ying; Rahaman, Shaik O; Gladson, Candece L; Ding, Qiang; Craik, Charles S; Chapman, Harold A; Olman, Mitchell A

2014-05-02

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

Development of a 3D origami multiplex electrochemical immunodevice using a nanoporous silver-paper electrode and metal ion functionalized nanoporous gold-chitosan.

PubMed

Li, Weiping; Li, Long; Li, Meng; Yu, Jinghua; Ge, Shenguang; Yan, Mei; Song, Xianrang

2013-10-25

A simple and sensitive 3D microfluidic origami multiplex electrochemical immunodevice was developed for the first time using a novel nanoporous silver modified paper working electrode as a sensor platform and different metal ion functionalized nanoporous gold-chitosan as a tracer.

Chemical synthesis of membrane proteins by the removable backbone modification method.

PubMed

Tang, Shan; Zuo, Chao; Huang, Dong-Liang; Cai, Xiao-Ying; Zhang, Long-Hua; Tian, Chang-Lin; Zheng, Ji-Shen; Liu, Lei

2017-12-01

Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

An enzyme-free flow cytometric bead assay for the sensitive detection of microRNAs based on click nucleic acid ligation-mediated signal amplification.

PubMed

Qi, Yan; Qiu, Liying; Fan, Wenjiao; Liu, Chenghui; Li, Zhengping

2017-08-07

A versatile flow cytometric bead assay (FCBA) coupled with a completely enzyme-free signal amplification mechanism is developed for the sensitive detection of microRNAs (miRNAs). This new strategy integrates click chemistry-mediated ligation chain reaction (CLCR) with hybridization chain reaction (HCR) for enzyme-free signal amplification on magnetic beads (MBs), and a flow cytometer for the robust fluorescence readout of the MBs. Firstly, target miRNA can initiate CLCR on the surface of MBs based on the click chemical ligation between dibenzocyclooctyne (DBCO)- and azide-modified single-stranded DNA (ssDNA) probes, and the amount of ligated ssDNA sequences on the MBs will be proportional to the dosage of target miRNA. Afterward, each of the ligated ssDNA products can trigger a cascade chain reaction of hybridization events between two alternating fluorophore-tagged hairpin probes, resulting in another signal amplification pathway with an amplified accumulation of fluorophores on the MBs. Finally, the fluorophore-anchored MBs are directly and rapidly analyzed by using a flow cytometer without any separation or elution processes. Herein, the click nucleic acid ligation only occurs on the surface of MBs, so the nonspecific ligations are greatly inhibited compared with that of ligation reaction performed in homogeneous solution. Furthermore, the signal amplification by CLCR-HCR is highly efficient but totally enzyme-free, which may overcome the potential drawbacks of conventional enzyme-catalyzed signal amplification protocols and lead to a high sensitivity. The CLCR-HCR-based FCBA has pushed the detection limit of let-7a miRNA down to the femtomolar (fM) level, showing great potential in miRNA-related biological studies and disease diagnosis.

Mode of Genetic Inheritance Modifies the Association of Head Circumference and Autism-Related Symptoms: A Cross-Sectional Study

PubMed Central

Davis, Jonathan M.; Keeney, Jonathon G.; Sikela, James M.; Hepburn, Susan

2013-01-01

Background Frequently individuals with autism spectrum disorder (ASD) have been noted with a larger head circumference (HC) than their typical developing peers. Biologic hypotheses suggest that an overly rapid brain growth leads to the core symptoms of ASD by impairing connectivity. Literature is divided however where deleterious, protective and null associations of HC with ASD symptoms in individuals with ASD have been found. Method Individuals (n = 1,416) from the Autism Genetic Resource Exchange with ASD were examined for associations of HC with ASD like symptoms. Mixed models controlling for sex, age, race/ethnicity, simplex/multiplex status and accounting for correlations between siblings were used. Interactions by simplex/multiplex were explored. Adjustments for height in a sub-population with available data were explored as well. Results A Significant interaction term (p = 0.03) suggested that the effect of HC was dependent on whether the individual was simplex or multiplex. In simplex individuals at mean age (8.9 years) 1 cm increase in head circumference was associated with a 24% increase in the odds of a high social diagnostic score from the Autism Diagnostic Interview – Revised (odds ratio  = 1.24, p = 0.01). There was no association in multiplex individuals. Additionally, individuals classified with a non-verbal IQ <70 were 90% simplex and had a significantly increased head circumference (0.7 cm p = 0.03) relative to a mid-range non-verbal IQ group. Interestingly, children classified with a >110 non-verbal IQ also had an increased HC (0.4 cm p = 0.04), relative to a mid-range non-verbal IQ group, and were 90% multiplex. HC effects do not appear to be confounded by height, however, larger samples with height information are needed. Conclusion The potential link between brain growth and autism like symptoms is complex and could depend on specific etiologies. Further investigations accounting for a likely mode of inheritance will help identify an ASD subtype related to HC. PMID:24058641

Antibody ligation of murine Ly-6G induces neutropenia, blood flow cessation, and death via complement-dependent and independent mechanisms.

PubMed

Abbitt, Katherine B; Cotter, Matthew J; Ridger, Victoria C; Crossman, David C; Hellewell, Paul G; Norman, Keith E

2009-01-01

Ly-6G is a member of the Ly-6 family of GPI-linked proteins, which is expressed on murine neutrophils. Antibodies against Ly-6G cause neutropenia, and fatal reactions also develop if mice are primed with TNF-alpha prior to antibody treatment. We have investigated the mechanisms behind these responses to Ly-6G ligation in the belief that similar mechanisms may be involved in neutropenia and respiratory disorders associated with alloantibody ligation of the related Ly-6 family member, NB1, in humans. Neutrophil adhesion, microvascular obstruction, breathing difficulties, and death initiated by anti-Ly-6G antibodies in TNF-alpha-primed mice were shown to be highly complement-dependent, partly mediated by CD11b, CD18, and FcgammaR and associated with clustering of Ly-6G. Neutrophil depletion, on the other hand, was only partly complement-dependent and was not altered by blockade of CD11b, CD18, or FcgammaR. Unlike other neutrophil-activating agents, Ly-6G ligation did not induce neutropenia via sequestration in the lungs. Cross-linking Ly-6G mimicked the responses seen with whole antibody in vivo and also activated murine neutrophils in vitro. Although this suggests that the responses are, in part, mediated by nonspecific properties of antibody ligation, neutrophil depletion requires an additional mechanism possibly specific to the natural function of Ly-6G.

Genetic analysis of an Indian family with members affected with Waardenburg syndrome and Duchenne muscular dystrophy

PubMed Central

Kapoor, Saketh; Bindu, Parayil Sankaran; Taly, Arun B.; Sinha, Sanjib; Gayathri, Narayanappa; Rani, S. Vasantha; Chandak, Giriraj Ratan

2012-01-01

Purpose Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS. Methods Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes. Results Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD. Conclusions This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3. PMID:22876130

New multiplex real-time PCR approach to detect gene mutations for spinal muscular atrophy.

PubMed

Liu, Zhidai; Zhang, Penghui; He, Xiaoyan; Liu, Shan; Tang, Shi; Zhang, Rong; Wang, Xinbin; Tan, Junjie; Peng, Bin; Jiang, Li; Hong, Siqi; Zou, Lin

2016-08-17

Spinal muscular atrophy (SMA) is the most common autosomal recessive disease in children, and the diagnosis is complicated and difficult, especially at early stage. Early diagnosis of SMA is able to improve the outcome of SMA patients. In our study, Real-time PCR was developed to measure the gene mutation or deletion of key genes for SMA and to further analyse genotype-phenotype correlation. The multiple real-time PCR for detecting the mutations of survival of motor neuron (SMN), apoptosis inhibitory protein (NAIP) and general transcription factor IIH, polypeptide 2 gene (GTF2H2) was established and confirmed by DNA sequencing and multiplex ligation-dependent probe amplification (MLPA). The diagnosis and prognosis of 141 hospitalized children, 100 normal children and further 2000 cases of dry blood spot (DBS) samples were analysed by this multiple real-time PCR. The multiple real-time PCR was established and the accuracy of it to detect the mutations of SMN, NAIP and GTF2H2 was at least 98.8 % comparing with DNA sequencing and MLPA. Among 141 limb movement disorders children, 75 cases were SMA. 71 cases of SMA (94.67 %) were with SMN c.840 mutation, 9 cases (12 %) with NAIP deletion and 3 cases (4 %) with GTF2H2 deletion. The multiple real-time PCR was able to diagnose and predict the prognosis of SMA patients. Simultaneously, the real-time PCR was applied to detect trace DNA from DBS and able to make an early diagnosis of SMA. The clinical and molecular characteristics of SMA in Southwest of China were presented. Our work provides a novel way for detecting SMA in children by using real-time PCR and the potential usage in newborn screening for early diagnosis of SMA.

Congenital diaphragmatic hernia (CDH) etiology as revealed by pathway genetics.

PubMed

Kantarci, Sibel; Donahoe, Patricia K

2007-05-15

Congenital diaphragmatic hernia (CDH) is a common birth defect with high mortality and morbidity. Two hundred seventy CDH patients were ascertained, carefully phenotyped, and classified as isolated (diaphragm defects alone) or complex (with additional anomalies) cases. We established different strategies to reveal CDH-critical chromosome loci and genes in humans. Candidate genes for sequencing analyses were selected from CDH animal models, genetic intervals of recurrent chromosomal aberration in humans, such as 15q26.1-q26.2 or 1q41-q42.12, as well as genes in the retinoic acid and related pathways and those known to be involved in embryonic lung development. For instance, FOG2, GATA4, and COUP-TFII are all needed for both normal diaphragm and lung development and are likely all in the same genetic and molecular pathway. Linkage analysis was applied first in a large inbred family and then in four multiplex families with Donnai-Barrow syndrome (DBS) associated with CDH. 10K SNP chip and microsatellite markers revealed a DBS locus on chromosome 2q23.3-q31.1. We applied array-based comparative genomic hybridization (aCGH) techniques to over 30, mostly complex, CDH patients and found a de novo microdeletion in a patient with Fryns syndrome related to CDH. Fluorescence in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA) techniques allowed us to further define the deletion interval. Our aim is to identify genetic intervals and, in those, to prioritize genes that might reveal molecular pathways, mutations in any step of which, might contribute to the same phenotype. More important, the elucidation of pathways may ultimately provide clues to treatment strategies. (c) 2007 Wiley-Liss, Inc.

Genetic analysis of an Indian family with members affected with Waardenburg syndrome and Duchenne muscular dystrophy.

PubMed

Kapoor, Saketh; Bindu, Parayil Sankaran; Taly, Arun B; Sinha, Sanjib; Gayathri, Narayanappa; Rani, S Vasantha; Chandak, Giriraj Ratan; Kumar, Arun

2012-01-01

Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS. Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes. Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD. This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3.

N-mustard analogs of S-adenosyl-L-methionine as biochemical probes of protein arginine methylation.

PubMed

Hymbaugh Bergman, Sarah J; Comstock, Lindsay R

2015-08-01

Nucleosomes, the fundamental building blocks of eukaryotic chromatin, undergo post-synthetic modifications and play a major role in the regulation of transcriptional processes. Combinations of these modifications, including methylation, regulate chromatin structure, determining its different functional states and playing a central role in differentiation. The biological significance of cellular methylation, particularly on chromatin, is widely recognized, yet we know little about the mechanisms that link biological methylation events. To characterize and fully understand protein methylation, we describe here novel N-mustard analogs of S-adenosyl-l-methionine (SAM) as biochemical tools to better understand protein arginine methylation events using protein arginine methyltransferase 1 (PRMT1). Specifically, azide- and alkyne-functionalized N-mustard analogs serve as cofactor mimics of SAM and are enzymatically transferred to a model peptide substrate in a PRMT1-dependent fashion. Once incorporated, the resulting alkynes and azides can be modified through chemoselective ligations, including click chemistry and the Staudinger ligation. These results readily demonstrate the feasibility of utilizing N-mustard analogs as biochemical tools to site-specifically label substrates of PRMT1 and serve as an alternative approach to study protein methylation events. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

PubMed

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

2017-08-01

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

PWS/AS MS-MLPA Confirms Maternal Origin of 15q11.2 Microduplication.

PubMed

Dawson, Angelika J; Cox, Janice; Hovanes, Karine; Spriggs, Elizabeth

2015-01-01

The proximal region of the long arm of chromosome 15q11.2-q13 is associated with various neurodevelopmental disorders, including Prader-Willi (PWS) and Angelman (AS) syndromes, autism, and other developmental abnormalities resulting from deletions and duplications. In addition, this region encompasses imprinted genes that cause PWS or AS, depending on the parent-of-origin. This imprinting allows for diagnosis of PWS or AS based on methylation status using methylation sensitive (MS) multiplex ligation dependent probe amplification (MLPA). Maternally derived microduplications at 15q11.2-q13 have been associated with autism and other neuropsychiatric disorders. Multiple methods have been used to determine the parent-of-origin for 15q11.2-q13 microdeletions and microduplications. In the present study, a four-year-old nondysmorphic female patient with developmental delay was found to have a de novo ~5 Mb duplication within 15q11.2 by oligonucleotide genomic array. In order to determine the significance of this microduplication to the clinical phenotype, the parent-of-origin needed to be identified. The PWS/AS MS-MLPA assay is generally used to distinguish between deletion and uniparental disomy (UPD) of 15q11.2-q13, resulting in either PWS or AS. However, our study shows that PWS/AS MS-MLPA can also efficiently distinguish the parental origin of duplications of 15q11.2-q13.

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

PubMed Central

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

2017-01-01

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

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization

PubMed Central

Girard, Laurie D.; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G.

2014-01-01

The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have a high complexity cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotidesequence-dependent segment and a unique “target sequence-independent” 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets. PMID:25489607

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization.

PubMed

Girard, Laurie D; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G

2015-02-07

The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have high complexity and cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotide sequence-dependent segment and a unique "target sequence-independent" 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets.

Accurate multiplex polony sequencing of an evolved bacterial genome.

PubMed

Shendure, Jay; Porreca, Gregory J; Reppas, Nikos B; Lin, Xiaoxia; McCutcheon, John P; Rosenbaum, Abraham M; Wang, Michael D; Zhang, Kun; Mitra, Robi D; Church, George M

2005-09-09

We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.

Probe-specific mixed-model approach to detect copy number differences using multiplex ligation-dependent probe amplification (MLPA)

PubMed Central

González, Juan R; Carrasco, Josep L; Armengol, Lluís; Villatoro, Sergi; Jover, Lluís; Yasui, Yutaka; Estivill, Xavier

2008-01-01

Background MLPA method is a potentially useful semi-quantitative method to detect copy number alterations in targeted regions. In this paper, we propose a method for the normalization procedure based on a non-linear mixed-model, as well as a new approach for determining the statistical significance of altered probes based on linear mixed-model. This method establishes a threshold by using different tolerance intervals that accommodates the specific random error variability observed in each test sample. Results Through simulation studies we have shown that our proposed method outperforms two existing methods that are based on simple threshold rules or iterative regression. We have illustrated the method using a controlled MLPA assay in which targeted regions are variable in copy number in individuals suffering from different disorders such as Prader-Willi, DiGeorge or Autism showing the best performace. Conclusion Using the proposed mixed-model, we are able to determine thresholds to decide whether a region is altered. These threholds are specific for each individual, incorporating experimental variability, resulting in improved sensitivity and specificity as the examples with real data have revealed. PMID:18522760

PubMed Central

Bello, Luca

2016-01-01

Accurate definition of genetic mutations causing Duchenne muscular dystrophy (DMD) has always been relevant in order to provide genetic counseling to patients and families, and helps to establish the prognosis in the case where the distinction between Duchenne, Becker, or intermediate muscular dystrophy is not obvious. As molecular treatments aimed at dystrophin restoration in DMD are increasingly available as commercialized drugs or within clinical trials, genetic diagnosis has become an indispensable tool in order to determine eligibility for these treatments. DMD patients in which multiplex ligation-dependent probe amplification (MLPA) or similar techniques show a deletion suitable to exon skipping of exons 44, 45, 51, or 53, may be currently treated with AONs targeting these exons, in the context of clinical trials, or, as is the case for exon 51 skipping in the United States, with the first commercialized drug (eteplirsen). Patients who test negative at MLPA, but in whom DMD gene sequencing shows a nonsense mutation, may be amenable for treatment with stop codon readthrough compounds such as ataluren. Novel molecular approaches such as CRISPR-Cas9 targeting of specific DMD mutations are still in the preclinical stages, but appear promising. In conclusion, an accurate genetic diagnosis represents the entrance into a new scenario of personalized medicine in DMD. PMID:28484312

Genetic Analyses of the NF1 Gene in Turkish Neurofibromatosis Type I Patients and Definition of three Novel Variants

PubMed Central

Ulusal, SD; Gürkan, H; Atlı, E; Özal, SA; Çiftdemir, M; Tozkır, H; Karal, Y; Güçlü, H; Eker, D; Görker, I

2017-01-01

Abstract Neurofibromatosis Type I (NF1) is a multi systemic autosomal dominant neurocutaneous disorder predisposing patients to have benign and/or malignant lesions predominantly of the skin, nervous system and bone. Loss of function mutations or deletions of the NF1 gene is responsible for NF1 disease. Involvement of various pathogenic variants, the size of the gene and presence of pseudogenes makes it difficult to analyze. We aimed to report the results of 2 years of multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) for genetic diagnosis of NF1 applied at our genetic diagnosis center. The MLPA, semiconductor sequencing and Sanger sequencing were performed in genomic DNA samples from 24 unrelated patients and their affected family members referred to our center suspected of having NF1. In total, three novel and 12 known pathogenic variants and a whole gene deletion were determined. We suggest that next generation sequencing is a practical tool for genetic analysis of NF1. Deletion/duplication analysis with MLPA may also be helpful for patients clinically diagnosed to carry NF1 but do not have a detectable mutation in NGS. PMID:28924536

Mutation spectrum of Chinese patients with Bartter syndrome

PubMed Central

Han, Yue; Lin, Yi; Sun, Qing; Wang, Shujuan; Gao, Yanxia; Shao, Leping

2017-01-01

Objective Bartter syndrome (BS) has been rarely reported in Chinese population except for a few case reports. This investigation was aimed to analyze the mutations of the causal genes in sixteen Chinese patients with BS, and review their followup and treatment. Methods Identify mutations by the next generation sequencing and the multiplex ligation-dependent probe amplification (MLPA). Clinical characteristics and biochemical findings at the first presentation as well as follow-up were reviewed. Results 15 different CLCNKB gene mutations were identified in fourteen patients with BS, including 11 novel ones. A novel missense mutation and a novel small deletion were found from SLC12A1 gene. A novel gross deletion was found in CLCNKA gene. A recurrent missense mutation was identified from BSND gene. We found that the whole gene deletion mutation of CLCNKB gene was the most frequent mutation (32%), and the rate of gross deletion was up to 50 percent in this group of Chinese patients. Conclusion The present study has found 19 mutations, including 14 novel ones, which would enrich the human gene mutation database (HGMD) and provide valuable references to the genetic counseling and diagnosis of the Chinese population. PMID:29254190

An epigenetically derived monoclonal origin for recurrent respiratory papillomatosis.

PubMed

Stephen, Josena Kunjoonju; Vaught, Lori E; Chen, Kang Mei; Shah, Veena; Schweitzer, Vanessa G; Gardner, Glendon; Benninger, Michael S; Worsham, Maria J

2007-07-01

To investigate the contribution of promoter methylation-mediated epigenetic events in recurrent respiratory papillomatosis tumorigenesis. Archival tissue DNA, extracted from microdissected papilloma lesions, was interrogated for methylation status by means of the novel, multigene methylation-specific multiplex ligation-dependent probe amplification assay. Fifteen subjects with recurrent respiratory papillomatosis, 3 females and 12 males, all with adult onset of illness (age range, 23-73 years) except for 1 female patient with juvenile onset (1 year old). Promoter hypermethylation was recorded in 14 of 15 cases, and 19 of 22 unique methylation-prone cancer genes in the multigene panel had altered DNA methylation in at least 1 laryngeal papilloma biopsy specimen. Identical abnormally methylated genes were found in 5 of 15 recurrent cases, of which the CDKN2B gene was hypermethylated in all 5 cases. Dissimilar epigenetic events were noted in the remaining cases. A clonal origin was derived for 5 of 15 recurrent respiratory papillomatosis biopsy specimens based on identical epigenetic events. The high frequency of epigenetic events, characterized by consistent promoter hypermethylation of multiple tumor suppressor genes, points to the use of gene silencing mechanisms in the pathogenesis of recurrent respiratory papillomatosis.

Novel deletions involving the USH2A gene in patients with Usher syndrome and retinitis pigmentosa.

PubMed

García-García, Gema; Aller, Elena; Jaijo, Teresa; Aparisi, Maria J; Larrieu, Lise; Faugère, Valérie; Blanco-Kelly, Fiona; Ayuso, Carmen; Roux, Anne-Francoise; Millán, José M

2014-01-01

The aim of the present work was to identify and characterize large rearrangements involving the USH2A gene in patients with Usher syndrome and nonsyndromic retinitis pigmentosa. The multiplex ligation-dependent probe amplification (MLPA) technique combined with a customized array-based comparative genomic hybridization (aCGH) analysis was applied to 40 unrelated patients previously screened for point mutations in the USH2A gene in which none or only one pathologic mutation was identified. We detected six large deletions involving USH2A in six out of the 40 cases studied. Three of the patients were homozygous for the deletion, and the remaining three were compound heterozygous with a previously identified USH2A point mutation. In five of these cases, the patients displayed Usher type 2, and the remaining case displayed nonsyndromic retinitis pigmentosa. The exact breakpoint junctions of the deletions found in USH2A in four of these cases were characterized. Our study highlights the need to develop improved efficient strategies of mutation screening based upon next generation sequencing (NGS) that reduce cost, time, and complexity and allow simultaneous identification of all types of disease-causing mutations in diagnostic procedures.

Partial USH2A deletions contribute to Usher syndrome in Denmark.

PubMed

Dad, Shzeena; Rendtorff, Nanna D; Kann, Erik; Albrechtsen, Anders; Mehrjouy, Mana M; Bak, Mads; Tommerup, Niels; Tranebjærg, Lisbeth; Rosenberg, Thomas; Jensen, Hanne; Møller, Lisbeth B

2015-12-01

Usher syndrome is an autosomal recessive disorder characterized by congenital hearing impairment, progressive visual loss owing to retinitis pigmentosa and in some cases vestibular dysfunction. Usher syndrome is divided into three subtypes, USH1, USH2 and USH3. Twelve loci and eleven genes have so far been identified. Duplications and deletions in PCDH15 and USH2A that lead to USH1 and USH2, respectively, have previously been identified in patients from United Kingdom, Spain and Italy. In this study, we investigate the proportion of exon deletions and duplications in PCDH15 and USH2A in 20 USH1 and 30 USH2 patients from Denmark using multiplex ligation-dependent probe amplification (MLPA). Two heterozygous deletions were identified in USH2A, but no deletions or duplications were identified in PCDH15. Next-generation mate-pair sequencing was used to identify the exact breakpoints of the two deletions identified in USH2A. Our results suggest that USH2 is caused by USH2A exon deletions in a small fraction of the patients, whereas deletions or duplications in PCDH15 might be rare in Danish Usher patients.

A Clinical and Molecular Genetic Study of 50 Families with Autosomal Recessive Parkinsonism Revealed Known and Novel Gene Mutations.

PubMed

Taghavi, Shaghayegh; Chaouni, Rita; Tafakhori, Abbas; Azcona, Luis J; Firouzabadi, Saghar Ghasemi; Omrani, Mir Davood; Jamshidi, Javad; Emamalizadeh, Babak; Shahidi, Gholam Ali; Ahmadi, Mona; Habibi, Seyed Amir Hassan; Ahmadifard, Azadeh; Fazeli, Atena; Motallebi, Marzieh; Petramfar, Peyman; Askarpour, Saeed; Askarpour, Shiva; Shahmohammadibeni, Hossein Ali; Shahmohammadibeni, Neda; Eftekhari, Hajar; Shafiei Zarneh, Amir Ehtesham; Mohammadihosseinabad, Saeed; Khorrami, Mehdi; Najmi, Safa; Chitsaz, Ahmad; Shokraeian, Parasto; Ehsanbakhsh, Hossein; Rezaeidian, Jalal; Ebrahimi Rad, Reza; Madadi, Faranak; Andarva, Monavvar; Alehabib, Elham; Atakhorrami, Minoo; Mortazavi, Seyed Erfan; Azimzadeh, Zahra; Bayat, Mahdis; Besharati, Amir Mohammad; Harati-Ghavi, Mohammad Ali; Omidvari, Samareh; Dehghani-Tafti, Zahra; Mohammadi, Faraz; Mohammad Hossein Pour, Banafsheh; Noorollahi Moghaddam, Hamid; Esmaili Shandiz, Ehsan; Habibi, Arman; Taherian-Esfahani, Zahra; Darvish, Hossein; Paisán-Ruiz, Coro

2018-04-01

In this study, the role of known Parkinson's disease (PD) genes was examined in families with autosomal recessive (AR) parkinsonism to assist with the differential diagnosis of PD. Some families without mutations in known genes were also subject to whole genome sequencing with the objective to identify novel parkinsonism-related genes. Families were selected from 4000 clinical files of patients with PD or parkinsonism. AR inheritance pattern, consanguinity, and a minimum of two affected individuals per family were used as inclusion criteria. For disease gene/mutation identification, multiplex ligation-dependent probe amplification, quantitative PCR, linkage, and Sanger and whole genome sequencing assays were carried out. A total of 116 patients (50 families) were examined. Fifty-four patients (46.55%; 22 families) were found to carry pathogenic mutations in known genes while a novel gene, not previously associated with parkinsonism, was found mutated in a single family (2 patients). Pathogenic mutations, including missense, nonsense, frameshift, and exon rearrangements, were found in Parkin, PINK1, DJ-1, SYNJ1, and VAC14 genes. In conclusion, variable phenotypic expressivity was seen across all families.

Ring chromosome 10: report on two patients and review of the literature.

PubMed

Guilherme, Roberta Santos; Kim, Chong Ae; Alonso, Luis Garcia; Honjo, Rachel S; Meloni, Vera Ayres; Christofolini, Denise Maria; Kulikowski, Leslie Domenici; Melaragno, Maria Isabel

2013-02-01

Ring chromosome 10--r(10)--is a rare disorder, with 14 cases reported in the literature, but only two with breakpoint determination by high-resolution techniques. We report here on two patients presenting a ring chromosome 10, studied by G-banding, fluorescent in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA) and SNP-array techniques, in order to investigate ring instability and determine breakpoints. Patient 1 showed a r(10)(p15.3q26.2) with a 7.9 Mb deletion in 10q26.2-q26.2, while patient 2 showed a r(10)(p15.3q26.13) with a 1.0 Mb deletion in 10p15.3 and a 8.8 Mb deletion in 10q26.13-q26.3, both unstable. While patient 1 presented with clinical features usually found in patients with r(10) and terminal 10q deletion, patient 2 presented characteristics so far not described in other patients with r(10), such as Dandy-Walker variant, osteopenia, semi-flexed legs, and dermal pigmentation regions. Our data and the data from literature show that there are no specific clinical findings to define a r(10) syndrome.

Duane retraction syndrome in a patient with Duchenne muscular dystrophy.

PubMed

Bosley, Thomas M; Salih, Mustafa A; Alkhalidi, Hisham; Oystreck, Darren T; El Khashab, Heba Y; Kondkar, Altaf A; Abu-Amero, Khaled K

2016-09-01

We describe the clinical features of a boy with bilateral Duane retraction syndrome (DRS), Duchenne muscular dystrophy (DMD), and other medical problems. The child was followed-up for five years; his chart was reviewed, including the results of a muscle biopsy and genetic testing. Multiplex ligation-dependent probe amplification (MLPA) was used to interrogate deletions/duplications in the dystrophin gene. The proband had bilateral DRS with otherwise normal ocular motility; he also had developmental delay, mild mental retardation, and seizures. Clinical diagnosis of DMD included progressive proximal weakness, highly elevated creatine kinase levels, and a muscle biopsy showing significant dystrophic changes including contracted, degenerative, and regenerative fibers, and negative dystrophin immunostaining. MLPA documented duplication of exons 3 and 4 of the dystrophin gene. This boy is the third patient to be reported with DRS and DMD, the second with bilateral DRS and the only one with other neurologic features. Mutated dystrophin is present in extraocular muscles and in the central nervous system (CNS) in DMD, leaving open the question of whether this co-occurrence is the result of the genetic muscle abnormality, CNS effects caused by dystrophin mutations, or chance.

Identification of 15 novel partial SHOX deletions and 13 partial duplications, and a review of the literature reveals intron 3 to be a hotspot region.

PubMed

Benito-Sanz, Sara; Belinchon-Martínez, Alberta; Aza-Carmona, Miriam; de la Torre, Carolina; Huber, Celine; González-Casado, Isabel; Ross, Judith L; Thomas, N Simon; Zinn, Andrew R; Cormier-Daire, Valerie; Heath, Karen E

2017-02-01

Short stature homeobox gene (SHOX) is located in the pseudoautosomal region 1 of the sex chromosomes. It encodes a transcription factor implicated in the skeletal growth. Point mutations, deletions or duplications of SHOX or its transcriptional regulatory elements are associated with two skeletal dysplasias, Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LMD), as well as in a small proportion of idiopathic short stature (ISS) individuals. We have identified a total of 15 partial SHOX deletions and 13 partial SHOX duplications in LWD, LMD and ISS patients referred for routine SHOX diagnostics during a 10 year period (2004-2014). Subsequently, we characterized these alterations using MLPA (multiplex ligation-dependent probe amplification assay), fine-tiling array CGH (comparative genomic hybridation) and breakpoint PCR. Nearly half of the alterations have a distal or proximal breakpoint in intron 3. Evaluation of our data and that in the literature reveals that although partial deletions and duplications only account for a small fraction of SHOX alterations, intron 3 appears to be a breakpoint hotspot, with alterations arising by non-allelic homologous recombination, non-homologous end joining or other complex mechanisms.

Current molecular genetics strategies for the diagnosis of lysosomal storage disorders.

PubMed

Giugliani, Roberto; Brusius-Facchin, Ana-Carolina; Pasqualim, Gabriela; Leistner-Segal, Sandra; Riegel, Mariluce; Matte, Ursula

2016-01-01

Lysosomal storage disorders (LSDs) are a group of almost 50 monogenic diseases characterized by mutations causing deficiency of lysosomal enzymes or non-enzyme proteins involved in transport across the lysosomal membrane, protein maturation or lysosomal biogenesis. Usually, affected patients are normal at birth and have a progressive and severe disease with high morbidity and reduced life expectancy. The overall incidence of LSDs is usually estimated as 1:5000, but newborn screening studies are indicating that it could be much higher. Specific therapies were already developed for selected LSDs, making the timely and correct diagnosis very important for successful treatment and also for genetic counseling. In most LSD cases the biochemical techniques provide a reliable diagnosis. However, the identification of pathogenic mutations by genetic analysis is being increasingly recommended to provide additional information. In this paper we discuss the conventional methods for genetic analysis used in the LSDs [restriction fragment length polymorphism (RFLP), amplification-refractory mutation system (ARMS), single strand conformation polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC), real-time polymerase chain reaction, high resolution melting (HRM), multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing] and also the newer approaches [massive parallel sequencing, array comparative genomic hybridization (CGH)].

[Copy number alterations in adult patients with mature B acute lymphoblastic leukemia treated with specific immunochemotherapy].

PubMed

Ribera, Jordi; Zamora, Lurdes; García, Olga; Hernández-Rivas, Jesús-María; Genescà, Eulàlia; Ribera, Josep-Maria

2016-12-02

Unlike Burkitt lymphoma, molecular abnormalities other than C-MYC rearrangements have scarcely been studied in patients with mature B acute lymphoblastic leukemia (B-ALL). The aim of this study was to analyze the frequency and prognostic significance of copy number alterations (CNA) in genes involved in lymphoid differentiation, cell cycle and tumor suppression in adult patients with B-ALL. We have analyzed by multiplex ligation-dependent probe amplification the genetic material from bone marrow at diagnosis from 25 adult B-ALL patients treated with rituximab and specific chemotherapy. The most frequent CNA were alterations in the 14q32.33 region (11 cases, 44%) followed by alterations in the cell cycle regulator genes CDKN2A/B and RB1 (16%). No correlation between the presence of specific CNA and the clinical-biologic features or the response to therapy was found. The high frequency of CNA in the 14q32.33 region, CDKN2A/B and RB1 found in our study could contribute to the aggressiveness and invasiveness of mature B-ALL. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

Search for copy number variants in chromosomes 15q11-q13 and 22q11.2 in obsessive compulsive disorder

PubMed Central

2010-01-01

Background Obsessive-compulsive disorder (OCD) is a clinically and etiologically heterogeneous syndrome. The high frequency of obsessive-compulsive symptoms reported in subjects with the 22q11.2 deletion syndrome (DiGeorge/velocardiofacial syndrome) or Prader-Willi syndrome (15q11-13 deletion of the paternally derived chromosome), suggests that gene dosage effects in these chromosomal regions could increase risk for OCD. Therefore, the aim of this study was to search for microrearrangements in these two regions in OCD patients. Methods We screened the 15q11-13 and 22q11.2 chromosomal regions for genomic imbalances in 236 patients with OCD using multiplex ligation-dependent probe amplification (MLPA). Results No deletions or duplications involving 15q11-13 or 22q11.2 were identified in our patients. Conclusions Our results suggest that deletions/duplications of chromosomes 15q11-13 and 22q11.2 are rare in OCD. Despite the negative findings in these two regions, the search for copy number variants in OCD using genome-wide array-based methods is a highly promising approach to identify genes of etiologic importance in the development of OCD. PMID:20565924

Search for copy number variants in chromosomes 15q11-q13 and 22q11.2 in obsessive compulsive disorder.

PubMed

Delorme, Richard; Moreno-De-Luca, Daniel; Gennetier, Aurélie; Maier, Wolfgang; Chaste, Pauline; Mössner, Rainald; Grabe, Hans Jörgen; Ruhrmann, Stephan; Falkai, Peter; Mouren, Marie-Christine; Leboyer, Marion; Wagner, Michael; Betancur, Catalina

2010-06-21

Obsessive-compulsive disorder (OCD) is a clinically and etiologically heterogeneous syndrome. The high frequency of obsessive-compulsive symptoms reported in subjects with the 22q11.2 deletion syndrome (DiGeorge/velocardiofacial syndrome) or Prader-Willi syndrome (15q11-13 deletion of the paternally derived chromosome), suggests that gene dosage effects in these chromosomal regions could increase risk for OCD. Therefore, the aim of this study was to search for microrearrangements in these two regions in OCD patients. We screened the 15q11-13 and 22q11.2 chromosomal regions for genomic imbalances in 236 patients with OCD using multiplex ligation-dependent probe amplification (MLPA). No deletions or duplications involving 15q11-13 or 22q11.2 were identified in our patients. Our results suggest that deletions/duplications of chromosomes 15q11-13 and 22q11.2 are rare in OCD. Despite the negative findings in these two regions, the search for copy number variants in OCD using genome-wide array-based methods is a highly promising approach to identify genes of etiologic importance in the development of OCD.

Copy number variation of GATA4 and NKX2-5 in Chinese fetuses with congenital heart disease.

PubMed

Liu, Zhen; Wang, Jing; Liu, Shanling; Deng, Ying; Liu, Hongqian; Li, Nana; Li, Shengli; Chen, Xinlin; Lin, Yuan; Wang, He; Zhu, Jun

2015-04-01

Congenital heart disease (CHD) is one of the most common birth defects in newborns. The etiology of CHD has remained largely unknown, but it is assumed to result from the combined effects of genetic and environmental factors. Recent investigations have detected potentially pathogenic copy number variations (CNV) in a proportion of patients with CHD. The present case-control study evaluated whether CNV in the GATA4 and NKX2-5 genes contribute to the pathogenesis of CHD in Chinese fetuses (n = 117), by comparing them with non-CHD control subjects (n = 100). Multiplex ligation-dependent probe amplification with the P311A probe mixture was used to detect CNV. The normalized signals were within the normal range for all exons in all CHD patients and non-CHD control subjects. Of the 117 CHD patients, three had a deletion of 22q11, and two had a duplication of 22q11. There was no evidence of a role for NKX2-5 and GATA4 CNV in fetal CHD; therefore, these CNV may not be common in fetal CHD in China. © 2014 Japan Pediatric Society.

[PAX3 gene mutation analysis for two Waardenburg syndrome type Ⅰ families and their prenatal diagnosis].

PubMed

Bai, Y; Liu, N; Kong, X D; Yan, J; Qin, Z B; Wang, B

2016-12-07

Objective: To analyze the mutations of PAX3 gene in two Waardenburg syndrome type Ⅰ (WS1) pedigrees and make prenatal diagnosis for the high-risk 18-week-old fetus. Methods: PAX3 gene was first analyzed by Sanger sequencing and multiplex ligation-dependent probe amplification(MLPA) for detecting pathogenic mutation of the probands of the two pedigrees. The mutations were confirmed by MLPA and Sanger in parents and unrelated healthy individuals.Prenatal genetic diagnosis for the high-risk fetus was performed by amniotic fluid cell after genotyping. Results: A heterozygous PAX3 gene gross deletion (E7 deletion) was identified in all patients from WS1-01 family, and not found in 20 healthy individuals.Prenatal diagnosis in WS1-01 family indicated that the fetus was normal. Molecular studies identified a novel deletion mutation c. 1385_1386delCT within the PAX3 gene in all affected WS1-02 family members, but in none of the unaffected relatives and 200 healthy individuals. Conclusions: PAX3 gene mutation is etiological for two WS1 families. Sanger sequencing plus MLPA is effective and accurate for making gene diagnosis and prenatal diagnosis.

A Pictet-Spengler ligation for protein chemical modification

PubMed Central

Agarwal, Paresh; van der Weijden, Joep; Sletten, Ellen M.; Rabuka, David; Bertozzi, Carolyn R.

2013-01-01

Aldehyde- and ketone-functionalized proteins are appealing substrates for the development of chemically modified biotherapeutics and protein-based materials. Their reactive carbonyl groups are typically conjugated with α-effect nucleophiles, such as substituted hydrazines and alkoxyamines, to generate hydrazones and oximes, respectively. However, the resulting C=N linkages are susceptible to hydrolysis under physiologically relevant conditions, which limits the utility of such conjugates in biological systems. Here we introduce a Pictet-Spengler ligation that is based on the classic Pictet-Spengler reaction of aldehydes and tryptamine nucleophiles. The ligation exploits the bioorthogonal reaction of aldehydes and alkoxyamines to form an intermediate oxyiminium ion; this intermediate undergoes intramolecular C–C bond formation with an indole nucleophile to form an oxacarboline product that is hydrolytically stable. We used the reaction for site-specific chemical modification of glyoxyl- and formylglycine-functionalized proteins, including an aldehyde-tagged variant of the therapeutic monoclonal antibody Herceptin. In conjunction with techniques for site-specific introduction of aldehydes into proteins, the Pictet-Spengler ligation offers a means to generate stable bioconjugates for medical and materials applications. PMID:23237853

Modified midi- and mini-multiplex PCR systems for mitochondrial DNA control region sequence analysis in degraded samples.

PubMed

Kim, Na Young; Lee, Hwan Young; Park, Sun Joo; Yang, Woo Ick; Shin, Kyoung-Jin

2013-05-01

Two multiplex polymerase chain reaction (PCR) systems (Midiplex and Miniplex) were developed for the amplification of the mitochondrial DNA (mtDNA) control region, and the efficiencies of the multiplexes for amplifying degraded DNA were validated using old skeletal remains. The Midiplex system consisted of two multiplex PCRs to amplify six overlapping amplicons ranging in length from 227 to 267 bp. The Miniplex system consisted of three multiplex PCRs to amplify 10 overlapping short amplicons ranging in length from 142 to 185 bp. Most mtDNA control region sequences of several 60-year-old and 400-500-year-old skeletal remains were successfully obtained using both PCR systems and consistent with those previously obtained by monoplex amplification. The multiplex system consisting of smaller amplicons is effective for mtDNA sequence analyses of ancient and forensic degraded samples, saving time, cost, and the amount of DNA sample consumed during analysis. © 2013 American Academy of Forensic Sciences.

Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection.

PubMed

Hernández, Marta; Rodríguez-Lázaro, David; Esteve, Teresa; Prat, Salomé; Pla, Maria

2003-12-15

Commercialization of several genetically modified crops has been approved worldwide to date. Uniplex polymerase chain reaction (PCR)-based methods to identify these different insertion events have been developed, but their use in the analysis of all commercially available genetically modified organisms (GMOs) is becoming progressively insufficient. These methods require a large number of assays to detect all possible GMOs present in the sample and thereby the development of multiplex PCR systems using combined probes and primers targeted to sequences specific to various GMOs is needed for detection of this increasing number of GMOs. Here we report on the development of a multiplex real-time PCR suitable for multiple GMO identification, based on the intercalating dye SYBR Green I and the analysis of the melting curves of the amplified products. Using this method, different amplification products specific for Maximizer 176, Bt11, MON810, and GA21 maize and for GTS 40-3-2 soybean were obtained and identified by their specific Tm. We have combined amplification of these products in a number of multiplex reactions and show the suitability of the methods for identification of GMOs with a sensitivity of 0.1% in duplex reactions. The described methods offer an economic and simple alternative to real-time PCR systems based on sequence-specific probes (i.e., TaqMan chemistry). These methods can be used as selection tests and further optimized for uniplex GMO quantification.

Detection of human papillomaviruses by polymerase chain reaction and ligation reaction on universal microarray.

PubMed

Ritari, Jarmo; Hultman, Jenni; Fingerroos, Rita; Tarkkanen, Jussi; Pullat, Janne; Paulin, Lars; Kivi, Niina; Auvinen, Petri; Auvinen, Eeva

2012-01-01

Sensitive and specific detection of human papillomaviruses (HPV) in cervical samples is a useful tool for the early diagnosis of epithelial neoplasia and anogenital lesions. Recent studies support the feasibility of HPV DNA testing instead of cytology (Pap smear) as a primary test in population screening for cervical cancer. This is likely to be an option in the near future in many countries, and it would increase the efficiency of screening for cervical abnormalities. We present here a microarray test for the detection and typing of 15 most important high-risk HPV types and two low risk types. The method is based on type specific multiplex PCR amplification of the L1 viral genomic region followed by ligation detection reaction where two specific ssDNA probes, one containing a fluorescent label and the other a flanking ZipCode sequence, are joined by enzymatic ligation in the presence of the correct HPV PCR product. Human beta-globin is amplified in the same reaction to control for sample quality and adequacy. The genotyping capacity of our approach was evaluated against Linear Array test using cervical samples collected in transport medium. Altogether 14 out of 15 valid samples (93%) gave concordant results between our test and Linear Array. One sample was HPV56 positive in our test and high-risk positive in Hybrid Capture 2 but remained negative in Linear Array. The preliminary results suggest that our test has accurate multiple HPV genotyping capability with the additional advantages of generic detection format, and potential for high-throughput screening.

Non-thiolate ligation of nickel by nucleotide-free UreG of Klebsiella aerogenes

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

Martin-Diaconescu, Vlad; Joseph, Crisjoe A.; Boer, Jodi L.

Nickel-dependent ureases are activated by a multiprotein complex that includes the GTPase UreG. Prior studies showed that nucleotide-free UreG from Klebsiella aerogenes is monomeric and binds one nickel or zinc ion with near-equivalent affinity using an undefined binding site, whereas nucleotide-free UreG from Helicobacter pylori selectively binds one zinc ion per dimer via a universally conserved Cys-Pro-His motif in each protomer. Iodoacetamide-treated K. aerogenes UreG was nearly unaffected in nickel binding compared to non-treated sample, suggesting the absence of thiolate ligands to the metal. X-ray absorption spectroscopy of nickel-bound UreG showed the metal possessed four-coordinate geometry with all O/N donormore » ligands including one imidazole, thus confirming the absence of thiolate ligation. The nickel site in Strep-tag II-modified protein possessed six-coordinate geometry, again with all O/N donor ligands, but now including two or three imidazoles. An identical site was noted for the Strep-tag II-modified H74A variant, substituted in the Cys-Pro-His motif, ruling out coordination by this His residue. These results are consistent with metal binding to both His6 and a His residue of the fusion peptide in Strep-tagged K. aerogenes UreG. We conclude that the nickel- and zinc-binding site in nucleotide-free K. aerogenes UreG is distinct from that of nucleotide-free H. pylori UreG and does not involve the Cys-Pro-His motif. Further, we show the Strep-tag II can perturb metal coordination of this protein.« less

Hypotension following patent ductus arteriosus ligation: the role of adrenal hormones.

PubMed

Clyman, Ronald I; Wickremasinghe, Andrea; Merritt, T Allen; Solomon, Tabitha; McNamara, Patrick; Jain, Amish; Singh, Jaideep; Chu, Alison; Noori, Shahab; Sekar, Krishnamurthy; Lavoie, Pascal M; Attridge, Joshua T; Swanson, Jonathan R; Gillam-Krakauer, Maria; Reese, Jeff; DeMauro, Sara; Poindexter, Brenda; Aucott, Sue; Satpute, Monique; Fernandez, Erika; Auchus, Richard J

2014-06-01

To test the hypothesis that an impaired adrenal response to stress might play a role in the hypotension that follows patent ductus arteriosus (PDA) ligation. We performed a multicenter study of infants born at <32 weeks' gestation who were about to undergo PDA ligation. Serum adrenal steroids were measured 3 times: before and after a cosyntropin (1.0 μg/kg) stimulation test (performed before the ligation), and at 10-12 hours after the ligation. A standardized approach for diagnosis and treatment of postoperative hypotension was followed at each site. A modified inotrope score (1 × dopamine [μg/kg/min] + 1 × dobutamine) was used to monitor the catecholamine support an infant received. Infants were considered to have catecholamine-resistant hypotension if their greatest inotrope score was >15. Of 95 infants enrolled, 43 (45%) developed hypotension and 14 (15%) developed catecholamine-resistant hypotension. Low postoperative cortisol levels were not associated with the overall incidence of hypotension after ligation. However, low cortisol levels were associated with the refractoriness of the hypotension to catecholamine treatment. In a multivariate analysis: the OR for developing catecholamine-resistant hypotension was OR 36.6, 95% CI 2.8-476, P = .006. Low cortisol levels (in infants with catecholamine-resistant hypotension) were not attributable to adrenal immaturity or impairment; their cortisol precursor concentrations were either low or unchanged, and their response to cosyntropin was similar to infants without catecholamine-resistant hypotension. Infants with low cortisol concentrations after PDA ligation are likely to develop postoperative catecholamine-resistant hypotension. We speculate that decreased adrenal stimulation, rather than an impaired adrenal response to stimulation, may account for the decreased production. Copyright © 2014 Elsevier Inc. All rights reserved.

Hypotension following patent ductus arteriosus ligation: the role of adrenal hormones

PubMed Central

Clyman, Ronald I.; Wickremasinghe, Andrea; Merritt, T. Allen; Solomon, Tabitha; McNamara, Patrick; Jain, Amish; Singh, Jaideep; Chu, Alison; Noori, Shahab; Sekar, Krishnamurthy; Lavoie, Pascal M.; Attridge, Joshua T.; Swanson, Jonathan R.; Gillam-Krakauer, Maria; Reese, Jeff; DeMauro, Sara; Poindexter, Brenda; Aucott, Sue; Satpute, Monique; Fernandez, Erika; Auchus, Richard J.

2014-01-01

Objective To test the hypothesis that an impaired adrenal response to stress might play a role in the hypotension that follows patent ductus arteriosus (PDA) ligation. Study design We performed a multicenter study of infants born at <32 weeks gestation who were about to undergo PDA ligation. Serum adrenal steroids were measured three times: before and after a cosyntropin (1.0 microgram/kg) stimulation test (performed prior to the ligation), and at 10–12 hours after the ligation. A standardized approach for diagnosis and treatment of postoperative hypotension was followed at each site. A modified Inotrope Score (1 x dopamine (μg/kg/min) + 1 x dobutamine) was used to monitor the catecholamine support an infant received. Infants were considered to have catecholamine-resistant hypotension if their highest Inotrope Score was >15. Results Of 95 infants enrolled, 43 (45%) developed hypotension and 14 (15%) developed catecholamine-resistant hypotension. Low post-operative cortisol levels were not associated with the overall incidence of hypotension following ligation. However, low cortisol levels were associated with the refractoriness of the hypotension to catecholamine treatment. In a multivariate analysis: the odds ratio for developing catecholamine-resistant hypotension was OR=36.6, CI=2.8–476, p=0.006. Low cortisol levels (in infants with catecholamine-resistant hypotension) were not due to adrenal immaturity or impairment; their cortisol precursor concentrations were either low or unchanged and their response to cosyntropin was similar to infants without catecholamine-resistant hypotension. Conclusion Infants with low cortisol concentrations following PDA ligation are likely to develop postoperative catecholamine-resistant hypotension. We speculate that decreased adrenal stimulation, rather than an impaired adrenal response to stimulation, may account for the decreased production. PMID:24636853

Angular multiplexing holograms of four images recorded on photopolymer films with recording-film-thickness-dependent holographic characteristics

NASA Astrophysics Data System (ADS)

Osabe, Keiichi; Kawai, Kotaro

2017-03-01

In this study, angular multiplexing hologram recording photopolymer films were studied experimentally. The films contained acrylamide as a monomer, eosin Y as a sensitizer, and triethanolamine as a promoter in a polyvinyl alcohol matrix. In order to determine the appropriate thickness of the photopolymer films for angular multiplexing, photopolymer films with thicknesses of 29-503 μm were exposed to two intersecting beams of a YVO laser at a wavelength of 532 nm to form a holographic grating with a spatial frequency of 653 line/mm. The diffraction efficiencies as a function of the incident angle of reconstruction were measured. A narrow angular bandwidth and high diffraction efficiency are required for angular multiplexing; hence, we define the Q value, which is the diffraction efficiency divided by half the bandwidth. The Q value of the films depended on the thickness of the films, and was calculated based on the measured diffraction efficiencies. The Q value of a 297-μm-thick film was the highest of the all films. Therefore, the angular multiplexing experiments were conducted using 300-μm-thick films. In the angular multiplexing experiments, the object beam transmitted by a square aperture was focused by a Fourier transform lens and interfered with a reference beam. The maximum order of angular multiplexing was four. The signal intensity that corresponds to the squared-aperture transmission and the noise intensity that corresponds to transmission without the square aperture were measured. The signal intensities decreased as the order of angular multiplexing increased, and the noise intensities were not dependent on the order of angular multiplexing.

Evaluation of High-Resolution Melting Curve Analysis of Ligation-Mediated Real-Time PCR, a Rapid Method for Epidemiological Typing of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter Species) Pathogens

PubMed Central

Ryberg, Anna; Billström, Hanna; Hällgren, Anita; Nilsson, Lennart E.; Marklund, Britt-Inger; Olsson-Liljequist, Barbro; Schön, Thomas

2014-01-01

A single-tube method, ligation-mediated real-time PCR high-resolution melt analysis (LMqPCR HRMA), was modified for the rapid typing of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens. A 97% agreement (60/62 isolates) was achieved in comparison to pulsed-field gel electrophoresis (PFGE) results, which indicates that LMqPCR HRMA is a rapid and accurate screening tool for monitoring nosocomial outbreaks. PMID:25232168

Screening for BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1 mutations in high-risk Finnish BRCA1/2-founder mutation-negative breast and/or ovarian cancer individuals.

PubMed

Kuusisto, Kirsi M; Bebel, Aleksandra; Vihinen, Mauno; Schleutker, Johanna; Sallinen, Satu-Leena

2011-02-28

Two major high-penetrance breast cancer genes, BRCA1 and BRCA2, are responsible for approximately 20% of hereditary breast cancer (HBC) cases in Finland. Additionally, rare mutations in several other genes that interact with BRCA1 and BRCA2 increase the risk of HBC. Still, a majority of HBC cases remain unexplained which is challenging for genetic counseling. We aimed to analyze additional mutations in HBC-associated genes and to define the sensitivity of our current BRCA1/2 mutation analysis protocol used in genetic counseling. Eighty-two well-characterized, high-risk hereditary breast and/or ovarian cancer (HBOC) BRCA1/2-founder mutation-negative Finnish individuals, were screened for germline alterations in seven breast cancer susceptibility genes, BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1. BRCA1/2 were analyzed by multiplex ligation-dependent probe amplification (MLPA) and direct sequencing. CHEK2 was analyzed by the high resolution melt (HRM) method and PALB2, RAD50, BRIP1 and CDH1 were analyzed by direct sequencing. Carrier frequencies between 82 (HBOC) BRCA1/2-founder mutation-negative Finnish individuals and 384 healthy Finnish population controls were compared by using Fisher's exact test. In silico prediction for novel missense variants effects was carried out by using Pathogenic-Or-Not -Pipeline (PON-P). Three previously reported breast cancer-associated variants, BRCA1 c.5095C > T, CHEK2 c.470T > C, and CHEK2 c.1100delC, were observed in eleven (13.4%) individuals. Ten of these individuals (12.2%) had CHEK2 variants, c.470T > C and/or c.1100delC. Fourteen novel sequence alterations and nine individuals with more than one non-synonymous variant were identified. One of the novel variants, BRCA2 c.72A > T (Leu24Phe) was predicted to be likely pathogenic in silico. No large genomic rearrangements were detected in BRCA1/2 by multiplex ligation-dependent probe amplification (MLPA). In this study, mutations in previously known breast cancer susceptibility genes can explain 13.4% of the analyzed high-risk BRCA1/2-negative HBOC individuals. CHEK2 mutations, c.470T > C and c.1100delC, make a considerable contribution (12.2%) to these high-risk individuals but further segregation analysis is needed to evaluate the clinical significance of these mutations before applying them in clinical use. Additionally, we identified novel variants that warrant additional studies. Our current genetic testing protocol for 28 Finnish BRCA1/2-founder mutations and protein truncation test (PTT) of the largest exons is sensitive enough for clinical use as a primary screening tool.

Screening for BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1 mutations in high-risk Finnish BRCA1/2-founder mutation-negative breast and/or ovarian cancer individuals

PubMed Central

2011-01-01

Introduction Two major high-penetrance breast cancer genes, BRCA1 and BRCA2, are responsible for approximately 20% of hereditary breast cancer (HBC) cases in Finland. Additionally, rare mutations in several other genes that interact with BRCA1 and BRCA2 increase the risk of HBC. Still, a majority of HBC cases remain unexplained which is challenging for genetic counseling. We aimed to analyze additional mutations in HBC-associated genes and to define the sensitivity of our current BRCA1/2 mutation analysis protocol used in genetic counseling. Methods Eighty-two well-characterized, high-risk hereditary breast and/or ovarian cancer (HBOC) BRCA1/2-founder mutation-negative Finnish individuals, were screened for germline alterations in seven breast cancer susceptibility genes, BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1. BRCA1/2 were analyzed by multiplex ligation-dependent probe amplification (MLPA) and direct sequencing. CHEK2 was analyzed by the high resolution melt (HRM) method and PALB2, RAD50, BRIP1 and CDH1 were analyzed by direct sequencing. Carrier frequencies between 82 (HBOC) BRCA1/2-founder mutation-negative Finnish individuals and 384 healthy Finnish population controls were compared by using Fisher's exact test. In silico prediction for novel missense variants effects was carried out by using Pathogenic-Or-Not -Pipeline (PON-P). Results Three previously reported breast cancer-associated variants, BRCA1 c.5095C > T, CHEK2 c.470T > C, and CHEK2 c.1100delC, were observed in eleven (13.4%) individuals. Ten of these individuals (12.2%) had CHEK2 variants, c.470T > C and/or c.1100delC. Fourteen novel sequence alterations and nine individuals with more than one non-synonymous variant were identified. One of the novel variants, BRCA2 c.72A > T (Leu24Phe) was predicted to be likely pathogenic in silico. No large genomic rearrangements were detected in BRCA1/2 by multiplex ligation-dependent probe amplification (MLPA). Conclusions In this study, mutations in previously known breast cancer susceptibility genes can explain 13.4% of the analyzed high-risk BRCA1/2-negative HBOC individuals. CHEK2 mutations, c.470T > C and c.1100delC, make a considerable contribution (12.2%) to these high-risk individuals but further segregation analysis is needed to evaluate the clinical significance of these mutations before applying them in clinical use. Additionally, we identified novel variants that warrant additional studies. Our current genetic testing protocol for 28 Finnish BRCA1/2-founder mutations and protein truncation test (PTT) of the largest exons is sensitive enough for clinical use as a primary screening tool. PMID:21356067

Evaluating Kinase ATP Uptake and Tyrosine Phosphorylation using Multiplexed Quantification of Chemically Labeled and Post-Translationally Modified Peptides

PubMed Central

Fang, Bin; Hoffman, Melissa A.; Mirza, Abu-Sayeef; Mishall, Katie M.; Li, Jiannong; Peterman, Scott M.; Smalley, Keiran S. M.; Shain, Kenneth H.; Weinberger, Paul M.; Wu, Jie; Rix, Uwe; Haura, Eric B.; Koomen, John M.

2015-01-01

Cancer biologists and other healthcare researchers face an increasing challenge in addressing the molecular complexity of disease. Biomarker measurement tools and techniques now contribute to both basic science and translational research. In particular, liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) for multiplexed measurements of protein biomarkers has emerged as a versatile tool for systems biology. Assays can be developed for specific peptides that report on protein expression, mutation, or post-translational modification; discovery proteomics data rapidly translated into multiplexed quantitative approaches. Complementary advances in affinity purification enrich classes of enzymes or peptides representing post-translationally modified or chemically labeled substrates. Here, we illustrate the process for the relative quantification of hundreds of peptides in a single LC-MRM experiment. Desthiobiotinylated peptides produced by activity-based protein profiling (ABPP) using ATP probes and tyrosine-phosphorylated peptides are used as examples. These targeted quantification panels can be applied to further understand the biology of human disease. PMID:25782629

Optofluidic wavelength division multiplexing for single-virus detection

PubMed Central

Ozcelik, Damla; Parks, Joshua W.; Wall, Thomas A.; Stott, Matthew A.; Cai, Hong; Parks, Joseph W.; Hawkins, Aaron R.; Schmidt, Holger

2015-01-01

Optical waveguides simultaneously transport light at different colors, forming the basis of fiber-optic telecommunication networks that shuttle data in dozens of spectrally separated channels. Here, we reimagine this wavelength division multiplexing (WDM) paradigm in a novel context––the differentiated detection and identification of single influenza viruses on a chip. We use a single multimode interference (MMI) waveguide to create wavelength-dependent spot patterns across the entire visible spectrum and enable multiplexed single biomolecule detection on an optofluidic chip. Each target is identified by its time-dependent fluorescence signal without the need for spectral demultiplexing upon detection. We demonstrate detection of individual fluorescently labeled virus particles of three influenza A subtypes in two implementations: labeling of each virus using three different colors and two-color combinatorial labeling. By extending combinatorial multiplexing to three or more colors, MMI-based WDM provides the multiplexing power required for differentiated clinical tests and the growing field of personalized medicine. PMID:26438840

Bacterial adhesion on conventional and self-ligating metallic brackets after surface treatment with plasma-polymerized hexamethyldisiloxane.

PubMed

Tupinambá, Rogerio Amaral; Claro, Cristiane Aparecida de Assis; Pereira, Cristiane Aparecida; Nobrega, Celestino José Prudente; Claro, Ana Paula Rosifini Alves

2017-01-01

Plasma-polymerized film deposition was created to modify metallic orthodontic brackets surface properties in order to inhibit bacterial adhesion. Hexamethyldisiloxane (HMDSO) polymer films were deposited on conventional (n = 10) and self-ligating (n = 10) stainless steel orthodontic brackets using the Plasma-Enhanced Chemical Vapor Deposition (PECVD) radio frequency technique. The samples were divided into two groups according to the kind of bracket and two subgroups after surface treatment. Scanning Electron Microscopy (SEM) analysis was performed to assess the presence of bacterial adhesion over samples surfaces (slot and wings region) and film layer integrity. Surface roughness was assessed by Confocal Interferometry (CI) and surface wettability, by goniometry. For bacterial adhesion analysis, samples were exposed for 72 hours to a Streptococcus mutans solution for biofilm formation. The values obtained for surface roughness were analyzed using the Mann-Whitney test while biofilm adhesion were assessed by Kruskal-Wallis and SNK test. Significant statistical differences (p< 0.05) for surface roughness and bacterial adhesion reduction were observed on conventional brackets after surface treatment and between conventional and self-ligating brackets; no significant statistical differences were observed between self-ligating groups (p> 0.05). Plasma-polymerized film deposition was only effective on reducing surface roughness and bacterial adhesion in conventional brackets. It was also noted that conventional brackets showed lower biofilm adhesion than self-ligating brackets despite the absence of film.

A multiplex microplatform for the detection of multiple DNA methylation events using gold-DNA affinity.

PubMed

Sina, Abu Ali Ibn; Foster, Matthew Thomas; Korbie, Darren; Carrascosa, Laura G; Shiddiky, Muhammad J A; Gao, Jing; Dey, Shuvashis; Trau, Matt

2017-10-07

We report a new multiplexed strategy for the electrochemical detection of regional DNA methylation across multiple regions. Using the sequence dependent affinity of bisulfite treated DNA towards gold surfaces, the method integrates the high sensitivity of a micro-fabricated multiplex device comprising a microarray of gold electrodes, with the powerful multiplexing capability of multiplex-PCR. The synergy of this combination enables the monitoring of the methylation changes across several genomic regions simultaneously from as low as 500 pg μl -1 of DNA with no sequencing requirement.

Vibrio cholerae ACE stimulates Ca(2+)-dependent Cl(-)/HCO(3)(-) secretion in T84 cells in vitro.

PubMed

Trucksis, M; Conn, T L; Wasserman, S S; Sears, C L

2000-09-01

ACE, accessory cholera enterotoxin, the third enterotoxin in Vibrio cholerae, has been reported to increase short-circuit current (I(sc)) in rabbit ileum and to cause fluid secretion in ligated rabbit ileal loops. We studied the ACE-induced change in I(sc) and potential difference (PD) in T84 monolayers mounted in modified Ussing chambers, an in vitro model of a Cl(-) secretory cell. ACE added to the apical surface alone stimulated a rapid increase in I(sc) and PD that was concentration dependent and immediately reversed when the toxin was removed. Ion replacement studies established that the current was dependent on Cl(-) and HCO(3)(-). ACE acted synergistically with the Ca(2+)-dependent acetylcholine analog, carbachol, to stimulate secretion in T84 monolayers. In contrast, the secretory response to cAMP or cGMP agonists was not enhanced by ACE. The ACE-stimulated secretion was dependent on extracellular and intracellular Ca(2+) but was not associated with an increase in intracellular cyclic nucleotides. We conclude that the mechanism of secretion by ACE involves Ca(2+) as a second messenger and that this toxin stimulates a novel Ca(2+)-dependent synergy.

Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.

PubMed

Kushnir, Susanna; Marsac, Yoann; Breitling, Reinhard; Granovsky, Igor; Brok-Volchanskaya, Vera; Goody, Roger S; Becker, Christian F W; Alexandrov, Kirill

2006-01-01

Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

Ligand induced structural isomerism in phosphine coordinated gold clusters revealed by ion mobility mass spectrometry

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

Ligare, Marshall R.; Baker, Erin S.; Laskin, Julia

Structural isomerism in ligated gold clusters is revealed using electrospray ionization ion mobility spectrometry mass spectrometry. Phosphine ligated Au8 clusters are shown to adopt more “extended” type structures with increasing exchange of methyldiphenylphosphine (MePPh2) for triphenylphosphine (PPh3). These ligand-dependant structure-property relationships are critical to applications of clusters in catalysis.

Screening DNA chip and event-specific multiplex PCR detection methods for biotech crops.

PubMed

Lee, Seong-Hun

2014-11-01

There are about 80 biotech crop events that have been approved by safety assessment in Korea. They have been controlled by genetically modified organism (GMO) and living modified organism (LMO) labeling systems. The DNA-based detection method has been used as an efficient scientific management tool. Recently, the multiplex polymerase chain reaction (PCR) and DNA chip have been developed as simultaneous detection methods for several biotech crops' events. The event-specific multiplex PCR method was developed to detect five biotech maize events: MIR604, Event 3272, LY 038, MON 88017 and DAS-59122-7. The specificity was confirmed and the sensitivity was 0.5%. The screening DNA chip was developed from four endogenous genes of soybean, maize, cotton and canola respectively along with two regulatory elements and seven genes: P35S, tNOS, pat, bar, epsps1, epsps2, pmi, cry1Ac and cry3B. The specificity was confirmed and the sensitivity was 0.5% for four crops' 12 events: one soybean, six maize, three cotton and two canola events. The multiplex PCR and DNA chip can be available for screening, gene-specific and event-specific analysis of biotech crops as efficient detection methods by saving on workload and time. © 2014 Society of Chemical Industry. © 2014 Society of Chemical Industry.

Biochemical and morphological changes in the liver after hepatic artery ligation in the presence or absence of extrahepatic cholestasis.

PubMed Central

Soares, A. F.; Castro e Silva Júnior, O.; Ceneviva, R.; Roselino, J. E.; Zucoloto, S.

1993-01-01

The present study was carried out to investigate the biochemical and morphological changes in the liver after ligation of the hepatic artery (HA) in the presence and in the absence of extrahepatic cholestasis (EHC). The study was conducted on 100 rats divided into four groups of 25 animals each: group 1, sham operation; group 2, hepatic artery ligation (HAL); group 3, bile duct ligation (BDL); and group 4, HAL plus BDL. All animals were sacrificed 7 days after surgery when total bilirubin and fractions, alkaline phosphatase (AP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in serum and on the inner hepatocyte mitochondrial membrane (IHMM); the incidence of necrosis and the volume fractions of vessels, bile ducts and hepatocytes in the liver were also determined. HAL reduces the relative volumes of bile ducts, with no changes in levels of bilirubin and fractions, AP, ALT, AST and IHMM, but HAL associated with EHC reduces duct proliferation and the liver becomes more vulnerable to necrosis. In conclusion, the normal liver depends on HA flow and this dependence is more evident in the presence of EHC. PMID:8398809

Genome-wide profiling of DNA-binding proteins using barcode-based multiplex Solexa sequencing.

PubMed

Raghav, Sunil Kumar; Deplancke, Bart

2012-01-01

Chromatin immunoprecipitation (ChIP) is a commonly used technique to detect the in vivo binding of proteins to DNA. ChIP is now routinely paired to microarray analysis (ChIP-chip) or next-generation sequencing (ChIP-Seq) to profile the DNA occupancy of proteins of interest on a genome-wide level. Because ChIP-chip introduces several biases, most notably due to the use of a fixed number of probes, ChIP-Seq has quickly become the method of choice as, depending on the sequencing depth, it is more sensitive, quantitative, and provides a greater binding site location resolution. With the ever increasing number of reads that can be generated per sequencing run, it has now become possible to analyze several samples simultaneously while maintaining sufficient sequence coverage, thus significantly reducing the cost per ChIP-Seq experiment. In this chapter, we provide a step-by-step guide on how to perform multiplexed ChIP-Seq analyses. As a proof-of-concept, we focus on the genome-wide profiling of RNA Polymerase II as measuring its DNA occupancy at different stages of any biological process can provide insights into the gene regulatory mechanisms involved. However, the protocol can also be used to perform multiplexed ChIP-Seq analyses of other DNA-binding proteins such as chromatin modifiers and transcription factors.

A high-throughput multiplex method adapted for GMO detection.

PubMed

Chaouachi, Maher; Chupeau, Gaëlle; Berard, Aurélie; McKhann, Heather; Romaniuk, Marcel; Giancola, Sandra; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

2008-12-24

A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

Evaluation of high-resolution melting curve analysis of ligation-mediated real-time PCR, a rapid method for epidemiological typing of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter Species) pathogens.

PubMed

Woksepp, Hanna; Ryberg, Anna; Billström, Hanna; Hällgren, Anita; Nilsson, Lennart E; Marklund, Britt-Inger; Olsson-Liljequist, Barbro; Schön, Thomas

2014-12-01

A single-tube method, ligation-mediated real-time PCR high-resolution melt analysis (LMqPCR HRMA), was modified for the rapid typing of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens. A 97% agreement (60/62 isolates) was achieved in comparison to pulsed-field gel electrophoresis (PFGE) results, which indicates that LMqPCR HRMA is a rapid and accurate screening tool for monitoring nosocomial outbreaks. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Template-Directed Ligation of Peptides to Oligonucleotides

NASA Technical Reports Server (NTRS)

Bruick, Richard K.; Dawson, Philip E.; Kent, Stephen BH; Usman, Nassim; Joyce, Gerald F.

1996-01-01

Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template.

Modification of two capripoxvirus quantitative real-time PCR assays to improve diagnostic sensitivity and include beta-actin as an internal positive control.

PubMed

Das, Amaresh; Deng, Ming Y; Babiuk, Shawn; McIntosh, Michael T

2017-05-01

Capripoxviruses (CaPVs), consisting of Sheeppox virus (SPV), Goatpox virus (GPV), and Lumpy skin disease virus (LSDV) species, cause economically significant diseases in sheep, goats, and cattle, respectively. Quantitative real-time polymerase chain reaction (qPCR) assays are routinely used for rapid detection of CaPVs in surveillance and outbreak management programs. We further modified and optimized 2 previously published CaPV qPCR assays, referred to as the Balinsky and Bowden assays, by changing commercial PCR reagents used in the tests. The modified assays displayed 100% analytical specificity and showed no apparent changes in analytical sensitivities for detection of CaPVs compared with the original assays. Diagnostic sensitivities, assessed using 50 clinical reference samples from experimentally infected sheep, goats, and cattle, improved from 82% to 92% for the modified Balinsky assay and from 58% to 82% for the modified Bowden assay. The modified qPCR assays were multiplexed for detection of beta-actin as an indicator for potential false-negative results. The multiplex modified qPCR assays exhibited the same diagnostic sensitivities as the singleplex assays suggesting their utility in the detection of CaPVs.

Protein tetrazinylation via diazonium coupling for covalent and catalyst-free bioconjugation.

PubMed

Zhang, Jie; Men, Yuwen; Lv, Shanshan; Yi, Long; Chen, Jian-Feng

2015-12-21

An efficient and bench-stable reagent was synthesized for direct and covalent introduction of tetrazines onto target protein or virus surfaces, which can be further modified based on tetrazine-ene ligation to achieve fluorescence labelling or PEGylation under mild conditions.

Modified multiplex PCR for identification of Bacillus Calmette-Guérin substrain Tokyo among clinical isolates.

PubMed

Seki, Masaaki; Sato, Akimasa; Honda, Ikuro; Yamazaki, Toshio; Yano, Ikuya; Koyama, Akira; Toida, Ichiro

2005-05-02

When an adverse reaction occurs and a mycobacterial species is isolated from a person vaccinated with Bacillus Calmette-Guérin (BCG) or a patient receiving BCG immunotherapy, it is essential to identify whether the isolate is BCG or another mycobacterial species. However, differentiation of BCG from other members of Mycobacterium tuberculosis complex has been very difficult. Using several specific primer-pairs, Bedwell et al. [Bedwell J, Kairo SK, Behr MA, Bygraves JA. Identification of substrains of BCG vaccine using multiplex PCR. Vaccine 2001; 19: 2146-51] recently reported that they could distinguish BCG substrains. We modified their method to improve differentiation of Tokyo 172 from other members of the M. tuberculosis complex, and examined whether this modified method could be applied to clinical isolates. Our method clearly identified BCG substrain (BCG Tokyo 172) among clinical isolates and easily distinguished between M. tuberculosis and wild-type Mycobacterium bovis.

Shape-coded silica nanotubes for multiplexed bioassay: rapid and reliable magnetic decoding protocols

PubMed Central

He, Bo; Kim, Sung Kyoung; Son, Sang Jun; Lee, Sang Bok

2010-01-01

Aims The recent development of 1D barcode arrays has proved their capabilities to be applicable to highly multiplexed bioassays. This article introduces two magnetic decoding protocols for suspension arrays of shape-coded silica nanotubes to process multiplexed assays rapidly and easily, which will benefit the minimization and automation of the arrays. Methods In the first protocol, the magnetic nanocrystals are incorporated into the inner voids of barcoded silica nanotubes in order to give the nanotubes magnetic properties. The second protocol is performed by trapping the barcoded silica nanotubes onto streptavidin-modified magnetic beads. Results The rapid and easy decoding process was demonstrated by applying the above two protocols to multiplexed assays, resulting in high selectivity. Furthermore, the magnetic bead-trapped barcode nanotubes provided a great opportunity to exclude the use of dye molecules in multiplexed assays by using barcode nanotubes as signals. Conclusion The rapid and easy manipulation of encoded carriers using magnetic properties could be used to develop promising suspension arrays for portable bioassays. PMID:20025466

[Numeric alterations in the dys gene and their association with clinical features].

PubMed

Mampel, Alejandra; Echeverría, María Inés; Vargas, Ana Lía; Roque, María

2011-01-01

The Duchenne/Becker muscular dystrophy is a hereditary miopathy with a recessive sex-linked pattern. The related gene is called DYS and the coded protein plays a crucial role in the anchorage between the cytoskeleton and the cellular membrane in muscle cells. Different clinical manifestations are observed depending on the impact of the genetic alteration on the protein. The global register of mutations reveals an enhanced frequency for deletions/duplications of one or more exons affecting the DYS gene. In the present work, numeric alterations have been studied in the 79 exons of the DYS gene. The study has been performed on 59 individuals, including 31 independent cases and 28 cases with a familial link. The applied methodology was Multiplex Ligation Dependent Probe Amplification (MLPA). In the 31 independent cases clinical data were established: i.e. the clinical score, the Raven test percentiles, and the creatininphosphokinase (CPK) blood values. Our results reveal a 61.3% frequency of numeric alterations affecting the DYS gene in our population, provoking all of them a reading frame shift. The rate for de novo mutations was identified as 35.2%. Alterations involving a specific region of one exon were observed with high frequency, affecting a specific region. A significant association was found between numeric alterations and a low percentile for the Raven test. These data contribute to the local knowledge of genetic alterations and their phenotypic impact for the Duchenne/Becker disease.

CDKL5 gene status in female patients with epilepsy and Rett-like features: two new mutations in the catalytic domain

PubMed Central

2012-01-01

Background Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) located in the Xp22 region have been shown to cause a subset of atypical Rett syndrome with infantile spasms or early seizures starting in the first postnatal months. Methods We performed mutation screening of CDKL5 in 60 female patients who had been identified as negative for the methyl CpG-binding protein 2 gene (MECP2) mutations, but who had current or past epilepsy, regardless of the age of onset, type, and severity. All the exons in the CDKL5 gene and their neighbouring sequences were examined, and CDKL5 rearrangements were studied by multiplex ligation-dependent probe amplification (MLPA). Results Six previously unidentified DNA changes were detected, two of which were disease-causing mutations in the catalytic domain: a frameshift mutation (c.509_510insGT; p.Glu170GlyfsX36) and a complete deletion of exon 10. Both were found in patients with seizures that started in the first month of life. Conclusions This study demonstrated the importance of CDKL5 mutations as etiological factors in neurodevelopmental disorders, and indicated that a thorough analysis of the CDKL5 gene sequence and its rearrangements should be considered in females with Rett syndrome-like phenotypes, severe encephalopathy and epilepsy with onset before 5 months of age. This study also confirmed the usefulness of MLPA as a diagnostic screening method for use in clinical practice. PMID:22867051

Xp22.3 genomic deletions involving the CDKL5 gene in girls with early onset epileptic encephalopathy.

PubMed

Mei, Davide; Marini, Carla; Novara, Francesca; Bernardina, Bernardo D; Granata, Tiziana; Fontana, Elena; Parrini, Elena; Ferrari, Anna R; Murgia, Alessandra; Zuffardi, Orsetta; Guerrini, Renzo

2010-04-01

Mutations of the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause an X-linked encephalopathy with early onset intractable epilepsy, including infantile spasms and other seizure types, and a Rett syndrome (RTT)-like phenotype. Very limited information is available on the frequency and phenotypic spectrum associated with CDKL5 deletions/duplications. We investigated the role of CDKL5 deletions/duplications in causing early onset intractable epilepsy of unknown etiology in girls. We studied 49 girls with early onset intractable epilepsy, with or without infantile spasms, and developmental impairment, for whom no etiologic factors were obvious after clinical examination, brain magnetic resonance imaging (MRI) and expanded screening for inborn errors of metabolism. We performed CDKL5 gene mutation analysis in all and multiplex ligation dependent probe amplification assay (MLPA) in those who were mutation negative. Custom Array-comparative genomic hybridization (CGH), breakpoint polymerase chain reaction (PCR) analysis, and X-inactivation studies were performed in patients in whom MLPA uncovered a genomic alteration. We found CDKL5 mutations in 8.2% (4 of 49) of patients and genomic deletions in 8.2% (4 of 49). Overall, abnormalities of the CDKL5 gene accounted for 16.3% (8 of 49) of patients. CDKL5 gene deletions are an under-ascertained cause of early onset intractable epilepsy in girls. Genetic testing of CDKL5, including both mutation and deletion/duplication analysis, should be considered in this clinical subgroup.

CDKL5 gene status in female patients with epilepsy and Rett-like features: two new mutations in the catalytic domain.

PubMed

Maortua, Hiart; Martínez-Bouzas, Cristina; Calvo, María-Teresa; Domingo, Maria-Rosario; Ramos, Feliciano; García-Ribes, Ainhoa; Martínez, María-Jesús; López-Aríztegui, María-Asunción; Puente, Nerea; Rubio, Izaskun; Tejada, María-Isabel

2012-08-06

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) located in the Xp22 region have been shown to cause a subset of atypical Rett syndrome with infantile spasms or early seizures starting in the first postnatal months. We performed mutation screening of CDKL5 in 60 female patients who had been identified as negative for the methyl CpG-binding protein 2 gene (MECP2) mutations, but who had current or past epilepsy, regardless of the age of onset, type, and severity. All the exons in the CDKL5 gene and their neighbouring sequences were examined, and CDKL5 rearrangements were studied by multiplex ligation-dependent probe amplification (MLPA). Six previously unidentified DNA changes were detected, two of which were disease-causing mutations in the catalytic domain: a frameshift mutation (c.509_510insGT; p.Glu170GlyfsX36) and a complete deletion of exon 10. Both were found in patients with seizures that started in the first month of life. This study demonstrated the importance of CDKL5 mutations as etiological factors in neurodevelopmental disorders, and indicated that a thorough analysis of the CDKL5 gene sequence and its rearrangements should be considered in females with Rett syndrome-like phenotypes, severe encephalopathy and epilepsy with onset before 5 months of age. This study also confirmed the usefulness of MLPA as a diagnostic screening method for use in clinical practice.

Novel CDKL5 Mutations in Czech Patients with Phenotypes of Atypical Rett Syndrome and Early-Onset Epileptic Encephalopathy.

PubMed

Záhoráková, D; Langová, M; Brožová, K; Laštůvková, J; Kalina, Z; Rennerová, L; Martásek, P

2016-01-01

The X-linked CDKL5 gene, which encodes cyclin-dependent kinase-like 5 protein, has been implicated in early-onset encephalopathy and atypical Rett syndrome with early-onset seizures. The CDKL5 protein is a kinase required for neuronal development and morphogenesis, but its precise functions are still largely unexplored. Individuals with CDKL5 mutations present with severe global developmental delay, intractable epilepsy, and Rett-like features. A clear genotype-phenotype correlation has not been established due to an insufficient number of reported cases. The aim of this study was to analyse the CDKL5 gene in Czech patients with early-onset seizures and Rett-like features. We performed mutation screening in a cohort of 83 individuals using high-resolution melting analysis, DNA sequencing and multiplex ligation- dependent probe amplification. Molecular analyses revealed heterozygous pathogenic mutations in three girls with severe intellectual disability and intractable epilepsy starting at the age of two months. All three identified mutations, c.637G>A, c.902_977+29del105, and c.1757_1758delCT, are novel, thus significantly extending the growing spectrum of known pathogenic CDKL5 sequence variants. Our results support the importance of genetic testing of the CDKL5 gene in patients with early-onset epileptic encephalopathy and Rett-like features with early-onset seizures. This is the first study referring to molecular defects of CDKL5 in Czech cases.

A ligation DNAzyme-induced magnetic nanoparticles assembly for ultrasensitive detection of copper ions.

PubMed

Yin, Honghong; Kuang, Hua; Liu, Liqiang; Xu, Liguang; Ma, Wei; Wang, Libing; Xu, Chuanlai

2014-04-09

A novel biosensor for ultrasensitive detection of copper (Cu(2+)) was established based on the assembly of magnetic nanoparticles induced by the Cu(2+)-dependent ligation DNAzyme. With a low limit of detection of 2.8 nM and high specificity, this method has the potential to serve as a general platform for the detection of heavy metal ions.

Detection and Serotyping of Dengue Virus in Serum Samples by Multiplex Reverse Transcriptase PCR-Ligase Detection Reaction Assay▿ †

PubMed Central

Das, S.; Pingle, M. R.; Muñoz-Jordán, J.; Rundell, M. S.; Rondini, S.; Granger, K.; Chang, G.-J. J.; Kelly, E.; Spier, E. G.; Larone, D.; Spitzer, E.; Barany, F.; Golightly, L. M.

2008-01-01

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV. PMID:18685000

The macrophage cytoskeleton acts as a contact sensor upon interaction with Entamoeba histolytica to trigger IL-1β secretion

PubMed Central

Moreau, France; Gorman, Hayley

2017-01-01

Entamoeba histolytica (Eh) is the causative agent of amebiasis, one of the major causes of dysentery-related morbidity worldwide. Recent studies have underlined the importance of the intercellular junction between Eh and host cells as a determinant in the pathogenesis of amebiasis. Despite the fact that direct contact and ligation between Eh surface Gal-lectin and EhCP-A5 with macrophage α5β1 integrin are absolute requirements for NLRP3 inflammasome activation and IL-1β release, many other undefined molecular events and downstream signaling occur at the interface of Eh and macrophage. In this study, we investigated the molecular events at the intercellular junction that lead to recognition of Eh through modulation of the macrophage cytoskeleton. Upon Eh contact with macrophages key cytoskeletal-associated proteins were rapidly post-translationally modified only with live Eh but not with soluble Eh proteins or fragments. Eh ligation with macrophages rapidly activated caspase-6 dependent cleavage of the cytoskeletal proteins talin, Pyk2 and paxillin and caused robust release of the pro-inflammatory cytokine, IL-1β. Macrophage cytoskeletal cleavages were dependent on Eh cysteine proteinases EhCP-A1 and EhCP-A4 but not EhCP-A5 based on pharmacological blockade of Eh enzyme inhibitors and EhCP-A5 deficient parasites. These results unravel a model where the intercellular junction between macrophages and Eh form an area of highly interacting proteins that implicate the macrophage cytoskeleton as a sensor for Eh contact that leads downstream to subsequent inflammatory immune responses. PMID:28837696

Design, fabrication and characterization of a 64 pixel metallic magnetic calorimeter array with integrated, on-chip microwave SQUID multiplexer

NASA Astrophysics Data System (ADS)

Kempf, S.; Wegner, M.; Deeg, L.; Fleischmann, A.; Gastaldo, L.; Herrmann, F.; Richter, D.; Enss, C.

2017-06-01

We report on the design, fabrication and characterization of a 64 pixel metallic magnetic calorimeter array that is read out by an integrated, on-chip microwave SQUID multiplexer. Based on the results of our comprehensive device characterization we refined the state-of-the-art multiplexer model which assumes each associated non-hysteretic rf-SQUID to purely behave as a flux-dependent inductor. In particular, we include the capacitance and the subgap resistance of the Josephson junction as well as screening effects and parasitic mutual couplings between different coils that show up only when a superconducting flux transformer is attached to the SQUID input. Thanks to these modifications, we are able to explain the occurrence of a magnetic flux dependence of the internal quality factor of the microwave resonators as well as to accurately calculate the characteristic multiplexer parameters. When combining the refined multiplexer model with the thermodynamical description of a metallic magnetic calorimeter, we find a reasonable agreement between our measurements and predictions.

Kinetic mechanism of human DNA ligase I reveals magnesium-dependent changes in the rate-limiting step that compromise ligation efficiency.

PubMed

Taylor, Mark R; Conrad, John A; Wahl, Daniel; O'Brien, Patrick J

2011-07-01

DNA ligase I (LIG1) catalyzes the ligation of single-strand breaks to complete DNA replication and repair. The energy of ATP is used to form a new phosphodiester bond in DNA via a reaction mechanism that involves three distinct chemical steps: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. We used steady state and pre-steady state kinetics to characterize the minimal mechanism for DNA ligation catalyzed by human LIG1. The ATP dependence of the reaction indicates that LIG1 requires multiple Mg(2+) ions for catalysis and that an essential Mg(2+) ion binds more tightly to ATP than to the enzyme. Further dissection of the magnesium ion dependence of individual reaction steps revealed that the affinity for Mg(2+) changes along the reaction coordinate. At saturating concentrations of ATP and Mg(2+) ions, the three chemical steps occur at similar rates, and the efficiency of ligation is high. However, under conditions of limiting Mg(2+), the nick-sealing step becomes rate-limiting, and the adenylylated DNA intermediate is prematurely released into solution. Subsequent adenylylation of enzyme prevents rebinding to the adenylylated DNA intermediate comprising an Achilles' heel of LIG1. These ligase-generated 5'-adenylylated nicks constitute persistent breaks that are a threat to genomic stability if they are not repaired. The kinetic and thermodynamic framework that we have determined for LIG1 provides a starting point for understanding the mechanism and specificity of mammalian DNA ligases.

Click nucleic acid ligation: applications in biology and nanotechnology.

PubMed

El-Sagheer, Afaf H; Brown, Tom

2012-08-21

Biochemical strategies that use a combination of synthetic oligonucleotides, thermostable DNA polymerases, and DNA ligases can produce large DNA constructs up to 1 megabase in length. Although these ambitious targets are feasible biochemically, comparable technologies for the chemical synthesis of long DNA strands lag far behind. The best available chemical approach is the solid-phase phosphoramidite method, which can be used to assemble DNA strands up to 150 bases in length. Beyond this point, deficiencies in the chemistry make it impossible to produce pure DNA. A possible alternative approach to the chemical synthesis of large DNA strands is to join together carefully purified synthetic oligonucleotides by chemical methods. Click ligation by the copper-catalyzed azide-alkyne (CuAAC) reaction could facilitate this process. In this Account, we describe the synthesis, characterization, and applications of oligonucleotides prepared by click ligation. The alkyne and azide oligonucleotide strands can be prepared by standard protocols, and the ligation reaction is compatible with a wide range of chemical modifications to DNA and RNA. We have employed click ligation to synthesize DNA constructs up to 300 bases in length and much longer sequences are feasible. When the resulting triazole linkage is placed in a PCR template, various DNA polymerases correctly copy the entire base sequence. We have also successfully demonstrated both in vitro transcription and rolling circle amplification through the modified linkage. This linkage has shown in vivo biocompatibility: an antibiotic resistance gene containing triazole linkages functions in E. coli . Using click ligation, we have synthesized hairpin ribozymes up to 100 nucleotides in length and a hammerhead ribozyme with the triazole linkage located at the substrate cleavage site. At the opposite end of the length scale, click-ligated, cyclic mini-DNA duplexes have been used as models to study base pairing. Cyclic duplexes have potential therapeutic applications. They have extremely high thermodynamic stability, have increased resistance to enzymatic degradation, and have been investigated as decoys for regulatory proteins. For potential nanotechnology applications, we have synthesized double stranded DNA catenanes by click ligation. Other researchers have studied covalently fixed multistranded DNA constructs including triplexes and quadruplexes.

Bacterial adhesion on conventional and self-ligating metallic brackets after surface treatment with plasma-polymerized hexamethyldisiloxane

PubMed Central

Tupinambá, Rogerio Amaral; Claro, Cristiane Aparecida de Assis; Pereira, Cristiane Aparecida; Nobrega, Celestino José Prudente; Claro, Ana Paula Rosifini Alves

2017-01-01

ABSTRACT Introduction: Plasma-polymerized film deposition was created to modify metallic orthodontic brackets surface properties in order to inhibit bacterial adhesion. Methods: Hexamethyldisiloxane (HMDSO) polymer films were deposited on conventional (n = 10) and self-ligating (n = 10) stainless steel orthodontic brackets using the Plasma-Enhanced Chemical Vapor Deposition (PECVD) radio frequency technique. The samples were divided into two groups according to the kind of bracket and two subgroups after surface treatment. Scanning Electron Microscopy (SEM) analysis was performed to assess the presence of bacterial adhesion over samples surfaces (slot and wings region) and film layer integrity. Surface roughness was assessed by Confocal Interferometry (CI) and surface wettability, by goniometry. For bacterial adhesion analysis, samples were exposed for 72 hours to a Streptococcus mutans solution for biofilm formation. The values obtained for surface roughness were analyzed using the Mann-Whitney test while biofilm adhesion were assessed by Kruskal-Wallis and SNK test. Results: Significant statistical differences (p< 0.05) for surface roughness and bacterial adhesion reduction were observed on conventional brackets after surface treatment and between conventional and self-ligating brackets; no significant statistical differences were observed between self-ligating groups (p> 0.05). Conclusion: Plasma-polymerized film deposition was only effective on reducing surface roughness and bacterial adhesion in conventional brackets. It was also noted that conventional brackets showed lower biofilm adhesion than self-ligating brackets despite the absence of film. PMID:28902253

Ultrasensitive Single Fluorescence-Labeled Probe-Mediated Single Universal Primer-Multiplex-Droplet Digital Polymerase Chain Reaction for High-Throughput Genetically Modified Organism Screening.

PubMed

Niu, Chenqi; Xu, Yuancong; Zhang, Chao; Zhu, Pengyu; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

2018-05-01

As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences ( 35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.

RNA-Catalyzed RNA Ligation on an External RNA Template

NASA Technical Reports Server (NTRS)

McGinness, Kathleen E.; Joyce, Gerald F.

2002-01-01

Variants of the hc ligase ribozyme, which catalyzes ligation of the 3' end of an RNA substrate to the 5' end of the ribozyme, were utilized to evolve a ribozyme that catalyzes ligation reactions on an external RNA template. The evolved ribozyme catalyzes the joining of an oligonucleotide 3'-hydroxyl to the 5'-triphosphate of an RNA hairpin molecule. The ribozyme can also utilize various substrate sequences, demonstrating a largely sequence-independent mechanism for substrate recognition. The ribozyme also carries out the ligation of two oligonucleotides that are bound at adjacent positions on a complementary template. Finally, it catalyzes addition of mononucleoside '5-triphosphates onto the '3 end of an oligonucleotide primer in a template-dependent manner. The development of ribozymes that catalyze polymerase-type reactions contributes to the notion that an RNA world could have existed during the early history of life on Earth.

Alu elements mediate large SPG11 gene rearrangements: further spatacsin mutations.

PubMed

Conceição Pereira, Maria; Loureiro, José Leal; Pinto-Basto, Jorge; Brandão, Eva; Margarida Lopes, Ana; Neves, Georgina; Dias, Pureza; Geraldes, Ruth; Martins, Isabel Pavão; Cruz, Vitor Tedim; Kamsteeg, Erik-Jan; Brunner, Han G; Coutinho, Paula; Sequeiros, Jorge; Alonso, Isabel

2012-01-01

Hereditary spastic paraplegias compose a group of neurodegenerative disorders with a large clinical and genetic heterogeneity. Among the autosomal recessive forms, spastic paraplegia type 11 is the most common. To better understand the spastic paraplegia type 11 mutation spectrum, we studied a group of 54 patients with hereditary spastic paraplegia. Mutation screening was performed by PCR amplification of SPG11 coding regions and intron boundaries, followed by sequencing. For the detection of large gene rearrangements, we performed multiplex ligation-dependent probe amplification. We report 13 families with spastic paraplegia type 11 carrying either novel or previously identified mutations. We describe a complex entire SPG11 rearrangement and show that large gene rearrangements are frequent among patients with spastic paraplegia type 11. Moreover, we mapped the deletion breakpoints of three different large SPG11 deletions and provide evidence for Alu microhomology-mediated exon deletion. Our analysis shows that the high number of repeated elements in SPG11 together with the presence of recombination hotspots and the high intrinsic instability of the 15q locus all contribute toward making this genomic region more prone to large gene rearrangements. These findings enlarge the amount of data relating repeated elements with neurodegenerative disorders and highlight their importance in human disease and genome evolution.

Constitutional MLH1 methylation presenting with colonic polyposis syndrome and not Lynch syndrome.

PubMed

Kidambi, Trilokesh D; Blanco, Amie; Van Ziffle, Jessica; Terdiman, Jonathan P

2016-04-01

At least one-third of patients meeting clinical criteria for Lynch syndrome will have no germline mutation and constitutional epimutations leading to promoter methylation of MLH1 have been identified in a subset of these patients. We report the first case of constitutional MLH1 promoter methylation associated with a colonic polyposis syndrome in a 39 year-old man with a family history of colorectal cancer (CRC) and a personal history of 21 polyps identified over 8 years as well as the development of two synchronous CRCs over 16 months who was evaluated for a hereditary cancer syndrome. Immunohistochemistry (IHC) of multiple tumors showed absent MLH1 and PMS2 expression, though germline testing with Sanger sequencing and multiplex ligation-dependent probe amplification of these mismatch repair genes (MMR) genes was negative. A next generation sequencing panel of 29 genes also failed to identify a pathogenic mutation. Hypermethylation was identified in MLH1 intron 1 in tumor specimens along with buccal cells and peripheral white blood cells, confirming the diagnosis of constitutional MLH1 promoter methylation. This case highlights that constitutional MLH1 methylation should be considered in the differential diagnosis for a polyposis syndrome if IHC staining shows absent MMR gene expression.

Intronic splicing mutations in PTCH1 cause Gorlin syndrome.

PubMed

Bholah, Zaynab; Smith, Miriam J; Byers, Helen J; Miles, Emma K; Evans, D Gareth; Newman, William G

2014-09-01

Gorlin syndrome is an autosomal dominant disorder characterized by multiple early-onset basal cell carcinoma, odontogenic keratocysts and skeletal abnormalities. It is caused by heterozygous mutations in the tumour suppressor PTCH1. Routine clinical genetic testing, by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to confirm a clinical diagnosis of Gorlin syndrome, identifies a mutation in 60-90 % of cases. We undertook RNA analysis on lymphocytes from ten individuals diagnosed with Gorlin syndrome, but without known PTCH1 mutations by exonic sequencing or MLPA. Two altered PTCH1 transcripts were identified. Genomic DNA sequence analysis identified an intron 7 mutation c.1068-10T>A, which created a strong cryptic splice acceptor site, leading to an intronic insertion of eight bases; this is predicted to create a frameshift p.(His358Alafs*12). Secondly, a deep intronic mutation c.2561-2057A>G caused an inframe insertion of 78 intronic bases in the cDNA transcript, leading to a premature stop codon p.(Gly854fs*3). The mutations are predicted to cause loss of function of PTCH1, consistent with its tumour suppressor function. The findings indicate the importance of RNA analysis to detect intronic mutations in PTCH1 not identified by routine screening techniques.

Use of next-generation sequencing to detect LDLR gene copy number variation in familial hypercholesterolemia[S

PubMed Central

Iacocca, Michael A.; Wang, Jian; Dron, Jacqueline S.; Robinson, John F.; McIntyre, Adam D.; Cao, Henian

2017-01-01

Familial hypercholesterolemia (FH) is a heritable condition of severely elevated LDL cholesterol, caused predominantly by autosomal codominant mutations in the LDL receptor gene (LDLR). In providing a molecular diagnosis for FH, the current procedure often includes targeted next-generation sequencing (NGS) panels for the detection of small-scale DNA variants, followed by multiplex ligation-dependent probe amplification (MLPA) in LDLR for the detection of whole-exon copy number variants (CNVs). The latter is essential because ∼10% of FH cases are attributed to CNVs in LDLR; accounting for them decreases false negative findings. Here, we determined the potential of replacing MLPA with bioinformatic analysis applied to NGS data, which uses depth-of-coverage analysis as its principal method to identify whole-exon CNV events. In analysis of 388 FH patient samples, there was 100% concordance in LDLR CNV detection between these two methods: 38 reported CNVs identified by MLPA were also successfully detected by our NGS method, while 350 samples negative for CNVs by MLPA were also negative by NGS. This result suggests that MLPA can be removed from the routine diagnostic screening for FH, significantly reducing associated costs, resources, and analysis time, while promoting more widespread assessment of this important class of mutations across diagnostic laboratories. PMID:28874442

Novel Variations of FANCA Gene Provokes Fanconi Anemia: Molecular Diagnosis in a Special Chinese Family.

PubMed

Li, Niu; Song, Aiyun; Ding, Lixia; Zhu, Hua; Li, Guoqiang; Miao, Yan; Wang, Jian; Li, Benshang; Chen, Jing

2018-07-01

Fanconi anemia (FA) is a rare autosomal recessive or X-linked disorder with highly variable clinical manifestations and an incidence of ∼1 to 5 in 1 million births. To date, 15 bona fide FA genes have been reported to be responsible for the known FA complementation groups and the FANCA gene accounts for almost 60%. In the present study, we report a special Chinese family, which has 2 children with classic FA characteristics. Via 2-step analysis of the whole-exome sequencing data and verification using multiplex ligation-dependent probe amplification test, one child was found to have a novel compound heterozygous mutation of a splicing variant (c.1471-1G>A) and a large intragenic deletion (exons 23-30 del) of the FANCA gene. The other child had the same splicing variant and another novel large deletion (exons 1-18 del) in the FANCA gene. Clone sequencing showed the c.1471-1G>A variant generate an altered transcript with 1 cryptic splice site in intron 15, resulting in a premature termination codon (p.Val490HisfsX6). This study not only shows the complexity of FA molecular diagnosis via comprehensively studying the FA pathogenic genes and the mutational spectrum, but also has significant reference value for the future molecular diagnosis of FA.

High frequency of exon 15 deletion in the FANCA gene in Tunisian patients affected with Fanconi anemia disease: implication for diagnosis.

PubMed

Amouri, Ahlem; Talmoudi, Faten; Messaoud, Olfa; d'Enghien, Catherine D; Rekaya, Mariem B; Allegui, Ines; Azaiez, Héla; Kefi, Rym; Abdelhak, Ahlem; Meseddi, Sondes H; Torjemane, Lamia; Ouederni, Monia; Mellouli, Fethi; Abid, Héla B; Aissaoui, Lamia; Bejaoui, Mohamed; Othmen, Tarek B; Lyonnet, Dominique S; Soulier, Jean; Hachicha, Mongia; Dellagi, Koussay; Abdelhak, Sonia; Fanconi, Tunisian

2014-03-01

Tunisian population is characterized by its heterogeneous ethnic background and high rate of consanguinity. In consequence, there is an increase in the frequency of recessive genetic disorders including Fanconi anemia (FA). The aim of this study was to confirm the existence of a founder haplotype among FA Tunisian patients and to identify the associated mutation in order to develop a simple tool for FA diagnosis. Seventy-four unrelated families with a total of 95 FA patients were investigated. All available family members were genotyped with four microsatellite markers flanking FANCA gene. Haplotype analysis and homozygosity mapping assigned 83 patients belonging to 62 families to the FA-A group. A common haplotype was shared by 42 patients from 26 families at a homozygous state while five patients from five families were heterozygous. Among them, 85% were from southern Tunisia suggesting a founder effect. Using multiplex ligation-dependent probe amplification (MLPA) technique, we have also demonstrated that this haplotype is associated with a total deletion of exon 15 in FANCA gene. Identification of a founder mutation allowed genetic counseling in relatives of these families, better bone marrow graft donor selection and prenatal diagnosis. This mutation should be investigated in priority for patients originating from North Africa and Middle East.

Diagnostics of SHOX gene rearrangement in 46,XX women with idiopathic short stature.

PubMed

Mitka, Magdalena; Bednarek, Michał; Kałużewski, Bogdan

2016-01-01

The SHOX gene has been mapped at the pseudoautosomal region 1 (PAR1) of chromosomes X (Xp22.33) and Y (Yp11.32). The loss of SHOX gene functionality is assumed to be responsible for the Leri-Weill syndrome formation and the disproportionate short stature (DSS). The SHOX gene rearrangements constitute the majority of cases of gene functionality loss. Therefore, a practical application of the method, which allows for the diagnostics of the gene rearrangements, becomes a primary issue. With such an assumption, the MLPA technique (multiplex ligation - dependent probe amplification) becomes the method of choice. DNA samples were evaluated in the study by means of the MLPA method. The DNA was isolated from peripheral blood of sixty-three (63) 46,XX patients with short stature. Out of the examined patients, deletions within the SHOX gene were found in five (5) patients, and duplication at the PAR1 regulatory region of the SHOX gene in one (1) case. The obtained results confirm the opinion that the MLPA method, while enabling the diagnostics of the etiopathogenetic factor of short stature, identified in approximately 9.5% of cases, is a useful tool in the diagnostics of SHOX gene deletion and duplication. (Endokrynol Pol 2016; 67 (4): 397-402).

Epileptic encephalopathy in a girl with an interstitial deletion of Xp22 comprising promoter and exon 1 of the CDKL5 gene.

PubMed

Bahi-Buisson, Nadia; Girard, Benoit; Gautier, Agnes; Nectoux, Juliette; Fichou, Yann; Saillour, Yoann; Poirier, Karine; Chelly, Jamel; Bienvenu, Thierry

2010-01-05

We report a 2-year-old girl with early onset seizures variant of Rett syndrome with a deletion at Xp22 detected by multiplex ligation-dependent probe amplification (MLPA) technique. This patient presented with tonic seizures at 7 days of life. Subsequently, she developed infantile spasms at three months and finally refractory myoclonic epilepsy. She demonstrated severe encephalopathy with hypotonia, deceleration of head growth, with eye gaze but limited eye pursuit, no language, limited hand use, and intermittent hand stereotypies. This combination of clinical features, suggestive of early onset variant of Rett syndrome led us to screen the CDKL5 gene. In a first step, screening of the whole coding sequence of the CDKL5 gene revealed no point mutations. In a second step, we searched gross rearrangements by MLPA and identified a microdeletion affecting both the promoter and exon 1 in CDKL5. Subsequent analysis on a Nimblegen HD2 microarray confirmed a deletion of approximately 300 kb at Xp22, including the BEND2, SCML2, and CDKL5 genes. In conclusion, our report suggests that searching for large rearrangements in CDKL5 should be considered in girls with early onset seizures and Rett-like features. (c) 2009 Wiley-Liss, Inc.

A strategy for molecular diagnostics of Fanconi anemia in Brazilian patients.

PubMed

Pilonetto, Daniela V; Pereira, Noemi F; Bonfim, Carmem M S; Ribeiro, Lisandro L; Bitencourt, Marco A; Kerkhoven, Lianne; Floor, Karijn; Ameziane, Najim; Joenje, Hans; Gille, Johan J P; Pasquini, Ricardo

2017-07-01

Fanconi anemia (FA) is a predominantly autosomal recessive disease with wide genetic heterogeneity resulting from mutations in several DNA repair pathway genes. To date, 21 genetic subtypes have been identified. We aimed to identify the FA genetic subtypes in the Brazilian population and to develop a strategy for molecular diagnosis applicable to routine clinical use. We screened 255 patients from Hospital de Clínicas, Universidade Federal do Paraná for 11 common FA gene mutations. Further analysis by multiplex ligation-dependent probe amplification (MLPA) for FANCA and Sanger sequencing of all coding exons of FANCA , -C , and - G was performed in cases who harbored a single gene mutation. We identified biallelic mutations in 128/255 patients (50.2%): 89, 11, and 28 carried FANCA , FANCC , and FANCG mutations, respectively. Of these, 71 harbored homozygous mutations, whereas 57 had compound heterozygous mutations. In 4/57 heterozygous patients, both mutations were identified by the initial screening, in 51/57 additional analyses was required for classification, and in 2/57 the second mutation remained unidentified. We found 52 different mutations of which 22 were novel. The proposed method allowed genetic subtyping of 126/255 (49.4%) patients at a significantly reduced time and cost, which makes molecular diagnosis of FA Brazilian patients feasible.

Short stature before puberty: which children should be screened for SHOX deficiency?

PubMed

Wolters, Barbara; Lass, Nina; Wunsch, Rainer; Böckmann, Beatrix; Austrup, Frank; Reinehr, Thomas

2013-01-01

We studied the prevalence of deficiency in the short stature homeobox containing gene (SHOX) in prepubertal short-statured children and analyzed the clinical and radiological signs. Screening for SHOX deficiency was performed in 449 prepubertal short-statured children (54% females, aged 4-10 years) by direct sequencing and multiplex ligation probe-dependent amplification. Children with SHOX deficiency were compared to 1:2 age- and gender-matched prepubertal children without SHOX deficiency with respect to left-hand radiographs and anthropometrics including different ratios to height and proposed scores. We identified 22 (4.9%) patients with SHOX deficiency (64% point mutations). Children with SHOX deficiency demonstrated a mesomelic shortening of extremities. Lower leg lengths but not forearm length was reduced in children <8 years with SHOX deficiency. 36% of all children and none of the children <8 years with SHOX deficiency demonstrated any typical radiologic sign. Increased sitting height-to-height ratio and decreased extremities-to-trunk ratio demonstrated the best positive and negative predictive values to identify SHOX deficiency. Screening for SHOX deficiency seems rational, especially in children with increased sitting height-to-height ratio or decreased extremities-to-trunk ratio. These criteria were also valid in young children. © 2013 S. Karger AG, Basel.

Diagnosis of Fanconi Anemia: Mutation Analysis by Multiplex Ligation-Dependent Probe Amplification and PCR-Based Sanger Sequencing

PubMed Central

Gille, Johan J. P.; Floor, Karijn; Kerkhoven, Lianne; Ameziane, Najim; Joenje, Hans; de Winter, Johan P.

2012-01-01

Fanconi anemia (FA) is a rare inherited disease characterized by developmental defects, short stature, bone marrow failure, and a high risk of malignancies. FA is heterogeneous: 15 genetic subtypes have been distinguished so far. A clinical diagnosis of FA needs to be confirmed by testing cells for sensitivity to cross-linking agents in a chromosomal breakage test. As a second step, DNA testing can be employed to elucidate the genetic subtype of the patient and to identify the familial mutations. This knowledge allows preimplantation genetic diagnosis (PGD) and enables prenatal DNA testing in future pregnancies. Although simultaneous testing of all FA genes by next generation sequencing will be possible in the near future, this technique will not be available immediately for all laboratories. In addition, in populations with strong founder mutations, a limited test using Sanger sequencing and MLPA will be a cost-effective alternative. We describe a strategy and optimized conditions for the screening of FANCA, FANCB, FANCC, FANCE, FANCF, and FANCG and present the results obtained in a cohort of 54 patients referred to our diagnostic service since 2008. In addition, the follow up with respect to genetic counseling and carrier screening in the families is discussed. PMID:22778927

High frequency of exon 15 deletion in the FANCA gene in Tunisian patients affected with Fanconi anemia disease: implication for diagnosis

PubMed Central

Amouri, Ahlem; Talmoudi, Faten; Messaoud, Olfa; d'Enghien, Catherine D; Rekaya, Mariem B; Allegui, Ines; Azaiez, Héla; Kefi, Rym; Abdelhak, Ahlem; Meseddi, Sondes H; Torjemane, Lamia; Ouederni, Monia; Mellouli, Fethi; Abid, Héla B; Aissaoui, Lamia; Bejaoui, Mohamed; Othmen, Tarek B; Lyonnet, Dominique S; Soulier, Jean; Hachicha, Mongia; Dellagi, Koussay; Abdelhak, Sonia; Fanconi, Tunisian

2014-01-01

Tunisian population is characterized by its heterogeneous ethnic background and high rate of consanguinity. In consequence, there is an increase in the frequency of recessive genetic disorders including Fanconi anemia (FA). The aim of this study was to confirm the existence of a founder haplotype among FA Tunisian patients and to identify the associated mutation in order to develop a simple tool for FA diagnosis. Seventy-four unrelated families with a total of 95 FA patients were investigated. All available family members were genotyped with four microsatellite markers flanking FANCA gene. Haplotype analysis and homozygosity mapping assigned 83 patients belonging to 62 families to the FA-A group. A common haplotype was shared by 42 patients from 26 families at a homozygous state while five patients from five families were heterozygous. Among them, 85% were from southern Tunisia suggesting a founder effect. Using multiplex ligation-dependent probe amplification (MLPA) technique, we have also demonstrated that this haplotype is associated with a total deletion of exon 15 in FANCA gene. Identification of a founder mutation allowed genetic counseling in relatives of these families, better bone marrow graft donor selection and prenatal diagnosis. This mutation should be investigated in priority for patients originating from North Africa and Middle East. PMID:24689079

Robust detection of EGFR copy number changes and EGFR variant III: technical aspects and relevance for glioma diagnostics.

PubMed

Jeuken, Judith; Sijben, Angelique; Alenda, Cristina; Rijntjes, Jos; Dekkers, Marieke; Boots-Sprenger, Sandra; McLendon, Roger; Wesseling, Pieter

2009-10-01

Epidermal growth factor receptor (EGFR) is commonly affected in cancer, generally in the form of an increase in DNA copy number and/or as mutation variants [e.g., EGFR variant III (EGFRvIII), an in-frame deletion of exons 2-7]. While detection of EGFR aberrations can be expected to be relevant for glioma patients, such analysis has not yet been implemented in a routine setting, also because feasible and robust assays were lacking. We evaluated multiplex ligation-dependent probe amplification (MLPA) for detection of EGFR amplification and EGFRvIII in DNA of a spectrum of 216 diffuse gliomas. EGFRvIII detection was verified at the protein level by immunohistochemistry and at the RNA level using the conventionally used endpoint RT-PCR as well as a newly developed quantitative RT-PCR. Compared to these techniques, the DNA-based MLPA assay for EGFR/EGFRvIII analysis tested showed 100% sensitivity and specificity. We conclude that MLPA is a robust assay for detection of EGFR/EGFRvIII aberrations. While the exact diagnostic, prognostic and predictive value of such EGFR testing remains to be seen, MLPA has great potential as it can reliably and relatively easily be performed on routinely processed (formalin-fixed, paraffin-embedded) tumor tissue in combination with testing for other relevant glioma markers.

A Deletion of More than 800 kb Is the Most Recurrent Mutation in Chilean Patients with SHOX Gene Defects.

PubMed

Poggi, Helena; Vera, Alejandra; Avalos, Carolina; Lagos, Marcela; Mellado, Cecilia; Aracena, Mariana; Aravena, Teresa; Garcia, Hernan; Godoy, Claudia; Cattani, Andreina; Reyes, Loreto; Lacourt, Patricia; Rumie, Hana; Mericq, Veronica; Arriaza, Marta; Martinez-Aguayo, Alejandro

2015-01-01

Deletions in the SHOX gene are the most frequent genetic cause of Leri-Weill syndrome and Langer mesomelic dysplasia, which are also present in idiopathic short stature. To describe the molecular and clinical findings observed in 23 of 45 non-consanguineous Chilean patients with different phenotypes related to SHOX deficiency. Multiplex ligation-dependent probe amplification was used to detect the deletions; the SHOX coding region and deletion-flanking areas were sequenced to identify point mutations and single-nucleotide polymorphisms (SNPs). The main genetic defects identified in 21 patients consisted of deletions; one of them, a large deletion of >800 kb, was found in 8 patients. Also, a smaller deletion of >350 kb was observed in 4 patients. Although we could not precisely determine the deletion breakpoint, we were able to identify a common haplotype in 7 of the 8 patients with the larger deletion based on 22 informative SNPs. These results suggest that the large deletion-bearing allele has a common ancestor and was either introduced by European immigrants or had originated in our Amerindian population. This study allowed us to identify one recurrent deletion in Chilean patients; also, it contributed to expanding our knowledge about the genetic background of our population. © 2015 S. Karger AG, Basel.

A Streamlined Protocol for Molecular Testing of the DMD Gene within a Diagnostic Laboratory: A Combination of Array Comparative Genomic Hybridization and Bidirectional Sequence Analysis

PubMed Central

Marquis-Nicholson, Renate; Lai, Daniel; Love, Jennifer M.; Love, Donald R.

2013-01-01

Purpose. The aim of this study was to develop a streamlined mutation screening protocol for the DMD gene in order to confirm a clinical diagnosis of Duchenne or Becker muscular dystrophy in affected males and to clarify the carrier status of female family members. Methods. Sequence analysis and array comparative genomic hybridization (aCGH) were used to identify mutations in the dystrophin DMD gene. We analysed genomic DNA from six individuals with a range of previously characterised mutations and from eight individuals who had not previously undergone any form of molecular analysis. Results. We successfully identified the known mutations in all six patients. A molecular diagnosis was also made in three of the four patients with a clinical diagnosis who had not undergone prior genetic screening, and testing for familial mutations was successfully completed for the remaining four patients. Conclusion. The mutation screening protocol described here meets best practice guidelines for molecular testing of the DMD gene in a diagnostic laboratory. The aCGH method is a superior alternative to more conventional assays such as multiplex ligation-dependent probe amplification (MLPA). The combination of aCGH and sequence analysis will detect mutations in 98% of patients with the Duchenne or Becker muscular dystrophy. PMID:23476807

Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy.

PubMed

Wojciechowska, Marzena; Sobczak, Krzysztof; Kozlowski, Piotr; Sedehizadeh, Saam; Wojtkowiak-Szlachcic, Agnieszka; Czubak, Karol; Markus, Robert; Lusakowska, Anna; Kaminska, Anna; Brook, J David

2018-04-12

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are human neuromuscular disorders associated with mutations of simple repetitive sequences in affected genes. The abnormal expansion of CTG repeats in the 3'-UTR of the DMPK gene elicits DM1, whereas elongated CCTG repeats in intron 1 of ZNF9/CNBP triggers DM2. Pathogenesis of both disorders is manifested by nuclear retention of expanded repeat-containing RNAs and aberrant alternative splicing. The precise determination of absolute numbers of mutant RNA molecules is important for a better understanding of disease complexity and for accurate evaluation of the efficacy of therapeutic drugs. We present two quantitative methods, Multiplex Ligation-Dependent Probe Amplification and droplet digital PCR, for studying the mutant DMPK transcript (DMPK exp RNA) and the aberrant alternative splicing in DM1 and DM2 human tissues and cells. We demonstrate that in DM1, the DMPK exp RNA is detected in higher copy number than its normal counterpart. Moreover, the absolute number of the mutant transcript indicates its low abundance with only a few copies per cell in DM1 fibroblasts. Most importantly, in conjunction with fluorescence in-situ hybridization experiments, our results suggest that in DM1 fibroblasts, the vast majority of nuclear RNA foci consist of a few molecules of DMPK exp RNA.

Genetic diagnosis as a tool for personalized treatment of Duchenne muscular dystrophy.

PubMed

Bello, Luca; Pegoraro, Elena

2016-12-01

Accurate definition of genetic mutations causing Duchenne muscular dystrophy (DMD) has always been relevant in order to provide genetic counseling to patients and families, and helps to establish the prognosis in the case where the distinction between Duchenne, Becker, or intermediate muscular dystrophy is not obvious. As molecular treatments aimed at dystrophin restoration in DMD are increasingly available as commercialized drugs or within clinical trials, genetic diagnosis has become an indispensable tool in order to determine eligibility for these treatments. DMD patients in which multiplex ligation-dependent probe amplification (MLPA) or similar techniques show a deletion suitable to exon skipping of exons 44, 45, 51, or 53, may be currently treated with AONs targeting these exons, in the context of clinical trials, or, as is the case for exon 51 skipping in the United States, with the first commercialized drug (eteplirsen). Patients who test negative at MLPA, but in whom DMD gene sequencing shows a nonsense mutation, may be amenable for treatment with stop codon readthrough compounds such as ataluren. Novel molecular approaches such as CRISPR-Cas9 targeting of specific DMD mutations are still in the preclinical stages, but appear promising. In conclusion, an accurate genetic diagnosis represents the entrance into a new scenario of personalized medicine in DMD.

Detection of SHOX gene aberrations in routine diagnostic practice and evaluation of phenotype scoring form effectiveness.

PubMed

Hirschfeldova, Katerina; Florianova, Martina; Kebrdlova, Vera; Urbanova, Marketa; Stekrova, Jitka

2017-02-01

Heterozygous aberrations of SHOX gene have been reported to be responsible for Léri-Weill dyschondrosteosis (LWD) and small portion of idiopathic short stature. The study was established to assess effectiveness of using phenotype 'scoring form' in patients indicated for SHOX gene defect analysis. The submitted study is based on a retrospective group of 352 unrelated patients enrolled as a part of the routine diagnostic practice and analyzed for aberrations affecting the SHOX gene. All participants were scanned for deletion/duplication within the main pseudoautosomal region (PAR1) using the multiplex ligation-dependent probe amplification (MLPA) method. The phenotype 'scoring form' is used in our laboratory practice to preselect patients for subsequent mutation analysis of SHOX gene-coding sequences. The overall detection rate was 11.1% but there was a significant increase in frequency of SHOX gene defect positive with increasing achieved score (P<0.0001). The most frequent aberration was a causal deletion within PAR1. In three probands, MLPA analysis indicated a more complex rearrangement. Madelung deformity or co-occurrence of disproportionate short stature, short forearm and muscular hypertrophy had represented the most potent markers to determine the likelihood of SHOX gene defect detection. We conclude that appliance of phenotype 'scoring form' had saved excessive sample analysis and enabled effective routine diagnostic testing.

Genotype-Phenotype Relationship in Patients and Relatives with SHOX Region Anomalies in the French Population.

PubMed

Auger, Julie; Baptiste, Amandine; Benabbad, Imane; Thierry, Gaëlle; Costa, Jean-Marc; Amouyal, Mélanie; Kottler, Marie-Laure; Leheup, Bruno; Touraine, Renaud; Schmitt, Sébastien; Lebrun, Marine; Cormier Daire, Valérie; Bonnefont, Jean-Paul; de Roux, Nicolas; Elie, Caroline; Rosilio, Myriam

2016-01-01

The aim of our study was to describe a large population with anomalies involving the SHOX region, responsible for idiopathic short stature and Léri-Weill dyschondrosteosis (LWD), and to identify a possible genotype/phenotype correlation. We performed a retrospective multicenter study on French subjects with a SHOX region anomaly diagnosed by multiplex ligation-dependent probe amplification or Sanger sequencing. Phenotypes were collected in each of the 7 genetic laboratories practicing this technique for SHOX analysis. Among 205 index cases and 100 related cases, 91.3% had LWD. For index cases, median age at evaluation was 11.7 (9.0; 15.9) years and mean height standard deviation score was -2.3 ± 1.1. A deletion of either SHOX or PAR1 or both was found in 74% of patients. Duplications and point mutations/indels affected 8 and 18% of the population, respectively. Genotype-phenotype correlation showed that deletions were more frequently associated with Madelung deformity and mesomelic shortening in girls, as well as with presence of radiologic anomalies, than duplications. Our results highlight genotype-phenotype relationships in the French population with a SHOX defect and provide new information showing that clinical expression is milder in cases of duplication compared to deletions. © 2016 S. Karger AG, Basel.

Usefulness of MLPA in the detection of SHOX deletions.

PubMed

Funari, Mariana F A; Jorge, Alexander A L; Souza, Silvia C A L; Billerbeck, Ana E C; Arnhold, Ivo J P; Mendonca, Berenice B; Nishi, Mirian Y

2010-01-01

SHOX haploinsufficiency causes a wide spectrum of short stature phenotypes, such as Leri-Weill dyschondrosteosis (LWD) and disproportionate short stature (DSS). SHOX deletions are responsible for approximately two thirds of isolated haploinsufficiency; therefore, it is important to determine the most appropriate methodology for detection of gene deletion. In this study, three methodologies for the detection of SHOX deletions were compared: the fluorescence in situ hybridization (FISH), microsatellite analysis and multiplex ligation-dependent probe amplification (MLPA). Forty-four patients (8 LWD and 36 DSS) were analyzed. The cosmid LLNOYCO3'M'34F5 was used as a probe for the FISH analysis and microsatellite analysis were performed using three intragenic microsatellite markers. MLPA was performed using commercial kits. Twelve patients (8 LWD and 4 DSS) had deletions in SHOX area detected by MLPA and 2 patients generated discordant results with the other methodologies. In the first case, the deletion was not detected by FISH. In the second case, both FISH and microsatellite analyses were unable to identify the intragenic deletion. In conclusion, MLPA was more sensitive, less expensive and less laborious; therefore, it should be used as the initial molecular method for the detection of SHOX gene deletion. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Identification and characterisation of mutations associated with von Willebrand disease in a Turkish patient cohort

PubMed Central

Hampshire, Daniel J.; Abuzenadah, Adel M.; Cartwright, Ashley; Al-Shammari, Nawal S.; Coyle, Rachael E.; Eckert, Michaela; Al-Buhairan, Ahlam M.; Messenger, Sarah L.; Budde, Ulrich; Gürsel, Türkiz; Ingerslev, Jørgen; Peake, Ian R.; Goodeve, Anne C.

2014-01-01

Summary Several cohort studies have investigated the molecular basis of von Willebrand disease (VWD); however these have mostly focused on European and North American populations. This study aimed to investigate mutation spectrum in 26 index cases (IC) from Turkey diagnosed with all three VWD types, the majority (73%) with parents who were knowingly related. IC were screened for mutations using multiplex ligation-dependent probe amplification and analysis of all von Willebrand factor gene (VWF) exons and exon/intron boundaries. Selected missense mutations were expressed in vitro. Candidate VWF mutations were identified in 25 of 26 IC and included propeptide missense mutations in four IC (two resulting in type 1 and two in recessive 2A), all influencing VWF expression in vitro. Four missense mutations, a nonsense mutation and a small in-frame insertion resulting in type 2A were also identified. Of 15 type 3 VWD IC, 13 were homozygous and two compound heterozygous for 14 candidate mutations predicted to result in lack of expression and two propeptide missense changes. Identification of intronic breakpoints of an exon 17–18 deletion suggested that the mutation resulted from non-homologous end joining. This study provides further insight into the pathogenesis of VWD in a population with a high degree of consanguineous partnerships. PMID:23702511

Evidence of metachronous development of ovarian teratomas: a case report of bilateral mature cystic teratomas of the ovaries and systematic literature review.

PubMed

Wang, Wen-Chung; Lai, Yen-Chein

2017-03-14

Mature cystic teratomas are usually found in the ovaries. They are bilateral in 10 to 15% of cases and multiple cystic teratomas may be present in one ovary. The aim of this study is to clarify if development of mature cystic teratomas of the ovaries in a single host is metachronous or due to autoimplant or recurrence. We report a woman with bilateral mature cystic teratomas of the ovaries. DNA profiles of these teratomas were investigated via short tandem repeat (STR) analysis and methylation statuses were determined via methylation sensitive multiplex ligation-dependent probe amplification methods. The results showed that the cystic teratomas originated from different stages of oogonia or primary oocyte before germinal vesicle stage failure of meiosis I in female gametogenesis. Potentially relevant literature was searched in PubMed database. Cases of bilateral or multiple mature cystic teratomas of the ovaries were analyzed. To date, there has been no reported case of multiple mature cystic teratomas in which clarification of the origin was achieved using molecular genetic methods. The results of this case study provide evidence of metachronous development of mature cystic teratomas of the ovaries and may serve as a reference in the management of patients following laparoscopic cystectomy.

Molecular analysis of holoprosencephaly in South America

PubMed Central

Savastano, Clarice Pagani; El-Jaick, Kênia Balbi; Costa-Lima, Marcelo Aguiar; Abath, Cristina Maria Batista; Bianca, Sebastiano; Cavalcanti, Denise Pontes; Félix, Têmis Maria; Scarano, Gioacchino; Llerena, Juan Clinton; Vargas, Fernando Regla; Moreira, Miguel Ângelo Martins; Seuánez, Hector N.; Castilla, Eduardo Enrique; Orioli, Iêda Maria

2014-01-01

Holoprosencephaly (HPE) is a spectrum of brain and facial malformations primarily reflecting genetic factors, such as chromosomal abnormalities and gene mutations. Here, we present a clinical and molecular analysis of 195 probands with HPE or microforms; approximately 72% of the patients were derived from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), and 82% of the patients were newborns. Alobar HPE was the predominant brain defect in almost all facial defect categories, except for patients without oral cleft and median or lateral oral clefts. Ethmocephaly, cebocephaly, and premaxillary agenesis were primarily observed among female patients. Premaxillary agenesis occurred in six of the nine diabetic mothers. Recurrence of HPE or microform was approximately 19%. The frequency of microdeletions, detected using Multiplex Ligation-dependant Probe Amplification (MLPA) was 17% in patients with a normal karyotype. Cytogenetics or QF-PCR analyses revealed chromosomal anomalies in 27% of the probands. Mutational analyses in genes SHH, ZIC2, SIX3 and TGIF were performed in 119 patients, revealing eight mutations in SHH, two mutations in SIX3 and two mutations in ZIC2. Thus, a detailed clinical description of new HPE cases with identified genetic anomalies might establish genotypic and phenotypic correlations and contribute to the development of additional strategies for the analysis of new cases. PMID:24764759

Wide clinical variability in cat eye syndrome patients: four non-related patients and three patients from the same family.

PubMed

Belangero, S I; Pacanaro, A N X; Bellucco, F T; Christofolini, D M; Kulikowski, L D; Guilherme, R S; Bortolai, A; Dutra, A R N; Piazzon, F B; Cernach, M C; Melaragno, M I

2012-01-01

A small supernumerary marker chromosome (sSMC) derived from chromosome 22 is a relatively common cytogenetic finding. This sSMC typically results in tetrasomy for a chromosomal region that spans the chromosome 22p arm and the proximal 2 Mb of 22q11.21. Using classical cytogenetics, fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, and array techniques, 7 patients with sSMCs derived from chromosome 22 were studied: 4 non-related and 3 from the same family (mother, daughter, and son). The sSMCs in all patients were dicentric and bisatellited chromosomes with breakpoints in the chromosome 22 low-copy repeat A region, resulting in cat eye syndrome (CES) due to chromosome 22 partial tetrasomy 22pter→q11.2 including the cat eye chromosome region. Although all subjects presented the same chromosomal abnormality, they showed a wide range of phenotypic differences, even in the 3 patients from the same family. There are no previous reports of CES occurring within 3 patients in the same family. Thus, the clinical and follow-up data presented here contribute to a better delineation of the phenotypes and outcomes of CES patients and will be useful for genetic counseling. Copyright © 2012 S. Karger AG, Basel.

Novel deletions involving the USH2A gene in patients with Usher syndrome and retinitis pigmentosa

PubMed Central

García-García, Gema; Jaijo, Teresa; Aparisi, Maria J.; Larrieu, Lise; Faugère, Valérie; Blanco-Kelly, Fiona; Ayuso, Carmen; Roux, Anne-Francoise; Millán, José M.

2014-01-01

Purpose The aim of the present work was to identify and characterize large rearrangements involving the USH2A gene in patients with Usher syndrome and nonsyndromic retinitis pigmentosa. Methods The multiplex ligation-dependent probe amplification (MLPA) technique combined with a customized array-based comparative genomic hybridization (aCGH) analysis was applied to 40 unrelated patients previously screened for point mutations in the USH2A gene in which none or only one pathologic mutation was identified. Results We detected six large deletions involving USH2A in six out of the 40 cases studied. Three of the patients were homozygous for the deletion, and the remaining three were compound heterozygous with a previously identified USH2A point mutation. In five of these cases, the patients displayed Usher type 2, and the remaining case displayed nonsyndromic retinitis pigmentosa. The exact breakpoint junctions of the deletions found in USH2A in four of these cases were characterized. Conclusions Our study highlights the need to develop improved efficient strategies of mutation screening based upon next generation sequencing (NGS) that reduce cost, time, and complexity and allow simultaneous identification of all types of disease-causing mutations in diagnostic procedures. PMID:25352746

Molecular defects of the CYP21A2 gene in Greek-Cypriot patients with congenital adrenal hyperplasia.

PubMed

Skordis, Nicos; Kyriakou, Andreas; Tardy, Véronique; Ioannou, Yiannis S; Varvaresou, Athanasia; Dracopoulou-Vabouli, Maria; Patsalis, Philippos C; Shammas, Christos; Neocleous, Vassos; Phylactou, Leonidas A

2011-01-01

To determine the mutations in the CYP21A2 gene in Greek-Cypriots with congenital adrenal hyperplasia (CAH) and attempt a genotype-phenotype correlation. Molecular analysis was performed by multiplex ligation-dependent probe amplification and direct sequencing of PCR products of the CYP21A2 gene in 32 CAH patients. The most frequent genetic defect in the classic salt-wasting and simple virilizing forms was the IVS2-13A/C>G (55%) mutation, followed by Large lesion (20%) and in the non-classical form, the p.V281L (79.5%). Genotypes were categorized in 4 mutation groups (null, A, B and C). All 3 patients in the null group manifested the salt-wasting form and all 6 patients in mutation group A presented with the classical form. One patient in group B had the simple virilizing form and 22 patients in group C exhibited the non-classical form. The spectrum of mutations of the CYP21A2 gene in our population is comparable to the most common reported in similar ethnic groups. The knowledge of the ethnic specificity of the CYP21A2 mutations represents a valuable diagnostic tool for all forms of CAH. Copyright © 2010 S. Karger AG, Basel.

Identification of a Novel De Novo Heterozygous Deletion in the SOX10 Gene in Waardenburg Syndrome Type II Using Next-Generation Sequencing.

PubMed

Li, Haonan; Jin, Peng; Hao, Qian; Zhu, Wei; Chen, Xia; Wang, Ping

2017-11-01

Waardenburg syndrome (WS) is a rare autosomal dominant disorder associated with pigmentation abnormalities and sensorineural hearing loss. In this study, we investigated the genetic cause of WSII in a patient and evaluated the reliability of the targeted next-generation exome sequencing method for the genetic diagnosis of WS. Clinical evaluations were conducted on the patient and targeted next-generation sequencing (NGS) was used to identify the candidate genes responsible for WSII. Multiplex ligation-dependent probe amplification (MLPA) and real-time quantitative polymerase chain reaction (qPCR) were performed to confirm the targeted NGS results. Targeted NGS detected the entire deletion of the coding sequence (CDS) of the SOX10 gene in the WSII patient. MLPA results indicated that all exons of the SOX10 heterozygous deletion were detected; no aberrant copy number in the PAX3 and microphthalmia-associated transcription factor (MITF) genes was found. Real-time qPCR results identified the mutation as a de novo heterozygous deletion. This is the first report of using a targeted NGS method for WS candidate gene sequencing; its accuracy was verified by using the MLPA and qPCR methods. Our research provides a valuable method for the genetic diagnosis of WS.

Electrochemical detection of aqueous Ag+ based on Ag+-assisted ligation reaction

NASA Astrophysics Data System (ADS)

Miao, Peng; Han, Kun; Wang, Bidou; Luo, Gangyin; Wang, Peng; Chen, Mingli; Tang, Yuguo

2015-03-01

In this work, a novel strategy to fabricate a highly sensitive and selective biosensor for the detection of Ag+ is proposed. Two DNA probes are designed and modified on a gold electrode surface by gold-sulfur chemistry and hybridization. In the presence of Ag+, cytosine-Ag+-cytosine composite forms and facilitates the ligation event on the electrode surface, which can block the release of electrochemical signals labeled on one of the two DNA probes during denaturation process. Ag+ can be sensitively detected with the detection limit of 0.1 nM, which is much lower than the toxicity level defined by U.S. Environmental Protection Agency. This biosensor can easily distinguish Ag+ from other interfering ions and the performances in real water samples are also satisfactory. Moreover, the two DNA probes are designed to contain the recognition sequences of a nicking endonuclease, and the ligated DNA can thus be cleaved at the original site. Therefore, the electrode can be regenerated, which allows the biosensor to be reused for additional tests.

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

NASA Astrophysics Data System (ADS)

Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

2016-10-01

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection.

PubMed

Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

2016-10-14

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

Topographic instructions, Book 3, multiplex procedure; Chapter 3 C7a-e

USGS Publications Warehouse

Loud, Edward I.

1952-01-01

By direct projection of overlapping photographs, printed on glass plates, the multiplex produces an exact optical model, in miniature, of the terrain to be mapped. To create the model, the multiplex projectors must be properly positioned and oriented so that they duplicate the orientation of the aerial camera at the instant of each exposure. By means of a floating mark, horizontal and vertical measurements can be made in the model, and planimetry and contours can be drawn. The applicability of the multiplex to a given mapping project depends largely on the contour interval and compilation scale required, and also depends, to a lesser extent, on the vegetation and terrain cover as it may affect accuracy requirements. The steps in multiplex procedure are orientation, stereotriangulation, and compilation of detail. In orientation, the projectors are arranged so that the projected images form a stereoscopic model which can be adjusted to fit horizontal and vertical control points. In stereotriangulation, three or more multiplex projectors are oriented so that the consecutive models fit existing control, permitting the establishment of additional or intermediate control. In compilation, the features appearing in the model are delineated on the map manuscript.

Novel multiplex qualitative detection using universal primer-multiplex-PCR combined with pyrosequencing.

PubMed

Shang, Ying; Xu, Wentao; Wang, Yong; Xu, Yuancong; Huang, Kunlun

2017-12-15

This study described a novel multiplex qualitative detection method using pyrosequencing. Based on the principle of the universal primer-multiplex-PCR, only one sequencing primer was employed to realize the detection of the multiple targets. Samples containing three genetically modified (GM) crops in different proportions were used to validate the method. The dNTP dispensing order was designed based on the product sequences. Only 12 rounds (ATCTGATCGACT) of dNTPs addition and, often, as few as three rounds (CAT) under ideal conditions, were required to detect the GM events qualitatively, and sensitivity was as low as 1% of a mixture. However, when considering a mixture, calculating signal values allowed the proportion of each GM to be estimated. Based on these results, we concluded that our novel method not only realized detection but also allowed semi-quantitative detection of individual events. Copyright © 2017. Published by Elsevier Ltd.

Multiplex PCR for Rapid Detection of Genes Encoding Class A Carbapenemases

PubMed Central

Hong, Sang Sook; Kim, Kyeongmi; Huh, Ji Young; Jung, Bochan; Kang, Myung Seo

2012-01-01

In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-β-lactamase nonproducers. PMID:22950072

Multiplex PCR for rapid detection of genes encoding class A carbapenemases.

PubMed

Hong, Sang Sook; Kim, Kyeongmi; Huh, Ji Young; Jung, Bochan; Kang, Myung Seo; Hong, Seong Geun

2012-09-01

In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-β-lactamase nonproducers.

New multiplex PCR methods for rapid screening of genetically modified organisms in foods

PubMed Central

Datukishvili, Nelly; Kutateladze, Tamara; Gabriadze, Inga; Bitskinashvili, Kakha; Vishnepolsky, Boris

2015-01-01

We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products. PMID:26257724

New multiplex PCR methods for rapid screening of genetically modified organisms in foods.

PubMed

Datukishvili, Nelly; Kutateladze, Tamara; Gabriadze, Inga; Bitskinashvili, Kakha; Vishnepolsky, Boris

2015-01-01

We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products.

Ligation-mediated PCR with a back-to-back adapter reduces amplification bias resulting from variations in GC content.

PubMed

Ishihara, Satoru; Kotomura, Naoe; Yamamoto, Naoki; Ochiai, Hiroshi

2017-08-15

Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence. The use of this adapter in LM-PCR reduced the impairment of PCR by substrate DNA with a high GC content, compared with the use of traditional LM-PCR adapters. This result suggested that our method has the potential to contribute to reduction of the amplification bias that is caused by an intrinsic property of the sequence context in substrate DNA. A DNA preparation obtained from a chromatin immunoprecipitation assay using pulldown of a specific form of histone H3 was successfully amplified using the modified LM-PCR, and the amplified products could be used as probes in a fluorescence in situ hybridization analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiplex Networks of Cortical and Hippocampal Neurons Revealed at Different Timescales

PubMed Central

Timme, Nicholas; Ito, Shinya; Myroshnychenko, Maxym; Yeh, Fang-Chin; Hiolski, Emma; Hottowy, Pawel; Beggs, John M.

2014-01-01

Recent studies have emphasized the importance of multiplex networks – interdependent networks with shared nodes and different types of connections – in systems primarily outside of neuroscience. Though the multiplex properties of networks are frequently not considered, most networks are actually multiplex networks and the multiplex specific features of networks can greatly affect network behavior (e.g. fault tolerance). Thus, the study of networks of neurons could potentially be greatly enhanced using a multiplex perspective. Given the wide range of temporally dependent rhythms and phenomena present in neural systems, we chose to examine multiplex networks of individual neurons with time scale dependent connections. To study these networks, we used transfer entropy – an information theoretic quantity that can be used to measure linear and nonlinear interactions – to systematically measure the connectivity between individual neurons at different time scales in cortical and hippocampal slice cultures. We recorded the spiking activity of almost 12,000 neurons across 60 tissue samples using a 512-electrode array with 60 micrometer inter-electrode spacing and 50 microsecond temporal resolution. To the best of our knowledge, this preparation and recording method represents a superior combination of number of recorded neurons and temporal and spatial recording resolutions to any currently available in vivo system. We found that highly connected neurons (“hubs”) were localized to certain time scales, which, we hypothesize, increases the fault tolerance of the network. Conversely, a large proportion of non-hub neurons were not localized to certain time scales. In addition, we found that long and short time scale connectivity was uncorrelated. Finally, we found that long time scale networks were significantly less modular and more disassortative than short time scale networks in both tissue types. As far as we are aware, this analysis represents the first systematic study of temporally dependent multiplex networks among individual neurons. PMID:25536059

Nonuniformity of axial and circumferential remodeling of large coronary veins in response to ligation.

PubMed

Choy, Jenny Susana; Dang, Quang; Molloi, Sabee; Kassab, Ghassan S

2006-04-01

The pressure-induced remodeling of coronary veins is important in coronary venous retroperfusion. Our hypothesis is that the response of the large coronary veins to pressure overload will depend on the degree of myocardial support. Eleven normal Yorkshire swine from either sex, weighing 31-39 kg, were studied. Five pigs underwent ligation of the left anterior descending (LAD) vein, and six served as sham-operated controls. The ligation of the coronary vein caused an increase in pressure intermediate to arterial and venous values. After 2 wk of ligation, the animals were euthanized and the coronary vessels were perfusion-fixed with glutaraldehyde. The LAD vein was sectioned, and detailed morphometric measurements were made along its length from the point of ligation near the base down to the apex of the heart. The structural remodeling of the vein was circumferentially nonuniform because the vein is partially embedded in the myocardium; it was also axially nonuniform because it is tethered to the myocardium to different degrees along its axial length. The wall area was significantly larger in the experimental group, whereas luminal area in the proximal LAD vein was significantly smaller in the same group compared with sham-operated controls. The wall thickness-to-radius ratio was also significantly larger in the experimental group in proportion to the increase in pressure. The major conclusion of this study is that the response of the vein depends on the local wall stress, which is, in part, determined by the surrounding tissue. Furthermore, the geometric remodeling of the coronary vein restores the circumferential stress to the homeostatic value.

Different effects of cytoprotective drugs on ethanol- and aspirin-induced gastric mucosal injury in pylorus-ligated rats

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

Takeuchi, K.; Nishiwaki, H.; Niida, H.

In anesthetized rats oral administration (2 ml) of both ethanol (50% in 150 mM HCl) and aspirin (80 mM in 150 mM HCl) produced bandlike lesions in the stomach, while more generalized lesions occurred in the pylorus-ligated stomach when the irritant was given intragastrically through the fistula prepared in the rumen and the mucosal folds were removed by stomach distension. The bandlike lesions induced in the intact stomach by both irritants were significantly and dose-dependently prevented by 16,16-dimethyl PGE2 (dmPGE2: 3 and 10 micrograms/kg, subcutaneously), cysteamine (30 and 100 mg/kg, subcutaneously) or timoprazole (10 and 30 mg/kg, per os) atmore » the doses which significantly inhibited gastric motility. In the pylorus-ligated stomach, however, neither of these agents showed any protection against the generalized lesions induced by ethanol, but such lesions caused by aspirin were significantly prevented only by dmPGE2. These agents also showed similar effects against the reduction of transmucosal PD in the pylorus-ligated stomach exposed to ethanol and aspirin. These results suggest that (1) the formation of bandlike lesions caused by ethanol and aspirin depends on the presence of mucosal folds and may be prevented by the agents that inhibit gastric motility, (2) the pathogenesis of the lesions induced by aspirin and ethanol may be different in the pylorus-ligated stomach, and (3) dmPGE2 has a unique protective ability that is not shared by usual cytoprotective agents.« less

Genetics of Type III Bartter Syndrome in Spain, Proposed Diagnostic Algorithm

PubMed Central

García Castaño, Alejandro; Pérez de Nanclares, Gustavo; Madariaga, Leire; Aguirre, Mireia; Madrid, Alvaro; Nadal, Inmaculada; Navarro, Mercedes; Lucas, Elena; Fijo, Julia; Espino, Mar; Espitaletta, Zilac; Castaño, Luis; Ariceta, Gema

2013-01-01

The p.Ala204Thr mutation (exon 7) of the CLCNKB gene is a "founder" mutation that causes most of type III Bartter syndrome cases in Spain. We performed genetic analysis of the CLCNKB gene, which encodes for the chloride channel protein ClC-Kb, in a cohort of 26 affected patients from 23 families. The diagnostic algorithm was: first, detection of the p.Ala204Thr mutation; second, detecting large deletions or duplications by Multiplex Ligation-dependent Probe Amplification and Quantitative Multiplex PCR of Short Fluorescent Fragments; and third, sequencing of the coding and flanking regions of the whole CLCNKB gene. In our genetic diagnosis, 20 families presented with the p.Ala204Thr mutation. Of those, 15 patients (15 families) were homozygous (57.7% of overall patients). Another 8 patients (5 families) were compound heterozygous for the founder mutation together with a second one. Thus, 3 patients (2 siblings) presented with the c. -19-?_2053+? del deletion (comprising the entire gene); one patient carried the p.Val170Met mutation (exon 6); and 4 patients (3 siblings) presented with the novel p.Glu442Gly mutation (exon 14). On the other hand, another two patients carried two novel mutations in compound heterozygosis: one presented the p.Ile398_Thr401del mutation (exon 12) associated with the c. -19-?_2053+? del deletion, and the other one carried the c.1756+1G>A splice-site mutation (exon 16) as well as the already described p.Ala210Val change (exon 7). One case turned out to be negative in our genetic screening. In addition, 51 relatives were found to be heterozygous carriers of the described CLCNKB mutations. In conclusion, different mutations cause type III Bartter syndrome in Spain. The high prevalence of the p.Ala204Thr in Spanish families thus justifies an initial screen for this mutation. However, should it not be detected further investigation of the CLCNKB gene is warranted in clinically diagnosed families. PMID:24058621

Genetics of type III Bartter syndrome in Spain, proposed diagnostic algorithm.

PubMed

García Castaño, Alejandro; Pérez de Nanclares, Gustavo; Madariaga, Leire; Aguirre, Mireia; Madrid, Alvaro; Nadal, Inmaculada; Navarro, Mercedes; Lucas, Elena; Fijo, Julia; Espino, Mar; Espitaletta, Zilac; Castaño, Luis; Ariceta, Gema

2013-01-01

The p.Ala204Thr mutation (exon 7) of the CLCNKB gene is a "founder" mutation that causes most of type III Bartter syndrome cases in Spain. We performed genetic analysis of the CLCNKB gene, which encodes for the chloride channel protein ClC-Kb, in a cohort of 26 affected patients from 23 families. The diagnostic algorithm was: first, detection of the p.Ala204Thr mutation; second, detecting large deletions or duplications by Multiplex Ligation-dependent Probe Amplification and Quantitative Multiplex PCR of Short Fluorescent Fragments; and third, sequencing of the coding and flanking regions of the whole CLCNKB gene. In our genetic diagnosis, 20 families presented with the p.Ala204Thr mutation. Of those, 15 patients (15 families) were homozygous (57.7% of overall patients). Another 8 patients (5 families) were compound heterozygous for the founder mutation together with a second one. Thus, 3 patients (2 siblings) presented with the c. -19-?_2053+? del deletion (comprising the entire gene); one patient carried the p.Val170Met mutation (exon 6); and 4 patients (3 siblings) presented with the novel p.Glu442Gly mutation (exon 14). On the other hand, another two patients carried two novel mutations in compound heterozygosis: one presented the p.Ile398_Thr401del mutation (exon 12) associated with the c. -19-?_2053+? del deletion, and the other one carried the c.1756+1G>A splice-site mutation (exon 16) as well as the already described p.Ala210Val change (exon 7). One case turned out to be negative in our genetic screening. In addition, 51 relatives were found to be heterozygous carriers of the described CLCNKB mutations. In conclusion, different mutations cause type III Bartter syndrome in Spain. The high prevalence of the p.Ala204Thr in Spanish families thus justifies an initial screen for this mutation. However, should it not be detected further investigation of the CLCNKB gene is warranted in clinically diagnosed families.

Significant clinical impact of recurrent BRCA1 and BRCA2 mutations in Mexico

PubMed Central

Villarreal-Garza, Cynthia; Alvarez-Gómez, Rosa María; Pérez-Plasencia, Carlos; Herrera, Luis A.; Herzog, Josef; Castillo, Danielle; Mohar, Alejandro; Castro, Clementina; Gallardo, Lenny N.; Gallardo, Dolores; Santibáñez, Miguel; Blazer, Kathleen R.; Weitzel, Jeffrey N.

2014-01-01

Background Frequent recurrent BRCA1 and BRCA2 gene (BRCA) mutations among Hispanics, including a large rearrangement Mexican founder mutation (BRCA1 ex9-12del), suggest that an ancestry-informed BRCA-testing strategy could reduce disparities and promote cancer prevention by enabling economical screening for hereditary breast and ovarian cancer in Mexico. Methods In a multistage approach, 188 cancer cases unselected for family cancer history (92 ovarian cancer and 96 breast cancer) were screened for BRCA mutations using a Hispanic mutation panel (HISPANEL®) of 115 recurrent mutations in a multiplex assay (114 on a mass spectroscopy platform, and a PCR assay for the BRCA1 ex9-12del mutation), followed by sequencing of all BRCA exons and adjacent intronic regions, and BRCA1 multiplex ligation-dependent probe amplification assay (MLPA) for HISPANEL negative cases. BRCA mutation prevalence was calculated and correlated with histology and tumor receptor status, and HISPANEL sensitivity was estimated. Results BRCA mutations were detected in 28% (26/92) of ovarian cancer cases and 15% (14/96) of breast cancer cases overall and 27% (9/33) of triple negative breast cancer. Most breast cancer cases were diagnosed with locally advanced disease. The Mexican founder mutation (BRCA1 ex9-12del) accounted for 35% of the BRCA-associated ovarian cancer cases and 29% of the BRCA-associated breast cancer cases. At 2% of the sequencing and MLPA cost, the HISPANEL detected 68% of all BRCA mutations. Conclusion In this study, we found a remarkably high prevalence of BRCA mutations among ovarian and breast cases not selected for family history, and BRCA1 ex9-12del explained one third of the total. The remarkable frequency of BRCA1 ex9-12del in Mexico City supports a nearby origin of this Mexican founder mutation and may constitute a regional public health problem. The HISPANEL presents a translational opportunity for cost-effective genetic testing to enable breast and ovarian cancer prevention. PMID:25236687

Multiplex ligation-dependent probe amplification for genetic screening in autism spectrum disorders: Efficient identification of known microduplications and identification of a novel microduplication in ASMT

PubMed Central

Cai, Guiqing; Edelmann, Lisa; Goldsmith, Juliet E; Cohen, Ninette; Nakamine, Alisa; Reichert, Jennifer G; Hoffman, Ellen J; Zurawiecki, Danielle M; Silverman, Jeremy M; Hollander, Eric; Soorya, Latha; Anagnostou, Evdokia; Betancur, Catalina; Buxbaum, Joseph D

2008-01-01

Background It has previously been shown that specific microdeletions and microduplications, many of which also associated with cognitive impairment (CI), can present with autism spectrum disorders (ASDs). Multiplex ligation-dependent probe amplification (MLPA) represents an efficient method to screen for such recurrent microdeletions and microduplications. Methods In the current study, a total of 279 unrelated subjects ascertained for ASDs were screened for genomic disorders associated with CI using MLPA. Fluorescence in situ hybridization (FISH), quantitative polymerase chain reaction (Q-PCR) and/or direct DNA sequencing were used to validate potential microdeletions and microduplications. Methylation-sensitive MLPA was used to characterize individuals with duplications in the Prader-Willi/Angelman (PWA) region. Results MLPA showed two subjects with typical ASD-associated interstitial duplications of the 15q11-q13 PWA region of maternal origin. Two additional subjects showed smaller, de novo duplications of the PWA region that had not been previously characterized. Genes in these two novel duplications include GABRB3 and ATP10A in one case, and MKRN3, MAGEL2 and NDN in the other. In addition, two subjects showed duplications of the 22q11/DiGeorge syndrome region. One individual was found to carry a 12 kb deletion in one copy of the ASPA gene on 17p13, which when mutated in both alleles leads to Canavan disease. Two subjects showed partial duplication of the TM4SF2 gene on Xp11.4, previously implicated in X-linked non-specific mental retardation, but in our subsequent analyses such variants were also found in controls. A partial duplication in the ASMT gene, located in the pseudoautosomal region 1 (PAR1) of the sex chromosomes and previously suggested to be involved in ASD susceptibility, was observed in 6–7% of the cases but in only 2% of controls (P = 0.003). Conclusion MLPA proves to be an efficient method to screen for chromosomal abnormalities. We identified duplications in 15q11-q13 and in 22q11, including new de novo small duplications, as likely contributing to ASD in the current sample by increasing liability and/or exacerbating symptoms. Our data indicate that duplications in TM4SF2 are not associated with the phenotype given their presence in controls. The results in PAR1/PAR2 are the first large-scale studies of gene dosage in these regions, and the findings at the ASMT locus indicate that further studies of the duplication of the ASMT gene are needed in order to gain insight into its potential involvement in ASD. Our studies also identify some limitations of MLPA, where single base changes in probe binding sequences alter results. In summary, our studies indicate that MLPA, with a focus on accepted medical genetic conditions, may be an inexpensive method for detection of microdeletions and microduplications in ASD patients for purposes of genetic counselling if MLPA-identified deletions are validated by additional methods. PMID:18925931

Identification of Malassezia species from pityriasis versicolor lesions with a new multiplex PCR method.

PubMed

Vuran, Emre; Karaarslan, Aydın; Karasartova, Djursun; Turegun, Buse; Sahin, Fikret

2014-02-01

Despite the fact that a range of molecular methods have been developed as tools for the diagnosis of Malassezia species, there are several drawbacks associated with them, such as inefficiency of differentiating all the species, high cost, and questionable reproducibility. In addition, most of the molecular methods require cultivation to enhance sensitivity. Therefore, alternative methods eliminating cultivation and capable of identifying species with high accuracy and reliability are needed. Herein, a multiplex polymerase chain reaction (PCR)-based method was especially developed for the detection of eleven Malassezia species. The multiplex PCR was standardized by incorporating a consensus forward primer, along with Malassezia species-specific reverse primers considering the sizes of the PCR products. In the method, the multiplex-PCR primer content is divided into three parts to circumvent the problem of increased nonspecific background resulting from the use of a large number of primers. DNA extraction protocol described by Harju and colleagues was modified using liquid nitrogen instead of -80 °C to break down the yeast membrane. By a modified extraction procedure followed by multiplex PCR and electrophoresis, the method enables identification and differentiation of Malassezia species from both of the samples obtained directly from skin and yeast colonies grown in culture. Fifty-five patients who were confirmed with pityriasis versicolor were enrolled in the study. Multiplex PCR detected and differentiated all 55 samples obtained directly from the patients' skin. However, 50 out of 55 samples yielded Malassezia colony in the culture. In addition, eight of 50 colonies were misdiagnosed or not completely differentiated by conventional methods based on the sequence analysis of eight colonies. The method is capable of identifying species with high accuracy and reliability. In addition, it is simple, quick, and cost-effective. More importantly, the method works efficiently for the diagnosis of Malassezia species obtained directly from patient samples.

Ultrafast FADC multiplexer

NASA Astrophysics Data System (ADS)

Mirzoyan, R.; Cortina, J.; Lorenz, E.; Martinez, M.; Ostankov, A.; Paneque, D.

2002-10-01

Ultrafast Flash amplitude-to-digital converters (FADCs) are still very expensive. Here we propose a multiplexing scheme allowing one in common trigger mode to read out multiple signal sources by using a single FADC channel. Usual coaxial cables can be used in the multiplexer as analog signal delay elements. The limited bandwidth of the coaxial cable, depending on its type and length will set an upper limit to the number of multiplexed channels. Better bandwidth and the correspondingly higher number of multiplexed channels one can obtain when using the technique of transmission of analog signals via optical fibers. Low-cost vertical cavity surface emitting laser (VCSEL) diodes can be used as converters of fast electrical signals into near infrared light. Multiplexing can be an economically priced solution when one needs ultrafast digitization of hundreds of fast signal channels.

Analysis on the propagation characteristics of two multiplexed groups of coaxial OAM beams in atmospheric turbulence

NASA Astrophysics Data System (ADS)

Zheng, Yongping; Tian, Qinghua; Zhang, Wei; Zhang, Qi; Zhu, Lei; Wang, Yongjun; Liu, Bo; Xin, Xiangjun

2018-01-01

Orbital angular momentum (OAM) as a new degree of freedom, greatly improves the spectrum efficiency and channel capacity of optical communication system. It has become the research focus in the field of optical communications. Some scholars have demonstrated that the feasibility of two multiplexed groups of concentric rings of Laguerre-Gaussian (LG) beams with OAM multiplexing transmission in free space. Based on the point, this paper makes the further research on the propagation characteristics of LG beams with this spatial multiplexing structure in atmospheric turbulence. The random phase screen is established by using the modified von Karman power spectrum and the received power and crosstalk power of OAM modes of LG beams are obtained under the Rytov approximation. We investigate the characteristic parameters of LG beams with this spatial multiplexing structure for mitigating turbulence. Simulation results show that the system exists an optimum beam waist related to wavelength in which the received power of OAM modes reaches the maximum. Meanwhile, the BER and aggregate capacity of the system with two multiplexed groups of concentric rings of LG beams with OAM multiplexing are simulated and analyzed under different intensities of atmospheric turbulence. The results reveal that the system with larger mode spacing generally has lower inter-modal crosstalk and larger aggregate capacity than that with the smaller mode spacing. Finally, on the basis of above the analysis and research, some suggestions for efficient OAM multiplexing detection scheme are proposed.

A Novel Universal Primer-Multiplex-PCR Method with Sequencing Gel Electrophoresis Analysis

PubMed Central

Huang, Kunlun; Zhang, Nan; Yuan, Yanfang; Shang, Ying; Luo, Yunbo

2012-01-01

In this study, a novel universal primer-multiplex-PCR (UP-M-PCR) method adding a universal primer (UP) in the multiplex PCR reaction system was described. A universal adapter was designed in the 5′-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP). PCR products were analyzed on sequencing gel electrophoresis (SGE) which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM) crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on. PMID:22272223

Clinically guided genetic screening in a large cohort of italian patients with pheochromocytomas and/or functional or nonfunctional paragangliomas.

PubMed

Mannelli, Massimo; Castellano, Maurizio; Schiavi, Francesca; Filetti, Sebastiano; Giacchè, Mara; Mori, Luigi; Pignataro, Viviana; Bernini, Gianpaolo; Giachè, Valentino; Bacca, Alessandra; Biondi, Bernadette; Corona, Giovanni; Di Trapani, Giuseppe; Grossrubatscher, Erika; Reimondo, Giuseppe; Arnaldi, Giorgio; Giacchetti, Gilberta; Veglio, Franco; Loli, Paola; Colao, Annamaria; Ambrosio, Maria Rosaria; Terzolo, Massimo; Letizia, Claudio; Ercolino, Tonino; Opocher, Giuseppe

2009-05-01

The aim of the study was to define the frequency of hereditary forms and the genotype/phenotype correlations in a large cohort of Italian patients with pheochromocytomas and/or functional or nonfunctional paragangliomas. We examined 501 consecutive patients with pheochromocytomas and/or paragangliomas (secreting or nonsecreting). Complete medical and family histories, as well as the results of clinical, laboratory, and imaging studies, were recorded in a database. Patients were divided into different groups according to their family history, the presence of lesions outside adrenals/paraganglia considered syndromic for VHL disease, MEN2, and NF1, and the number and types of pheochromocytomas and/or paragangliomas. Germ-line mutations in known susceptibility genes were investigated by gene sequencing (VHL, RET, SDHB, SDHC, SDHD) or diagnosed according to phenotype (NF1). In 160 patients younger than 50 yr with a wild-type profile, multiplex ligation-dependent probe amplification assays were performed to detect genomic rearrangements. Germline mutations were detected in 32.1% of cases, but frequencies varied widely depending on the classification criteria and ranged from 100% in patients with associated syndromic lesions to 11.6% in patients with a single tumor and a negative family history. The types and number of pheochromocytomas/paragangliomas as well as age at presentation and malignancy suggest which gene should be screened first. Genomic rearrangements were found in two of 160 patients (1.2%). The frequency of the hereditary forms of pheochromocytoma/paraganglioma varies depending on the family history and the clinical presentation. A positive family history and an accurate clinical evaluation of patients are strong indicators of which genes should be screened first.

Multiplexed DNA detection using spectrally encoded porous SiO2 photonic crystal particles.

PubMed

Meade, Shawn O; Chen, Michelle Y; Sailor, Michael J; Miskelly, Gordon M

2009-04-01

A particle-based multiplexed DNA assay based on encoded porous SiO(2) photonic crystal disks is demonstrated. A "spectral barcode" is generated by electrochemical etch of a single-crystal silicon wafer using a programmed current-time waveform. A lithographic procedure is used to isolate cylindrical microparticles 25 microm in diameter and 10 microm thick, which are then oxidized, modified with a silane linker, and conjugated to various amino-functionalized oligonucleotide probes via cyanuric chloride. It is shown that the particles can be decoded based on their reflectivity spectra and that a multiple analyte assay can be performed in a single sample with a modified fluorescence microscope. The homogeneity of the reflectivity and fluorescence spectra, both within and across the microparticles, is also reported.

Two Distinct Types of E3 Ligases Work in Unison to Regulate Substrate Ubiquitylation.

PubMed

Scott, Daniel C; Rhee, David Y; Duda, David M; Kelsall, Ian R; Olszewski, Jennifer L; Paulo, Joao A; de Jong, Annemieke; Ovaa, Huib; Alpi, Arno F; Harper, J Wade; Schulman, Brenda A

2016-08-25

Hundreds of human cullin-RING E3 ligases (CRLs) modify thousands of proteins with ubiquitin (UB) to achieve vast regulation. Current dogma posits that CRLs first catalyze UB transfer from an E2 to their client substrates and subsequent polyubiquitylation from various linkage-specific E2s. We report an alternative E3-E3 tagging cascade: many cellular NEDD8-modified CRLs associate with a mechanistically distinct thioester-forming RBR-type E3, ARIH1, and rely on ARIH1 to directly add the first UB and, in some cases, multiple additional individual monoubiquitin modifications onto CRL client substrates. Our data define ARIH1 as a component of the human CRL system, demonstrate that ARIH1 can efficiently and specifically mediate monoubiquitylation of several CRL substrates, and establish principles for how two distinctive E3s can reciprocally control each other for simultaneous and joint regulation of substrate ubiquitylation. These studies have broad implications for CRL-dependent proteostasis and mechanisms of E3-mediated UB ligation. Copyright © 2016 Elsevier Inc. All rights reserved.

EndoU is a novel regulator of AICD during peripheral B cell selection

PubMed Central

Poe, Jonathan C.; Kountikov, Evgueni I.; Lykken, Jacquelyn M.; Natarajan, Abirami; Marchuk, Douglas A.

2014-01-01

Balanced transmembrane signals maintain a competent peripheral B cell pool limited in self-reactive B cells that may produce pathogenic autoantibodies. To identify molecules regulating peripheral B cell survival and tolerance to self-antigens (Ags), a gene modifier screen was performed with B cells from CD22-deficient C57BL/6 (CD22−/−[B6]) mice that undergo activation-induced cell death (AICD) and fail to up-regulate c-Myc expression after B cell Ag receptor ligation. Likewise, lysozyme auto-Ag–specific B cells in IgTg hen egg lysozyme (HEL) transgenic mice inhabit the spleen but undergo AICD after auto-Ag encounter. This gene modifier screen identified EndoU, a single-stranded RNA-binding protein of ancient origin, as a major regulator of B cell survival in both models. EndoU gene disruption prevents AICD and normalizes c-Myc expression. These findings reveal that EndoU is a critical regulator of an unexpected and novel RNA-dependent pathway controlling peripheral B cell survival and Ag responsiveness that may contribute to peripheral B cell tolerance. PMID:24344237

EndoU is a novel regulator of AICD during peripheral B cell selection.

PubMed

Poe, Jonathan C; Kountikov, Evgueni I; Lykken, Jacquelyn M; Natarajan, Abirami; Marchuk, Douglas A; Tedder, Thomas F

2014-01-13

Balanced transmembrane signals maintain a competent peripheral B cell pool limited in self-reactive B cells that may produce pathogenic autoantibodies. To identify molecules regulating peripheral B cell survival and tolerance to self-antigens (Ags), a gene modifier screen was performed with B cells from CD22-deficient C57BL/6 (CD22(-/-[B6])) mice that undergo activation-induced cell death (AICD) and fail to up-regulate c-Myc expression after B cell Ag receptor ligation. Likewise, lysozyme auto-Ag-specific B cells in Ig(Tg) hen egg lysozyme (HEL) transgenic mice inhabit the spleen but undergo AICD after auto-Ag encounter. This gene modifier screen identified EndoU, a single-stranded RNA-binding protein of ancient origin, as a major regulator of B cell survival in both models. EndoU gene disruption prevents AICD and normalizes c-Myc expression. These findings reveal that EndoU is a critical regulator of an unexpected and novel RNA-dependent pathway controlling peripheral B cell survival and Ag responsiveness that may contribute to peripheral B cell tolerance.

[Rubber band ligation in treatment of hemorrhoids: our experience].

PubMed

Gaj, F; Biviano, I; Sportelli, G; Candeloro, L

2015-01-01

Hemorrhoids are a very common condition. The treatment depends upon persistence and severity of symptoms. For hemorrhoids of II and III grade the rubber band ligation may be therapeutic. Our aim is to report the outcomes of rubber band ligation of hemorrhoids, with a follow up of 6 months. A total of 50 patients underwent rubber band ligation for symptomatic hemorrhoids (grade II and III) without prolapse, between June 2012 and June 2014. All patients underwent plug test to rule out presence of rectal mucosal prolapse and were classified according to PATE classification (1). Each hemorrhoid was ligated with one rubber band through a ligator. All patients were evaluated immediately at the end of the procedure, after ten days and six months after the treatment. Patient's demographic and operative data were collected and analyzed. The mean patients age was 47.6±12.3 years (range 24-72). All procedures were performed without complications. Before rubber band ligation, 42 patients had rectal bleeding, 38 had perineal discomfort and 27 had itching. Ten days after the treatment, 12 patients presented self-limited rectal bleeding, but 10 of these had more hemorrhoids and underwent a second rubber band ligation. No patients complained perineal discomfort, and 8 patients had itching; 78% and 16% of patients respectively, experienced feeling of a foreign body inside the canal anal and anal pain. After 6 months, only 13 patients were occasionally symptomatic: 4 patients had rectal bleeding, 2 had perineal discomfort and 4 had itching. Three more patients presented both perineal discomfort and hitching. None had the feeling of a foreign body in anal canal or anal pain. Rubber band ligation is an efficacious, cost-effective and simple treatment for the second and third degree hemorrhoids without rectal mucosal prolapsed. In our hands, no severe complications developed and minor complications could be handled with ease.

On-column trypsinization allows for re-use of matrix in modified multiplexed inhibitor beads assay.

PubMed

Petrovic, Voin; Olaisen, Camilla; Sharma, Animesh; Nepal, Anala; Bugge, Steffen; Sundby, Eirik; Hoff, Bård Helge; Slupphaug, Geir; Otterlei, Marit

2017-04-15

The Multiplexed Inhibitor Bead (MIB) assay is a previously published quantitative proteomic MS-based approach to study cellular kinomes. A rather extensive procedure, need for multiple custom-made kinase inhibitors and an inability to re-use the MIB-columns, has limited its applicability. Here we present a modified MIB assay in which elution of bound proteins is facilitated by on-column trypsinization. We tested the modified MIB assay by analyzing extract from three human cancer cell lines treated with the cytotoxic drugs cisplatin or docetaxel. Using only three immobilized kinase inhibitors, we were able to detect about 6000 proteins, including ∼40% of the kinome, as well as other signaling, metabolic and structural proteins. The method is reproducible and the MIB-columns are re-usable without loss of performance. This makes the MIB assay a simple, affordable, and rapid assay for monitoring changes in cellular signaling. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Invited Article: Generation of one-million-mode continuous-variable cluster state by unlimited time-domain multiplexing

NASA Astrophysics Data System (ADS)

Yoshikawa, Jun-ichi; Yokoyama, Shota; Kaji, Toshiyuki; Sornphiphatphong, Chanond; Shiozawa, Yu; Makino, Kenzo; Furusawa, Akira

2016-09-01

In recent quantum optical continuous-variable experiments, the number of fully inseparable light modes has drastically increased by introducing a multiplexing scheme either in the time domain or in the frequency domain. Here, modifying the time-domain multiplexing experiment reported in the work of Yokoyama et al. [Nat. Photonics 7, 982 (2013)], we demonstrate the successive generation of fully inseparable light modes for more than one million modes. The resulting multi-mode state is useful as a dual-rail continuous variable cluster state. We circumvent the previous problem of optical phase drifts, which has limited the number of fully inseparable light modes to around ten thousands, by continuous feedback control of the optical system.

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

PubMed Central

Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

2016-01-01

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets. PMID:27739510

Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR.

PubMed

Villalobos, Michael J; Betti, Christopher J; Vaughan, Andrew T M

2006-01-01

Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks introduced into any identifiable genomic location.

Control of Angular Intervals for Angle-Multiplexed Holographic Memory

NASA Astrophysics Data System (ADS)

Kinoshita, Nobuhiro; Muroi, Tetsuhiko; Ishii, Norihiko; Kamijo, Koji; Shimidzu, Naoki

2009-03-01

In angle-multiplexed holographic memory, the full width at half maximum of the Bragg selectivity curves is dependent on the angle formed between the medium and incident laser beams. This indicates the possibility of high density and high multiplexing number by varying the angular intervals between adjacent holograms. We propose an angular interval scheduling for closely stacking holograms into medium even when the angle range is limited. We obtained bit error rates of the order of 10-4 under the following conditions: medium thickness of 1 mm, laser beam wavelength of 532 nm, and angular multiplexing number of 300.

The role of the GABRA2 polymorphism in multiplex alcohol dependence families with minimal comorbidity: within-family association and linkage analyses.

PubMed

Matthews, Abigail G; Hoffman, Eric K; Zezza, Nicholas; Stiffler, Scott; Hill, Shirley Y

2007-09-01

The genes encoding the gamma-aminobutyric acid(A) (GABA(A)) receptor have been the focus of several recent studies investigating the genetic etiology of alcohol dependence. Analyses of multiplex families found a particular gene, GABRA2, to be highly associated with alcohol dependence, using within-family association tests and other methods. Results were confirmed in three case-control studies. The objective of this study was to investigate the GABRA2 gene in another collection of multiplex families. Analyses were based on phenotypic and genotypic data available for 330 individuals from 65 bigenerational pedigrees with a total of 232 alcohol-dependent subjects. A proband pair of same-sex siblings meeting Diagnostic and Statistical Manual of Mental Disorders, Third Edition, criteria for alcohol dependence was required for entry of a family into the study. One member of the proband pair was identified while in treatment for alcohol dependence. Linkage and association of GABRA2 and alcohol dependence were evaluated using SIBPAL (a nonparametric linkage package) and both the Pedigree Disequilibrium Test and the Family-Based Association Test, respectively. We find no evidence of a relationship between GABRA2 and alcohol dependence. Linkage analyses exhibited no linkage using affected/affected, affected/unaffected, and unaffected/unaffected sib pairs (all p's < .13). There was no evidence of a within-family association (all p's > .39). Comorbidity may explain why our results differ from those in the literature. The presence of primary drug dependence and/or other psychiatric disorders is minimal in our pedigrees, although several of the other previously published multiplex family analyses exhibit a greater degree of comorbidity.

Chemoselective ligation

DOEpatents

Saxon, Eliana [Albany, CA; Bertozzi, Carolyn Ruth [Berkeley, CA

2011-12-13

The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

Chemoselective ligation

DOEpatents

Saxon, Eliana; Bertozzi, Carolyn

2006-10-17

The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

Chemoselective ligation

DOEpatents

Saxon, Eliana [Albany, CA; Bertozzi, Carolyn R [Berkeley, CA

2011-05-10

The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

Chemoselective ligation

DOEpatents

Saxon, Eliana; Bertozzi, Carolyn Ruth

2010-11-23

The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

Chemoselective ligation

DOEpatents

Saxon, Eliana [Albany, CA; Bertozzi, Carolyn R [Berkeley, CA

2011-04-12

The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

Chemoselective ligation

DOEpatents

Saxon, Eliana; Bertozzi, Carolyn R.

2010-02-23

The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g. on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

Chemoselective ligation

DOEpatents

Saxon, Eliana [Albany, CA; Bertozzi, Carolyn [Berkeley, CA

2003-05-27

The present invention features a chemoselective ligation reaction that can be carried out under physiological conditions. In general, the invention involves condensation of a specifically engineered phosphine, which can provide for formation of an amide bond between the two reactive partners resulting in a final product comprising a phosphine moiety, or which can be engineered to comprise a cleavable linker so that a substituent of the phosphine is transferred to the azide, releasing an oxidized phosphine byproduct and producing a native amide bond in the final product. The selectivity of the reaction and its compatibility with aqueous environments provides for its application in vivo (e.g., on the cell surface or intracellularly) and in vitro (e.g., synthesis of peptides and other polymers, production of modified (e.g., labeled) amino acids).

Comparative Evaluation of Multiplex PCR and Routine Laboratory Phenotypic Methods for Detection of Carbapenemases among Gram Negative Bacilli.

PubMed

Solanki, Rachana; Vanjari, Lavanya; Subramanian, Sreevidya; B, Aparna; E, Nagapriyanka; Lakshmi, Vemu

2014-12-01

Carbapenem resistant pathogens cause infections associated with significant morbidity and mortality. This study evaluates the use of Multiplex PCR for rapid detection of carbapenemase genes among carbapenem resistant Gram negative bacteria in comparison with the existing phenotypic methods like modified Hodge test (MHT), combined disc test (CDT) and automated methods. A total of 100 Carbapenem resistant clinical isolates, [Escherichia coli (25), Klebsiella pneumoniae (35) P. aeruginosa (18) and Acinetobacter baumannii (22)] were screened for the presence of carbapenemases (bla NDM-1, bla VIM , blaIMP and blaKPC genes) by phenotype methods such as the modified Hodge test (MHT) and combined disc test (CDT) and the molecular methods such as Multiplex PCR. Seventy of the 100 isolates were MHT positive while, 65 isolates were positive by CDT. All the CDT positive isolates with EDTA and APB were Metallo betalactamase (MBL) and K. pneumoniae carbapenemase (KPC) producers respectively. bla NDM-1 was present as a lone gene in 44 isolates. In 14 isolates bla NDM-1 gene was present with blaKPC gene, and in one isolate bla NDM-1 gene was present with blaVIM , gene. Only one E. coli isolate had a lone blaKPC gene. We didn't find bla IMP gene in any of the isolates. Neither of the genes could be detected in 35 isolates. Accurate detection of the genes related with carbapenemase production by Molecular methods like Multiplex PCR overcome the limitations of the phenotypic methods and Automated systems.

Multiplex PCR for the detection of genes encoding aminoglycoside modifying enzymes and methicillin resistance among Staphylococcus species.

PubMed Central

Choi, Su Mi; Kim, Seung-Han; Kim, Hee-Jung; Lee, Dong-Gun; Choi, Jung-Hyun; Yoo, Jin-Hong; Kang, Jin-Han; Shin, Wan-Shik; Kang, Moon-Won

2003-01-01

We developed multiplex polymerase chain reaction (PCR) to detect aac(6 ')/aph(2 "), aph(3 ')-IIIa, and ant(4 ')-Ia, the genes encoding the most clinically relevant amino-glycoside modifying enzymes (AME), and simultaneously, the methicillin resistant gene, mecA, in Staphylococcus species. Clinical isolates of 45 S. aureus and 47 coagulase negative staphylococci (CNS) from tertiary university hospitals were tested by conventional susceptibility testing, using the agar dilution method and by multiplex PCR. Of a total of 92 isolates, 61 isolates were found to be methicillin-resistant. Of these, 54 isolates (89%) were found to be harboring mecA. Seventy-five percent of the 92 isolates demonstrated resistance to at least one of the aminoglycosides tested. Moreover, resistance to aminoglycosides was closely associated with methicillin-resistance (p<0.05). The most prevalent AME gene was aac(6 ')/aph(2 ") which was found in 65% of the isolates, and ant(4 ')-Ia and aph(3 ')-IIIa were present in 41% and 9% of the isolates, respectively. The concordance between methicillin-resistance and the presence of mecA gene was 98% in S. aureus and 81% in CNS. The concordance between gentamicin resistance and the presence of aac(6 ')/aph(2 ") gene was 100% in S. aureus and 85% in CNS. The multiplex PCR method that we developed appears to be both a more rapid and reliable than conventional method. PMID:14555812

Minimally Invasive Treatment for a Sacral Tarlov Cyst Through Tubular Retractors.

PubMed

Del Castillo-Calcáneo, Juan D; Navarro-Ramírez, Rodrigo; Nakhla, Jonathan; Kim, Eliana; Härtl, Roger

2017-12-01

Tarlov cysts (TC) are focal dilations of arachnoid and dura mater of the spinal posterior nerve root sheath that appear as cystic lesions of the nerve roots typically in the lower spine, especially in the sacrum, which can cause radicular symptoms when they increase in size and compress the nerve roots. Different open procedures have been described to treat TCs, but no minimally invasive procedures have been described to effectively address this pathology. A 29-year-old woman presented with right lower extremity pain and weakness. A magnetic resonance imaging scan demonstrated a lumbosacral TC that protruded through the right L5-S1 foramina. Through a small laminotomy, cyst drainage followed by neck ligation using a Scanlan modified technique through tubular retractors was performed. The patient recovered full motor function within the first days postoperatively and showed no signs of relapse at 6-month follow-up. Minimally invasive spine surgery through tubular retractors can be safely performed for successful excision and ligation of TC using a Scanlan modified technique. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparison of Multiplex Suspension Array Large-Panel Kits for Profiling Cytokines and Chemokines in Rheumatoid Arthritis Patients

PubMed Central

Khan, Imran H.; Krishnan, V.V.; Ziman, Melanie; Janatpour, Kim; Wun, Ted; Luciw, Paul A.; Tuscano, Joseph

2015-01-01

Background Multiplex analysis allows measurements of a large number of analytes simultaneously in each sample. Based on the Luminex multiplex technology (xMAP), kits for measuring multiple cytokines and chemokines (immunomodulators) are commercially available and are useful in investigations on inflammatory diseases. This study evaluated four multiplex kits (Bio-Plex, LINCOplex, Fluorokine, and Beadlyte) that contained 27, 29, 20 and 22 analytes each, respectively, for the analysis of immunomodulators in plasma of rheumatoid arthritis (RA) patients who underwent treatment with antibody against CD20 (rituximab), a B-cell reductive therapy. Methods Multiplex kits were tested on serial plasma samples obtained from six RA patients at baseline and multiple time points (3, 6, and 9 months) post-treatment with rituximab. The RA patients included in this study had previously failed therapy with disease modifying anti-arthritis drugs (DMARD) and treatment with anti-TNFα antibody (infliximab). Results Computer modeling and hierarchical cluster analysis of the multiplex data allowed a comparison of the performance of multiplex assay kits and revealed profiles of immunomodulators in the RA patients. Conclusions In plasma of RA patients who appeared to have benefited from rituximab treatment the profile of significantly elevated immunomodulators by at least two of the three kits (BioPlex, LINCOplex, Beadlyte), is as follows: IL-12p70, Eotaxin, IL-4, TNFα, Il-9, IL-1β, IFNγ, IL-10, IL-6, and IL-13. Immunomodulator profiling by multiplex analysis may provide useful plasma biomarkers for monitoring response to B-cell reductive therapy in RA patients. PMID:18823005

Cost and efficacy comparison of in vitro fertilization and tubal anastomosis for women after tubal ligation

PubMed Central

Messinger, Lauren B.; Alford, Connie E.; Csokmay, John M.; Henne, Melinda B.; Mumford, Sunni L.; Segars, James H.; Armstrong, Alicia Y.

2016-01-01

Objective To compare cost and efficacy of tubal anastomosis to in vitro fertilization (IVF) in women who desired fertility after a tubal ligation. Design Cost-effectiveness analysis. Setting Not applicable. Patient(s) Not applicable. Intervention(s) Not applicable. Main Outcome Measure(s) Cost per ongoing pregnancy. Result(s) Cost per ongoing pregnancy for women after tubal anastomosis ranged from $16,446 to $223,482 (2014 USD), whereas IVF ranged from $32,902 to $111,679 (2014 USD). Across maternal age groups <35 and 35–40, years tubal anastomosis was more cost effective than IVF for ongoing pregnancy. Sensitivity analyses validated these findings across a wide range of ongoing pregnancy probabilities as well as costs per procedure. Conclusion(s) Tubal anastomosis was the most cost-effective approach for most women less than 41 years of age, whereas IVF was the most cost-effective approach for women aged ≥41 years who desired fertility after tubal ligation. A model was created that can be modified based on cost and success rates in individual clinics for improved patient counseling. PMID:26006734

Cost and efficacy comparison of in vitro fertilization and tubal anastomosis for women after tubal ligation.

PubMed

Messinger, Lauren B; Alford, Connie E; Csokmay, John M; Henne, Melinda B; Mumford, Sunni L; Segars, James H; Armstrong, Alicia Y

2015-07-01

To compare cost and efficacy of tubal anastomosis to in vitro fertilization (IVF) in women who desired fertility after a tubal ligation. Cost-effectiveness analysis. Not applicable. Not applicable. Not applicable. Cost per ongoing pregnancy. Cost per ongoing pregnancy for women after tubal anastomosis ranged from $16,446 to $223,482 (2014 USD), whereas IVF ranged from $32,902 to $111,679 (2014 USD). Across maternal age groups <35 and 35-40, years tubal anastomosis was more cost effective than IVF for ongoing pregnancy. Sensitivity analyses validated these findings across a wide range of ongoing pregnancy probabilities as well as costs per procedure. Tubal anastomosis was the most cost-effective approach for most women less than 41 years of age, whereas IVF was the most cost-effective approach for women aged ≥41 years who desired fertility after tubal ligation. A model was created that can be modified based on cost and success rates in individual clinics for improved patient counseling. Copyright © 2015 American Society for Reproductive Medicine. All rights reserved.

Development of a qualitative, multiplex real-time PCR kit for screening of genetically modified organisms (GMOs).

PubMed

Dörries, Hans-Henno; Remus, Ivonne; Grönewald, Astrid; Grönewald, Cordt; Berghof-Jäger, Kornelia

2010-03-01

The number of commercially available genetically modified organisms (GMOs) and therefore the diversity of possible target sequences for molecular detection techniques are constantly increasing. As a result, GMO laboratories and the food production industry currently are forced to apply many different methods to reliably test raw material and complex processed food products. Screening methods have become more and more relevant to minimize the analytical effort and to make a preselection for further analysis (e.g., specific identification or quantification of the GMO). A multiplex real-time PCR kit was developed to detect the 35S promoter of the cauliflower mosaic virus, the terminator of the nopaline synthase gene of Agrobacterium tumefaciens, the 35S promoter from the figwort mosaic virus, and the bar gene of the soil bacterium Streptomyces hygroscopicus as the most widely used sequences in GMOs. The kit contains a second assay for the detection of plant-derived DNA to control the quality of the often processed and refined sample material. Additionally, the plant-specific assay comprises a homologous internal amplification control for inhibition control. The determined limits of detection for the five assays were 10 target copies/reaction. No amplification products were observed with DNAs of 26 bacterial species, 25 yeasts, 13 molds, and 41 not genetically modified plants. The specificity of the assays was further demonstrated to be 100% by the specific amplification of DNA derived from reference material from 22 genetically modified crops. The applicability of the kit in routine laboratory use was verified by testing of 50 spiked and unspiked food products. The herein described kit represents a simple and sensitive GMO screening method for the reliable detection of multiple GMO-specific target sequences in a multiplex real-time PCR reaction.

An A-T linker adapter polymerase chain reaction method for chromosome walking without restriction site cloning bias.

PubMed

Trinh, Quoclinh; Xu, Wentao; Shi, Hui; Luo, Yunbo; Huang, Kunlun

2012-06-01

A-T linker adapter polymerase chain reaction (PCR) was modified and employed for the isolation of genomic fragments adjacent to a known DNA sequence. The improvements in the method focus on two points. The first is the modification of the PO(4) and NH(2) groups in the adapter to inhibit the self-ligation of the adapter or the generation of nonspecific products. The second improvement is the use of the capacity of rTaq DNA polymerase to add an adenosine overhang at the 3' ends of digested DNA to suppress self-ligation in the digested DNA and simultaneously resolve restriction site clone bias. The combination of modifications in the adapter and in the digested DNA leads to T/A-specific ligation, which enhances the flexibility of this method and makes it feasible to use many different restriction enzymes with a single adapter. This novel A-T linker adapter PCR overcomes the inherent limitations of the original ligation-mediated PCR method such as low specificity and a lack of restriction enzyme choice. Moreover, this method also offers higher amplification efficiency, greater flexibility, and easier manipulation compared with other PCR methods for chromosome walking. Experimental results from 143 Arabidopsis mutants illustrate that this method is reliable and efficient in high-throughput experiments. Copyright © 2012 Elsevier Inc. All rights reserved.

Development of genomic microsatellite multiplex PCR using dye-labeled universal primer and its validation in pedigree analysis of Pacific oyster ( Crassostrea gigas)

NASA Astrophysics Data System (ADS)

Liu, Ting; Li, Qi; Song, Junlin; Yu, Hong

2017-02-01

There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.

Interlaboratory transfer of a PCR multiplex method for simultaneous detection of four genetically modified maize lines: Bt11, MON810, T25, and GA21.

PubMed

Hernández, Marta; Rodríguez-Lázaro, David; Zhang, David; Esteve, Teresa; Pla, Maria; Prat, Salomé

2005-05-04

The number of cultured hectares and commercialized genetically modified organisms (GMOs) has increased exponentially in the past 9 years. Governments in many countries have established a policy of labeling all food and feed containing or produced by GMOs. Consequently, versatile, laboratory-transferable GMO detection methods are in increasing demand. Here, we describe a qualitative PCR-based multiplex method for simultaneous detection and identification of four genetically modified maize lines: Bt11, MON810, T25, and GA21. The described system is based on the use of five primers directed to specific sequences in these insertion events. Primers were used in a single optimized multiplex PCR reaction, and sequences of the amplified fragments are reported. The assay allows amplification of the MON810 event from the 35S promoter to the hsp intron yielding a 468 bp amplicon. Amplification of the Bt11 and T25 events from the 35S promoter to the PAT gene yielded two different amplicons of 280 and 177 bp, respectively, whereas amplification of the 5' flanking region of the GA21 gave rise to an amplicon of 72 bp. These fragments are clearly distinguishable in agarose gels and have been reproduced successfully in a different laboratory. Hence, the proposed method comprises a rapid, simple, reliable, and sensitive (down to 0.05%) PCR-based assay, suitable for detection of these four GM maize lines in a single reaction.

Establishment of a Novel Murine Model of Ischemic Cardiomyopathy with Multiple Diffuse Coronary Lesions

PubMed Central

Nakaoka, Hajime; Nakagawa-Toyama, Yumiko; Nishida, Makoto; Okada, Takeshi; Kawase, Ryota; Yamashita, Taiji; Yuasa-Kawase, Miyako; Nakatani, Kazuhiro; Masuda, Daisaku; Ohama, Tohru; Sonobe, Takashi; Shirai, Mikiyasu; Komuro, Issei; Yamashita, Shizuya

2013-01-01

Objectives Atherosclerotic lesions of the coronary arteries are the pathological basis for myocardial infarction and ischemic cardiomyopathy. Progression of heart failure after myocardial infarction is associated with cardiac remodeling, which has been studied by means of coronary ligation in mice. However, this ligation model requires excellent techniques. Recently, a new murine model, HypoE mouse was reported to exhibit atherogenic Paigen diet-induced coronary atherosclerosis and myocardial infarction; however, the HypoE mice died too early to make possible investigation of cardiac remodeling. Therefore, we aimed to modify the HypoE mouse model to establish a novel model for ischemic cardiomyopathy caused by atherosclerotic lesions, which the ligation model does not exhibit. Methods and Results In our study, the sustained Paigen diet for the HypoE mice was shortened to 7 or 10 days, allowing the mice to survive longer. The 7-day Paigen diet intervention starting when the mice were 8 weeks old was adequate to permit the mice to survive myocardial infarction. Our murine model, called the “modified HypoE mouse”, was maintained until 8 weeks, with a median survival period of 36 days, after the dietary intervention (male, n = 222). Echocardiography demonstrated that the fractional shortening 2 weeks after the Paigen diet (n = 14) significantly decreased compared with that just before the Paigen diet (n = 6) (31.4±11.9% vs. 54.4±2.6%, respectively, P<0.01). Coronary angiography revealed multiple diffuse lesions. Cardiac remodeling and fibrosis were identified by serial analyses of cardiac morphological features and mRNA expression levels in tissue factors such as MMP-2, MMP-9, TIMP-1, collagen-1, and TGF-β. Conclusion Modified HypoE mice are a suitable model for ischemic cardiomyopathy with multiple diffuse lesions and may be considered as a novel and convenient model for investigations of cardiac remodeling on a highly atherogenic background. PMID:23950999

Electrochemical sensor for multiplex screening of genetically modified DNA: identification of biotech crops by logic-based biomolecular analysis.

PubMed

Liao, Wei-Ching; Chuang, Min-Chieh; Ho, Ja-An Annie

2013-12-15

Genetically modified (GM) technique, one of the modern biomolecular engineering technologies, has been deemed as profitable strategy to fight against global starvation. Yet rapid and reliable analytical method is deficient to evaluate the quality and potential risk of such resulting GM products. We herein present a biomolecular analytical system constructed with distinct biochemical activities to expedite the computational detection of genetically modified organisms (GMOs). The computational mechanism provides an alternative to the complex procedures commonly involved in the screening of GMOs. Given that the bioanalytical system is capable of processing promoter, coding and species genes, affirmative interpretations succeed to identify specified GM event in terms of both electrochemical and optical fashions. The biomolecular computational assay exhibits detection capability of genetically modified DNA below sub-nanomolar level and is found interference-free by abundant coexistence of non-GM DNA. This bioanalytical system, furthermore, sophisticates in array fashion operating multiplex screening against variable GM events. Such a biomolecular computational assay and biosensor holds great promise for rapid, cost-effective, and high-fidelity screening of GMO. Copyright © 2013 Elsevier B.V. All rights reserved.

Crosstalk mitigation using pilot assisted least square algorithm in OFDM-carrying orbital angular momentum multiplexed free-space-optical communication links.

PubMed

Sun, Tengfen; Liu, Minwen; Li, Yingchun; Wang, Min

2017-10-16

In this paper, we experimentally investigate the performance of crosstalk mitigation for 16-ary quadrature amplitude modulation orthogonal frequency division multiplexing (16QAM-OFDM) signals carrying orbital angular momentum (OAM) multiplexed free-space-optical communication (FSO) links using the pilot assisted Least Square (LS) algorithm. At the demodulating spatial light modulators (SLMs), we launch the distorted phase holograms which have the information of atmospheric turbulence obeying the modified Hill spectrum. And crosstalk can be introduced by these holograms with the experimental verification. The pilot assisted LS algorithm can efficiently improve the quality of system performance, the points of constellations get closer to the reference points and around two orders of magnitude improvement of bit-error rate (BER) is obtained.

A Liquid Array Platform For the Multiplexed Analysis of Synthetic Molecule-Protein Interactions

PubMed Central

Doran, Todd M.; Kodadek, Thomas

2014-01-01

Synthetic molecule microarrays, consisting of many different compounds spotted onto a planar surface such as modified glass or cellulose, have proven to be useful tools for the multiplexed analysis of small molecule- and peptide-protein interactions. However, these arrays are technically difficult to manufacture and use with high reproducibility and require specialized equipment. Here we report a more convenient alternative comprised of color-encoded beads that display a small molecule protein ligand on the surface. Quantitative, multiplexed assay of protein binding to up to 24 different ligands can be achieved using a common flow cytometer for the readout. This technology should be useful for evaluating hits from library screening efforts, the determination of structure activity relationships and for certain types of serological analyses. PMID:24245981

Large object investigation by digital holography with effective spectrum multiplexing under single-exposure approach

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

Liu, Ning, E-mail: coolboy006@sohu.com; Zhang, Yingying; Xie, Jun

2014-10-13

We present a method to investigate large object by digital holography with effective spectrum multiplexing under single-exposure approach. This method splits the original reference beam and redirects one of its branches as a second object beam. Through the modified Mach-Zehnder interferometer, the two object beams can illuminate different parts of the large object and create a spectrum multiplexed hologram onto the focal plane array of the charge-coupled device/complementary metal oxide semiconductor camera. After correct spectrum extraction and image reconstruction, the large object can be fully observed within only one single snap-shot. The flexibility and great performance make our method amore » very attractive and promising technique for large object investigation under common 632.8 nm illumination.« less

Helicase dependent OnChip-amplification and its use in multiplex pathogen detection.

PubMed

Andresen, Dennie; von Nickisch-Rosenegk, Markus; Bier, Frank F

2009-05-01

The need for fast, specific and sensitive multiparametric detection methods is an ever growing demand in molecular diagnostics. Here we report on a newly developed method, the helicase dependent OnChip amplification (OnChip-HDA). This approach integrates the analysis and detection in one single reaction thus leading to time and cost savings in multiparametric analysis. HDA is an isothermal amplification method that is not depending on thermocycling as known from PCR due to the helicases' ability to unwind DNA double-strands. We have combined the HDA with microarray based detection, making it suitable for multiplex detection. As an example we used the OnChip HDA in single and multiplex amplifications for the detection of the two pathogens N. gonorrhoeae and S. aureus directly on surface bound primers. We have successfully shown the OnChip-HDA and applied it for single- and duplex-detection of the pathogens N. gonorrhoeae and S. aureus. We have developed a new method, the OnChip-HDA for the multiplex detection of pathogens. Its simplicity in reaction setup and potential for miniaturization and multiparametric analysis is advantageous for the integration in miniaturized Lab on Chip systems, e.g. needed in point of care diagnostics.

Doubling the referral rate of monogenic diabetes through a nationwide information campaign--update on glucokinase gene mutations in a Polish cohort.

PubMed

Borowiec, M; Fendler, W; Antosik, K; Baranowska, A; Gnys, P; Zmyslowska, A; Malecki, M; Mlynarski, W

2012-12-01

In order to improve recruitment efficiency of patients with monogenic diabetes in Poland, in September 2010 a nationwide advertising campaign was launched to inform multiple target groups interested or participating in pediatric diabetologic care. Promotional actions aimed at informing physicians, patients, parents and educators were carried out through nationwide newspapers, medical and patient-developed websites and educational conference presentations. Recruitment efficiency was compared between September 2010 (publication of the first report on project's results) and the following 12 months. The number of families and patients referred to genetic screening was increased by 92% and 96% respectively nearly reaching the numbers recruited throughout the initial 4 years of the project. Participation of non-academic centers was also significantly increased from 2.3% to 7.5% (p = 0.0005). DNA sequencing and Multiplex Ligation-dependant Probe Amplification of the glucokinase gene resulted in finding 50 different mutations. Among those mutations, 19 were novel variants, which included: 17 missense mutations (predicted to be pathogenic according to bioinformatic analysis), 1 nonsense mutation and 1 mutation affecting a consensus intronic splice site. Advertising actions directed at increasing recruitment efficiency are a powerful and possibly neglected tool in screening for rare genetic disorders with a clinically defined phenotype. © 2011 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Use of next-generation sequencing to detect LDLR gene copy number variation in familial hypercholesterolemia.

PubMed

Iacocca, Michael A; Wang, Jian; Dron, Jacqueline S; Robinson, John F; McIntyre, Adam D; Cao, Henian; Hegele, Robert A

2017-11-01

Familial hypercholesterolemia (FH) is a heritable condition of severely elevated LDL cholesterol, caused predominantly by autosomal codominant mutations in the LDL receptor gene ( LDLR ). In providing a molecular diagnosis for FH, the current procedure often includes targeted next-generation sequencing (NGS) panels for the detection of small-scale DNA variants, followed by multiplex ligation-dependent probe amplification (MLPA) in LDLR for the detection of whole-exon copy number variants (CNVs). The latter is essential because ∼10% of FH cases are attributed to CNVs in LDLR ; accounting for them decreases false negative findings. Here, we determined the potential of replacing MLPA with bioinformatic analysis applied to NGS data, which uses depth-of-coverage analysis as its principal method to identify whole-exon CNV events. In analysis of 388 FH patient samples, there was 100% concordance in LDLR CNV detection between these two methods: 38 reported CNVs identified by MLPA were also successfully detected by our NGS method, while 350 samples negative for CNVs by MLPA were also negative by NGS. This result suggests that MLPA can be removed from the routine diagnostic screening for FH, significantly reducing associated costs, resources, and analysis time, while promoting more widespread assessment of this important class of mutations across diagnostic laboratories. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

A case report of Fanconi anemia diagnosed by genetic testing followed by prenatal diagnosis.

PubMed

Lee, Hwa Jeen; Park, Seungman; Kang, Hyoung Jin; Jun, Jong Kwan; Lee, Jung Ae; Lee, Dong Soon; Park, Sung Sup; Seong, Moon-Woo

2012-09-01

Fanconi anemia (FA) is a rare genetic disorder affecting multiple body systems. Genetic testing, including prenatal testing, is a prerequisite for the diagnosis of many clinical conditions. However, genetic testing is complicated for FA because there are often many genes that are associated with its development, and large deletions, duplications, or sequence variations are frequently found in some of these genes. This study describes successful genetic testing for molecular diagnosis, and subsequent prenatal diagnosis, of FA in a patient and his family in Korea. We analyzed all exons and flanking regions of the FANCA, FANCC, and FANCG genes for mutation identification and subsequent prenatal diagnosis. Multiplex ligation-dependent probe amplification analysis was performed to detect large deletions or duplications in the FANCA gene. Molecular analysis revealed two mutations in the FANCA gene: a frameshift mutation c.2546delC and a novel splice-site mutation c.3627-1G>A. The FANCA mutations were separately inherited from each parent, c.2546delC was derived from the father, whereas c.3627-1G>A originated from the mother. The amniotic fluid cells were c.3627-1G>A heterozygotes, suggesting that the fetus was unaffected. This is the first report of genetic testing that was successfully applied to molecular diagnosis of a patient and subsequent prenatal diagnosis of FA in a family in Korea.

FANCA and FANCG are the major Fanconi anemia genes in the Korean population.

PubMed

Park, J; Chung, N-G; Chae, H; Kim, M; Lee, S; Kim, Y; Lee, J-W; Cho, B; Jeong, D C; Park, I Y

2013-09-01

Fanconi anemia (FA) is a rare disorder characterized by physical abnormalities, bone marrow failure (BMF), increased risk of malignancies, and cellular hypersensitivity to DNA cross-linking agents. This study evaluated the genetic alterations in three major Fanconi genes (FANCA, FANCC, and FANCG) in 30 FA patients using multiplex ligation-dependent probe amplification and direct sequencing. Thirteen BMF patients were genetically classified as FA-A (n = 6, 46%) and FA-G (n = 7, 54%). Four common founder mutations were identified and included two FANCA mutations (c.2546delC and c.3720_3724delAAACA) and two FANCG mutations (c.307+1G>C and c.1066C>T), which had previously been commonly observed in a Japanese FA population. We also detected four novel deleterious mutations: c.2778+1G>C and c.3627-1G>A of FANCA, and c.1589_1591delATA and c.1761-1G>A of FANCG. This study shows that mutations in FANCA and FANCG are common in Korean FA patients and the existence of four common founder mutations in an East Asian FA population. Mutation screening workflow that includes these common mutations may be useful in the creation of an international database, and to better understand the ethnic characteristics of FA. © 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Update on Novel CCM Gene Mutations in Patients with Cerebral Cavernous Malformations.

PubMed

Scimone, Concetta; Bramanti, Placido; Alafaci, Concetta; Granata, Francesca; Piva, Francesco; Rinaldi, Carmela; Donato, Luigi; Greco, Federica; Sidoti, Antonina; D'Angelo, Rosalia

2017-02-01

Cerebral cavernous malformations (CCMs) are lesions affecting brain microvessels. The pathogenesis is not clearly understood. Conventional classification criterion is based on genetics, and thus, familial and sporadic forms can be distinguished; however, classification of sporadic cases with multiple lesions still remains uncertain. To date, three CCM causative genes have been identified: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. In our previous mutation screening, performed in a cohort of 95 Italian patients, with both sporadic and familial cases, we identified several mutations in CCM genes. This study represents further molecular screening in a cohort of 19 Italian patients enrolled by us in the few last years and classified into familial, sporadic and sporadic with multiple lesions cases. Direct sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis were performed to detect point mutations and large genomic rearrangements, respectively. Effects of detected mutations and single-nucleotide polymorphisms (SNPs) were evaluated by an in silico approach and by western blot analysis. A novel nonsense mutation in CCM1 and a novel missense mutation in CCM2 were detected; moreover, several CCM2 gene polymorphisms in sporadic CCM patients were reported. We believe that these data enrich the mutation spectrum of CCM genes, which is useful for genetic counselling to identify both familial and sporadic CCM cases, as early as possible.

Duplication of SOX9 associated with 46,XX ovotesticular disorder of sex development.

PubMed

López-Hernández, Berenice; Méndez, Juan Pablo; Coral-Vázquez, Ramón Mauricio; Benítez-Granados, Jesús; Zenteno, Juan Carlos; Villegas-Ruiz, Vanessa; Calzada-León, Raúl; Soderlund, Daniela; Canto, Patricia

2018-04-04

The purpose of the present study was to investigate whether ten unrelated SRY-negative individuals with this sex differentiation disorder presented a double dose of SOX9 as the cause of their disease. Ten unrelated SRY-negative 46,XX ovotesticular disorder of sexual development (DSD) subjects were molecularly studied. Multiplex-ligation dependent probe amplification (MLPA) and quantitative real-time PCR analysis (qRT-PCR) for SOX9 were performed. The MLPA analysis demonstrated that one patient presented a heterozygous duplication of the entire SOX9 coding region (above 1.3 value of peak ratio), as well as at least a ~ 483 kb upstream duplication. Moreover, no duplication of other SOX9 probes was observed corresponding to the region between -1007 and -1500 kb upstream. A qRT-PCR analysis showed a duplication of at least -581 kb upstream and ~1.63 kb of the coding region that encompasses exon 3. The limits of the duplication were mapped approximately from ~71539762 to 72122741 of Chr17. No molecular abnormalities were found in the remaining nine patients. This study is thought to be the first report regarding a duplication of SOX9 that is associated with the presence of 46,XX ovotesticular DSD, encompassing at least -581 kb upstream, and the almost entire coding region of the gene. Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

First Report of a Single Exon Deletion in TCOF1 Causing Treacher Collins Syndrome

PubMed Central

Beygo, J.; Buiting, K.; Seland, S.; Lüdecke, H.-J.; Hehr, U.; Lich, C.; Prager, B.; Lohmann, D.R.; Wieczorek, D.

2012-01-01

Treacher Collins syndrome (TCS) is a rare craniofacial disorder characterized by facial anomalies and ear defects. TCS is caused by mutations in the TCOF1 gene and follows autosomal dominant inheritance. Recently, mutations in the POLR1D and POLR1C genes have also been identified to cause TCS. However, in a subset of patients no causative mutation could be found yet. Inter- and intrafamilial phenotypic variability is high as is the variety of mainly family-specific mutations identified throughout TCOF1. No obvious correlation between pheno- and genotype could be observed. The majority of described point mutations, small insertions and deletions comprising only a few nucleotides within TCOF1 lead to a premature termination codon. We investigated a cohort of 112 patients with a tentative clinical diagnosis of TCS by multiplex ligation-dependent probe amplification (MLPA) to search for larger deletions not detectable with other methods used. All patients were selected after negative screening for mutations in TCOF1, POLR1D and POLR1C. In 1 patient with an unequivocal clinical diagnosis of TCS, we identified a 3.367 kb deletion. This deletion abolishes exon 3 and is the first described single exon deletion within TCOF1. On RNA level we observed loss of this exon which supposedly leads to haploinsufficiency of TREACLE, the nucleolar phosphoprotein encoded by TCOF1. PMID:22712005

First Report of a Single Exon Deletion in TCOF1 Causing Treacher Collins Syndrome.

PubMed

Beygo, J; Buiting, K; Seland, S; Lüdecke, H-J; Hehr, U; Lich, C; Prager, B; Lohmann, D R; Wieczorek, D

2012-01-01

Treacher Collins syndrome (TCS) is a rare craniofacial disorder characterized by facial anomalies and ear defects. TCS is caused by mutations in the TCOF1 gene and follows autosomal dominant inheritance. Recently, mutations in the POLR1D and POLR1C genes have also been identified to cause TCS. However, in a subset of patients no causative mutation could be found yet. Inter- and intrafamilial phenotypic variability is high as is the variety of mainly family-specific mutations identified throughout TCOF1. No obvious correlation between pheno- and genotype could be observed. The majority of described point mutations, small insertions and deletions comprising only a few nucleotides within TCOF1 lead to a premature termination codon. We investigated a cohort of 112 patients with a tentative clinical diagnosis of TCS by multiplex ligation-dependent probe amplification (MLPA) to search for larger deletions not detectable with other methods used. All patients were selected after negative screening for mutations in TCOF1, POLR1D and POLR1C. In 1 patient with an unequivocal clinical diagnosis of TCS, we identified a 3.367 kb deletion. This deletion abolishes exon 3 and is the first described single exon deletion within TCOF1. On RNA level we observed loss of this exon which supposedly leads to haploinsufficiency of TREACLE, the nucleolar phosphoprotein encoded by TCOF1.

Clinical and genetic characteristics of chinese patients with Birt-Hogg-Dubé syndrome.

PubMed

Liu, Yaping; Xu, Zhiyan; Feng, Ruie; Zhan, Yongzhong; Wang, Jun; Li, Guozhen; Li, Xue; Zhang, Weihong; Hu, Xiaowen; Tian, Xinlun; Xu, Kai-Feng; Zhang, Xue

2017-05-30

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder, the main manifestations of which are fibrofolliculomas, renal tumors, pulmonary cysts and recurrent pneumothorax. The known causative gene for BHD syndrome is the folliculin (FLCN) gene on chromosome 17p11.2. Studies of the FLCN mutation for BHD syndrome are less prevalent in Chinese populations than in Caucasian populations. Our study aims to investigate the genotype spectrum in a group of Chinese patients with BHD. We enrolled 51 patients with symptoms highly suggestive of BHD from January 2014 to February 2017. The FLCN gene was examined using PCR and Sanger sequencing in every patient, for those whose Sanger sequencing showed negative mutation results, multiplex ligation-dependent probe amplification (MLPA) testing was conducted to detect any losses of large segments. Among the 51 patients, 27 had FLCN germline mutations. In total, 20 mutations were identified: 14 were novel mutations, including 3 splice acceptor site mutations, 2 different deletions, 6 nonsense mutations, 1 missense mutation, 1 small insertion, and 1 deletion of the whole exon 8. We found a similar genotype spectrum but different mutant loci in Chinese patients with BHD compared with European and American patients, thus providing stronger evidence for the clinical molecular diagnosis of BHD in China. It suggests that mutation analysis of the FLCN gene should be systematically conducted in patients with cystic lung diseases.

Co-segregation of Norrie disease and idiopathic pulmonary hypertension in a family with a microdeletion of the NDP region at Xp11.3-p11.4.

PubMed

Staropoli, John F; Xin, Winnie; Sims, Katherine B

2010-11-01

Norrie disease is a rare X-linked congenital retinal vasculopathy that may be accompanied by sensorineural deafness, mental retardation, and other neurological deficits. Here we present a family in which Norrie disease co-segregated with either early-onset idiopathic pulmonary hypertension or sudden death preceded by a period of progressive dyspnea. Neither Norrie disease, nor its atypical variants described to date, have been associated with this extended clinical phenotype. Molecular analysis of the Norrie disease gene (NDP) and adjacent loci was performed by multiplex ligation-dependent probe amplification and comparative genomic hybridisation. Affected males in this family showed an inherited hemizygous deletion restricted to NDP and two immediately telomeric genes, monoamine oxidase-B (MAO-B) and monoamine oxidase-A (MAO-A), which encode closely related enzymes that metabolize biogenic amines including serotonin, dopamine, and norepinephrine. Sequencing of the deletion junction showed an unusual pattern in which a region of microhomology flanked intervening genomic sequence. Because abnormalities of biogenic amines, particularly serotonin, have been implicated in the pathophysiology of pulmonary hypertension, we propose that presumed MAO deficiency in these patients may represent a novel risk factor for pulmonary hypertension, particularly forms with very early onset. Fine-mapping of other microdeletions at this locus may provide insights into additional mechanisms for nonrecurrent genomic rearrangements at this and other chromosomal loci.

Craniosynostosis in 10q26 deletion patients: A consequence of brain underdevelopment or altered suture biology?

PubMed

Faria, Ágatha Cristhina; Rabbi-Bortolini, Eliete; Rebouças, Maria R G O; de S Thiago Pereira, Andréia L A; Frasson, Milena G Tonini; Atique, Rodrigo; Lourenço, Naila Cristina V; Rosenberg, Carla; Kobayashi, Gerson S; Passos-Bueno, Maria Rita; Errera, Flávia Imbroisi Valle

2016-02-01

Approximately a hundred patients with terminal 10q deletions have been described. They present with a wide range of clinical features always accompanied by delayed development, intellectual disability and craniofacial dysmorphisms. Here, we report a girl and a boy with craniosynostosis, developmental delay and other congenital anomalies. Karyotyping and molecular analysis including Multiplex Ligation dependent probe amplification (MLPA) and Array Comparative Genomic Hybridization (aCGH) were performed in both patients. We detected a 13.1 Mb pure deletion at 10q26.12-q26.3 in the girl and a 10.9 Mb pure deletion at 10q26.13-q26.3 in the boy, both encompassing about 100 genes. The clinical and molecular findings in these patients reinforce the importance of the DOCK1 smallest region of overlap I (SRO I), previously suggested to explain the clinical signs, and together with a review of the literature suggest a second 3.5 Mb region important for the phenotype (SRO II). Genotype-phenotype correlations and literature data suggest that the craniosynostosis is not directly related to dysregulated signaling in suture development, but may be secondary to alterations in brain development instead. Further, genes at 10q26 may be involved in the molecular crosstalk between brain and cranial vault. © 2015 Wiley Periodicals, Inc.

PAX6 molecular analysis and genotype–phenotype correlations in families with aniridia from Australasia and Southeast Asia

PubMed Central

Rudkin, Adam K.; Dubowsky, Andrew; Casson, Robert J.; Muecke, James S.; Mancel, Erica; Whiting, Mark; Mills, Richard A.D.; Burdon, Kathryn P.; Craig, Jamie E.

2018-01-01

Purpose Aniridia is a congenital disorder caused by variants in the PAX6 gene. In this study, we assessed the involvement of PAX6 in patients with aniridia from Australasia and Southeast Asia. Methods Twenty-nine individuals with aniridia from 18 families originating from Australia, New Caledonia, Cambodia, Sri Lanka, and Bhutan were included. The PAX6 gene was investigated for sequence variants and analyzed for deletions with multiplex ligation-dependent probe amplification. Results We identified 11 sequence variants and six chromosomal deletions, including one in mosaic. Four deleterious sequence variants were novel: p.(Pro81HisfsTer12), p.(Gln274Ter), p.(Ile29Thr), and p.(Met1?). Ocular complications were associated with a progressive loss of visual function as shown by a visual acuity ≤ 1.00 logMAR reported in 65% of eyes. The prevalence of keratopathy was statistically significantly higher in the Australasian cohort (78.6%) compared with the Southeast Asian cohort (9.1%, p=0.002). Variants resulting in protein truncating codons displayed limited genotype–phenotype correlations compared with other variants. Conclusions PAX6 variants and deletions were identified in 94% of patients with aniridia from Australasia and Southeast Asia. This study is the first report of aniridia and variations in PAX6 in individuals from Cambodia, Sri Lanka, Bhutan, and New Caledonia, and the largest cohort from Australia. PMID:29618921

Longitudinal molecular characterization of endoscopic specimens from colorectal lesions

PubMed Central

Minarikova, Petra; Benesova, Lucie; Halkova, Tereza; Belsanova, Barbora; Suchanek, Stepan; Cyrany, Jiri; Tuckova, Inna; Bures, Jan; Zavoral, Miroslav; Minarik, Marek

2016-01-01

AIM: To compare molecular profiles of proximal colon, distal colon and rectum in large adenomas, early and late carcinomas. To assess feasibility of testing directed at molecular markers from this study in routine clinical practice. METHODS: A prospective 3-year study has resulted in the acquisition of samples from 159 large adenomas and 138 carcinomas along with associated clinical parameters including localization, grade and histological type for adenomas and localization and stage for carcinomas. A complex molecular phenotyping has been performed using multiplex ligation-dependent probe amplification technique for the evaluation of CpG-island methylator phenotype (CIMP), PCR fragment analysis for detection of microsatellite instability and denaturing capillary electrophoresis for sensitive detection of somatic mutations in KRAS, BRAF, TP53 and APC genes. RESULTS: Molecular types according to previously introduced Jass classification have been evaluated for large adenomas and early and late carcinomas. An increase in CIMP+ type, eventually accompanied with KRAS mutations, was notable between large adenomas and early carcinomas. As expected, the longitudinal observations revealed a correlation of the CIMP+/BRAF+ type with proximal location. CONCLUSION: Prospective molecular classification of tissue specimens is feasible in routine endoscopy practice. Increased frequency of some molecular types corresponds to the developmental stages of colorectal tumors. As expected, a clear distinction is notable for tumors located in proximal colon supposedly arising from the serrated (methylation) pathway. PMID:27239120

Recognizing the tenascin-X deficient type of Ehlers-Danlos syndrome: a cross-sectional study in 17 patients.

PubMed

Demirdas, S; Dulfer, E; Robert, L; Kempers, M; van Beek, D; Micha, D; van Engelen, B G; Hamel, B; Schalkwijk, J; Loeys, B; Maugeri, A; Voermans, N C

2017-03-01

The tenascin-X (TNX) deficient type Ehlers-Danlos syndrome (EDS) is similar to the classical type of EDS. Because of the limited awareness among geneticists and the challenge of the molecular analysis of the TNXB gene, the TNX-deficient type EDS is probably to be under diagnosed. We therefore performed an observational, cross-sectional study. History and physical examination were performed. Results of serum TNX measurements were collected and mutation analysis was performed by a combination of next-generation sequencing (NGS), Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Included were 17 patients of 11 families with autosomal recessive inheritance and childhood onset. All patients had hyperextensible skin without atrophic scarring. Hypermobility of the joints was observed in 16 of 17 patients. Deformities of the hands and feet were observed frequently. TNX serum level was tested and absent in 11 patients (seven families). Genetic testing was performed in all families; 12 different mutations were detected, most of which are suspected to lead to non-sense mRNA mediated decay. In short, patients with the TNX-deficient type EDS typically have generalized joint hypermobility, skin hyperextensibility and easy bruising. In contrast to the classical type, the inheritance pattern is autosomal recessive and atrophic scarring is absent. Molecular analysis of TNXB in a diagnostic setting is challenging. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Atypical multifocal Dravet syndrome lacks generalized seizures and may show later cognitive decline.

PubMed

Kim, Young Ok; Bellows, Susannah; McMahon, Jacinta M; Iona, Xenia; Damiano, John; Dibbens, Leanne; Kelley, Kent; Gill, Deepak; Cross, J Helen; Berkovic, Samuel F; Scheffer, Ingrid E

2014-01-01

To show that atypical multifocal Dravet syndrome is a recognizable, electroclinical syndrome associated with sodium channel gene (SCN1A) mutations that readily escapes diagnosis owing to later cognitive decline and tonic seizures. Eight patients underwent electroclinical characterization. SCN1A was sequenced and copy number variations sought by multiplex ligation-dependent probe amplification. All patients were female (age range at assessment 5-26y) with median seizure onset at 6.5 months (range 4-19mo). The initial seizure was brief in seven and status epilepticus only occurred in one; three were febrile. Focal seizures occurred in four patients and bilateral convulsion in the other four. All patients developed multiple focal seizure types and bilateral convulsions, with seizure clusters in six. The most common focal seizure semiology (six out of eight) comprised unilateral clonic activity. Five also had focal or asymmetric tonic seizures. Rare or transient myoclonic seizures occurred in six individuals, often triggered by specific antiepileptic drugs. Developmental slowing occurred in all: six between 3 years and 8 years, and two around 1 year 6 months. Cognitive outcome varied from severe to mild intellectual disability. Multifocal epileptiform discharges were seen on electroencephalography. Seven out of eight patients had SCN1A mutations. Atypical, multifocal Dravet syndrome with SCN1A mutations may not be recognized because of later cognitive decline and frequent tonic seizures. © 2013 Mac Keith Press.

Tooth agenesis in osteogenesis imperfecta related to mutations in the collagen type I genes.

PubMed

Malmgren, B; Andersson, K; Lindahl, K; Kindmark, A; Grigelioniene, G; Zachariadis, V; Dahllöf, G; Åström, E

2017-01-01

Osteogenesis imperfecta (OI) is a heterogeneous group of disorders of connective tissue, mainly caused by mutations in the collagen type I genes (COL1A1 and COL1A2). Tooth agenesis is a common feature of OI. We investigated the association between tooth agenesis and collagen type I mutations in individuals with OI. In this cohort study, 128 unrelated individuals with OI were included. Panoramic radiographs were analyzed regarding dentinogenesis imperfecta (DGI) and congenitally missing teeth. The collagen I genes were sequenced in all individuals, and in 25, multiplex ligation-dependent probe amplification was performed. Mutations in the COL1A1 and COL1A2 genes were found in 104 of 128 individuals. Tooth agenesis was diagnosed in 17% (hypodontia 11%, oligodontia 6%) and was more frequent in those with DGI (P = 0.016), and in those with OI type III, 47%, compared to those with OI types I, 12% (P = 0.003), and IV, 13% (P = 0.017). Seventy-five percent of the individuals with oligodontia (≥6 missing teeth) had qualitative mutations, but there was no association with OI type, gender, or presence of DGI. The prevalence of tooth agenesis is high (17%) in individuals with OI, and OI caused by a qualitative collagen I mutation is associated with oligodontia. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Assessment of the role of copy-number variants in 150 patients with congenital heart defects.

PubMed

Derwińska, Katarzyna; Bartnik, Magdalena; Wiśniowiecka-Kowalnik, Barbara; Jagła, Mateusz; Rudziński, Andrzej; Pietrzyk, Jacek J; Kawalec, Wanda; Ziółkowska, Lidia; Kutkowska-Kaźmierczak, Anna; Gambin, Tomasz; Sykulski, Maciej; Shaw, Chad A; Gambin, Anna; Mazurczak, Tadeusz; Obersztyn, Ewa; Bocian, Ewa; Stankiewicz, Paweł

2012-01-01

Congenital heart defects are the most common group of major birth anomalies and one of the leading causes of infant deaths. Mendelian and chromosomal syndromes account for about 20% of congenital heart defects and in some cases are associated with other malformations, intellectual disability, and/or dysmorphic features. The remarkable conservation of genetic pathways regulating heart development in animals suggests that genetic factors can be responsible for a significantly higher percentage of cases. Assessment of the role of CNVs in the etiology of congenital heart defects using microarray studies. Genome-wide array comparative genomic hybridization, targeting genes known to play an important role in heart development or responsible for abnormal cardiac phenotype was used in the study on 150 patients. In addition, we have used multiplex ligation-dependent probe amplification specific for chromosome 22q11.2 region. We have identified 21 copy-number variants, including 13 known causative recurrent rearrangements (12 deletions 22q11.2 and one deletion 7q11.23), three potentially pathogenic duplications (5q14.2, 15q13.3, and 22q11.2), and five variants likely benign for cardiac anomalies. We suggest that abnormal copy-number of the ARRDC3 and KLF13 genes can be responsible for heart defects. Our study demonstrates that array comparative genomic hybridization enables detection of clinically significant chromosomal imbalances in patients with congenital heart defects.

Copy number abnormality of acute lymphoblastic leukemia cell lines based on their genetic subtypes.

PubMed

Tomoyasu, Chihiro; Imamura, Toshihiko; Tomii, Toshihiro; Yano, Mio; Asai, Daisuke; Goto, Hiroaki; Shimada, Akira; Sanada, Masashi; Iwamoto, Shotaro; Takita, Junko; Minegishi, Masayoshi; Inukai, Takeshi; Sugita, Kanji; Hosoi, Hajime

2018-05-21

In this study, we performed genetic analysis of 83 B cell precursor acute lymphoblastic leukemia (B-ALL) cell lines. First, we performed multiplex ligation-dependent probe amplification analysis to identify copy number abnormalities (CNAs) in eight genes associated with B-ALL according to genetic subtype. In Ph + B-ALL cell lines, the frequencies of IKZF1, CDKN2A/2B, BTG1, and PAX5 deletion were significantly higher than those in Ph - B-ALL cell lines. The frequency of CDKN2A/2B deletion in KMT2A rearranged cell lines was significantly lower than that in non-KMT2A rearranged cell lines. These findings suggest that CNAs are correlated with genetic subtype in B-ALL cell lines. In addition, we determined that three B-other ALL cell lines had IKZF1 deletions (YCUB-5, KOPN49, and KOPN75); we therefore performed comprehensive genetic analysis of these cell lines. YCUB-5, KOPN49, and KOPN75 had P2RY8-CRLF2, IgH-CRLF2, and PAX5-ETV6 fusions, respectively. Moreover, targeted capture sequencing revealed that YCUB-5 had JAK2 R683I and KRAS G12D, and KOPN49 had JAK2 R683G and KRAS G13D mutations. These data may contribute to progress in the field of leukemia research.

Germline rearrangements in families with strong family history of glioma and malignant melanoma, colon, and breast cancer

PubMed Central

Andersson, Ulrika; Wibom, Carl; Cederquist, Kristina; Aradottir, Steina; Borg, Åke; Armstrong, Georgina N.; Shete, Sanjay; Lau, Ching C.; Bainbridge, Matthew N.; Claus, Elizabeth B.; Barnholtz-Sloan, Jill; Lai, Rose; Il'yasova, Dora; Houlston, Richard S.; Schildkraut, Joellen; Bernstein, Jonine L.; Olson, Sara H.; Jenkins, Robert B.; Lachance, Daniel H.; Wrensch, Margaret; Davis, Faith G.; Merrell, Ryan; Johansen, Christoffer; Sadetzki, Siegal; Bondy, Melissa L.; Melin, Beatrice S.; Adatto, Phyllis; Morice, Fabian; Payen, Sam; McQuinn, Lacey; McGaha, Rebecca; Guerra, Sandra; Paith, Leslie; Roth, Katherine; Zeng, Dong; Zhang, Hui; Yung, Alfred; Aldape, Kenneth; Gilbert, Mark; Weinberger, Jeffrey; Colman, Howard; Conrad, Charles; de Groot, John; Forman, Arthur; Groves, Morris; Levin, Victor; Loghin, Monica; Puduvalli, Vinay; Sawaya, Raymond; Heimberger, Amy; Lang, Frederick; Levine, Nicholas; Tolentino, Lori; Saunders, Kate; Thach, Thu-Trang; Iacono, Donna Dello; Sloan, Andrew; Gerson, Stanton; Selman, Warren; Bambakidis, Nicholas; Hart, David; Miller, Jonathan; Hoffer, Alan; Cohen, Mark; Rogers, Lisa; Nock, Charles J; Wolinsky, Yingli; Devine, Karen; Fulop, Jordonna; Barrett, Wendi; Shimmel, Kristen; Ostrom, Quinn; Barnett, Gene; Rosenfeld, Steven; Vogelbaum, Michael; Weil, Robert; Ahluwalia, Manmeet; Peereboom, David; Staugaitis, Susan; Schilero, Cathy; Brewer, Cathy; Smolenski, Kathy; McGraw, Mary; Naska, Theresa; Rosenfeld, Steven; Ram, Zvi; Blumenthal, Deborah T.; Bokstein, Felix; Umansky, Felix; Zaaroor, Menashe; Cohen, Avi; Tzuk-Shina, Tzeela; Voldby, Bo; Laursen, René; Andersen, Claus; Brennum, Jannick; Henriksen, Matilde Bille; Marzouk, Maya; Davis, Mary Elizabeth; Boland, Eamon; Smith, Marcel; Eze, Ogechukwu; Way, Mahalia; Lada, Pat; Miedzianowski, Nancy; Frechette, Michelle; Paleologos, Nina; Byström, Gudrun; Svedberg, Eva; Huggert, Sara; Kimdal, Mikael; Sandström, Monica; Brännström, Nikolina; Hayat, Amina; Tihan, Tarik; Zheng, Shichun; Berger, Mitchel; Butowski, Nicholas; Chang, Susan; Clarke, Jennifer; Prados, Michael; Rice, Terri; Sison, Jeannette; Kivett, Valerie; Duo, Xiaoqin; Hansen, Helen; Hsuang, George; Lamela, Rosito; Ramos, Christian; Patoka, Joe; Wagenman, Katherine; Zhou, Mi; Klein, Adam; McGee, Nora; Pfefferle, Jon; Wilson, Callie; Morris, Pagan; Hughes, Mary; Britt-Williams, Marlin; Foft, Jessica; Madsen, Julia; Polony, Csaba; McCarthy, Bridget; Zahora, Candice; Villano, John; Engelhard, Herbert; Borg, Ake; Chanock, Stephen K; Collins, Peter; Elston, Robert; Kleihues, Paul; Kruchko, Carol; Petersen, Gloria; Plon, Sharon; Thompson, Patricia; Johansen, C.; Sadetzki, S.; Melin, B.; Bondy, Melissa L.; Lau, Ching C.; Scheurer, Michael E.; Armstrong, Georgina N.; Liu, Yanhong; Shete, Sanjay; Yu, Robert K.; Aldape, Kenneth D.; Gilbert, Mark R.; Weinberg, Jeffrey; Houlston, Richard S.; Hosking, Fay J.; Robertson, Lindsay; Papaemmanuil, Elli; Claus, Elizabeth B.; Claus, Elizabeth B.; Barnholtz-Sloan, Jill; Sloan, Andrew E.; Barnett, Gene; Devine, Karen; Wolinsky, Yingli; Lai, Rose; McKean-Cowdin, Roberta; Il'yasova, Dora; Schildkraut, Joellen; Sadetzki, Siegal; Yechezkel, Galit Hirsh; Bruchim, Revital Bar-Sade; Aslanov, Lili; Sadetzki, Siegal; Johansen, Christoffer; Kosteljanetz, Michael; Broholm, Helle; Bernstein, Jonine L.; Olson, Sara H.; Schubert, Erica; DeAngelis, Lisa; Jenkins, Robert B.; Yang, Ping; Rynearson, Amanda; Andersson, Ulrika; Wibom, Carl; Henriksson, Roger; Melin, Beatrice S.; Cederquist, Kristina; Aradottir, Steina; Borg, Åke; Merrell, Ryan; Lada, Patricia; Wrensch, Margaret; Wiencke, John; Wiemels, Joe; McCoy, Lucie; McCarthy, Bridget J.; Davis, Faith G.

2014-01-01

Background Although familial susceptibility to glioma is known, the genetic basis for this susceptibility remains unidentified in the majority of glioma-specific families. An alternative approach to identifying such genes is to examine cancer pedigrees, which include glioma as one of several cancer phenotypes, to determine whether common chromosomal modifications might account for the familial aggregation of glioma and other cancers. Methods Germline rearrangements in 146 glioma families (from the Gliogene Consortium; http://www.gliogene.org/) were examined using multiplex ligation-dependent probe amplification. These families all had at least 2 verified glioma cases and a third reported or verified glioma case in the same family or 2 glioma cases in the family with at least one family member affected with melanoma, colon, or breast cancer.The genomic areas covering TP53, CDKN2A, MLH1, and MSH2 were selected because these genes have been previously reported to be associated with cancer pedigrees known to include glioma. Results We detected a single structural rearrangement, a deletion of exons 1-6 in MSH2, in the proband of one family with 3 cases with glioma and one relative with colon cancer. Conclusions Large deletions and duplications are rare events in familial glioma cases, even in families with a strong family history of cancers that may be involved in known cancer syndromes. PMID:24723567

Quantification of the methylation status of the PWS/AS imprinted region: comparison of two approaches based on bisulfite sequencing and methylation-sensitive MLPA.

PubMed

Dikow, Nicola; Nygren, Anders Oh; Schouten, Jan P; Hartmann, Carolin; Krämer, Nikola; Janssen, Bart; Zschocke, Johannes

2007-06-01

Standard methods used for genomic methylation analysis allow the detection of complete absence of either methylated or non-methylated alleles but are usually unable to detect changes in the proportion of methylated and unmethylated alleles. We compare two methods for quantitative methylation analysis, using the chromosome 15q11-q13 imprinted region as model. Absence of the non-methylated paternal allele in this region leads to Prader-Willi syndrome (PWS) whilst absence of the methylated maternal allele results in Angelman syndrome (AS). A proportion of AS is caused by mosaic imprinting defects which may be missed with standard methods and require quantitative analysis for their detection. Sequence-based quantitative methylation analysis (SeQMA) involves quantitative comparison of peaks generated through sequencing reactions after bisulfite treatment. It is simple, cost-effective and can be easily established for a large number of genes. However, our results support previous suggestions that methods based on bisulfite treatment may be problematic for exact quantification of methylation status. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) avoids bisulfite treatment. It detects changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in one simple reaction. Once established in a laboratory setting, the method is more accurate, reliable and less time consuming.

Germline PMS2 mutation screened by mismatch repair protein immunohistochemistry of colorectal cancer in Japan.

PubMed

Sugano, Kokichi; Nakajima, Takeshi; Sekine, Shigeki; Taniguchi, Hirokazu; Saito, Shinya; Takahashi, Masahiro; Ushiama, Mineko; Sakamoto, Hiromi; Yoshida, Teruhiko

2016-11-01

Germline PMS2 gene mutations were detected by RT-PCR/direct sequencing of total RNA extracted from puromycin-treated peripheral blood lymphocytes (PBL) and multiplex ligation-dependent probe amplification (MLPA) analyses of Japanese patients with colorectal cancer (CRC) fulfilling either the revised Bethesda Guidelines or being an age at disease onset of younger than 70 years, and screened by mismatch repair protein immunohistochemistry of formalin-fixed paraffin embedded sections. Of the 501 subjects examined, 7 (1.40%) showed the downregulated expression of the PMS2 protein alone and were referred to the genetic counseling clinic. Germline PMS2 mutations were detected in 6 (85.7%), including 3 nonsense and 1 frameshift mutations by RT-PCR/direct sequencing and 2 genomic deletions by MLPA. No mutations were identified in the other MMR genes (i.e. MSH2, MLH1 and MSH6). The prevalence of the downregulated expression of the PMS2 protein alone was 1.40% among the subjects examined and IHC results predicted the presence of PMS2 germline mutations. RT-PCR from puromycin-treated PBL and MLPA may be employed as the first screening step to detect PMS2 mutations without pseudogene interference, followed by the long-range PCR/nested PCR validation using genomic DNA. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Avoidance of pseudogene interference in the detection of 3' deletions in PMS2.

PubMed

Vaughn, Cecily P; Hart, Kimberly J; Samowitz, Wade S; Swensen, Jeffrey J

2011-09-01

Lynch syndrome is characterized by mutations in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2. In PMS2, detection of mutations is confounded by numerous pseudogenes. Detection of 3' deletions is particularly complicated by the pseudogene PMS2CL, which has strong similarity to PMS2 exons 9 and 11-15, due to extensive gene conversion. A newly designed multiplex ligation-dependent probe amplification (MLPA) kit incorporates probes for variants found in both PMS2 and PMS2CL. This provides detection of deletions, but does not allow localization of deletions to the gene or pseudogene. To address this, we have developed a methodology incorporating reference samples with known copy numbers of variants, and paired MLPA results with sequencing of PMS2 and PMS2CL. We tested a subset of clinically indicated samples for which mutations were either unidentified or not fully characterized using existing methods. We identified eight unrelated patients with deletions encompassing exons 9-15, 11-15, 13-15, 14-15, and 15. By incorporating specific, characterized reference samples and sequencing the gene and pseudogene it is possible to identify deletions in this region of PMS2 and provide clinically relevant results. This methodology represents a significant advance in the diagnosis of patients with Lynch syndrome caused by PMS2 mutations. © 2011 Wiley-Liss, Inc.

The importance of biochemical and genetic findings in the diagnosis of atypical Norrie disease.

PubMed

Rodríguez-Muñoz, Ana; García-García, Gema; Menor, Francisco; Millán, José M; Tomás-Vila, Miguel; Jaijo, Teresa

2018-01-26

Norrie disease (ND) is a rare X-linked disorder characterized by bilateral congenital blindness. ND is caused by a mutation in the Norrie disease pseudoglioma (NDP) gene, which encodes a 133-amino acid protein called norrin. Intragenic deletions including NDP and adjacent genes have been identified in ND patients with a more severe neurologic phenotype. We report the biochemical, molecular, clinical and radiological features of two unrelated affected males with a deletion including NDP and MAO genes. Biochemical and genetic analyses were performed to understand the atypical phenotype and radiological findings. Biogenic amines in cerebrospinal fluid (CSF) were measured by high-performance liquid chromatography. The coding exons of NDP gene were amplified by polymerase chain reaction. Multiplex ligation-dependent probe amplification and chromosomal microarray were carried out on both affected males. Computed tomography and magnetic resonance imaging were performed on the two patients. In one patient, the serotonin and catecholamine metabolite levels in CSF were virtually undetectable. In both patients, genetic studies revealed microdeletions in the Xp11.3 region, involving the NDP, MAOA and MAOB genes. Radiological examination demonstrated brain and cerebellar atrophy. We suggest that alterations caused by MAO deficit may remain during the first years of life. Clinical phenotype, biochemical findings and neuroimaging can guide the genetic study in patients with atypical ND and help us to a better understanding of this disease.

Heterozygous deletion at the SOX10 gene locus in two patients from a Chinese family with Waardenburg syndrome type II.

PubMed

Wenzhi, He; Ruijin, Wen; Jieliang, Li; Xiaoyan, Ma; Haibo, Liu; Xiaoman, Wang; Jiajia, Xian; Shaoying, Li; Shuanglin, Li; Qing, Li

2015-10-01

Waardenburg syndrome (WS) is a rare disease characterized by sensorineural deafness and pigment disturbance. To date, almost 100 mutations have been reported, but few reports on cases with SOX10 gene deletion. The inheritance pattern of SOX10 gene deletion is still unclear. Our objective was to identify the genetic causes of Waardenburg syndrome type II in a two-generation Chinese family. Clinical evaluations were conducted in both of the patients. Microarray analysis and multiplex ligation-dependent probe amplification (MLPA) were performed to identify disease-related copy number variants (CNVs). DNA sequencing of the SOX10, MITF and SNAI2 genes was performed to identify the pathogenic mutation responsible for WS2. A 280kb heterozygous deletion at the 22q13.1 chromosome region (including SOX10) was detected in both of the patients. No mutation was found in the patients, unaffected family members and 30 unrelated healthy controls. This report is the first to describe SOX10 heterozygous deletions in Chinese WS2 patients. Our result conform the thesis that heterozygous deletions at SOX10 is an important pathogenicity for WS, and present as autosomal dominant inheritance. Nevertheless, heterozygous deletion of the SOX10 gene would be worth investigating to understand their functions and contributions to neurologic phenotypes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Focal cortical malformations in children with early infantile epilepsy and PCDH19 mutations: case report.

PubMed

Kurian, Mary; Korff, Christian M; Ranza, Emmanuelle; Bernasconi, Andrea; Lübbig, Anja; Nangia, Srishti; Ramelli, Gian Paolo; Wohlrab, Gabriele; Nordli, Douglas R; Bast, Thomas

2018-01-01

In this case report we assess the occurrence of cortical malformations in children with early infantile epilepsy associated with variants of the gene protocadherin 19 (PCDH19). We describe the clinical course, and electrographic, imaging, genetic, and neuropathological features in a cohort of female children with pharmacoresistant epilepsy. All five children (mean age 10y) had an early onset of epilepsy during infancy and a predominance of fever sensitive seizures occurring in clusters. Cognitive impairment was noted in four out of five patients. Radiological evidence of cortical malformations was present in all cases and, in two patients, validated by histology. Sanger sequencing and Multiplex Ligation-dependent Probe Amplification analysis of PCDH19 revealed pathogenic variants in four patients. In one patient, array comparative genomic hybridization showed a microdeletion encompassing PCDH19. We propose molecular testing and analysis of PCDH19 in patients with pharmacoresistant epilepsy, with onset in early infancy, seizures in clusters, and fever sensitivity. Structural lesions are to be searched in patients with PCDH19 pathogenic variants. Further, PCDH19 analysis should be considered in epilepsy surgery evaluation even in the presence of cerebral structural lesions. Focal cortical malformations and monogenic epilepsy syndromes may coexist. Structural lesions are to be searched for in patients with protocadherin 19 (PCDH19) pathogenic variants with refractory focal seizures. © 2017 Mac Keith Press.

Scheduling Operative Surgical Services to Recover CHAMPUS Surgical Procedures at Blanchfield Army Community Hospital, Fort Campbell, Kentucky

DTIC Science & Technology

1993-08-01

use since World War II. The facility’s medical service region encompasses the entire state of Tennessee and the twelve southwestern counties of... 8545 RT MODIFIED RADICAL MASTECTOMY 10 34:40 3:28 4576 SIGMOIDECTOMY 6 25:00 4:10 4610 COLOSTOMY CLOSURE 6 26:25 4:24 5732 CYSTOLITHOLAPAXY, BLADDER BX...2:59 3859 LIGATION/STRIPPING OF VARICOSE VEINS 21 45:50 2:10 5310 BILATERAL INGUINAL HERNIA REPAIR 13 29:30 2:16 8545 RT MODIFIED RADICAL MASTECTOMY

The HubBLe trial: haemorrhoidal artery ligation (HAL) versus rubber band ligation (RBL) for haemorrhoids.

PubMed

Tiernan, Jim; Hind, Daniel; Watson, Angus; Wailoo, Allan J; Bradburn, Michael; Shephard, Neil; Biggs, Katie; Brown, Steven

2012-10-25

Haemorrhoids (piles) are a very common condition seen in surgical clinics. After exclusion of more sinister causes of haemorrhoidal symptoms (rectal bleeding, perianal irritation and prolapse), the best option for treatment depends upon persistence and severity of the symptoms. Minor symptoms often respond to conservative treatment such as dietary fibre and reassurance. For more severe symptoms treatment such as rubber band ligation may be therapeutic and is a very commonly performed procedure in the surgical outpatient setting. Surgery is usually reserved for those who have more severe symptoms, as well as those who do not respond to non-operative therapy; surgical techniques include haemorrhoidectomy and haemorrhoidopexy. More recently, haemorrhoidal artery ligation has been introduced as a minimally invasive, non destructive surgical option.There are substantial data in the literature concerning efficacy and safety of 'rubber band ligation including multiple comparisons with other interventions, though there are no studies comparing it to haemorrhoidal artery ligation. A recent overview has been carried out by the National Institute for Health and Clinical Excellence which concludes that current evidence shows haemorrhoidal artery ligation to be a safe alternative to haemorrhoidectomy and haemorrhoidopexy though it also highlights the lack of good quality data as evidence for the advantages of the technique. The aim of this study is to establish the clinical effectiveness and cost effectiveness of haemorrhoidal artery ligation compared with conventional rubber band ligation in the treatment of people with symptomatic second or third degree (Grade II or Grade III) haemorrhoids. A multi-centre, parallel group randomised controlled trial. The primary outcome is patient-reported symptom recurrence twelve months following the intervention. Secondary outcome measures relate to symptoms, complications, health resource use, health related quality of life and cost effectiveness following the intervention. 350 patients with grade II or grade III haemorrhoids will be recruited in surgical departments in up to 14 NHS hospitals. A multi-centre, parallel group randomised controlled trial. Block randomisation by centre will be used, with 175 participants randomised to each group. The results of the research will help inform future practice for the treatment of grade II and III haemorrhoids. ISRCTN41394716.

Multiplexing of Radio-Frequency Single Electron Transistors

NASA Technical Reports Server (NTRS)

Stevenson, Thomas R.; Pellerano, F. A.; Stahle, C. M.; Aidala, K.; Schoelkopf, R. J.; Krebs, Carolyn (Technical Monitor)

2001-01-01

We present results on wavelength division multiplexing of radio-frequency single electron transistors. We use a network of resonant impedance matching circuits to direct applied rf carrier waves to different transistors depending on carrier frequency. A two-channel demonstration of this concept using discrete components successfully reconstructed input signals with small levels of cross coupling. A lithographic version of the rf circuits had measured parameters in agreement with electromagnetic modeling, with reduced cross capacitance and inductance, and should allow 20 to 50 channels to be multiplexed.

Digital quantitative analysis of microRNA in single cell based on ligation-depended polymerase colony (Polony).

PubMed

Wang, Hui; Wang, Honghong; Duan, Xinrui; Liu, Chenghui; Li, Zhengping

2017-09-15

The ability to dissect cell-to-cell variations of microRNA (miRNA) expression with single-cell resolution has become a powerful tool to investigate the regulatory function of miRNAs in biological processes and the pathogenesis of miRNA-related diseases. Herein, we have developed a novel scheme for digital detection of miRNA in single cell by using the ligation-depended DNA polymerase colony (polony). Firstly, two simply designed target-specific DNA probes were ligated by using individual miRNA as the template. Then the ligated DNA probe acted as polony template that was amplified by PCR process in the thin polyacrylamide hydrogel. Due to the covalent attachment of a PCR primer on polyacrylamide matrix and the retarding effect of the polyacrylamide hydrogel matrix itself, as the polony reaction proceeds, the PCR products diffused radially near individual template molecule to form a bacteria colony-like spots of DNA molecules. The spots can be counted after staining the polyacrylamide gel with SYBR Green I and imaging with a microarray scanner. Our polony-based method is sensitive enough to detect 60 copies of miRNA molecules. Meanwhile, the new strategy has the capability of distinguishing singe-base difference. Due to its high sensitivity and specificity, the proposed method has been successfully applied to analysis of the expression profiling of miRNA in single cell. Copyright © 2017 Elsevier B.V. All rights reserved.

Optimizing diffusion in multiplexes by maximizing layer dissimilarity

NASA Astrophysics Data System (ADS)

Serrano, Alfredo B.; Gómez-Gardeñes, Jesús; Andrade, Roberto F. S.

2017-05-01

Diffusion in a multiplex depends on the specific link distribution between the nodes in each layer, but also on the set of the intralayer and interlayer diffusion coefficients. In this work we investigate, in a quantitative way, the efficiency of multiplex diffusion as a function of the topological similarity among multiplex layers. This similarity is measured by the distance between layers, taken among the pairs of layers. Results are presented for a simple two-layer multiplex, where one of the layers is held fixed, while the other one can be rewired in a controlled way in order to increase or decrease the interlayer distance. The results indicate that, for fixed values of all intra- and interlayer diffusion coefficients, a large interlayer distance generally enhances the global multiplex diffusion, providing a topological mechanism to control the global diffusive process. For some sets of networks, we develop an algorithm to identify the most sensitive nodes in the rewirable layer, so that changes in a small set of connections produce a drastic enhancement of the global diffusion of the whole multiplex system.

Association of Patent Ductus Arteriosus Ligation With Death or Neurodevelopmental Impairment Among Extremely Preterm Infants

PubMed Central

Mirea, Lucia; Rosenberg, Erin; Jang, Maximus; Ly, Linh; Church, Paige T.; Kelly, Edmond; Kim, S. Joseph; Jain, Amish; McNamara, Patrick J.; Shah, Prakesh S.

2017-01-01

Importance Observational studies have associated patent ductus arteriosus (PDA) ligation among preterm infants with adverse neonatal outcomes and neurodevelopmental impairment in early childhood, with a resultant secular trend away from surgical treatment. However, to our knowledge, studies have inadequately addressed sources of residual bias, including survival bias and major neonatal morbidities arising before exposure to ligation. Objective Evaluate the association between PDA ligation vs medical management and neonatal and neurodevelopmental outcomes. Design, Setting, and Participants This retrospective cohort study of preterm infants younger than 28 weeks gestational age born between January 1, 2006, and December 31, 2012, with clinical and echocardiography diagnoses of hemodynamically significant PDA was conducted at 3 tertiary neonatal intensive care units and affiliated follow-up programs. Exposure Surgical ligation vs medical management. Main Outcomes and Measures The primary outcome was a composite of death or neurodevelopmental impairment (NDI) at 18 to 24 months corrected age. Secondary outcomes included death before discharge, NDI, moderate-severe chronic lung disease, and severe retinopathy of prematurity. Multivariable logistic regression analysis was used to adjust for perinatal and postnatal confounders. Results Of 754 infants with hemodynamically significant PDA (mean [standard deviation] gestational age 25.7 [1.2] weeks and birth weight 813 [183] grams), 184 (24%) underwent ligation. Infants who underwent ligation had a higher frequency of morbidities before PDA closure, including sepsis, necrotizing enterocolitis, and a dependence on mechanical ventilation. After adjusting for perinatal characteristics and preligation morbidities, there was no difference in the odds of death or NDI (adjusted odds ratio (aOR), 0.83; 95% CI, 0.52-1.32), NDI (aOR, 1.27; 95% CI, 0.78-2.06), chronic lung disease (aOR, 1.36; 95% CI, 0.78-2.39) or severe retinopathy of prematurity (aOR, 1.61; 95% CI, 0.85-3.06). Ligation was associated with lower odds of mortality (aOR, 0.09; 95% CI, 0.04-0.21). Conclusions and Relevance Patent ductus arteriosus ligation among preterm neonates younger than 28 weeks gestational age was not associated with the composite outcome of death or NDI, and there were no differences in chronic lung disease, retinopathy of prematurity, or NDI among survivors. Mortality was lower among infants who underwent ligation, though residual survival bias could not be excluded. Previously reported associations of ligation with increased morbidity may be because of bias from confounding by indication. PMID:28264088

Simple methods for the 3' biotinylation of RNA.

PubMed

Moritz, Bodo; Wahle, Elmar

2014-03-01

Biotinylation of RNA allows its tight coupling to streptavidin and is thus useful for many types of experiments, e.g., pull-downs. Here we describe three simple techniques for biotinylating the 3' ends of RNA molecules generated by chemical or enzymatic synthesis. First, extension with either the Schizosaccharomyces pombe noncanonical poly(A) polymerase Cid1 or Escherichia coli poly(A) polymerase and N6-biotin-ATP is simple, efficient, and generally applicable independently of the 3'-end sequences of the RNA molecule to be labeled. However, depending on the enzyme and the reaction conditions, several or many biotinylated nucleotides are incorporated. Second, conditions are reported under which splint-dependent ligation by T4 DNA ligase can be used to join biotinylated and, presumably, other chemically modified DNA oligonucleotides to RNA 3' ends even if these are heterogeneous as is typical for products of enzymatic synthesis. Third, we describe the use of 29 DNA polymerase for a template-directed fill-in reaction that uses biotin-dUTP and, thanks to the enzyme's proofreading activity, can cope with more extended 3' heterogeneities.

Development and inter-laboratory assessment of droplet digital PCR assays for multiplex quantification of 15 genetically modified soybean lines.

PubMed

Košir, Alexandra Bogožalec; Spilsberg, Bjørn; Holst-Jensen, Arne; Žel, Jana; Dobnik, David

2017-08-17

Quantification of genetically modified organisms (GMOs) in food and feed products is often required for their labelling or for tolerance thresholds. Standard-curve-based simplex quantitative polymerase chain reaction (qPCR) is the prevailing technology, which is often combined with screening analysis. With the rapidly growing number of GMOs on the world market, qPCR analysis becomes laborious and expensive. Innovative cost-effective approaches are therefore urgently needed. Here, we report the development and inter-laboratory assessment of multiplex assays to quantify GMO soybean using droplet digital PCR (ddPCR). The assays were developed to facilitate testing of foods and feed for compliance with current GMO regulations in the European Union (EU). Within the EU, the threshold for labelling is 0.9% for authorised GMOs per ingredient. Furthermore, the EU has set a technical zero tolerance limit of 0.1% for certain unauthorised GMOs. The novel multiplex ddPCR assays developed target 11 GMO soybean lines that are currently authorised, and four that are tolerated, pending authorisation in the EU. Potential significant improvements in cost efficiency are demonstrated. Performance was assessed for the critical parameters, including limits of detection and quantification, and trueness, repeatability, and robustness. Inter-laboratory performance was also determined on a number of proficiency programme and real-life samples.

A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize.

PubMed

Matsuoka, T; Kuribara, H; Akiyama, H; Miura, H; Goda, Y; Kusakabe, Y; Isshiki, K; Toyoda, M; Hino, A

2001-02-01

Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system.

Modified rib pivot lateral thoracotomy: a case series.

PubMed

Appelgrein, C; Hosgood, G

2018-01-01

To describe a modified rib pivot thoracotomy and its clinical application in client-owned dogs and cats. Case series of 24 dogs and 1 cat requiring a thoracotomy. A lateral thoracic incision over the required thoracic segment was made. The latissimus dorsi muscle was reflected dorsally. The predetermined rib was identified and the periosteum overlying the rib was elevated circumferentially, avoiding the intercostal neurovascular structures. Holes were pre-placed above and below the proposed osteotomy site. The rib was osteotomised and pivoted cranially. The pleura was incised and the required intrathoracic procedure was then performed. The thoracic cavity was closed by pre-placement of a suture through the pre-placed holes within the osteotomised rib. The pleura and intercostal musculature were closed, avoiding the intercostal neurovascular structures. The rib was re-apposed and the lateral approach was closed. The cases included were reviewed for both the rib pivoted as per the procedure required and postoperative complications. The study group comprised 8 Staffordshire Bull Terriers, 2 Poodles, 2 German Shepherd Dogs, 1 each of Basset Hound, Rhodesian Ridgeback, Golden Retriever, Australian Shepherd, Vizsla, Bull Mastiff, Schnauzer, Jack Russell Terrier, Bulldog, Deerhound, Labrador Retriever and Australian Terrier, and 1 cat. A modified rib pivot thoracotomy was performed for lung lobectomy (n = 11), oesophagectomy (7), subtotal pericardectomy (5), patent ductus arteriosus ligation (1) and thoracic duct ligation (1). Follow-up ranged from 2 to 40 weeks postoperatively. Postoperative complications included seroma formation in two dogs. A modified rib pivot thoracotomy should be considered as an alternative lateral thoracic approach with good exposure, minimal complications and low morbidity. © 2018 Australian Veterinary Association.

In vitro assessment of competency for different lingual brackets in sliding mechanics.

PubMed

Lalithapriya, S; Kumaran, N Kurunji; Rajasigamani, K

2015-01-01

To determine the static frictional resistance of different lingual brackets at different second order angulations when coupled with stainless steel (SS) archwire in dry and wet conditions. Using a modified jig, frictional resistance was evaluated under different conditions for a total of 270 upper premolar lingual brackets (0.018″ × 0.025″ - conventional - 7(th) generation and STb, self-ligating - evolution) with no in-built tip or torque together with 0.016″ × 0.022″ straight length SS archwires. For conventional brackets, the archwire was secured with 0.008″ preformed SS short ligature ties. One way analysis of variance with Tukey HSD as post-hoc test was applied for degree wise and bracket wise comparison within dry condition and wet condition. For pair wise comparison Student's t-test was used. Under both conditions the static frictional resistance is significantly higher for self-ligating brackets at 0°, while at 5° and 10° it is higher for 7(th) generation brackets. Statistically, significant difference does not exist at 0° between conventional brackets and the same was found at 5° and 10° between STb and self-ligating brackets. With an increase in second order angulations, all the evaluated samples exhibited an increased frictional value. Wet condition samples obtained a higher value than their corresponding dry condition. The self-ligating bracket evaluated in this in vitro study is not beneficial in reducing friction during en-mass retraction due to its interactive clip type.

Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes

PubMed Central

Seguin-Orlando, Andaine; Schubert, Mikkel; Clary, Joel; Stagegaard, Julia; Alberdi, Maria T.; Prado, José Luis; Prieto, Alfredo; Willerslev, Eske; Orlando, Ludovic

2013-01-01

Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries. PMID:24205269

Continuous in vitro evolution of a ribozyme ligase: a model experiment for the evolution of a biomolecule.

PubMed

Ledbetter, Michael P; Hwang, Tony W; Stovall, Gwendolyn M; Ellington, Andrew D

2013-01-01

Evolution is a defining criterion of life and is central to understanding biological systems. However, the timescale of evolutionary shifts in phenotype limits most classroom evolution experiments to simple probability simulations. In vitro directed evolution (IVDE) frequently serves as a model system for the study of Darwinian evolution but produces noticeable phenotypic shifts in a matter of hours. An IVDE demonstration lab would serve to both directly demonstrate how Darwinian selection can act on a pool of variants and introduce students to an essential method of modern molecular biology. To produce an IVDE demonstration lab, continuous IVDE of a T500 ribozyme ligase population has been paired with a fluorescent strand displacement reporter system to visualize the selection of improved catalytic function. A ribozyme population is taken through rounds of isothermal amplification dependent on the self-ligation of a T7 promoter. As the population is selectively enriched with better ligase activity, the strand displacement system allows for the monitoring of the population's ligation rate. The strand displacement reporter system permits the detection of ligated ribozyme. Once ligated with the T7 promoter, the 5' end of the ribozyme displaces paired fluorophore-quencher oligonucleotides, in turn, generating visible signal upon UV light excitation. As the ligation rate of the population increases, due to the selection for faster ligating species, the fluorescent signal develops more rapidly. The pairing of the continuous isothermal system with the fluorescent reporting scheme allows any user, provided with minimal materials, to model the continuous directed evolution of a biomolecule. Copyright © 2013 Wiley-Liss, Inc.

Multiplexity and multireciprocity in directed multiplexes.

PubMed

Gemmetto, Valerio; Squartini, Tiziano; Picciolo, Francesco; Ruzzenenti, Franco; Garlaschelli, Diego

2016-10-01

Real-world multilayer networks feature nontrivial dependencies among links of different layers. Here we argue that if links are directed, then dependencies are twofold. Besides the ordinary tendency of links of different layers to align as the result of "multiplexity," there is also a tendency to antialign as a result of what we call "multireciprocity," i.e., the fact that links in one layer can be reciprocated by opposite links in a different layer. Multireciprocity generalizes the scalar definition of single-layer reciprocity to that of a square matrix involving all pairs of layers. We introduce multiplexity and multireciprocity matrices for both binary and weighted multiplexes and validate their statistical significance against maximum-entropy null models that filter out the effects of node heterogeneity. We then perform a detailed empirical analysis of the world trade multiplex (WTM), representing the import-export relationships between world countries in different commodities. We show that the WTM exhibits strong multiplexity and multireciprocity, an effect which is, however, largely encoded into the degree or strength sequences of individual layers. The residual effects are still significant and allow us to classify pairs of commodities according to their tendency to be traded together in the same direction and/or in opposite ones. We also find that the multireciprocity of the WTM is significantly lower than the usual reciprocity measured on the aggregate network. Moreover, layers with low (high) internal reciprocity are embedded within sets of layers with comparably low (high) mutual multireciprocity. This suggests that, in the WTM, reciprocity is inherent to groups of related commodities rather than to individual commodities. We discuss the implications for international trade research focusing on product taxonomies, the product space, and fitness and complexity metrics.

Multiplex analysis of DNA

DOEpatents

Church, George M.; Kieffer-Higgins, Stephen

1992-01-01

This invention features vectors and a method for sequencing DNA. The method includes the steps of: a) ligating the DNA into a vector comprising a tag sequence, the tag sequence includes at least 15 bases, wherein the tag sequence will not hybridize to the DNA under stringent hybridization conditions and is unique in the vector, to form a hybrid vector, b) treating the hybrid vector in a plurality of vessels to produce fragments comprising the tag sequence, wherein the fragments differ in length and terminate at a fixed known base or bases, wherein the fixed known base or bases differs in each vessel, c) separating the fragments from each vessel according to their size, d) hybridizing the fragments with an oligonucleotide able to hybridize specifically with the tag sequence, and e) detecting the pattern of hybridization of the tag sequence, wherein the pattern reflects the nucleotide sequence of the DNA.

Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray.

PubMed

Xu, Xiaodan; Li, Yingcong; Zhao, Heng; Wen, Si-yuan; Wang, Sheng-qi; Huang, Jian; Huang, Kun-lun; Luo, Yun-bo

2005-05-18

To devise a rapid and reliable method for the detection and identification of genetically modified (GM) events, we developed a multiplex polymerase chain reaction (PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a single reaction. The system included probes for screening gene, species reference gene, specific gene, construct-specific gene, event-specific gene, and internal and negative control genes. 18S rRNA was combined with species reference genes as internal controls to assess the efficiency of all reactions and to eliminate false negatives. Two sets of the multiplex PCR system were used to amplify four and five targets, respectively. Eight different structure genes could be detected and identified simultaneously for Roundup Ready soybean in a single microarray. The microarray specificity was validated by its ability to discriminate two GM maizes Bt176 and Bt11. The advantages of this method are its high specificity and greatly reduced false-positives and -negatives. The multiplex PCR coupled with microarray technology presented here is a rapid and reliable tool for the simultaneous detection of GM organism ingredients.

Methods for detection of GMOs in food and feed.

PubMed

Marmiroli, Nelson; Maestri, Elena; Gullì, Mariolina; Malcevschi, Alessio; Peano, Clelia; Bordoni, Roberta; De Bellis, Gianluca

2008-10-01

This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application.

Multiplexed SNP genotyping using the Qbead™ system: a quantum dot-encoded microsphere-based assay

PubMed Central

Xu, Hongxia; Sha, Michael Y.; Wong, Edith Y.; Uphoff, Janet; Xu, Yanzhang; Treadway, Joseph A.; Truong, Anh; O’Brien, Eamonn; Asquith, Steven; Stubbins, Michael; Spurr, Nigel K.; Lai, Eric H.; Mahoney, Walt

2003-01-01

We have developed a new method using the Qbead™ system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot™ semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral ‘barcodes’ are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein–protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications. PMID:12682378

Coordination-Assisted Bioorthogonal Chemistry: Orthogonal Tetrazine Ligation with Vinylboronic Acid and a Strained Alkene.

PubMed

Eising, Selma; Xin, Bo-Tao; Kleinpenning, Fleur; Heming, Juriaan; Florea, Bogdan; Overkleeft, Herman; Bonger, Kimberly Michelle

2018-05-28

Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality present. Recent developments in the field provided orthogonal bioorthogonal reactions for modification of multiple biomolecules simultaneously. During our research, we have observed exceptional high reaction rates in the bioorthogonal inverse electron-demand Diels-Alder (iEDDA) reaction between non-strained vinylboronic acids (VBAs) and dipyridyl-s-tetrazines relative to that of tetrazines bearing a methyl or phenyl substituent. As VBAs are mild Lewis acids, we hypothesize that coordination of the pyridyl nitrogen to the boronic acid promotes the tetrazine ligation. Here, we explore the molecular basis and scope of the VBA-tetrazine ligation in more detail and benefit from its unique reactivity in the simultaneous orthogonal tetrazine labelling of two proteins modified with VBA and norbornene, a widely used strained alkene. We further show that the two orthogonal iEDDA reactions can be carried out in living cells by labelling of the proteasome using a non-selective probe equipped with a VBA and a subunit-selective one bearing a norbornene. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Wavelength Division Multiplexing Scheme for Radio-Frequency Single Electron Transistors

NASA Technical Reports Server (NTRS)

Stevenson, Thomas R.; Pellerano, F. A.; Stahle, C. M.; Aidala, K.; Schoelkopf, R. J.; Krebs, Carolyn (Technical Monitor)

2001-01-01

We describe work on a wavelength division multiplexing scheme for radio-frequency single electron transistors. We use a network of resonant impedance matching circuits to direct applied rf carrier waves to different transistors depending on carrier frequency. Using discrete components, we made a two-channel demonstration of this concept and successfully reconstructed input signals with small levels of cross coupling. A lithographic version of the rf circuits had measured parameters in agreement with electromagnetic modeling, with reduced cross capacitance and inductance, and should allow 20 to 50 channels to be multiplexed.

Digital image compression for a 2f multiplexing optical setup

NASA Astrophysics Data System (ADS)

Vargas, J.; Amaya, D.; Rueda, E.

2016-07-01

In this work a virtual 2f multiplexing system was implemented in combination with digital image compression techniques and redundant information elimination. Depending on the image type to be multiplexed, a memory-usage saving of as much as 99% was obtained. The feasibility of the system was tested using three types of images, binary characters, QR codes, and grey level images. A multiplexing step was implemented digitally, while a demultiplexing step was implemented in a virtual 2f optical setup following real experimental parameters. To avoid cross-talk noise, each image was codified with a specially designed phase diffraction carrier that would allow the separation and relocation of the multiplexed images on the observation plane by simple light propagation. A description of the system is presented together with simulations that corroborate the method. The present work may allow future experimental implementations that will make use of all the parallel processing capabilities of optical systems.

Improved Efficiency and Robustness in qPCR and Multiplex End-Point PCR by Twisted Intercalating Nucleic Acid Modified Primers

PubMed Central

Schneider, Uffe Vest; Mikkelsen, Nikolaj Dam; Lindqvist, Anja; Okkels, Limei Meng; Jøhnk, Nina; Lisby, Gorm

2012-01-01

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5′-end. In qPCR, the 5′-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5′-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5′-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5′-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5′-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5′-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions. PMID:22701644

Germline mutations in SUFU cause Gorlin syndrome-associated childhood medulloblastoma and redefine the risk associated with PTCH1 mutations.

PubMed

Smith, Miriam J; Beetz, Christian; Williams, Simon G; Bhaskar, Sanjeev S; O'Sullivan, James; Anderson, Beverley; Daly, Sarah B; Urquhart, Jill E; Bholah, Zaynab; Oudit, Deemesh; Cheesman, Edmund; Kelsey, Anna; McCabe, Martin G; Newman, William G; Evans, D Gareth R

2014-12-20

Heterozygous germline PTCH1 mutations are causative of Gorlin syndrome (naevoid basal cell carcinoma), but detection rates > 70% have rarely been reported. We aimed to define the causative mutations in individuals with Gorlin syndrome without PTCH1 mutations. We undertook exome sequencing on lymphocyte DNA from four unrelated individuals from families with Gorlin syndrome with no PTCH1 mutations found by Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), or RNA analysis. A germline heterozygous nonsense mutation in SUFU was identified in one of four exomes. Sanger sequencing of SUFU in 23 additional PTCH1-negative Gorlin syndrome families identified a SUFU mutation in a second family. Copy-number analysis of SUFU by MLPA revealed a large heterozygous deletion in a third family. All three SUFU-positive families fulfilled diagnostic criteria for Gorlin syndrome, although none had odontogenic jaw keratocysts. Each SUFU-positive family included a single case of medulloblastoma, whereas only two (1.7%) of 115 individuals with Gorlin syndrome and a PTCH1 mutation developed medulloblastoma. We demonstrate convincing evidence that SUFU mutations can cause classical Gorlin syndrome. Our study redefines the risk of medulloblastoma in Gorlin syndrome, dependent on the underlying causative gene. Previous reports have found a 5% risk of medulloblastoma in Gorlin syndrome. We found a < 2% risk in PTCH1 mutation-positive individuals, with a risk up to 20× higher in SUFU mutation-positive individuals. Our data suggest childhood brain magnetic resonance imaging surveillance is justified in SUFU-related, but not PTCH1-related, Gorlin syndrome. © 2014 by American Society of Clinical Oncology.

Defining new guidelines for screening the 22q11.2 deletion based on a clinical and dysmorphologic evaluation of 194 individuals and review of the literature.

PubMed

Monteiro, Fabíola P; Vieira, Társis P; Sgardioli, Ilária C; Molck, Miriam C; Damiano, Ana Paula; Souza, Josiane; Monlleó, Isabella L; Fontes, Marshall I B; Fett-Conte, Agnes C; Félix, Têmis M; Leal, Gabriela F; Ribeiro, Erlane M; Banzato, Claudio E M; Dantas, Clarissa de R; Lopes-Cendes, Iscia; Gil-da-Silva-Lopes, Vera Lúcia

2013-07-01

The 22q11.2 deletion is the most frequent interstitial deletion in humans and presents a wide phenotypic spectrum, with over 180 clinical manifestations described. Distinct studies have detected frequencies of the deletion ranging from 0 % to 75 %, depending on the studied population and selection criteria adopted. Due to the lack of consensus in this matter, several studies have been conducted aiming to define which patients would be eligible for screening; however, the issue is still up for debate. In order to contribute to the delineation of possible clinical and dysmorphologic guidelines to optimize decision making in the clinical setting, 194 individuals with variable features of the 22q11.2 deletion syndromes (22q11.2DS) were evaluated. Group I, clinical suspicion of 22q11.2DS with palatal anomalies; Group II, clinical suspicion without palatal anomalies; Group III, cardiac malformations associated with the 22q11.2DS; and Group IV, juvenile-onset schizophrenia. Multiplex ligation-dependent probe amplification was used for screening the 22q11.2 deletion, which was detected in 45 patients (23.2 %), distributed as such: Group I, 35/101 (34.7 %); Group II, 4/18 (22.2 %); Group III, 6/52 (11.5 %); and Group IV, 0/23 (0 %). Clinical data were analyzed by frequency distribution and statistically. Based on the present results and on the review of the literature, we propose a set of guidelines for screening patients with distinct manifestations of the 22q11.2DS in order to maximize resources. In addition, we report the dysmorphic features which we found to be statistically correlated with the presence of the 22q11.2DS.

Identification of 47 novel mutations in patients with Alport syndrome and thin basement membrane nephropathy.

PubMed

Weber, Stefanie; Strasser, Katja; Rath, Sabine; Kittke, Achim; Beicht, Sonja; Alberer, Martin; Lange-Sperandio, Bärbel; Hoyer, Peter F; Benz, Marcus R; Ponsel, Sabine; Weber, Lutz T; Klein, Hanns-Georg; Hoefele, Julia

2016-06-01

Alport syndrome (ATS) is a progressive hereditary nephropathy characterized by hematuria and proteinuria. It can be associated with extrarenal manifestations. In contrast, thin basement membrane nephropathy (TBMN) is characterized by microscopic hematuria, is largely asymptomatic, and is rarely associated with proteinuria and end-stage renal disease. Mutations have been identified in the COL4A5 gene in ATS and in the COL4A3 and COL4A4 genes in ATS and TBMN. To date, more than 1000 different mutations in COL4A5, COL4A3, and COL4A4 are known. In this study mutational analysis by exon sequencing and multiplex ligation-dependent probe amplification was performed in a large European cohort of families with ATS and TBMN. Molecular diagnostic testing of 216 individuals led to the detection of 47 novel mutations, thereby expanding the spectrum of known mutations causing ATS and TBMN by up to 10 and 6%, respectively, depending on the database. Remarkably, a high number of ATS patients with only single mutations in COL4A3 and COL4A4 were identified. Additionally, three ATS patients presented with synonymous sequence variants that possible affect correct mRNA splicing, as suggested by in silico analysis. The results of this study clearly broaden the genotypic spectrum of known mutations for ATS and TBMN, which will in turn now facilitate future studies into genotype-phenotype correlations. Further studies should also examine the significance of single heterozygous mutations in COL4A3 and COL4A4 and of synonymous sequence variants associated with ATS.

MLPA based detection of mutations in the dystrophin gene of 180 Polish families with Duchenne/Becker muscular dystrophy.

PubMed

Zimowski, Janusz G; Massalska, Diana; Holding, Mariola; Jadczak, Sylwia; Fidziańska, Elżbieta; Lusakowska, Anna; Kostera-Pruszczyk, Anna; Kamińska, Anna; Zaremba, Jacek

2014-01-01

Duchenne/Becker muscular dystrophy (DMD/BMD) is a recessive, X-linked disorder caused by a mutation in the dystrophin gene. Deletions account for approximately 60-65% of mutations, duplications for 5-10%. The remaining cases are mainly point mutations. According to Monaco theory clinical form of the disease depends on maintaining or disrupting the reading frame. The purpose of the study was to determine frequency and location of deletions and duplications in the dystrophin gene, to determine the compliance between maintaining/disrupting the reading frame and clinical form of the disease and to check the effectiveness of MLPA (multiplex ligation-dependent probe amplification) in the detection of these mutations in hemizygous patients and heterozygous female carriers. The material is composed of combined results of molecular diagnosis carried out in years 2009-2012 in 180 unrelated patients referred with the diagnosis of DMD/BMD tested by use of MLPA. We identified 110 deletions, 22 duplication (in one patient two different duplications were detected) and 2 point mutations. Deletions involved mainly exons 45-54 and 3-21, whereas most duplications involved exons 3-18. The compliance with Monaco theory was 95% for deletions and 76% for duplications. Most of mutations in the dystrophin gene were localized in the hot spots - different for deletions and duplications. MLPA enabled their quick identification, exact localization and determination whether or not they maintained or disrupted the reading frame. MLPA was also effective in detection of deletions and duplications in female carriers. Copyright © 2014 Polish Neurological Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Total chemical synthesis of proteins without HPLC purification† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc01883a Click here for additional data file.

PubMed Central

Loibl, S. F.; Harpaz, Z.; Zitterbart, R.

2016-01-01

The total chemical synthesis of proteins is a tedious and time-consuming endeavour. The typical steps involve solid phase synthesis of peptide thioesters and cysteinyl peptides, native chemical ligation (NCL) in solution, desulfurization or removal of ligation auxiliaries in the case of extended NCL as well as many intermediary and final HPLC purification steps. With an aim to facilitate and improve the throughput of protein synthesis we developed the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step. The method relies on the combination of three orthogonal protein tags that allow sequential immobilization (via the N-terminal and C-terminal ends), extended native chemical ligation and release reactions. The peptide fragments to be ligated are prepared by conventional solid phase synthesis and used as crude materials in the subsequent steps. An N-terminal His6 unit permits selective immobilization of the full length peptide thioester onto Ni-NTA agarose beads. The C-terminal peptide fragment carries a C-terminal peptide hydrazide and an N-terminal 2-mercapto-2-phenyl-ethyl ligation auxiliary, which serves as a reactivity tag for the full length peptide. As a result, only full length peptides, not truncation products, react in the subsequent on-bead extended NCL. After auxiliary removal the ligation product is liberated into solution upon treatment with mild acid, and is concomitantly captured by an aldehyde-modified resin. This step allows the removal of the most frequently observed by-product in NCL chemistry, i.e. the hydrolysed peptide thioester (which does not contain a C-terminal peptide hydrazide). Finally, the target protein is released with diluted hydrazine or acid. We applied the method in the synthesis of 46 to 126 amino acid long MUC1 proteins comprising 2–6 copies of a 20mer tandem repeat sequence. Only three days were required for the parallel synthesis of 9 MUC1 proteins which were obtained in 8–33% overall yield with 90–98% purity despite the omission of HPLC purification. PMID:28451120

Growing multiplex networks with arbitrary number of layers

NASA Astrophysics Data System (ADS)

Momeni, Naghmeh; Fotouhi, Babak

2015-12-01

This paper focuses on the problem of growing multiplex networks. Currently, the results on the joint degree distribution of growing multiplex networks present in the literature pertain to the case of two layers and are confined to the special case of homogeneous growth and are limited to the state state (that is, the limit of infinite size). In the present paper, we first obtain closed-form solutions for the joint degree distribution of heterogeneously growing multiplex networks with arbitrary number of layers in the steady state. Heterogeneous growth means that each incoming node establishes different numbers of links in different layers. We consider both uniform and preferential growth. We then extend the analysis of the uniform growth mechanism to arbitrary times. We obtain a closed-form solution for the time-dependent joint degree distribution of a growing multiplex network with arbitrary initial conditions. Throughout, theoretical findings are corroborated with Monte Carlo simulations. The results shed light on the effects of the initial network on the transient dynamics of growing multiplex networks and takes a step towards characterizing the temporal variations of the connectivity of growing multiplex networks, as well as predicting their future structural properties.

Development of multiplex PCR assay for authentication of Cornu Cervi Pantotrichum in traditional Chinese medicine based on cytochrome b and C oxidase subunit 1 genes.

PubMed

Gao, Lijun; Xia, Wei; Ai, Jinxia; Li, Mingcheng; Yuan, Guanxin; Niu, Jiamu; Fu, Guilian; Zhang, Lihua

2016-07-01

This study describes a method for discriminating the true Cervus antlers from its counterfeits using multiplex PCR. Bioinformatics were carried out to design the specific alleles primers for mitochondrial (mt) cytochrome b (Cyt b) and cytochrome C oxidase subunit 1 (Cox 1) genes. The mt DNA and genomic DNA were extracted from Cervi Cornu Pantotrichum through the modified alkaline and the salt-extracting method in addition to its counterfeits, respectively. Sufficient DNA templates were extracted from all samples used in two methods, and joint fragments of 354 bp and 543 bp that were specifically amplified from both of true Cervus antlers served as a standard control. The data revealed that the multiplex PCR-based assays using two primer sets can be used for forensic and quantitative identification of original Cervus deer products from counterfeit antlers in a single step.

A wearable multiplexed silicon nonvolatile memory array using nanocrystal charge confinement

PubMed Central

Kim, Jaemin; Son, Donghee; Lee, Mincheol; Song, Changyeong; Song, Jun-Kyul; Koo, Ja Hoon; Lee, Dong Jun; Shim, Hyung Joon; Kim, Ji Hoon; Lee, Minbaek; Hyeon, Taeghwan; Kim, Dae-Hyeong

2016-01-01

Strategies for efficient charge confinement in nanocrystal floating gates to realize high-performance memory devices have been investigated intensively. However, few studies have reported nanoscale experimental validations of charge confinement in closely packed uniform nanocrystals and related device performance characterization. Furthermore, the system-level integration of the resulting devices with wearable silicon electronics has not yet been realized. We introduce a wearable, fully multiplexed silicon nonvolatile memory array with nanocrystal floating gates. The nanocrystal monolayer is assembled over a large area using the Langmuir-Blodgett method. Efficient particle-level charge confinement is verified with the modified atomic force microscopy technique. Uniform nanocrystal charge traps evidently improve the memory window margin and retention performance. Furthermore, the multiplexing of memory devices in conjunction with the amplification of sensor signals based on ultrathin silicon nanomembrane circuits in stretchable layouts enables wearable healthcare applications such as long-term data storage of monitored heart rates. PMID:26763827

A wearable multiplexed silicon nonvolatile memory array using nanocrystal charge confinement.

PubMed

Kim, Jaemin; Son, Donghee; Lee, Mincheol; Song, Changyeong; Song, Jun-Kyul; Koo, Ja Hoon; Lee, Dong Jun; Shim, Hyung Joon; Kim, Ji Hoon; Lee, Minbaek; Hyeon, Taeghwan; Kim, Dae-Hyeong

2016-01-01

Strategies for efficient charge confinement in nanocrystal floating gates to realize high-performance memory devices have been investigated intensively. However, few studies have reported nanoscale experimental validations of charge confinement in closely packed uniform nanocrystals and related device performance characterization. Furthermore, the system-level integration of the resulting devices with wearable silicon electronics has not yet been realized. We introduce a wearable, fully multiplexed silicon nonvolatile memory array with nanocrystal floating gates. The nanocrystal monolayer is assembled over a large area using the Langmuir-Blodgett method. Efficient particle-level charge confinement is verified with the modified atomic force microscopy technique. Uniform nanocrystal charge traps evidently improve the memory window margin and retention performance. Furthermore, the multiplexing of memory devices in conjunction with the amplification of sensor signals based on ultrathin silicon nanomembrane circuits in stretchable layouts enables wearable healthcare applications such as long-term data storage of monitored heart rates.

Multiplex Detection of IgG and IgM to Rift Valley Fever Virus Nucleoprotein, Nonstructural Proteins, and Glycoprotein in Ovine and Bovine.

PubMed

Hossain, Mohammad M; Wilson, William C; Faburay, Bonto; Richt, Jürgen; McVey, David S; Rowland, Raymond R

2016-08-01

A multiplex fluorescence microsphere immunoassay (FMIA) was used to detect bovine and ovine IgM and IgG antibodies to several Rift Valley fever virus (RVFV) proteins, including the major surface glycoprotein, Gn; the nonstructural proteins, NSs and NSm; and the nucleoprotein, N. Target antigens were assembled into a multiplex and tested in serum samples from infected wild-type RVFV or MP12, a modified live virus vaccine. As expected, the N protein was immunodominant and the best target for early detection of infection. Antibody activity against the other targets was also detected. The experimental results demonstrate the capabilities of FMIA for the detection of antibodies to RVFV structural and nonstructural proteins, which can be applied to future development and validation of diagnostic tests that can be used to differentiate vaccinated from infected animals.

IRF4 haploinsufficiency in a family with Whipple’s disease

PubMed Central

Guérin, Antoine; Kerner, Gaspard; Marr, Nico; Markle, Janet G; Fenollar, Florence; Wong, Natalie; Boughorbel, Sabri; Avery, Danielle T; Ma, Cindy S; Bougarn, Salim; Bouaziz, Matthieu; Béziat, Vivien; Della Mina, Erika; Oleaga-Quintas, Carmen; Lazarov, Tomi; Worley, Lisa; Nguyen, Tina; Patin, Etienne; Deswarte, Caroline; Martinez-Barricarte, Rubén; Boucherit, Soraya; Ayral, Xavier; Edouard, Sophie; Boisson-Dupuis, Stéphanie; Rattina, Vimel; Bigio, Benedetta; Vogt, Guillaume; Geissmann, Frédéric; Quintana-Murci, Lluis; Chaussabel, Damien; Tangye, Stuart G; Raoult, Didier; Abel, Laurent; Bustamante, Jacinta

2018-01-01

Most humans are exposed to Tropheryma whipplei (Tw). Whipple’s disease (WD) strikes only a small minority of individuals infected with Tw (<0.01%), whereas asymptomatic chronic carriage is more common (<25%). We studied a multiplex kindred, containing four WD patients and five healthy Tw chronic carriers. We hypothesized that WD displays autosomal dominant (AD) inheritance, with age-dependent incomplete penetrance. We identified a single very rare non-synonymous mutation in the four patients: the private R98W variant of IRF4, a transcription factor involved in immunity. The five Tw carriers were younger, and also heterozygous for R98W. We found that R98W was loss-of-function, modified the transcriptome of heterozygous leukocytes following Tw stimulation, and was not dominant-negative. We also found that only six of the other 153 known non-synonymous IRF4 variants were loss-of-function. Finally, we found that IRF4 had evolved under purifying selection. AD IRF4 deficiency can underlie WD by haploinsufficiency, with age-dependent incomplete penetrance. PMID:29537367

Rapid genetic typing of diarrheagenic Escherichia coli using a two-tube modified molecular beacon based multiplex real-time PCR assay and its clinical application

PubMed Central

2014-01-01

Background Diarrheagenic Escherichia coli (DEC), including Enterotoxigenic E.coli (ETEC), Enteroaggregative E.coli (EAEC), Enteropathogenic E.coli (EPEC), Enterohemolysin E.coli (EHEC) and Enteroinvasive E.coli (EIEC) causes diarrhea or hemolytic uremic syndromes among infants and travelers around the world. A rapid, reliable and repeatable method is urgent for identifying DEC so as to provide the reference for responding to diarrheal disease outbreak and the treatment of the diarrheal patients associated with DEC. Methods In this study, specific primers and modified molecular beacon probes of nine specific virulence genes, whose 5′end were added with homo tail sequence, were designed; and a two-tube modified molecular beacon based multiplex real–time PCR (rtPCR) assay for the identification of five Escherichia coli pathotypes, including ETEC, EAEC, EPEC, EHEC and EIEC was developed and optimized. Totally 102 bacterial strains, including 52 reference bacterial strains and 50 clinical strains were detected to confirm whether the target genes selected were specific. Then detection limits of the assay were tested. Lastly, the assay was applied to the detection of 11860 clinical samples to evaluate the specificity and sensitivity of the developed assay compared with the conventional PCR. Results The target genes were 100% specific as assessed on 102 bacterial strains since no cross-reactions were observed. The detection limits ranged from 88 CFU/mL (EHEC) to 880 CFU/mL (EPEC). Compared with the conventional PCR, the specificity and sensitivity of the multiplex rtPCR was 100% and over 99%, respectively. The coefficient of variation (CV) for each target gene ranged from 0.45% to 1.53%. 171 positive clinical samples were mostly identified as ETEC (n = 111, 64.9%) and EPEC (n = 38, 22.2%), which were the dominating pathotypes of DEC strains. Conclusion The developed multiplex rtPCR assay for the identification of DEC was high sensitive and specific and could be applied to the rapid identification of DEC in clinical and public health laboratories. PMID:25023669

CD22-Binding Synthetic Sialosides Regulate B Lymphocyte Proliferation Through CD22 Ligand-Dependent and Independent Pathways, and Enhance Antibody Production in Mice

PubMed Central

Matsubara, Naoko; Imamura, Akihiro; Yonemizu, Tatsuya; Akatsu, Chizuru; Yang, Hongrui; Ueki, Akiharu; Watanabe, Natsuki; Abdu-Allah, Hajjaj; Numoto, Nobutaka; Takematsu, Hiromu; Kitazume, Shinobu; Tedder, Thomas F.; Marth, Jamey D.; Ito, Nobutoshi; Ando, Hiromune; Ishida, Hideharu; Kiso, Makoto; Tsubata, Takeshi

2018-01-01

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are expressed in various immune cells and most of them carry signaling functions. High-affinity synthetic sialoside ligands have been developed for various Siglecs. Therapeutic potentials of the nanoparticles and compounds that contain multiple numbers of these sialosides and other reagents such as toxins and antigens have been demonstrated. However, whether immune responses can be regulated by monomeric sialoside ligands has not yet been known. CD22 (also known as Siglec-2) is an inhibitory molecule preferentially expressed in B lymphocytes (B cells) and is constitutively bound and functionally regulated by α2,6 sialic acids expressed on the same cell (cis-ligands). Here, we developed synthetic sialosides GSC718 and GSC839 that bind to CD22 with high affinity (IC50 ~100 nM), and inhibit ligand binding of CD22. When B cells are activated by B cell antigen receptor (BCR) ligation, both GSC718 and GSC839 downregulate proliferation of B cells, and this regulation requires both CD22 and α2,6 sialic acids. This result suggests that these sialosides regulate BCR ligation-induced B cell activation by reversing endogenous ligand-mediated regulation of CD22. By contrast, GSC718 and GSC839 augment B cell proliferation induced by TLR ligands or CD40 ligation, and this augmentation requires CD22 but not α2,6 sialic acids. Thus, these sialosides appear to enhance B cell activation by directly suppressing the inhibitory function of CD22 independently of endogenous ligand-mediated regulation. Moreover, GSC839 augments B cell proliferation that depends on both BCR ligation and CD40 ligation as is the case for in vivo B cell responses to antigens, and enhanced antibody production to the extent comparable to CpG oligonuleotides or a small amount of alum. Although these known adjuvants induce production of the inflammatory cytokines or accumulation of inflammatory cells, CD22-binding sialosides do not. Thus, synthetic sialosides that bind to CD22 with high-affinity modulate B cell activation through endogenous ligand-dependent and independent pathways, and carry an adjuvant activity without inducing inflammation. PMID:29725338

CD22-Binding Synthetic Sialosides Regulate B Lymphocyte Proliferation Through CD22 Ligand-Dependent and Independent Pathways, and Enhance Antibody Production in Mice.

PubMed

Matsubara, Naoko; Imamura, Akihiro; Yonemizu, Tatsuya; Akatsu, Chizuru; Yang, Hongrui; Ueki, Akiharu; Watanabe, Natsuki; Abdu-Allah, Hajjaj; Numoto, Nobutaka; Takematsu, Hiromu; Kitazume, Shinobu; Tedder, Thomas F; Marth, Jamey D; Ito, Nobutoshi; Ando, Hiromune; Ishida, Hideharu; Kiso, Makoto; Tsubata, Takeshi

2018-01-01

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are expressed in various immune cells and most of them carry signaling functions. High-affinity synthetic sialoside ligands have been developed for various Siglecs. Therapeutic potentials of the nanoparticles and compounds that contain multiple numbers of these sialosides and other reagents such as toxins and antigens have been demonstrated. However, whether immune responses can be regulated by monomeric sialoside ligands has not yet been known. CD22 (also known as Siglec-2) is an inhibitory molecule preferentially expressed in B lymphocytes (B cells) and is constitutively bound and functionally regulated by α2,6 sialic acids expressed on the same cell (cis-ligands). Here, we developed synthetic sialosides GSC718 and GSC839 that bind to CD22 with high affinity (IC 50 ~100 nM), and inhibit ligand binding of CD22. When B cells are activated by B cell antigen receptor (BCR) ligation, both GSC718 and GSC839 downregulate proliferation of B cells, and this regulation requires both CD22 and α2,6 sialic acids. This result suggests that these sialosides regulate BCR ligation-induced B cell activation by reversing endogenous ligand-mediated regulation of CD22. By contrast, GSC718 and GSC839 augment B cell proliferation induced by TLR ligands or CD40 ligation, and this augmentation requires CD22 but not α2,6 sialic acids. Thus, these sialosides appear to enhance B cell activation by directly suppressing the inhibitory function of CD22 independently of endogenous ligand-mediated regulation. Moreover, GSC839 augments B cell proliferation that depends on both BCR ligation and CD40 ligation as is the case for in vivo B cell responses to antigens, and enhanced antibody production to the extent comparable to CpG oligonuleotides or a small amount of alum. Although these known adjuvants induce production of the inflammatory cytokines or accumulation of inflammatory cells, CD22-binding sialosides do not. Thus, synthetic sialosides that bind to CD22 with high-affinity modulate B cell activation through endogenous ligand-dependent and independent pathways, and carry an adjuvant activity without inducing inflammation.

Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR.

PubMed

Singh, Sheetal; Shih, Shyh-Jen; Vaughan, Andrew T M

2014-01-01

Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter- and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks and rearrangements introduced into any identifiable genomic location. We have also applied parallel sequencing for the high-throughput analysis of inverse PCR products to facilitate the unbiased recording of all rearrangements located at a specific genomic location.

Modulation of human Th17 cell responses through complement receptor 3 (CD11 b/CD18) ligation on monocyte-derived dendritic cells.

PubMed

Nowatzky, Johannes; Manches, Olivier; Khan, Shaukat Ali; Godefroy, Emmanuelle; Bhardwaj, Nina

2018-06-13

Apoptotic cell receptors contribute to the induction of tolerance by modulating dendritic cell function following the uptake of apoptotic cells or microparticles. Dendritic cells that have bound or ingested apoptotic cells produce only low amounts of pro-inflammatory cytokines and fail to prime effector T cell responses. Specifically, ligation of the apoptotic cell receptor CR3 (CD11 b/CD18) on human monocyte-derived dendritic cells (moDC) down-modates proinflammatory cytokine secretion, but the consequences for human Th17 cell homeostasis and effector responses remain unknown. Here, we aimed to establish whether CD11b-ligated moDC modulate Th17 cell effector reponses to assess their potential for future use in moDC-based suppressive immunotherapy. We generated a bead-based surrogate system to target CD11b on monocyte-derived human dendritic cells and examined the effects of CD11b ligation on Th17-skewing cytokine secretion, priming, expansion and functional plasticity in DC/T cell co-culture systems at the poly- and monoclonal level. We show that Th17 cell expansion within the human memory CD4 + T cell compartment was efficiently constricted by targeting the CD11b receptor on moDC. This tolerogenic capacity was primarily dependent on cytokine skewing. Furthermore, ligation of CD11b on healthy homozygous carriers of the rs11143679 (ITGAM) variant - a strong genetic susceptibility marker for human systemic lupus erythematosus - also down-modulated the secretion of Th17-skewing cytokines. Overall, our findings underline the potential of targeted CD11b ligation on human dendritic cells for the engineering of suppressive immunotherapy for Th17-related autoimmune disorders. Copyright © 2018 Elsevier Ltd. All rights reserved.

A ligation-triggered DNAzyme cascade for amplified fluorescence detection of biological small molecules with zero-background signal.

PubMed

Lu, Li-Min; Zhang, Xiao-Bing; Kong, Rong-Mei; Yang, Bin; Tan, Weihong

2011-08-03

Many types of fluorescent sensing systems have been reported for biological small molecules. Particularly, several methods have been developed for the recognition of ATP or NAD(+), but they only show moderate sensitivity, and they cannot discriminate either ATP or NAD(+) from their respective analogues. We have addressed these limitations and report here a dual strategy which combines split DNAzyme-based background reduction with catalytic and molecular beacon (CAMB)-based amplified detection to develop a ligation-triggered DNAzyme cascade, resulting in ultrahigh sensitivity. First, the 8-17 DNAzyme is split into two separate oligonucleotide fragments as the building blocks for the DNA ligation reaction, thereby providing a zero-background signal to improve overall sensitivity. Next, a CAMB strategy is further employed for amplified signal detection achieved through cycling and regenerating the DNAzyme to realize the true enzymatic multiple turnover (one enzyme catalyzes the cleavage of several substrates) of catalytic beacons. This combination of zero-background signal and signal amplification significantly improves the sensitivity of the sensing systems, resulting in detection limits of 100 and 50 pM for ATP and NAD(+), respectively, much lower than those of previously reported biosensors. Moreover, by taking advantage of the highly specific biomolecule-dependence of the DNA ligation reaction, the developed DNAzyme cascades show significantly high selectivity toward the target cofactor (ATP or NAD(+)), and the target biological small molecule can be distinguished from its analogues. Therefore, as a new and universal platform for the design of DNA ligation reaction-based sensing systems, this novel ligation-triggered DNAzyme cascade method may find a broad spectrum of applications in both environmental and biomedical fields.

An in vitro approach for prioritization and evaluation of chemical effects on glucocorticoid receptor mediated adipogenesis.

PubMed

Hartman, Jessica K; Beames, Tyler; Parks, Bethany; Doheny, Daniel; Song, Gina; Efremenko, Alina; Yoon, Miyoung; Foley, Briana; Deisenroth, Chad; McMullen, Patrick D; Clewell, Rebecca A

2018-05-18

Rising obesity rates worldwide have socio-economic ramifications. While genetics, diet, and lack of exercise are major contributors to obesity, environmental factors may enhance susceptibility through disruption of hormone homeostasis and metabolic processes. The obesogen hypothesis contends that chemical exposure early in development may enhance adipocyte differentiation, thereby increasing the number of adipocytes and predisposing for obesity and metabolic disease. We previously developed a primary human adipose stem cell (hASC) assay to evaluate the effect of environmental chemicals on PPARG-dependent adipogenesis. Here, the assay was modified to determine the effects of chemicals on the glucocorticoid receptor (GR) pathway. In differentiation cocktail lacking the glucocorticoid agonist dexamethasone (DEX), hASCs do not differentiate into adipocytes. In the presence of GR agonists, adipocyte maturation was observed using phenotypic makers for lipid accumulation, adipokine secretion, and expression of key genes. To evaluate the role of environmental compounds on adipocyte differentiation, progenitor cells were treated with 19 prioritized compounds previously identified by ToxPi as having GR-dependent bioactivity, and multiplexed assays were used to confirm a GR-dependent mode of action. Five chemicals were found to be strong agonists. The assay was also modified to evaluate GR-antagonists, and 8/10 of the hypothesized antagonists inhibited adipogenesis. The in vitro bioactivity data was put into context with extrapolated human steady state concentrations (Css) and clinical exposure data (Cmax). These data support using a human adipose-derived stem cell differentiation assay to test the potential of chemicals to alter human GR-dependent adipogenesis. Copyright © 2017. Published by Elsevier Inc.

A new PCR-CGE (size and color) method for simultaneous detection of genetically modified maize events.

PubMed

Nadal, Anna; Coll, Anna; La Paz, Jose-Luis; Esteve, Teresa; Pla, Maria

2006-10-01

We present a novel multiplex PCR assay for simultaneous detection of multiple transgenic events in maize. Initially, five PCR primers pairs specific to events Bt11, GA21, MON810, and NK603, and Zea mays L. (alcohol dehydrogenase) were included. The event specificity was based on amplification of transgene/plant genome flanking regions, i.e., the same targets as for validated real-time PCR assays. These short and similarly sized amplicons were selected to achieve high and similar amplification efficiency for all targets; however, its unambiguous identification was a technical challenge. We achieved a clear distinction by a novel CGE approach that combined the identification by size and color (CGE-SC). In one single step, all five targets were amplified and specifically labeled with three different fluorescent dyes. The assay was specific and displayed an LOD of 0.1% of each genetically modified organism (GMO). Therefore, it was adequate to fulfill legal thresholds established, e.g., in the European Union. Our CGE-SC based strategy in combination with an adequate labeling design has the potential to simultaneously detect higher numbers of targets. As an example, we present the detection of up to eight targets in a single run. Multiplex PCR-CGE-SC only requires a conventional sequencer device and enables automation and high throughput. In addition, it proved to be transferable to a different laboratory. The number of authorized GMO events is rapidly growing; and the acreage of genetically modified (GM) varieties cultivated and commercialized worldwide is rapidly increasing. In this context, our multiplex PCR-CGE-SC can be suitable for screening GM contents in food.

A screening method for the detection of the 35S promoter and the nopaline synthase terminator in genetically modified organisms in a real-time multiplex polymerase chain reaction using high-resolution melting-curve analysis.

PubMed

Akiyama, Hiroshi; Nakamura, Fumi; Yamada, Chihiro; Nakamura, Kosuke; Nakajima, Osamu; Kawakami, Hiroshi; Harikai, Naoki; Furui, Satoshi; Kitta, Kazumi; Teshima, Reiko

2009-11-01

To screen for unauthorized genetically modified organisms (GMO) in the various crops, we developed a multiplex real-time polymerase chain reaction high-resolution melting-curve analysis method for the simultaneous qualitative detection of 35S promoter sequence of cauliflower mosaic virus (35SP) and the nopaline synthase terminator (NOST) in several crops. We selected suitable primer sets for the simultaneous detection of 35SP and NOST and designed the primer set for the detection of spiked ColE1 plasmid to evaluate the validity of the polymerase chain reaction (PCR) analyses. In addition, we optimized the multiplex PCR conditions using the designed primer sets and EvaGreen as an intercalating dye. The contamination of unauthorized GMO with single copy similar to NK603 maize can be detected as low as 0.1% in a maize sample. Furthermore, we showed that the present method would be applicable in identifying GMO in various crops and foods like authorized GM soybean, authorized GM potato, the biscuit which is contaminated with GM soybeans and the rice which is contaminated with unauthorized GM rice. We consider this method to be a simple and reliable assay for screening for unauthorized GMO in crops and the processing food products.

One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

PubMed

Malandraki, Ioanna; Varveri, Christina; Olmos, Antonio; Vassilakos, Nikon

2015-03-01

A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys. Copyright © 2014 Elsevier B.V. All rights reserved.

Cross talk and diffraction efficiency in angular multiplexed memories using improved polypeptide

NASA Astrophysics Data System (ADS)

Ramenah, Harry K.; Bertrand, Paul; Soubari, E. H.; Meyrueis, Patrick

1996-12-01

We studied energy coupling between gratings and angularly multiplexed 20 gratings with a uniform diffraction efficiency within 25 micrometer layer thickness of dichromated gelatin. The dependence of diffraction efficiency on beam ratio is given. We recorded a matrix form memory of nxmxp elements, where n and m are the rows and columns and p the number of multiplexes. For indication only, n equals m equals 10, p equals 20, the surface area of the matrix is 1 cm2. Color diffractive images and digital data are illustrated as well as video, cartography and medical applications.

Intracellular recordings of action potentials by an extracellular nanoscale field-effect transistor.

PubMed

Duan, Xiaojie; Gao, Ruixuan; Xie, Ping; Cohen-Karni, Tzahi; Qing, Quan; Choe, Hwan Sung; Tian, Bozhi; Jiang, Xiaocheng; Lieber, Charles M

2011-12-18

The ability to make electrical measurements inside cells has led to many important advances in electrophysiology. The patch clamp technique, in which a glass micropipette filled with electrolyte is inserted into a cell, offers both high signal-to-noise ratio and temporal resolution. Ideally, the micropipette should be as small as possible to increase the spatial resolution and reduce the invasiveness of the measurement, but the overall performance of the technique depends on the impedance of the interface between the micropipette and the cell interior, which limits how small the micropipette can be. Techniques that involve inserting metal or carbon microelectrodes into cells are subject to similar constraints. Field-effect transistors (FETs) can also record electric potentials inside cells, and because their performance does not depend on impedance, they can be made much smaller than micropipettes and microelectrodes. Moreover, FET arrays are better suited for multiplexed measurements. Previously, we have demonstrated FET-based intracellular recording with kinked nanowire structures, but the kink configuration and device design places limits on the probe size and the potential for multiplexing. Here, we report a new approach in which a SiO2 nanotube is synthetically integrated on top of a nanoscale FET. This nanotube penetrates the cell membrane, bringing the cell cytosol into contact with the FET, which is then able to record the intracellular transmembrane potential. Simulations show that the bandwidth of this branched intracellular nanotube FET (BIT-FET) is high enough for it to record fast action potentials even when the nanotube diameter is decreased to 3 nm, a length scale well below that accessible with other methods. Studies of cardiomyocyte cells demonstrate that when phospholipid-modified BIT-FETs are brought close to cells, the nanotubes can spontaneously penetrate the cell membrane to allow the full-amplitude intracellular action potential to be recorded, thus showing that a stable and tight seal forms between the nanotube and cell membrane. We also show that multiple BIT-FETs can record multiplexed intracellular signals from both single cells and networks of cells.

Intracellular recordings of action potentials by an extracellular nanoscale field-effect transistor

NASA Astrophysics Data System (ADS)

Duan, Xiaojie; Gao, Ruixuan; Xie, Ping; Cohen-Karni, Tzahi; Qing, Quan; Choe, Hwan Sung; Tian, Bozhi; Jiang, Xiaocheng; Lieber, Charles M.

2012-03-01

The ability to make electrical measurements inside cells has led to many important advances in electrophysiology. The patch clamp technique, in which a glass micropipette filled with electrolyte is inserted into a cell, offers both high signal-to-noise ratio and temporal resolution. Ideally, the micropipette should be as small as possible to increase the spatial resolution and reduce the invasiveness of the measurement, but the overall performance of the technique depends on the impedance of the interface between the micropipette and the cell interior, which limits how small the micropipette can be. Techniques that involve inserting metal or carbon microelectrodes into cells are subject to similar constraints. Field-effect transistors (FETs) can also record electric potentials inside cells, and because their performance does not depend on impedance, they can be made much smaller than micropipettes and microelectrodes. Moreover, FET arrays are better suited for multiplexed measurements. Previously, we have demonstrated FET-based intracellular recording with kinked nanowire structures, but the kink configuration and device design places limits on the probe size and the potential for multiplexing. Here, we report a new approach in which a SiO2 nanotube is synthetically integrated on top of a nanoscale FET. This nanotube penetrates the cell membrane, bringing the cell cytosol into contact with the FET, which is then able to record the intracellular transmembrane potential. Simulations show that the bandwidth of this branched intracellular nanotube FET (BIT-FET) is high enough for it to record fast action potentials even when the nanotube diameter is decreased to 3 nm, a length scale well below that accessible with other methods. Studies of cardiomyocyte cells demonstrate that when phospholipid-modified BIT-FETs are brought close to cells, the nanotubes can spontaneously penetrate the cell membrane to allow the full-amplitude intracellular action potential to be recorded, thus showing that a stable and tight seal forms between the nanotube and cell membrane. We also show that multiple BIT-FETs can record multiplexed intracellular signals from both single cells and networks of cells.

Identification of transcription coactivator OCA-B-dependent genes involved in antigen-dependent B cell differentiation by cDNA array analyses.

PubMed

Kim, Unkyu; Siegel, Rachael; Ren, Xiaodi; Gunther, Cary S; Gaasterland, Terry; Roeder, Robert G

2003-07-22

The tissue-specific transcriptional coactivator OCA-B is required for antigen-dependent B cell differentiation events, including germinal center formation. However, the identity of OCA-B target genes involved in this process is unknown. This study has used large-scale cDNA arrays to monitor changes in gene expression patterns that accompany mature B cell differentiation. B cell receptor ligation alone induces many genes involved in B cell expansion, whereas B cell receptor and helper T cell costimulation induce genes associated with B cell effector function. OCA-B expression is induced by both B cell receptor ligation alone and helper T cell costimulation, suggesting that OCA-B is involved in B cell expansion as well as B cell function. Accordingly, several genes involved in cell proliferation and signaling, such as Lck, Kcnn4, Cdc37, cyclin D3, B4galt1, and Ms4a11, have been identified as OCA-B-dependent genes. Further studies on the roles played by these genes in B cells will contribute to an understanding of B cell differentiation.

Design of multiplex calibrant plasmids, their use in GMO detection and the limit of their applicability for quantitative purposes owing to competition effects.

PubMed

Debode, Frédéric; Marien, Aline; Janssen, Eric; Berben, Gilbert

2010-03-01

Five double-target multiplex plasmids to be used as calibrants for GMO quantification were constructed. They were composed of two modified targets associated in tandem in the same plasmid: (1) a part of the soybean lectin gene and (2) a part of the transgenic construction of the GTS40-3-2 event. Modifications were performed in such a way that each target could be amplified with the same primers as those for the original target from which they were derived but such that each was specifically detected with an appropriate probe. Sequence modifications were done to keep the parameters of the new target as similar as possible to those of its original sequence. The plasmids were designed to be used either in separate reactions or in multiplex reactions. Evidence is given that with each of the five different plasmids used in separate wells as a calibrant for a different copy number, a calibration curve can be built. When the targets were amplified together (in multiplex) and at different concentrations inside the same well, the calibration curves showed that there was a competition effect between the targets and this limits the range of copy numbers for calibration over a maximum of 2 orders of magnitude. Another possible application of multiplex plasmids is discussed.

Anatomical description of the umbilical arteries and impact of their ligation on pelvic and perineal vascular supply after cystectomy in women.

PubMed

Chantalat, E; Vaysse, C; Delchier, M C; Bordier, B; Game, X; Chaynes, P; Cavaignac, E; Roumiguié, M

2018-03-27

In radical cystectomy, the surgeon generally ligates the umbilical artery at its origin. This artery may give rise to several arteries that supply the sexual organs. Our aim was to evaluate pelvic and perineal devascularisation in women after total cystectomy. We carried out a prospective anatomical and radiological study. We performed bilateral pelvic dissections of fresh adult female cadavers to identify the dividing branches of the umbilical artery. In parallel, we examined and compared the pre- and postoperative imaging investigations [magnetic resonance imaging (MRI) angiography] in patients undergoing cystectomy for benign disease to quantify the loss of pelvic vascularisation on the postoperative images by identifying the occluded arteries. The anatomical study together with the radiological study visualised 35 umbilical arteries (n = 70) with their branching patterns and collateral arteries. The uterine artery originated from the umbilical artery in more than 75% of cases (n = 54) of the internal pudendal artery in 34% (n = 24) and the vaginal artery in 43% (n = 30). The postoperative MRI angiograms showed pelvic devascularisation in four patients. Devascularisation was dependent on the level of surgical ligation. In the four patients with loss of pelvic vascular supply, the umbilical artery had been ligated at its origin. The umbilical artery gives rise to various branches that supply the pelvis and perineum. If the surgeon ligates the umbilical artery at its origin during total cystectomy, there is a significant risk of pelvic and perineal devascularisation.

Diversity of genetic events associated with MLH1 promoter methylation in Lynch syndrome families with heritable constitutional epimutation.

PubMed

Leclerc, Julie; Flament, Cathy; Lovecchio, Tonio; Delattre, Lucie; Ait Yahya, Emilie; Baert-Desurmont, Stéphanie; Burnichon, Nelly; Bronner, Myriam; Cabaret, Odile; Lejeune, Sophie; Guimbaud, Rosine; Morin, Gilles; Mauillon, Jacques; Jonveaux, Philippe; Laurent-Puig, Pierre; Frébourg, Thierry; Porchet, Nicole; Buisine, Marie-Pierre

2018-04-12

PurposeConstitutional epimutations are an alternative to genetic mutations in the etiology of genetic diseases. Some of these epimutations, termed secondary, correspond to the epigenetic effects of cis-acting genetic defects transmitted to the offspring following a Mendelian inheritance pattern. In Lynch syndrome, a few families with such apparently heritable MLH1 epimutations have been reported so far.MethodsWe designed a long-range polymerase chain reaction next-generation sequencing strategy to screen MLH1 entire gene and applied it to 4 French families with heritable epimutations and 10 additional patients with no proven transmission of their epimutations.ResultsThis strategy successfully detected the insertion of an Alu element in MLH1 coding sequence in one family. Two previously unreported MLH1 variants were also identified in other epimutation carriers: a nucleotide substitution within intron 1 and a single-nucleotide deletion in the 5'-UTR. Detection of a partial MLH1 duplication in another family required multiplex ligation-dependent probe amplification technology. We demonstrated the segregation of these variants with MLH1 methylation and studied the functional consequences of these defects on transcription.ConclusionThis is the largest cohort of patients with MLH1 secondary epimutations associated with a broad spectrum of genetic defects. This study provides further insight into the complexity of molecular mechanisms leading to secondary epimutations.GENETICS in MEDICINE advance online publication, 12 April 2018; doi:10.1038/gim.2018.47.

Investigation of FANCA gene in Fanconi anaemia patients in Iran

PubMed Central

Saffar Moghadam, Ali Akbar; Mahjoubi, Frouzandeh; Reisi, Nahid; Vosough, Parvaneh

2016-01-01

Background & objectives: Fanconi anaemia (FA) is a syndrome with a predisposition to bone marrow failure, congenital anomalies and malignancies. It is characterized by cellular hypersensitivity to cross-linking agents such as mitomycin C (MMC). In the present study, a new approach was selected to investigate FANCA (Fanconi anaemia complementation group A) gene in patients clinically diagnosed with cellular hypersensitivity to DNA cross-linking agent MMC. Methods: Chromosomal breakage analysis was performed to prove the diagnosis of Fanconi anaemia in 318 families. Of these, 70 families had a positive result. Forty families agreed to molecular genetic testing. In total, there were 27 patients with unknown complementary types. Genomic DNA was extracted and total RNA was isolated from fresh whole blood of the patients. The first-strand cDNA was synthesized and the cDNA of each patient was then tested with 21 pairs of overlapping primers. High resolution melting curve analysis was used to screen FANCA, and LinReg software version 1.7 was utilized for analysis of expression. Results: In total, six sequence alterations were identified, which included two stop codons, two frames-shift mutations, one large deletion and one amino acid exchange. FANCA expression was downregulated in patients who had sequence alterations. Interpretation & conclusions: The results of the present study show that high resolution melting (HRM) curve analysis may be useful in the detection of sequence alteration. It is simpler and more costeffective than the multiplex ligation-dependent probe amplification (MLPA) procedure. PMID:27121516

Investigation of FANCA gene in Fanconi anaemia patients in Iran.

PubMed

Moghadam, Ali Akbar Saffar; Mahjoubi, Frouzandeh; Reisi, Nahid; Vosough, Parvaneh

2016-02-01

Fanconi anaemia (FA) is a syndrome with a predisposition to bone marrow failure, congenital anomalies and malignancies. It is characterized by cellular hypersensitivity to cross-linking agents such as mitomycin C (MMC). In the present study, a new approach was selected to investigate FANCA (Fanconi anaemia complementation group A) gene in patients clinically diagnosed with cellular hypersensitivity to DNA cross-linking agent MMC. Chromosomal breakage analysis was performed to prove the diagnosis of Fanconi anaemia in 318 families. Of these, 70 families had a positive result. Forty families agreed to molecular genetic testing. In total, there were 27 patients with unknown complementary types. Genomic DNA was extracted and total RNA was isolated from fresh whole blood of the patients. The first-strand cDNA was synthesized and the cDNA of each patient was then tested with 21 pairs of overlapping primers. High resolution melting curve analysis was used to screen FANCA, and LinReg software version 1.7 was utilized for analysis of expression. In total, six sequence alterations were identified, which included two stop codons, two frames-shift mutations, one large deletion and one amino acid exchange. FANCA expression was downregulated in patients who had sequence alterations. The results of the present study show that high resolution melting (HRM) curve analysis may be useful in the detection of sequence alteration. It is simpler and more cost-effective than the multiplex ligation-dependent probe amplification (MLPA) procedure.

Spectrum of SMARCB1/INI1 Mutations in Familial and Sporadic Rhabdoid Tumors

PubMed Central

Eaton, Katherine W.; Tooke, Laura S.; Wainwright, Luanne M.; Judkins, Alexander R.; Biegel, Jaclyn A.

2011-01-01

Background Germline mutations and deletions of SMARCB1/INI1 in chromosome band 22q11.2 predispose patients to rhabdoid tumor and schwannomatosis. Previous estimates suggested that 15–20% of rhabdoid tumors were caused by an underlying germline abnormality of SMARCB1. However, these studies were limited by case selection and an inability to detect intragenic deletions and duplications. Procedure One hundred matched tumor and blood samples from patients with rhabdoid tumors of the brain, kidney, or soft tissues were analyzed for mutations and deletions of SMARCB1 by FISH, multiplex ligation-dependent probe amplification (MLPA), sequence analysis and high resolution Illumina 610K SNP based oligonucleotide array studies. Results Thirty-five of 100 patients were found to have a germline SMARCB1 abnormality. These abnormalities included point and frameshift mutations, intragenic deletions and duplications, and larger deletions including regions both proximal and distal to SMARCB1. There were 9 cases that demonstrated parent to child transmission of a mutated copy of SMARCB1. In 8 of the 9 cases, one or more family members were also diagnosed with rhabdoid tumor or schwannoma, and 2 of the 8 families presented with multiple affected children in a manner consistent with gonadal mosaicism. Conclusions Approximately one third of newly diagnosed patients with rhabdoid tumor have an underlying genetic predisposition to tumors due to a germline SMARCB1 alteration. Families may demonstrate incomplete penetrance and gonadal mosaicism, which must be considered when counseling families of patients with rhabdoid tumor. PMID:21108436

SHOX duplications found in some cases with type I Mayer-Rokitansky-Kuster-Hauser syndrome.

PubMed

Gervasini, Cristina; Grati, Francesca Romana; Lalatta, Faustina; Tabano, Silvia; Gentilin, Barbara; Colapietro, Patrizia; De Toffol, Simona; Frontino, Giada; Motta, Francesca; Maitz, Silvia; Bernardini, Laura; Dallapiccola, Bruno; Fedele, Luigi; Larizza, Lidia; Miozzo, Monica

2010-10-01

The Mayer-Rokitansky-Küster-Hauser syndrome is defined as congenital aplasia of müllerian ducts derived structures in females with a normal female chromosomal and gonadal sex. Most cases with Mayer-Rokitansky-Küster-Hauser syndrome are sporadic, although familial cases have been reported. The genetic basis of Mayer-Rokitansky-Küster-Hauser syndrome is largely unknown and seems heterogeneous, and a small number of cases were found to have mutations in the WNT4 gene. The aim of this study was to identify possible recurrent submicroscopic imbalances in a cohort of familial and sporadic cases with Mayer-Rokitansky-Küster-Hauser syndrome. Multiplex ligation-dependent probe amplification was used to screen the subtelomeric sequences of all chromosomes in 30 patients with Mayer-Rokitansky-Küster-Hauser syndrome (sporadic, n = 27 and familial, n = 3). Segregation analysis and pyrosequencing were applied to validate the MLPA results in the informative family. Partial duplication of the Xpter pseudoautosomal region 1 containing the short stature homeobox (SHOX) gene was detected in five patients with Mayer-Rokitansky-Küster-Hauser syndrome (familial, n = 3 and sporadic, n = 2) and not in 53 healthy controls. The duplications were not overlapping, and SHOX was never entirely duplicated. Haplotyping in the informative family revealed that SHOX gene duplication was inherited from the unaffected father and was absent in two healthy sisters. Partial duplication of SHOX gene is found in some cases with both familial and sporadic Mayer-Rokitansky-Küster-Hauser type I syndrome.

Application of a molecular diagnostic algorithm for haemophilia A and B using next-generation sequencing of entire F8, F9 and VWF genes.

PubMed

Bastida, Jose Maria; González-Porras, Jose Ramon; Jiménez, Cristina; Benito, Rocio; Ordoñez, Gonzalo R; Álvarez-Román, Maria Teresa; Fontecha, M Elena; Janusz, Kamila; Castillo, David; Fisac, Rosa María; García-Frade, Luis Javier; Aguilar, Carlos; Martínez, María Paz; Bermejo, Nuria; Herrero, Sonia; Balanzategui, Ana; Martin-Antorán, Jose Manuel; Ramos, Rafael; Cebeiro, Maria Jose; Pardal, Emilia; Aguilera, Carmen; Pérez-Gutierrez, Belen; Prieto, Manuel; Riesco, Susana; Mendoza, Maria Carmen; Benito, Ana; Hortal Benito-Sendin, Ana; Jiménez-Yuste, Víctor; Hernández-Rivas, Jesus Maria; García-Sanz, Ramon; González-Díaz, Marcos; Sarasquete, Maria Eugenia

2017-01-05

Currently, molecular diagnosis of haemophilia A and B (HA and HB) highlights the excess risk-inhibitor development associated with specific mutations, and enables carrier testing of female relatives and prenatal or preimplantation genetic diagnosis. Molecular testing for HA also helps distinguish it from von Willebrand disease (VWD). Next-generation sequencing (NGS) allows simultaneous investigation of several complete genes, even though they may span very extensive regions. This study aimed to evaluate the usefulness of a molecular algorithm employing an NGS approach for sequencing the complete F8, F9 and VWF genes. The proposed algorithm includes the detection of inversions of introns 1 and 22, an NGS custom panel (the entire F8, F9 and VWF genes), and multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 102 samples (97 FVIII- and FIX-deficient patients, and five female carriers) were studied. IVS-22 screening identified 11 out of 20 severe HA patients and one female carrier. IVS-1 analysis did not reveal any alterations. The NGS approach gave positive results in 88 cases, allowing the differential diagnosis of mild/moderate HA and VWD in eight cases. MLPA confirmed one large exon deletion. Only one case did have no pathogenic variants. The proposed algorithm had an overall success rate of 99 %. In conclusion, our evaluation demonstrates that this algorithm can reliably identify pathogenic variants and diagnose patients with HA, HB or VWD.

Spectrum of BRCA1/2 variants in 940 patients from Argentina including novel, deleterious and recurrent germline mutations: impact on healthcare and clinical practice.

PubMed

Solano, Angela Rosaria; Cardoso, Florencia Cecilia; Romano, Vanesa; Perazzo, Florencia; Bas, Carlos; Recondo, Gonzalo; Santillan, Francisco Bernardo; Gonzalez, Eduardo; Abalo, Eduardo; Viniegra, María; Michel, José Davalos; Nuñez, Lina María; Noblia, Cristina Maria; Mc Lean, Ignacio; Canton, Enrique Diaz; Chacon, Reinaldo Daniel; Cortese, Gustavo; Varela, Eduardo Beccar; Greco, Martín; Barrientos, María Laura; Avila, Silvia Adela; Vuotto, Hector Daniel; Lorusso, Antonio; Podesta, Ernesto Jorge; Mando, Oscar Gaspar

2017-09-01

BRCA1/2 mutations in Latin America are scarcely documented and in serious need of knowledge about the spectrum of BRCA pathogenic variants, information which may alter clinical practice and subsequently improve patient outcome. In addition, the search for data on testing policies in different regions constitutes a fundamental strength for the present study, which analyzes BRCA1/2 gene sequences and large rearrangements in 940 probands with familial and/or personal history of breast/ovary cancer (BOC). In non-mutated DNA samples, Multiplex Ligation-dependent Probe Amplification assays (MLPA) were used for the analysis of large rearrangements. Our studies detected 179 deleterious mutations out of 940 (19.04%) probands, including 5 large rearrangements and 22 novel mutations. The recurrent mutations accounted for 15.08% of the total and only 2.87% of the probands analyzed, very different from a Hispanic panel previously described. a) this first comprehensive description of the spectrum in BRCA1/2 sheds light on the low frequency of recurrent mutations; b) this information is key in clinical practice to select adequate sequencing studies in our population, subsequently improve patient outcome and prevent damage associated to false normal reports resulting from the use of invalid population panels; c) panels of mutations from other populations should be cautiously validated before imported, even those of apparently similar origin, a concept to be considered beyond significance in Argentina.

Mutation screening of the CDKL5 gene in cryptogenic infantile intractable epilepsy and review of clinical sensitivity.

PubMed

Intusoma, Utcharee; Hayeeduereh, Fadell; Plong-On, Oradawan; Sripo, Thanya; Vasiknanonte, Punnee; Janjindamai, Supachai; Lusawat, Apasri; Thammongkol, Sasipa; Visudtibhan, Anannit; Limprasert, Pornprot

2011-09-01

To perform CDKL5 mutation screening in Thai children with cryptogenic infantile intractable epilepsy and to determine the clinical sensitivity of CDKL5 screening when different inclusion criteria were applied. Children with cryptogenic infantile intractable epilepsy were screened for CDKL5 mutation using multiplex ligation-dependent probe amplification and DNA sequencing. The clinical sensitivity was reviewed by combining the results of studies using similar inclusion screening criteria. Thirty children (19 girls and 11 boys) with a median seizure onset of 7 months were screened. Almost a half had infantile spasms and one fifth had stereotypic hand movements. A novel c.2854C>T (p.R952X) was identified in an ambulatory girl who had severe mental retardation, multiple types of seizures without Rett-like features. Her mother had a mild intellectual disability, yet her grandmother and half sister were normal despite having the same genetic alteration (random X-inactivation patterns). The pathogenicity of p.R952X identified here was uncertain since healthy relatives and 6 female controls also harbor this alteration. The clinical sensitivity of CDKL5 mutation screening among females with Rett-like features and negative MECP2 screening was 7.8% while the clinical sensitivity among females having cryptogenic intractable seizures with an onset before the ages of 12, 6 and 3 months were 4.7, 11.6 and 14.3%, respectively. Copyright © 2011 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

Rare genomic rearrangement in a boy with Williams-Beuren syndrome associated to XYY syndrome and intriguing behavior.

PubMed

Dutra, Roberta L; Piazzon, Flavia B; Zanardo, Évelin A; Costa, Thais Virginia Moura Machado; Montenegro, Marília M; Novo-Filho, Gil M; Dias, Alexandre T; Nascimento, Amom M; Kim, Chong Ae; Kulikowski, Leslie D

2015-12-01

Williams-Beuren syndrome (WBS) is caused by a hemizygous contiguous gene microdeletion of 1.55-1.84 Mb at 7q11.23 region. Approximately, 28 genes have been shown to contribute to classical phenotype of SWB with presence of dysmorphic facial features, supravalvular aortic stenosis (SVAS), intellectual disability, and overfriendliness. With the use of Microarray-based comparative genomic hybridization and other molecular cytogenetic techniques, is possible define with more accuracy partial or atypical deletion and refine the genotype-phenotype correlation. Here, we report on a rare genomic structural rearrangement in a boy with atypical deletion in 7q11.23 and XYY syndrome with characteristic clinical signs, but not sufficient for the diagnosis of WBS. Cytogenetic analysis of G-banding showed a karyotype 47,XYY. Analysis of DNA with the technique of MLPA (Multiplex Ligation-dependent Probe Amplification) using kits a combination of kits (P064, P036, P070, and P029) identified an atypical deletion on 7q11.23. In addition, high resolution SNP Oligonucleotide Microarray Analysis (SNP-array) confirmed the alterations found by MLPA and revealed others pathogenic CNVs, in the chromosomes 7 and X. The present report demonstrates an association not yet described in literature, between Williams-Beuren syndrome and 47,XYY. The identification of atypical deletion in 7q11.23 concomitant to additional pathogenic CNVs in others genomic regions allows a better comprehension of clinical consequences of atypical genomic rearrangements. © 2015 Wiley Periodicals, Inc.

Mutational analysis using Sanger and next generation sequencing in sporadic spindle cell hemangiomas: A study of 19 cases.

PubMed

Ten Broek, Roel W; Bekers, Elise M; de Leng, Wendy W J; Strengman, Eric; Tops, Bastiaan B J; Kutzner, Heinz; Leeuwis, Jan Willem; van Gorp, Joost M; Creytens, David H; Mentzel, Thomas; van Diest, Paul J; Eijkelenboom, Astrid; Flucke, Uta

2017-12-01

Spindle cell hemangioma (SCH) is a distinct vascular soft-tissue lesion characterized by cavernous blood vessels and a spindle cell component mainly occurring in the distal extremities of young adults. The majority of cases harbor heterozygous mutations in IDH1/2 sporadically or rarely in association with Maffucci syndrome. However, based on mosaicism and accordingly a low percentage of lesional cells harboring a mutant allele, detection can be challenging. We tested 19 sporadic SCHs by Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), conventional next generation sequencing (NGS), and NGS using a single molecule molecular inversion probes (smMIP)-based library preparation to compare their diagnostic value. Out of 10 cases tested by Sanger sequencing and 2 analyzed using MLPA, 4 and 1, respectively, revealed a mutation in IDH1 (p.R132C). The 7 remaining negative cases and additional 6 cases were investigated using smMIP/NGS, showing hot spot mutations in IDH1 (p.R132C) (8 cases) and IDH2 (3 cases; twice p.R172S and once p.R172G, respectively). One case was negative. Owing to insufficient DNA quality and insufficient coverage, 2 cases were excluded. In total, in 16 out of 17 cases successfully tested, an IDH1/2 mutation was found. Given that IDH1/2 mutations were absent in 161 other vascular lesions tested by smMIP/NGS, the mutation can be considered as highly specific for SCH. © 2017 Wiley Periodicals, Inc.

Familial cases of Norrie disease detected by copy number analysis.

PubMed

Arai, Eisuke; Fujimaki, Takuro; Yanagawa, Ai; Fujiki, Keiko; Yokoyama, Toshiyuki; Okumura, Akihisa; Shimizu, Toshiaki; Murakami, Akira

2014-09-01

Norrie disease (ND, MIM#310600) is an X-linked disorder characterized by severe vitreoretinal dysplasia at birth. We report the results of causative NDP gene analysis in three male siblings with Norrie disease and describe the associated phenotypes. Three brothers with suspected Norrie disease and their mother presented for clinical examination. After obtaining informed consent, DNA was extracted from the peripheral blood of the proband, one of his brothers and his unaffected mother. Exons 1-3 of the NDP gene were amplified by polymerase chain reaction (PCR), and direct sequencing was performed. Multiplex ligation-dependent probe amplification (MLPA) was also performed to search for copy number variants in the NDP gene. The clinical findings of the three brothers included no light perception, corneal opacity, shallow anterior chamber, leukocoria, total retinal detachment and mental retardation. Exon 2 of the NDP gene was not amplified in the proband and one brother, even when the PCR primers for exon 2 were changed, whereas the other two exons showed no mutations by direct sequencing. MLPA analysis showed deletion of exon 2 of the NDP gene in the proband and one brother, while there was only one copy of exon 2 in the mother. Norrie disease was diagnosed in three patients from a Japanese family by clinical examination and was confirmed by genetic analysis. To localize the defect, confirmation of copy number variation by the MLPA method was useful in the present study.

Familial Breast/Ovarian Cancer and BRCA1/2 Genetic Screening: The Role of Immunohistochemistry as an Additional Method in the Selection of Patients

PubMed Central

Vaz, Fátima H.; Machado, Patrícia M.; Brandão, Rita D.; Laranjeira, Cátia T.; Eugénio, Joana S.; Fernandes, Aires H.; André, Saudade P.

2007-01-01

Only 20–25% of families screened for BRCA1/2 mutations are found positive. Because only a positive result is informative, we studied the role of BRCA1/2 immunohistochemistry as an additional method for patient selection. From 53 high-risk-affected probands, 18 (34%) had available paraffin blocks of their tumors and were selected for this study. Mutation screening was done by conformation-sensitive gel electrophoresis and multiplex ligation-dependent probe amplification. For immunohistochemistry, 21 neoplastic specimens (15 breast carcinomas, 5 ovary neoplasms, and 1 rectal adenocarcinoma) were analyzed with BRCA1 (monoclonal antibody, Ab-1, oncogene) and BRCA2 (polyclonal antibody, Ab-2, oncogene) antibodies. Absence of the BRCA1 protein was confirmed in negative tumors by Western blotting. Seven patients were positive for BRCA1/2 mutations: 5 for BRCA1 and 2 for BRCA2. Four out of five positive patients had tumors negative for BRCA1 immunostaining, and the remaining 13 BRCA1-negative patients had positive BRCA1 immunostaining in all tumor samples. Sensitivity to predict for BRCA1 mutation carriers was 80%, and specificity was 100%, with a positive predictive value of 100% and a negative predictive value of 93%. This correlation was statistically significant (p=0.001). No correlation was observed for BRCA2. If larger studies confirm these results, high-risk patients with BRCA1-negative tumors should be screened first for this gene. PMID:17625228

CNV analysis in Tourette syndrome implicates large genomic rearrangements in COL8A1 and NRXN1.

PubMed

Nag, Abhishek; Bochukova, Elena G; Kremeyer, Barbara; Campbell, Desmond D; Muller, Heike; Valencia-Duarte, Ana V; Cardona, Julio; Rivas, Isabel C; Mesa, Sandra C; Cuartas, Mauricio; Garcia, Jharley; Bedoya, Gabriel; Cornejo, William; Herrera, Luis D; Romero, Roxana; Fournier, Eduardo; Reus, Victor I; Lowe, Thomas L; Farooqi, I Sadaf; Mathews, Carol A; McGrath, Lauren M; Yu, Dongmei; Cook, Ed; Wang, Kai; Scharf, Jeremiah M; Pauls, David L; Freimer, Nelson B; Plagnol, Vincent; Ruiz-Linares, Andrés

2013-01-01

Tourette syndrome (TS) is a neuropsychiatric disorder with a strong genetic component. However, the genetic architecture of TS remains uncertain. Copy number variation (CNV) has been shown to contribute to the genetic make-up of several neurodevelopmental conditions, including schizophrenia and autism. Here we describe CNV calls using SNP chip genotype data from an initial sample of 210 TS cases and 285 controls ascertained in two Latin American populations. After extensive quality control, we found that cases (N = 179) have a significant excess (P = 0.006) of large CNV (>500 kb) calls compared to controls (N = 234). Amongst 24 large CNVs seen only in the cases, we observed four duplications of the COL8A1 gene region. We also found two cases with ∼400 kb deletions involving NRXN1, a gene previously implicated in neurodevelopmental disorders, including TS. Follow-up using multiplex ligation-dependent probe amplification (and including 53 more TS cases) validated the CNV calls and identified additional patients with rearrangements in COL8A1 and NRXN1, but none in controls. Examination of available parents indicates that two out of three NRXN1 deletions detected in the TS cases are de-novo mutations. Our results are consistent with the proposal that rare CNVs play a role in TS aetiology and suggest a possible role for rearrangements in the COL8A1 and NRXN1 gene regions.

Digital detection of multiple minority mutants and expression levels of multiple colorectal cancer-related genes using digital-PCR coupled with bead-array.

PubMed

Huang, Huan; Li, Shuo; Sun, Lizhou; Zhou, Guohua

2015-01-01

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed "multiplex ligation-dependent probe amplification-digital amplification coupled with hydrogel bead-array" (MLPA-DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA-DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA-DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC.

Whole exome sequencing and array-based molecular karyotyping as aids to prenatal diagnosis in fetuses with suspected Simpson-Golabi-Behmel syndrome.

PubMed

Kehrer, Christina; Hoischen, Alexander; Menkhaus, Ralf; Schwab, Eva; Müller, Andreas; Kim, Sarah; Kreiß, Martina; Weitensteiner, Valerie; Hilger, Alina; Berg, Christoph; Geipel, Anne; Reutter, Heiko; Gembruch, Ulrich

2016-10-01

Simpson-Golabi-Behmel (SGBS) syndrome type 1 and type 2 represent rare X-linked prenatal overgrowth disorders. The aim of our study is to describe the prenatal sonographic features as well as the genetic work-up. Retrospective analysis of four cases with a pre- or postnatal diagnosis of SGBS in a single tertiary referral center within a period of 4 years. In the study period, four male fetuses with SGBS were detected. The final diagnosis was made prenatally in three cases. In all cases the second trimester anomaly scan revealed left sided congenital diaphragmatic hernia (CDH) with additional anomalies; three fetuses with SGBS type 1 showed fetal overgrowth. In two of these, whole exome sequencing showed a possible frameshift mutation and a point mutation in the gene GPC3, respectively. In the third case, multiplex ligation-dependent probe amplification (MLPA) revealed a hemizygous duplication of exon 3-7 in the gene GPC3. In the fourth case, SGBS type 2 was confirmed by array comparative genomic hybridization (CGH) of amniotic fluid cells showing a deletion of the gene OFD1. We could demonstrate, that in the presence of a CDH, syndromes of the fetus can be increasingly differentiated by detailed sonography followed by a selective and graded molecular diagnostic using microarray techniques and whole exome sequencing. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

Post-mortem cytogenomic investigations in patients with congenital malformations.

PubMed

Dias, Alexandre Torchio; Zanardo, Évelin Aline; Dutra, Roberta Lelis; Piazzon, Flavia Balbo; Novo-Filho, Gil Monteiro; Montenegro, Marilia Moreira; Nascimento, Amom Mendes; Rocha, Mariana; Madia, Fabricia Andreia Rosa; Costa, Thais Virgínia Moura Machado; Milani, Cintia; Schultz, Regina; Gonçalves, Fernanda Toledo; Fridman, Cintia; Yamamoto, Guilherme Lopes; Bertola, Débora Romeo; Kim, Chong Ae; Kulikowski, Leslie Domenici

2016-08-01

Congenital anomalies are the second highest cause of infant deaths, and, in most cases, diagnosis is a challenge. In this study, we characterize patterns of DNA copy number aberrations in different samples of post-mortem tissues from patients with congenital malformations. Twenty-eight patients undergoing autopsy were cytogenomically evaluated using several methods, specifically, Multiplex Ligation-dependent Probe Amplification (MLPA), microsatellite marker analysis with a MiniFiler kit, FISH, a cytogenomic array technique and bidirectional Sanger sequencing, which were performed on samples of different tissues (brain, heart, liver, skin and diaphragm) preserved in RNAlater, in formaldehyde or by paraffin-embedding. The results identified 13 patients with pathogenic copy number variations (CNVs). Of these, eight presented aneuploidies involving chromosomes 13, 18, 21, X and Y (two presented inter- and intra-tissue mosaicism). In addition, other abnormalities were found, including duplication of the TYMS gene (18p11.32); deletion of the CHL1 gene (3p26.3); deletion of the HIC1 gene (17p13.3); and deletion of the TOM1L2 gene (17p11.2). One patient had a pathogenic missense mutation of g.8535C>G (c.746C>G) in exon 7 of the FGFR3 gene consistent with Thanatophoric Dysplasia type I. Cytogenomic techniques were reliable for the analysis of autopsy material and allowed the identification of inter- and intra-tissue mosaicism and a better understanding of the pathogenesis of congenital malformations. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of large rearrangements in autosomal dominant polycystic kidney disease and the PKD1/TSC2 contiguous gene syndrome

PubMed Central

Consugar, Mark B.; Wong, Wai C.; Lundquist, Patrick A.; Rossetti, Sandro; Kubly, Vickie J.; Walker, Denise L.; Rangel, Laureano J.; Aspinwall, Richard; Niaudet, W. Patrick; Özen, Seza; David, Albert; Velinov, Milen; Bergstralh, Eric J.; Bae, Kyongtae T.; Chapman, Arlene B.; Guay-Woodford, Lisa M.; Grantham, Jared J.; Torres, Vicente E.; Sampson, Julian R.; Dawson, Brian D.; Harris, Peter C.

2009-01-01

Large DNA rearrangements account for about 8% of disease mutations and are more common in duplicated genomic regions, where they are difficult to detect. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2. PKD1 is located in an intrachromosomally duplicated region. A tuberous sclerosis gene, TSC2, lies immediately adjacent to PKD1 and large deletions can result in the PKD1/TSC2 contiguous gene deletion syndrome. To rapidly identify large rearrangements, a multiplex ligation-dependent probe amplification assay was developed employing base-pair differences between PKD1 and the six pseudogenes to generate PKD1-specific probes. All changes in a set of 25 previously defined deletions in PKD1, PKD2 and PKD1/TSC2 were detected by this assay and we also found 14 new mutations at these loci. About 4% of the ADPKD patients in the CRISP study were found to have gross rearrangements, and these accounted for about a third of base-pair mutation negative families. Sensitivity of the assay showed that about 40% of PKD1/TSC contiguous gene deletion syndrome families contained mosaic cases. Characterization of a family found to be mosaic for a PKD1 deletion is discussed here to illustrate family risk and donor selection considerations. Our assay improves detection levels and the reliability of molecular testing of patients with ADPKD. PMID:18818683

Phelan-McDermid syndrome presenting with developmental delays and facial dysmorphisms.

PubMed

Kim, Yoon-Myung; Choi, In-Hee; Kim, Jun Suk; Kim, Ja Hye; Cho, Ja Hyang; Lee, Beom Hee; Kim, Gu-Hwan; Choi, Jin-Ho; Seo, Eul-Ju; Yoo, Han-Wook

2016-11-01

Phelan-McDermid syndrome is a rare genetic disorder caused by the terminal or interstitial deletion of the chromosome 22q13.3. Patients with this syndrome usually have global developmental delay, hypotonia, and speech delays. Several putative genes such as the SHANK3 , RAB , RABL2B , and IB2 are responsible for the neurological features. This study describes the clinical features and outcomes of Korean patients with Phelan-McDermid syndrome. Two patients showing global developmental delay, hypotonia, and speech delay were diagnosed with Phelan-McDermid syndrome via chromosome analysis, fluorescent in situ hybridization, and multiplex ligation-dependent probe amplification analysis. Brain magnetic resonance imaging of Patients 1 and 2 showed delayed myelination and severe communicating hydrocephalus, respectively. Electroencephalography in patient 2 showed high amplitude spike discharges from the left frontotemporoparietal area, but neither patient developed seizures. Kidney ultrasonography of both the patients revealed multicystic kidney disease and pelviectasis, respectively. Patient 2 experienced recurrent respiratory infections, and chest computed tomography findings demonstrated laryngotracheomalacia and bronchial narrowing. He subsequently died because of heart failure after a ventriculoperitoneal shunt operation at 5 months of age. Patient 1, who is currently 20 months old, has been undergoing rehabilitation therapy. However, global developmental delay was noted, as determines using the Korean Infant and Child Development test, the Denver developmental test, and the Bayley developmental test. This report describes the clinical features, outcomes, and molecular genetic characteristics of two Korean patients with Phelan-McDermid syndrome.

Detection of BRCA1 gross rearrangements by droplet digital PCR.

PubMed

Preobrazhenskaya, Elena V; Bizin, Ilya V; Kuligina, Ekatherina Sh; Shleykina, Alla Yu; Suspitsin, Evgeny N; Zaytseva, Olga A; Anisimova, Elena I; Laptiev, Sergey A; Gorodnova, Tatiana V; Belyaev, Alexey M; Imyanitov, Evgeny N; Sokolenko, Anna P

2017-10-01

Large genomic rearrangements (LGRs) constitute a significant share of pathogenic BRCA1 mutations. Multiplex ligation-dependent probe amplification (MLPA) is a leading method for LGR detection; however, it is entirely based on the use of commercial kits, includes relatively time-consuming hybridization step, and is not convenient for large-scale screening of recurrent LGRs. We developed and validated the droplet digital PCR (ddPCR) assay, which covers the entire coding region of BRCA1 gene and is capable to precisely quantitate the copy number for each exon. 141 breast cancer (BC) patients, who demonstrated evident clinical features of hereditary BC but turned out to be negative for founder BRCA1/2 mutations, were subjected to the LGR analysis. Four patients with LGR were identified, with three cases of exon 8 deletion and one women carrying the deletion of exons 5-7. Excellent concordance with MLPA test was observed. Exon 8 copy number was tested in additional 720 BC and 184 ovarian cancer (OC) high-risk patients, and another four cases with the deletion were revealed; MLPA re-analysis demonstrated that exon 8 loss was a part of a larger genetic alteration in two cases, while the remaining two patients had isolated defect of exon 8. Long-range PCR and next generation sequencing of DNA samples carrying exon 8 deletion revealed two types of recurrent LGRs. Droplet digital PCR is a reliable tool for the detection of large genomic rearrangements.

Copy number profiling of adult relapsed B-cell precursor acute lymphoblastic leukemia reveals potential leukemia progression mechanisms.

PubMed

Ribera, Jordi; Zamora, Lurdes; Morgades, Mireia; Mallo, Mar; Solanes, Neus; Batlle, Montserrat; Vives, Susana; Granada, Isabel; Juncà, Jordi; Malinverni, Roberto; Genescà, Eulàlia; Guàrdia, Ramon; Mercadal, Santiago; Escoda, Lourdes; Martinez-Lopez, Joaquín; Tormo, Mar; Esteve, Jordi; Pratcorona, Marta; Martinez-Losada, Carmen; Solé, Francesc; Feliu, Evarist; Ribera, Josep-Maria

2017-11-01

The outcome of relapsed adult acute lymphoblastic leukemia (ALL) remains dismal despite new therapeutic approaches. Previous studies analyzing relapse samples have shown a high degree of heterogeneity regarding gene alterations without an evident relapse signature. Bone marrow or peripheral blood samples from 31 adult B-cell precursor ALL patients at first relapse, and 21 paired diagnostic samples were analyzed by multiplex ligation probe-dependent amplification (MLPA). Nineteen paired diagnostic and relapse samples of these 21 patients were also analyzed by SNP arrays. A trend to acquire homozygous CDKN2A/B deletions and a significant increase in the number of copy number alterations (CNA) was observed from diagnosis to first relapse. Evolution from an ancestral clone was the main pattern of clonal evolution. Relapse samples were extremely heterogeneous regarding CNA frequencies. However, CDKN2A/B, PAX5, ETV6, ATM, IKZF1, VPREB1, and TP53 deletions and duplications of 1q, 8q, 17q, 21, X/Y PAR1, and Xp were frequently detected at relapse. Duplications of genes involved in cell proliferation, drug resistance and stem cell homeostasis regulation, as well as deletions of KDM6A and STAG2 genes emerged as specific alterations at relapse. Genomics of relapsed adult B-cell precursor ALL is highly heterogeneous, although some recurrent lesions involved in essential pathways deregulation were frequently observed. Selective and simultaneous targeting of these deregulated pathways may improve the results of current salvage therapies. © 2017 Wiley Periodicals, Inc.

Methylation status as a predictor of intravesical Bacillus Calmette-Guérin (BCG) immunotherapy response of high grade non-muscle invasive bladder tumor.

PubMed

Husek, Petr; Pacovsky, Jaroslav; Chmelarova, Marcela; Podhola, Miroslav; Brodak, Milos

2017-06-01

Genetic and epigenetic alterations play an important role in urothelial cancer pathogenesis. Deeper understanding of these processes could help us achieve better diagnosis and management of this life-threatening disease. The aim of this research was to evaluate the methylation status of selected tumor suppressor genes for predicting BCG response in patients with high grade non-muscle-invasive bladder tumor (NMIBC). We retrospectively evaluated 82 patients with high grade non-muscle-invasive bladder tumor (stage Ta, T1, CIS) who had undergone BCG instillation therapy. We compared epigenetic methylation status in BCG-responsive and BCG-failure groups. We used the MS-MLPA (Methylation-Specific Multiplex Ligation-Dependent Probe Amplification probe sets ME001 and ME004. The control group was 13 specimens of normal urotel (bladder tissue)). Newly identified methylations in high grade NMIBC were found in MUS81a, NTRK1 and PCCA. The methylation status of CDKN2B (P=0.00312 ** ) and MUS81a (P=0.0191 * ) is associated with clinical outcomes of BCG instillation therapy response. CDKN2B and MUS81a unmethylation was found in BCG failure patients. The results show that the methylation status of selected tumor suppressor genes (TSGs) has the potential for predicting BCG response in patients with NMIBC high grade tumors. Tumor suppressor genes such as CDKN2b, MUS81a, PFM-1, MSH6 and THBS1 are very promising for future research.

LARG at chromosome 11q23 has functional characteristics of a tumor suppressor in human breast cancer

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

Ong, Danny C.T.; Rudduck, Christina; Chin, Koei

2008-05-06

Deletion of 11q23-q24 is frequent in a diverse variety of malignancies, including breast and colorectal carcinoma, implicating the presence of a tumor suppressor gene at that chromosomal region. We show here that LARG, from 11q23, has functional characteristics of a tumor suppressor. We examined a 6-Mb region on 11q23 by high-resolution deletion mapping, utilizing both loss of heterozygosity (LOH) analysis and microarray comparative genomic hybridization (CGH). LARG (also called ARHGEF12), identified from the analyzed region, was underexpressed in 34% of primary breast carcinomas and 80% of breast cancer cell lines including the MCF-7 line. Multiplex ligation-dependent probe amplification on 30more » primary breast cancers and six breast cancer cell lines showed that LARG had the highest frequency of deletion compared to the BCSC-1 and TSLC1 genes, two known candidate tumor suppressor genes from 11q. In vitro analysis of breast cancer cell lines that underexpress LARG showed that LARG could be reactivated by trichostatin A, a histone deacetylase inhibitor, but not by 5-Aza-2{prime}-deoxycytidine, a demethylating agent. Bisulfite sequencing and quantitative high-throughput analysis of DNA methylation confirmed the lack of CpG island methylation in LARG in breast cancer. Restoration of LARG expression in MCF-7 cells by stable transfection resulted in reduced proliferation and colony formation, suggesting that LARG has functional characteristics of a tumor suppressor gene.« less

Spectrum of BRCA1/2 variants in 940 patients from Argentina including novel, deleterious and recurrent germline mutations: impact on healthcare and clinical practice

PubMed Central

Solano, Angela Rosaria; Cardoso, Florencia Cecilia; Romano, Vanesa; Perazzo, Florencia; Bas, Carlos; Recondo, Gonzalo; Santillan, Francisco Bernardo; Gonzalez, Eduardo; Abalo, Eduardo; Viniegra, María; Michel, José Davalos; Nuñez, Lina María; Noblia, Cristina Maria; Mc Lean, Ignacio; Canton, Enrique Diaz; Chacon, Reinaldo Daniel; Cortese, Gustavo; Varela, Eduardo Beccar; Greco, Martín; Barrientos, María Laura; Avila, Silvia Adela; Vuotto, Hector Daniel; Lorusso, Antonio; Podesta, Ernesto Jorge; Mando, Oscar Gaspar

2017-01-01

BRCA1/2 mutations in Latin America are scarcely documented and in serious need of knowledge about the spectrum of BRCA pathogenic variants, information which may alter clinical practice and subsequently improve patient outcome. In addition, the search for data on testing policies in different regions constitutes a fundamental strength for the present study, which analyzes BRCA1/2 gene sequences and large rearrangements in 940 probands with familial and/or personal history of breast/ovary cancer (BOC). In non-mutated DNA samples, Multiplex Ligation-dependent Probe Amplification assays (MLPA) were used for the analysis of large rearrangements. Our studies detected 179 deleterious mutations out of 940 (19.04%) probands, including 5 large rearrangements and 22 novel mutations. The recurrent mutations accounted for 15.08% of the total and only 2.87% of the probands analyzed, very different from a Hispanic panel previously described. In conclusion: a) this first comprehensive description of the spectrum in BRCA1/2 sheds light on the low frequency of recurrent mutations; b) this information is key in clinical practice to select adequate sequencing studies in our population, subsequently improve patient outcome and prevent damage associated to false normal reports resulting from the use of invalid population panels; c) panels of mutations from other populations should be cautiously validated before imported, even those of apparently similar origin, a concept to be considered beyond significance in Argentina. PMID:28947987

CNV Analysis in Tourette Syndrome Implicates Large Genomic Rearrangements in COL8A1 and NRXN1

PubMed Central

Nag, Abhishek; Bochukova, Elena G.; Kremeyer, Barbara; Campbell, Desmond D.; Muller, Heike; Valencia-Duarte, Ana V.; Cardona, Julio; Rivas, Isabel C.; Mesa, Sandra C.; Cuartas, Mauricio; Garcia, Jharley; Bedoya, Gabriel; Cornejo, William; Herrera, Luis D.; Romero, Roxana; Fournier, Eduardo; Reus, Victor I.; Lowe, Thomas L.; Farooqi, I. Sadaf; Mathews, Carol A.; McGrath, Lauren M.; Yu, Dongmei; Cook, Ed; Wang, Kai; Scharf, Jeremiah M.; Pauls, David L.; Freimer, Nelson B.; Plagnol, Vincent; Ruiz-Linares, Andrés

2013-01-01

Tourette syndrome (TS) is a neuropsychiatric disorder with a strong genetic component. However, the genetic architecture of TS remains uncertain. Copy number variation (CNV) has been shown to contribute to the genetic make-up of several neurodevelopmental conditions, including schizophrenia and autism. Here we describe CNV calls using SNP chip genotype data from an initial sample of 210 TS cases and 285 controls ascertained in two Latin American populations. After extensive quality control, we found that cases (N = 179) have a significant excess (P = 0.006) of large CNV (>500 kb) calls compared to controls (N = 234). Amongst 24 large CNVs seen only in the cases, we observed four duplications of the COL8A1 gene region. We also found two cases with ∼400kb deletions involving NRXN1, a gene previously implicated in neurodevelopmental disorders, including TS. Follow-up using multiplex ligation-dependent probe amplification (and including 53 more TS cases) validated the CNV calls and identified additional patients with rearrangements in COL8A1 and NRXN1, but none in controls. Examination of available parents indicates that two out of three NRXN1 deletions detected in the TS cases are de-novo mutations. Our results are consistent with the proposal that rare CNVs play a role in TS aetiology and suggest a possible role for rearrangements in the COL8A1 and NRXN1 gene regions. PMID:23533600

Maturity Onset Diabetes of the Young (MODY) in Tunisia: Low frequencies of GCK and HNF1A mutations.

PubMed

Ben Khelifa, S; Martinez, R; Dandana, A; Khochtali, I; Ferchichi, S; Castaño, L

2018-04-20

Maturity Onset Diabetes of the Young (MODY) is a monogenic form of diabetes characterized by autosomal dominant inheritance, an early clinical onset and a primary defect in β-cell function. Mutations in the GCK and HNF1A genes are the most common cause of MODY among Caucasians. The etiology of MODY in Tunisia stills a challenge for researchers. The aim of this study was to screen for mutations in GCK, HNF1A, HNF4A and INS genes in North African Tunisians subjects, in whom the clinical profile was very suggestive of MODY. A total of 23 unrelated patients, with clinical presentation of MODY were tested for mutations in GCK, HNF1A, HNF4A and INS genes, using Denaturing High Performance Liquid Chromatography (DHPLC), Multiplex Ligation-depend Probe Amplification (MLPA) and sequencing analysis. We identified the previously reported mutation c-169C > T in one patient as well as a new mutation c-457C > T in two unrelated patients. No mutations were detected in the HNF1A and INS genes. Despite restrictive clinical criteria used for selecting patients in this study, the most common genes known for MODY do not explain the majority of cases in Tunisians. This suggests that there are others candidate or unidentified genes contributing to the etiology of MODY in Tunisians families. Copyright © 2018 Elsevier B.V. All rights reserved.

Genetic analysis results of mature cystic teratomas of the ovary in Taiwan disagree with the previous origin theory of this tumor.

PubMed

Wang, Wen-Chung; Lai, Yen-Chein

2016-06-01

The most accepted theory regarding mature cystic teratomas of the ovary is that they are of parthenogenetic origin from oocyte after the completion of first division. Our previous study demonstrated that the origin of mature cystic teratoma of the uterus is not related to the parthenogenetic process, but is most likely pluripotential stem cell or primordial germ cell before meiosis I. Further studies are needed to clarify the origin of benign mature cystic teratomas of the ovary in Taiwan. In the present study, we investigated the DNA profiles of 9 mature cystic teratomas of the ovary using short tandem repeat analysis with AmpFLSTR SGM Plus, Profiler PCR amplification kits. The methylation statuses of the HhaI sites in the SNRPN, H19DMR, and KvDMR regions were determined on methylation-sensitive multiplex ligation-dependent probe amplification analysis. DNA profiling data from the 9 mature cystic teratomas of the ovary excluded parthenogenetic origin, as most of the 15 short tandem repeat loci were heterozygous on genotyping. There were varying degrees of hypermethylation of SNRPN gene and KvDMR locus in the presence of maternal uniparental disomy in all 9 mature cystic teratomas of the ovary. In light of these results, we further postulated that the origin of these mature cystic teratomas of the ovary is oogonia or primary oocyte before germinal vesicle stage failure of meiosis I. Copyright © 2016 Elsevier Inc. All rights reserved.

Clinical Validation of Copy Number Variant Detection from Targeted Next-Generation Sequencing Panels.

PubMed

Kerkhof, Jennifer; Schenkel, Laila C; Reilly, Jack; McRobbie, Sheri; Aref-Eshghi, Erfan; Stuart, Alan; Rupar, C Anthony; Adams, Paul; Hegele, Robert A; Lin, Hanxin; Rodenhiser, David; Knoll, Joan; Ainsworth, Peter J; Sadikovic, Bekim

2017-11-01

Next-generation sequencing (NGS) technology has rapidly replaced Sanger sequencing in the assessment of sequence variations in clinical genetics laboratories. One major limitation of current NGS approaches is the ability to detect copy number variations (CNVs) approximately >50 bp. Because these represent a major mutational burden in many genetic disorders, parallel CNV assessment using alternate supplemental methods, along with the NGS analysis, is normally required, resulting in increased labor, costs, and turnaround times. The objective of this study was to clinically validate a novel CNV detection algorithm using targeted clinical NGS gene panel data. We have applied this approach in a retrospective cohort of 391 samples and a prospective cohort of 2375 samples and found a 100% sensitivity (95% CI, 89%-100%) for 37 unique events and a high degree of specificity to detect CNVs across nine distinct targeted NGS gene panels. This NGS CNV pipeline enables stand-alone first-tier assessment for CNV and sequence variants in a clinical laboratory setting, dispensing with the need for parallel CNV analysis using classic techniques, such as microarray, long-range PCR, or multiplex ligation-dependent probe amplification. This NGS CNV pipeline can also be applied to the assessment of complex genomic regions, including pseudogenic DNA sequences, such as the PMS2CL gene, and to mitochondrial genome heteroplasmy detection. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

The Spectrum of CFTR Variants in Nonwhite Cystic Fibrosis Patients: Implications for Molecular Diagnostic Testing.

PubMed

Schrijver, Iris; Pique, Lynn; Graham, Steve; Pearl, Michelle; Cherry, Athena; Kharrazi, Martin

2016-01-01

Despite the implementation of cystic fibrosis (CF) newborn screening programs across the United States, the identification of CFTR gene variants in nonwhite populations compared with whites remains suboptimal. Our objective was to establish the spectrum of CFTR variants and their frequencies in CF patients in the United States with African, Native American, Asian, East Indian, or Middle Eastern backgrounds. By using direct DNA sequencing, we identified two CFTR variants in 89 of 140 affected nonwhite individuals with uncharacterized genotypes. Seven variants were novel. Multiplex ligation-dependent probe amplification detected 14 rearrangements in the remaining 51 patients, 6 of which were novel. Deletions and duplications accounted for 17% of unidentified alleles. A cross-sectional analysis of genotyping data from the CF Foundation Patient Registry was performed, comparing 3496 nonwhite patients with 22,206 white CF patients. Patients of Hispanic, black, or Asian ancestry were less likely to have two identified CFTR variants (P < 0.0001 for Hispanics and blacks, P = 0.003 for Asians), and more likely to carry no mutations on the commonly used 23 mutation carrier screening panel (P < 0.0001). We analyzed the mutations recorded for each ancestry and summarized the most frequent ones. This research could facilitate equity in mutation detection between white and nonwhite or mixed-ethnicity CF patients, enabling an earlier diagnosis improving their quality of life. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

High incidence of large deletions in the PMS2 gene in Spanish Lynch syndrome families.

PubMed

Brea-Fernández, A J; Cameselle-Teijeiro, J M; Alenda, C; Fernández-Rozadilla, C; Cubiella, J; Clofent, J; Reñé, J M; Anido, U; Milá, M; Balaguer, F; Castells, A; Castellvi-Bel, S; Jover, R; Carracedo, A; Ruiz-Ponte, C

2014-06-01

Lynch syndrome (LS) is caused by germline mutations in one of the four mismatch repair (MMR) genes. Defects in this pathway lead to microsatellite instability (MSI) in DNA tumors, which constitutes the molecular hallmark of this disease. Selection of patients for genetic testing in LS is usually based on fulfillment of diagnostic clinical criteria (i.e. Amsterdam criteria or the revised Bethesda guidelines). However, following these criteria PMS2 mutations have probably been underestimated as their penetrances appear to be lower than those of the other MMR genes. The use of universal MMR study-based strategies, using MSI testing and immunohistochemical (IHC) staining, is being one proposed alternative. Besides, germline mutation detection in PMS2 is complicated by the presence of highly homologous pseudogenes. Nevertheless, specific amplification of PMS2 by long-range polymerase chain reaction (PCR) and the improvement of the analysis of large deletions/duplications by multiplex ligation-dependent probe amplification (MLPA) overcome this difficulty. By using both approaches, we analyzed 19 PMS2-suspected carriers who have been selected by clinical or universal strategies and found five large deletions and one frameshift mutation in PMS2 in six patients (31%). Owing to the high incidence of large deletions found in our cohort, we recommend MLPA analysis as the first-line method for searching germline mutations in PMS2. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Design and Validation of a New MLPA-Based Assay for the Detection of RS1 Gene Deletions and Application in a Large Family with X-Linked Juvenile Retinoschisis.

PubMed

Nicoletti, Annalisa; Ziccardi, Lucia; Maltese, Paolo Enrico; Benedetti, Sabrina; Palumbo, Orazio; Rendina, Michelina; D'Agruma, Leonardo; Falsini, Benedetto; Wang, Xinjing; Bertelli, Matteo

2017-02-01

X-linked juvenile retinoschisis (XLRS) is a severe ocular disorder that can evolve to blindness. More than 200 different disease-causing mutations have been reported in the RS1 gene and approximately 10% of these are deletions. Since transmission is X-linked, males are always affected and females are usually carriers. The identification of female carriers is always important and poses a technical challenge. Therefore, we sought to develop a multiplex ligation dependent probe amplification (MLPA)-based method to identify deletions or duplications in this gene. We then used our assay to study a large XLRS family. We designed six probes specific for each RS1 exon and then optimized and validated our method using control samples with known gene deletions. In the XLRS family, RS1 gene copy number variation was assessed by "home-made" MLPA analysis and by single nucleotide polymorphism (SNP) array analysis using the CytoScan HD Array. Direct sequencing was used for deletion breakpoint mapping. Our assay detected all deletions in control samples. All affected males of the family were positive for a deletion of exon 2 of the RS1 gene (RS1:NM_000330:c.53-?_78+?del). Carrier females were also identified. Our method is easily replicated, reliable, and inexpensive and allows female carriers to be detected. This is the first report of deep characterization of a whole exon deletion in the RS1 gene.

Detection of Protein SUMOylation In Situ by Proximity Ligation Assays.

PubMed

Sahin, Umut; Jollivet, Florence; Berthier, Caroline; de Thé, Hugues; Lallemand-Breitenbach, Valérie

2016-01-01

Sumoylation is a posttranslational process essential for life and concerns a growing number of crucial proteins. Understanding the influence of this phenomenon on individual proteins or on cellular pathways in which they function has become an intense area of research. A critical step in studying protein sumoylation is to detect sumoylated forms of a particular protein. This has proven to be a challenging task for a number of reasons, especially in the case of endogenous proteins and in vivo studies or when studying rare cells such as stem cells. Proximity ligation assays that allow detection of closely interacting protein partners can be adapted for initial detection of endogenous sumoylation or ubiquitination in a rapid, ultrasensitive, and cheap manner. In addition, modified forms of a given protein can be detected in situ in various cellular compartments. Finally, the flexibility of this technique may allow rapid screening of drugs and stress signals that may modulate protein sumoylation.

Improvement for identification of heterophile antibody interference and AFP hook effect in immunoassays with multiplex suspension bead array system.

PubMed

Wang, Yajie; Yu, Jinsheng; Ren, Yuan; Liu, Li; Li, Haowen; Guo, Anchen; Shi, Congning; Fang, Fang; Juehne, Twyla; Yao, Jianer; Yang, Enhuan; Zhou, Xuelei; Kang, Xixiong

2013-11-15

A variety of immunoassays including multiplex suspension bead array have been developed for tumor marker detections; however, these assays could be compromised in their sensitivity and specificity by well-known heterophile antibody interference and hook effect. Using Luminex® multiplex suspension bead arrays, we modified protocols with two newly-developed solutions that can identify heterophile antibody interference and AFP hook effect. Effectiveness of the two solutions was assessed in serum samples from patients. Concentrations of 9 tumor markers in heterophile antibody positive samples assayed with Solution A, containing murine monoclonal antibodies and mouse serum, were significantly reduced when compared with those false high signals assayed without Solution A (all p<0.01). With incorporation of Solution H (fluorescent beads linked with AFP antigen), a new strategy for identification of AFP hook effect was established, and with this strategy AFP hook effect was identified effectively in serum samples with very high levels of AFP. Two proprietary solutions improve the identification of heterophile antibody interference and AFP hook effect. With these solutions, multiplex suspension bead arrays provide more reliable testing results in tumor marker detection where complex clinical serum samples are used. © 2013.

Prenatal programming of pulmonary hypertension induced by chronic hypoxia or ductal ligation in sheep.

PubMed

Papamatheakis, Demosthenes G; Chundu, Madalitso; Blood, Arlin B; Wilson, Sean M

2013-12-01

Pulmonary hypertension of the newborn is caused by a spectrum of functional and structural abnormalities of the cardiopulmonary circuit. The existence of multiple etiologies and an incomplete understanding of the mechanisms of disease progression have hindered the development of effective therapies. Animal models offer a means of gaining a better understanding of the fundamental basis of the disease. To that effect, a number of experimental animal models are being used to generate pulmonary hypertension in the fetus and newborn. In this review, we compare the mechanisms associated with pulmonary hypertension caused by two such models: in utero ligation of the ductus arteriosus and chronic perinatal hypoxia in sheep fetuses and newborns. In this manner, we make direct comparisons between ductal ligation and chronic hypoxia with respect to the associated mechanisms of disease, since multiple studies have been performed with both models in a single species. We present evidence that the mechanisms associated with pulmonary hypertension are dependent on the type of stress to which the fetus is subjected. Such an analysis allows for a more thorough evaluation of the disease etiology, which can help focus clinical treatments. The final part of the review provides a clinical appraisal of current treatment strategies and lays the foundation for developing individualized therapies that depend on the causative factors.

Integrated-Optic Wavelength Multiplexer In Glass Fabricated By A Charge Controlled Ion Exchange

NASA Astrophysics Data System (ADS)

Klein, R.; Jestel, D.; Lilienhof, H. J.; Rottman, F.; Voges, E.

1989-02-01

Integrated-optic wavelength division multiplexing (WDM) is commonly used in communication systems. These WDM-devices are also well suited to build up optical fiber networks for both intensity and interferometric sensor types. The operation principle of our wavelength division multiplexing devise is based on the wavelength dependent two-mode interference in a two-moded waveguide, which is coupled adiabatically to the single-mode input and output strip waveguides. The single-mode input and output waveguides are connected via two Y-branches ( "'kJ- 1° branching angle ) with a two-moded intersection region. The ratio of the light powers in the single-mode output waveguides depends on wavelength . The two-mode interference within the two-moded center waveguide leads to an almost wavelength periodic transmission caracteristic . Dual-channel multiplexers/demultiplexers were fabricated by a charge controlled field assisted pottasium exchange in B-270 glass (Desag). The devices have a typical channel separation of 30 - 40 nm and a far-end crosstalk attenuation of better than 16 dB. The operation wavelength regions of the fabricated devices are 0.6 - 0.8 µm and 1.3 - 1.6 µm, respectively.

Evidence of variable bacterial colonization on coloured elastomeric ligatures during orthodontic treatment: An intermodular comparative study.

PubMed

Sharma, Ravish; Sharma, Kavita; Sawhney, Rajesh

2018-03-01

Besides, other factors, the choice of materials used as orthodontic ligatures could be one of the many tools to counter the effects of microbial adhesion, that culminates into dental ailments. Therefore, we assessed bacterial adhesion on elastomeric ligatures with special reference to coloured elastomeric rings during orthodontic treatment. A split mouth study, involving 240 samples of different elastomeric ligatures from forty orthodontic patients possessing good oral hygiene was carried out. The archwire was ligated to the brackets on both arches with elastomeric rings (superslick, clear transparent , blue and pink) at predetermined quadrants. After six weeks, ligatures from second premolars were removed and processed for bacterial enumeration using standard techniques. Bacterial counts were also determined for stimulated saliva samples taken at 0 and 6 weeks. A statistically significant difference in bacterial counts was obtained amongst different elastomeric modules used. Maximum bacterial counts were found on conventional pigmented elastomeric modules, followed by Superslick module and clear module. More number of bacteria associated with the conventional pink as compared to the conventional blue pigmented modules, however it was not statistically significant. The three bacterial genera Streptococcus Staphylococcus and Aerobic Lactobacilli adhered to elastomeric modules in following predominant pattern i.e. Conventional pink>Conventional Blue>Superslick>Clear. The studies evidenced colour and material dependent bacterial colonization on orthodontic modules and could be an indicator of bacterial biofilm forming potential based on surface chemistries and a clinically efficacious tool to redesign conventional and modified elastomeric rings as orthodontic ligation accessories. Key words: Bacterial colonization, biofilm, coloured elastomers, orthodontic ligatures.

Extent of Vascular Remodeling Is Dependent on the Balance Between Estrogen Receptor α and G-Protein-Coupled Estrogen Receptor.

PubMed

Gros, Robert; Hussain, Yasin; Chorazyczewski, Jozef; Pickering, J Geoffrey; Ding, Qingming; Feldman, Ross D

2016-11-01

Estrogens are important regulators of cardiovascular function. Some of estrogen's cardiovascular effects are mediated by a G-protein-coupled receptor mechanism, namely, G-protein-coupled estrogen receptor (GPER). Estradiol-mediated regulation of vascular cell programmed cell death reflects the balance of the opposing actions of GPER versus estrogen receptor α (ERα). However, the significance of these opposing actions on the regulation of vascular smooth muscle cell proliferation or migration in vitro is unclear, and the significance in vivo is unknown. To determine the effects of GPER activation in vitro, we studied rat aortic vascular smooth muscle cells maintained in primary culture. GPER was reintroduced using adenoviral gene transfer. Both estradiol and G1, a GPER agonist, inhibited both proliferation and cell migration effects that were blocked by the GPER antagonist, G15. To determine the importance of the GPER-ERα balance in regulating vascular remodeling in a rat model of carotid ligation, we studied the effects of upregulation of GPER expression versus downregulation of ERα. Reintroduction of GPER significantly attenuated the extent of medial hypertrophy and attenuated the extent of CD45 labeling. Downregulation of ERα expression comparably attenuated the extent of medial hypertrophy and inflammation after carotid ligation. These studies demonstrate that the balance between GPER and ERα regulates vascular remodeling. Receptor-specific modulation of estrogen's effects may be an important new approach in modifying vascular remodeling in both acute settings like vascular injury and perhaps in longer term regulation like in hypertension. © 2016 American Heart Association, Inc.

Semi-synthesis of murine prion protein by native chemical ligation and chemical activation for preparation of polypeptide-α-thioester.

PubMed

Shi, Lei; Chen, Huai; Zhang, Si-Yu; Chu, Ting-Ting; Zhao, Yu-Fen; Chen, Yong-Xiang; Li, Yan-Mei

2017-06-01

Prions are suspected as pathogen of the fatal transmissible spongiform encephalopathies. Strategies to access homogenous prion protein (PrP) are required to fully comprehend the molecular mechanism of prion diseases. However, the polypeptide fragments from PrP show a high tendency to form aggregates, which is a gigantic obstacle of protein synthesis and purification. In this study, murine prion sequence 90 to 230 that is the core three-dimensional structure domain was constructed from three segments murine PrP (mPrP)(90-177), mPrP(178-212), and mPrP(213-230) by combining protein expression, chemical synthesis and chemical ligation. The protein sequence 90 to 177 was obtained from expression and finally converted into the polypeptide hydrazide by chemical activation of a cysteine in the tail. The other two polypeptide fragments of the C-terminal were obtained by chemical synthesis, which utilized the strategies of isopeptide and pseudoproline building blocks to complete the synthesis of such difficult sequences. The three segments were finally assembled by sequentially using native chemical ligation. This strategy will allow more straightforward access to homogeneously modified PrP variants. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Multiplex PageRank.

PubMed

Halu, Arda; Mondragón, Raúl J; Panzarasa, Pietro; Bianconi, Ginestra

2013-01-01

Many complex systems can be described as multiplex networks in which the same nodes can interact with one another in different layers, thus forming a set of interacting and co-evolving networks. Examples of such multiplex systems are social networks where people are involved in different types of relationships and interact through various forms of communication media. The ranking of nodes in multiplex networks is one of the most pressing and challenging tasks that research on complex networks is currently facing. When pairs of nodes can be connected through multiple links and in multiple layers, the ranking of nodes should necessarily reflect the importance of nodes in one layer as well as their importance in other interdependent layers. In this paper, we draw on the idea of biased random walks to define the Multiplex PageRank centrality measure in which the effects of the interplay between networks on the centrality of nodes are directly taken into account. In particular, depending on the intensity of the interaction between layers, we define the Additive, Multiplicative, Combined, and Neutral versions of Multiplex PageRank, and show how each version reflects the extent to which the importance of a node in one layer affects the importance the node can gain in another layer. We discuss these measures and apply them to an online multiplex social network. Findings indicate that taking the multiplex nature of the network into account helps uncover the emergence of rankings of nodes that differ from the rankings obtained from one single layer. Results provide support in favor of the salience of multiplex centrality measures, like Multiplex PageRank, for assessing the prominence of nodes embedded in multiple interacting networks, and for shedding a new light on structural properties that would otherwise remain undetected if each of the interacting networks were analyzed in isolation.

Age- and sex-dependent susceptibility to phenobarbital-resistant neonatal seizures: role of chloride co-transporters

PubMed Central

Kang, Seok Kyu; Markowitz, Geoffrey J.; Kim, Shin Tae; Johnston, Michael V.; Kadam, Shilpa D.

2015-01-01

Ischemia in the immature brain is an important cause of neonatal seizures. Temporal evolution of acquired neonatal seizures and their response to anticonvulsants are of great interest, given the unreliability of the clinical correlates and poor efficacy of first-line anti-seizure drugs. The expression and function of the electroneutral chloride co-transporters KCC2 and NKCC1 influence the anti-seizure efficacy of GABAA-agonists. To investigate ischemia-induced seizure susceptibility and efficacy of the GABAA-agonist phenobarbital (PB), with NKCC1 antagonist bumetanide (BTN) as an adjunct treatment, we utilized permanent unilateral carotid-ligation to produce acute ischemic-seizures in post-natal day 7, 10, and 12 CD1 mice. Immediate post-ligation video-electroencephalograms (EEGs) quantitatively evaluated baseline and post-treatment seizure burdens. Brains were examined for stroke-injury and western blot analyses to evaluate the expression of KCC2 and NKCC1. Severity of acute ischemic seizures post-ligation was highest at P7. PB was an efficacious anti-seizure agent at P10 and P12, but not at P7. BTN failed as an adjunct, at all ages tested and significantly blunted PB-efficacy at P10. Significant acute post-ischemic downregulation of KCC2 was detected at all ages. At P7, males displayed higher age-dependent seizure susceptibility, associated with a significant developmental lag in their KCC2 expression. This study established a novel neonatal mouse model of PB-resistant seizures that demonstrates age/sex-dependent susceptibility. The age-dependent profile of KCC2 expression and its post-insult downregulation may underlie the PB-resistance reported in this model. Blocking NKCC1 with low-dose BTN following PB treatment failed to improve PB-efficacy. PMID:26029047

Age- and sex-dependent susceptibility to phenobarbital-resistant neonatal seizures: role of chloride co-transporters.

PubMed

Kang, Seok Kyu; Markowitz, Geoffrey J; Kim, Shin Tae; Johnston, Michael V; Kadam, Shilpa D

2015-01-01

Ischemia in the immature brain is an important cause of neonatal seizures. Temporal evolution of acquired neonatal seizures and their response to anticonvulsants are of great interest, given the unreliability of the clinical correlates and poor efficacy of first-line anti-seizure drugs. The expression and function of the electroneutral chloride co-transporters KCC2 and NKCC1 influence the anti-seizure efficacy of GABAA-agonists. To investigate ischemia-induced seizure susceptibility and efficacy of the GABAA-agonist phenobarbital (PB), with NKCC1 antagonist bumetanide (BTN) as an adjunct treatment, we utilized permanent unilateral carotid-ligation to produce acute ischemic-seizures in post-natal day 7, 10, and 12 CD1 mice. Immediate post-ligation video-electroencephalograms (EEGs) quantitatively evaluated baseline and post-treatment seizure burdens. Brains were examined for stroke-injury and western blot analyses to evaluate the expression of KCC2 and NKCC1. Severity of acute ischemic seizures post-ligation was highest at P7. PB was an efficacious anti-seizure agent at P10 and P12, but not at P7. BTN failed as an adjunct, at all ages tested and significantly blunted PB-efficacy at P10. Significant acute post-ischemic downregulation of KCC2 was detected at all ages. At P7, males displayed higher age-dependent seizure susceptibility, associated with a significant developmental lag in their KCC2 expression. This study established a novel neonatal mouse model of PB-resistant seizures that demonstrates age/sex-dependent susceptibility. The age-dependent profile of KCC2 expression and its post-insult downregulation may underlie the PB-resistance reported in this model. Blocking NKCC1 with low-dose BTN following PB treatment failed to improve PB-efficacy.

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

Nagino, Ko; Yokozawa, Junji; Sasaki, Yu

Highlights: Black-Right-Pointing-Pointer Insulin secretion was increased during the OGTT or IVGTT in mesenteric lymph duct-ligated rats. Black-Right-Pointing-Pointer Proliferation of islet {beta}-cells was upregulated in lymph duct-ligated rats. Black-Right-Pointing-Pointer Mesenteric lymph duct flow has a role in glucose metabolism. -- Abstract: Background and aims: It has been suggested that intestinal lymph flow plays an important role in insulin secretion and glucose metabolism after meals. In this study, we investigated the influence of ligation of the mesenteric lymph duct on glucose metabolism and islet {beta}-cells in rats. Methods: Male Sprague-Dawley rats (10 weeks old) were divided into two groups: one underwent ligationmore » of the mesenteric lymph duct above the cistern (ligation group), and the other underwent a sham operation (sham group). After 1 and 2 weeks, fasting plasma concentrations of glucose, insulin, triglyceride, glucose-dependent insulinotropic polypeptide (GIP), and the active form of glucagon-like peptide-1 (GLP-1) were measured. At 2 weeks after the operation, the oral glucose tolerance test (OGTT) and intravenous glucose tolerance test (IVGTT) were performed. After the rats had been sacrificed, the insulin content of the pancreas was measured and the proliferation of {beta}-cells was assessed immunohistochemically using antibodies against insulin and Ki-67. Results: During the OGTT, the ligation group showed a significant decrease in the plasma glucose concentration at 120 min (p < 0.05) and a significant increase in the plasma insulin concentration by more than 2-fold at 15 min (p < 0.01). On the other hand, the plasma GIP concentration was significantly decreased at 60 min (p < 0.01) in the ligated group, while the active form of GLP-1 showed a significantly higher level at 90 min (1.7-fold; p < 0.05) and 120 min (2.5-fold; p < 0.01). During the IVGTT, the plasma insulin concentration in the ligation group was significantly higher at 2 min (more than 1.4-fold; p < 0.05). Immunohistochemistry showed that the ratios of {beta}-cell area/acinar cell area and {beta}-cell area/islet area, and also {beta}-cell proliferation, were significantly higher in the ligation group than in the sham group (p < 0.05, p < 0.01 and p < 0.01, respectively). The insulin content per unit wet weight of pancreas was also significantly increased in the ligation group (p < 0.05). Conclusions: In rats with ligation of the mesenteric lymph duct, insulin secretion during the OGTT or IVGTT was higher, and the insulin content and {beta}-cell proliferation in the pancreas were also increased. Our data show that mesenteric lymph duct flow has a role in glucose metabolism.« less

Rapid diagnosis of sepsis with TaqMan-Based multiplex real-time PCR.

PubMed

Liu, Chang-Feng; Shi, Xin-Ping; Chen, Yun; Jin, Ye; Zhang, Bing

2018-02-01

The survival rate of septic patients mainly depends on a rapid and reliable diagnosis. A rapid, broad range, specific and sensitive quantitative diagnostic test is the urgent need. Thus, we developed a TaqMan-Based Multiplex real-time PCR assays to identify bloodstream pathogens within a few hours. Primers and TaqMan probes were designed to be complementary to conserved regions in the 16S rDNA gene of different kinds of bacteria. To evaluate accurately, sensitively, and specifically, the known bacteria samples (Standard strains, whole blood samples) are determined by TaqMan-Based Multiplex real-time PCR. In addition, 30 blood samples taken from patients with clinical symptoms of sepsis were tested by TaqMan-Based Multiplex real-time PCR and blood culture. The mean frequency of positive for Multiplex real-time PCR was 96% at a concentration of 100 CFU/mL, and it was 100% at a concentration greater than 1000 CFU/mL. All the known blood samples and Standard strains were detected positively by TaqMan-Based Multiplex PCR, no PCR products were detected when DNAs from other bacterium were used in the multiplex assay. Among the 30 patients with clinical symptoms of sepsis, 18 patients were confirmed positive by Multiplex real-time PCR and seven patients were confirmed positive by blood culture. TaqMan-Based Multiplex real-time PCR assay with highly sensitivity, specificity and broad detection range, is a rapid and accurate method in the detection of bacterial pathogens of sepsis and should have a promising usage in the diagnosis of sepsis. © 2017 Wiley Periodicals, Inc.

Sensitive detection of EML4-ALK fusion oncoprotein of lung cancer by in situ proximity ligation assay.

PubMed

Rho, Jin Kyung; Lee, Hyangsin; Park, Chan-Sik; Choi, Chang-Min; Lee, Jae Cheol

2013-09-01

EML4-ALK fusion oncogene has emerged as a novel molecular target in non-small cell lung cancer (NSCLC). Although break-apart fluorescent in situ hybridization (FISH) is the standard method for diagnosis, it is expensive, not readily available and sometimes difficult to interpret. In addition, ALK immunohistochemistry (IHC) may miss the diagnosis because of relatively low level of ALK transcription. In situ proximity ligation assay (PLA) originally developed for precise detection and quantification of proteins by dual recognition and amplification process was used for sensitive detection of EML4-ALK fusion oncoprotein in NSCLC cell lines (ALK negative cell: PC-9 and H460, ALK positive cell: H3122 and H2228). EML4-ALK oncogene and protein in lung cancer cells were confirmed by multiplex RT-PCR and Western blots. We detected 117 kDa variant 1 of EML4-ALK in H3122 and 90 kDa variant 3 of EML4-ALK in H2228. These cells were more sensitive to crizotinib, an ALK inhibitor compared with PC-9 and H460 cells without EML4-ALK rearrangement. After fixing on glass slides by cytospin centrifuge, in situ PLA test was performed. Among four cell lines, distinct, tiny spots were visible only in H3122 and H2228 cell lines with ALK rearrangement. The same results were also obtained when paraffin-embedded cell blocks were used. Highly specific and sensitive detection of EML4-ALK fusion oncoprotein is possible by in situ PLA method suggesting its clinical application.

Ferromagnetic transition in a simple variant of the Ising model on multiplex networks

NASA Astrophysics Data System (ADS)

Krawiecki, A.

2018-02-01

Multiplex networks consist of a fixed set of nodes connected by several sets of edges which are generated separately and correspond to different networks ("layers"). Here, a simple variant of the Ising model on multiplex networks with two layers is considered, with spins located in the nodes and edges corresponding to ferromagnetic interactions between them. Critical temperatures for the ferromagnetic transition are evaluated for the layers in the form of random Erdös-Rényi graphs or heterogeneous scale-free networks using the mean-field approximation and the replica method, from the replica symmetric solution. Both methods require the use of different "partial" magnetizations, associated with different layers of the multiplex network, and yield qualitatively similar results. If the layers are strongly heterogeneous the critical temperature differs noticeably from that for the Ising model on a network being a superposition of the two layers, evaluated in the mean-field approximation neglecting the effect of the underlying multiplex structure on the correlations between the degrees of nodes. The critical temperature evaluated from the replica symmetric solution depends sensitively on the correlations between the degrees of nodes in different layers and shows satisfactory quantitative agreement with that obtained from Monte Carlo simulations. The critical behavior of the magnetization for the model with strongly heterogeneous layers can depend on the distributions of the degrees of nodes and is then determined by the properties of the most heterogeneous layer.

Assembly of Oriented Virus Arrays by Chemo-Selective Ligation Methods and Nanolithography Techniques

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

Camarero, J A; Cheung, C L; Lin, T

2002-12-02

The present work describes our ongoing efforts towards the creation of nano-scaled ordered arrays of protein/virus covalently attached to site-specific chemical linkers patterned by different nanolithograpy techniques. We will present a new and efficient solid-phase approach for the synthesis of chemically modified long alkyl-thiols. These compounds can be used to introduce chemoselective reacting groups onto gold and silicon-based surfaces. Furthermore, these modified thiols have been used to create nanometric patterns by using different nanolithography techniques. We will show that these patterns can react chemoselectively with proteins and/or virus which have been chemically or recombinantly modified to contain complementary chemical groupsmore » at specific positions thus resulting in the oriented attachment of the protein or virus to the surface.« less

Total chemical synthesis of modified histones

NASA Astrophysics Data System (ADS)

Qi, Yun-Kun; Ai, Hua-Song; Li, Yi-Ming; Yan, Baihui

2018-02-01

In the post-genome era, epigenetics has received increasing attentions in recent years. The post-translational modifications (PTMs) of four core histones play central roles in epigenetic regulation of eukaryotic genome by either directly altering the biophysical properties of nucleosomes or by recruiting other effector proteins. In order to study the biological functions and structural mechanisms of these histone PTMs, an obligatory step is to prepare a sufficient amount of homogeneously modified histones. This task cannot be fully accomplished either by recombinant technology or enzymatic modification. In this context, synthetic chemists have developed novel protein synthetic tools and state-of-the-art chemical ligation strategies for the preparation of homologous modified histones. In this review, we summarize the recent advances in the preparation of modified histones, focusing on the total chemical synthesis strategies. The importance and potential of synthetic chemistry for the study of histone code will be also discussed.

Asynchronous updates can promote the evolution of cooperation on multiplex networks

NASA Astrophysics Data System (ADS)

Allen, James M.; Hoyle, Rebecca B.

2017-04-01

We study the importance to the frequency of cooperation of the choice of updating strategies in a game played asynchronously or synchronously across layers in a multiplex network. Updating asynchronously in the public goods game leads to higher frequencies of cooperation compared to synchronous updates. How large this effect is depends on the sensitivity of the game dynamics to changes in the number of cooperators surrounding a player, with the largest effect observed when players payoffs are small. The discovery of this effect enhances understanding of cooperation on multiplex networks, and demonstrates a new way to maintain cooperation in these systems.

Optimizing complex phenotypes through model-guided multiplex genome engineering

DOE PAGES

Kuznetsov, Gleb; Goodman, Daniel B.; Filsinger, Gabriel T.; ...

2017-05-25

Here, we present a method for identifying genomic modifications that optimize a complex phenotype through multiplex genome engineering and predictive modeling. We apply our method to identify six single nucleotide mutations that recover 59% of the fitness defect exhibited by the 63-codon E. coli strain C321.ΔA. By introducing targeted combinations of changes in multiplex we generate rich genotypic and phenotypic diversity and characterize clones using whole-genome sequencing and doubling time measurements. Regularized multivariate linear regression accurately quantifies individual allelic effects and overcomes bias from hitchhiking mutations and context-dependence of genome editing efficiency that would confound other strategies.

Optimizing complex phenotypes through model-guided multiplex genome engineering

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

Kuznetsov, Gleb; Goodman, Daniel B.; Filsinger, Gabriel T.

Here, we present a method for identifying genomic modifications that optimize a complex phenotype through multiplex genome engineering and predictive modeling. We apply our method to identify six single nucleotide mutations that recover 59% of the fitness defect exhibited by the 63-codon E. coli strain C321.ΔA. By introducing targeted combinations of changes in multiplex we generate rich genotypic and phenotypic diversity and characterize clones using whole-genome sequencing and doubling time measurements. Regularized multivariate linear regression accurately quantifies individual allelic effects and overcomes bias from hitchhiking mutations and context-dependence of genome editing efficiency that would confound other strategies.

Real-Time NMR Studies of Oxyamine Ligations of Reducing Carbohydrates under Equilibrium Conditions.

PubMed

Baudendistel, Oliver R; Wieland, Daniel E; Schmidt, Magnus S; Wittmann, Valentin

2016-11-21

Ligation reactions at the anomeric center of carbohydrates have gained increasing importance in the field of glycobiology. Oxyamines are frequently used in labeling, immobilization, and bioconjugation of reducing carbohydrates. Herein, we present a systematic investigation of these ligation reactions under aqueous conditions. A series of four unprotected monosaccharides (glucose, N-acetylglucosamine, mannose, and 2-deoxyglucose) and one disaccharide (N,N'-diacetylchitobiose) was reacted with three primary and one secondary oxyamine. We monitored the concentrations of the starting materials and products by 1 H NMR spectroscopy and determined reaction times and equilibrium yields. Our experiments show that the outcome of the ligation reaction is not only dependent on the sugar and oxyamine used but also strongly on the reaction conditions. In the case of glucose, lowering the pH from 6 to 3 led to steadily increasing reaction rates, whereas the yields were decreasing at the same time. Variation of the temperature did not only influence the product ratio in equilibrium but can also have a strong impact on the equilibrium yield. In the case of reactions of a primary oxyamine, increased temperatures led to a higher proportion of acyclic products. Reaction of the secondary oxyamine with glucose unexpectedly led to lower yields at higher temperatures. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Vasodilator responses to nitric oxide are enhanced in mesenteric arteries of portal hypertensive rats.

PubMed

Heinemann, A; Stauber, R E

1996-09-01

Nitric oxide (NO) is discussed as a mediator of the splanchnic hyperaemia in portal hypertension. We assessed the vasorelaxation by the NO-dependent vasodilator acetylcholine, the NO donor 3-morpholino-sydnonimine (SIN-1) and forskolin, a stimulator of the adenylate cyclase pathway in potassium-preconstricted isolated perfused mesenteric arteries of portal vein-ligated and sham-operated rats. Dilator responses to acetylcholine and SIN-1 were significantly enhanced in vessels of portal vein-ligated rats as compared to sham-operated rats, whereas no difference was found in forskolin-induced vasodilatation. This suggests enhanced reactivity of the vasculature to NO in experimental portal hypertension.

Integrated Semiconductor-based Diagnostics System for Multiplexed Genomic Amplification and Electrochemical Detection of Biothreat Agents

DTIC Science & Technology

2009-01-01

microporous disc with adhesive backing. Figure 6. Illustration of the bottom polypropylene layer with the PCR chamber. PCR Chamber UNCLASSIFIED...consistent heating in the PCR chamber. Using insulation and a modified commercial temperature sensor, consistent thermal cycling was achieved with this

Mincle suppresses Toll-like receptor 4 activation.

PubMed

Greco, Stephanie H; Mahmood, Syed Kashif; Vahle, Anne-Kristin; Ochi, Atsuo; Batel, Jennifer; Deutsch, Michael; Barilla, Rocky; Seifert, Lena; Pachter, H Leon; Daley, Donnele; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu R; Miller, George

2016-07-01

Regulation of Toll-like receptor responses is critical for limiting tissue injury and autoimmunity in both sepsis and sterile inflammation. We found that Mincle, a C-type lectin receptor, regulates proinflammatory Toll-like receptor 4 signaling. Specifically, Mincle ligation diminishes Toll-like receptor 4-mediated inflammation, whereas Mincle deletion or knockdown results in marked hyperresponsiveness to lipopolysaccharide in vitro, as well as overwhelming lipopolysaccharide-mediated inflammation in vivo. Mechanistically, Mincle deletion does not up-regulate Toll-like receptor 4 expression or reduce interleukin 10 production after Toll-like receptor 4 ligation; however, Mincle deletion decreases production of the p38 mitogen-activated protein kinase-dependent inhibitory intermediate suppressor of cytokine signaling 1, A20, and ABIN3 and increases expression of the Toll-like receptor 4 coreceptor CD14. Blockade of CD14 mitigates the increased sensitivity of Mincle(-/-) leukocytes to Toll-like receptor 4 ligation. Collectively, we describe a major role for Mincle in suppressing Toll-like receptor 4 responses and implicate its importance in nonmycobacterial models of inflammation. © Society for Leukocyte Biology.

Fuzzy-logic based strategy for validation of multiplex methods: example with qualitative GMO assays.

PubMed

Bellocchi, Gianni; Bertholet, Vincent; Hamels, Sandrine; Moens, W; Remacle, José; Van den Eede, Guy

2010-02-01

This paper illustrates the advantages that a fuzzy-based aggregation method could bring into the validation of a multiplex method for GMO detection (DualChip GMO kit, Eppendorf). Guidelines for validation of chemical, bio-chemical, pharmaceutical and genetic methods have been developed and ad hoc validation statistics are available and routinely used, for in-house and inter-laboratory testing, and decision-making. Fuzzy logic allows summarising the information obtained by independent validation statistics into one synthetic indicator of overall method performance. The microarray technology, introduced for simultaneous identification of multiple GMOs, poses specific validation issues (patterns of performance for a variety of GMOs at different concentrations). A fuzzy-based indicator for overall evaluation is illustrated in this paper, and applied to validation data for different genetically modified elements. Remarks were drawn on the analytical results. The fuzzy-logic based rules were shown to be applicable to improve interpretation of results and facilitate overall evaluation of the multiplex method.

fMRI BOLD response to the eyes task in offspring from multiplex alcohol dependence families.

PubMed

Hill, Shirley Y; Kostelnik, Bryan; Holmes, Brian; Goradia, Dhruman; McDermott, Michael; Diwadkar, Vaibhav; Keshavan, Matcheri

2007-12-01

Increased susceptibility for developing alcohol dependence (AD) may be related to structural and functional differences in brain circuits that influence social cognition and more specifically, theory of mind (ToM). Alcohol dependent individuals have a greater likelihood of having deficits in social skills and greater social alienation. These characteristics may be related to inherited differences in the neuroanatomical network that comprises the social brain. Adolescent/young adult participants from multiplex AD families and controls (n = 16) were matched for gender, age, IQ, education, and handedness and administered the Eyes Task of Baron-Cohen during functional magnetic resonance imaging (fMRI). High-risk (HR) subjects showed significantly diminished blood oxygen level dependent (BOLD) response in comparison with low-risk control young adults in the right middle temporal gyrus (RMTG) and the left inferior frontal gyrus (LIFG), areas that have previously been implicated in ToM tasks. Offspring from multiplex families for AD may manifest one aspect of their genetic susceptibility by having a diminished BOLD response in brain regions associated with performance of ToM tasks. These results suggest that those at risk for developing AD may have reduced ability to empathize with others' state of mind, possibly resulting in diminished social skill.

Flow cytometric detection method for DNA samples

DOEpatents

Nasarabadi, Shanavaz [Livermore, CA; Langlois, Richard G [Livermore, CA; Venkateswaran, Kodumudi S [Round Rock, TX

2011-07-05

Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM.TM. on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA.TM., on the 5' end.

Flow cytometric detection method for DNA samples

DOEpatents

Nasarabadi, Shanavaz [Livermore, CA; Langlois, Richard G [Livermore, CA; Venkateswaran, Kodumudi S [Livermore, CA

2006-08-01

Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM, on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA, on the 5' end.

Trinary Encoder, Decoder, Multiplexer and Demultiplexer Using Savart Plate and Spatial Light Modulator

NASA Astrophysics Data System (ADS)

Ghosh, Amal K.; Singha Roy, Souradip; Mandal, Sudipta; Basuray, Amitabha

Optoelectronic processors have already been developed with the strong potentiality of optics in information and data processing. Encoder, Decoder, Multiplexers and Demultiplexers are the most important components in modern system designs and in communications. We have implemented the same using trinary logic gates with signed magnitude defined as Modified Trinary Number (MTN). The Spatial Light Modulator (SLM) based optoelectronic circuit is suitable for high speed data processing and communications using photon as carrier. We also presented here a possible method of implementing the same using light with photon as carrier of information. The importance of the method is that all the basic gates needed may be fabricated based on basic building block.

Multiplexed interfacial transduction of nucleic acid hybridization using a single color of immobilized quantum dot donor and two acceptors in fluorescence resonance energy transfer.

PubMed

Algar, W Russ; Krull, Ulrich J

2010-01-01

A multiplexed solid-phase assay for the detection of nucleic acid hybridization was developed on the basis of a single color of immobilized CdSe/ZnS quantum dot (QD) as a donor in fluorescence resonance energy transfer (FRET). This work demonstrated that two channels of detection did not necessitate two different QD donors. Two probe oligonucleotides were coimmobilized on optical fibers modified with QDs, and a sandwich assay was used to associate the acceptor dyes with interfacial hybridization events without target labeling. FRET-sensitized acceptor emission provided an analytical signal that was concentration dependent down to 10 nM. Changes in the ratio of coimmobilized probe oligonucleotides were found to yield linear changes in the relative amounts of acceptor emission. These changes were compared to previous studies that used mixed films of two QD donors for two detection channels. The analysis indicated that probe dilution effects were primarily driven by changes in acceptor number density and that QD dilution effects or changes in mean donor-acceptor distance were secondary. Hybridization kinetics were found to be consistent between different ratios of coimmobilized probes, suggesting that hybridization in this type of system occurred via the accepted model for solid-phase hybridization, where adsorption and then diffusion at the solid interface drove hybridization.

Tc-99m-galactosyl-neoglycoalbumin (Tc-NGA) liver imaging: Potential application in liver transplantation

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

Woodle, E.S.; Vera, D.R.; Ward, R.E.

1984-01-01

Tc-NGA is a hepatocyte receptor-specific imaging agent whose uptake by the liver has been shown to be dependent upon blood flow and receptor concentration. The combination of anatomic and physiologic information obtained with Tc-NGA may provide a new tool for studying hepatic function in liver transplant recipients. To evaluate the potential role of Tc-NGA in liver transplant recipients, studies were performed in four groups of pigs: controls (n=18); common bile duct (CBD) ligation (n=8); orthotopic liver transplant (n=9); and acute hepatic artery ligation (n=1). Serial studies performed in two animals with CBD ligation demonstrated normal imaging anatomy with minor changesmore » in the hepatic time-activity curves when compared to control studies. Studies in liver-transplanted animals showed significant changes in the hepatic time-activity curves during acute rejection and in preservation-related ischemic injury. Tc-NGA also demonstrated focal areas of hepatic infarction in a hepatic allograft within 24 hours of transplantation. The hepatic artery ligation study showed massive changes in the hepatic time-activity curve within two hours after ligation, with a diffuse decrease in hepatic activity. These results indicate that: (1) extrahepatic biliary tract obstruction causes only minor changes in Tc-NGA uptake; (2) Tc-NGA uptake by the liver is very sensitive to acute hepatic ischemia; (3) Tc-NGA may indicate the presence of preservation damage in the early postoperative period; and (4) Tc-NGA hepatic time-activity curves demonstrate significant changes during acute rejection.« less

A Modified Method for Creating Elastase-Induced Aneurysms by Ligation of Common Carotid Arteries in Rabbits and Its Effect on Surrounding Arteries.

PubMed

Kainth, Daraspreet; Salazar, Pascal; Safinia, Cyrus; Chow, Ricky; Bachour, Ornina; Andalib, Sasan; McKinney, Alexander M; Divani, Afshin A

2017-01-01

Rabbit models of intracranial aneurysms are frequently used in pre-clinical settings. This study aimed to demonstrate an alternative, extravascular method for creating elastase-induced aneurysms, and how ligation of the right common carotid arteries (RCCA) can impact flow redistribution into left CCA (LCCA). Elastase-induced aneurysms in 18 New Zealand rabbits (4.14 ± 0.314 kg) were created by applying 3-5 U of concentrated elastase solution to the exterior of the right and left CCA roots (RCCA and LCCA). After the induction of the aneurysm, the aneurysm was either kept intact to the rest of the corresponding CCA, severed from the rest of the CCA to allow for a free standing aneurysm, or was anchored to nearby tissue to influence the angle and orientation of the aneurysm with respect to the parent vessel. Ultrasound studies were performed before and after creation of aneurysms to collect blood flow measurements inside the aneurysm pouch and surrounding arteries. Prior to sacrificing the animals, computed tomography angiography studies were performed. Harvested aneurysmal tissues were used for histological analysis. Elastase-induced aneurysms were successfully created by the extravascular approach. Histological studies showed that the biological response was similar to human cerebral aneurysms and previously published elastase-induced rabbit aneurysm models. Ultrasound measurements indicated that after the RCCA was ligated, blood flow significantly increased in the LCCA at one-month follow-up. An alternate method for creating elastase-induced aneurysms has been demonstrated. The novel aspects of our method allow for ligation of one or both common carotid arteries to create a single or bilateral aneurysm with an ability to control the orientation of the induced aneurysm.

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer.

PubMed

Noor, M Omair; Tavares, Anthony J; Krull, Ulrich J

2013-07-25

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1. Copyright © 2013 Elsevier B.V. All rights reserved.

On-site detection of stacked genetically modified soybean based on event-specific TM-LAMP and a DNAzyme-lateral flow biosensor.

PubMed

Cheng, Nan; Shang, Ying; Xu, Yuancong; Zhang, Li; Luo, Yunbo; Huang, Kunlun; Xu, Wentao

2017-05-15

Stacked genetically modified organisms (GMO) are becoming popular for their enhanced production efficiency and improved functional properties, and on-site detection of stacked GMO is an urgent challenge to be solved. In this study, we developed a cascade system combining event-specific tag-labeled multiplex LAMP with a DNAzyme-lateral flow biosensor for reliable detection of stacked events (DP305423× GTS 40-3-2). Three primer sets, both event-specific and soybean species-specific, were newly designed for the tag-labeled multiplex LAMP system. A trident-like lateral flow biosensor displayed amplified products simultaneously without cross contamination, and DNAzyme enhancement improved the sensitivity effectively. After optimization, the limit of detection was approximately 0.1% (w/w) for stacked GM soybean, which is sensitive enough to detect genetically modified content up to a threshold value established by several countries for regulatory compliance. The entire detection process could be shortened to 120min without any large-scale instrumentation. This method may be useful for the in-field detection of DP305423× GTS 40-3-2 soybean on a single kernel basis and on-site screening tests of stacked GM soybean lines and individual parent GM soybean lines in highly processed foods. Copyright © 2017 Elsevier B.V. All rights reserved.

Loop-Mediated Isothermal Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection of Enterococcus faecalis and Staphylococcus aureus

PubMed Central

Wang, Yi; Li, Hui; Wang, Yan; Zhang, Lu; Xu, Jianguo; Ye, Changyun

2017-01-01

The report describes a simple, rapid and sensitive assay for visual and multiplex detection of Enterococcus faecalis and Staphylococcus aureus based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow biosensor (LFB). Detection and differentiation of the Ef0027 gene (E. faecalis-specific gene) and nuc gene (S. aureus-specific gene) were determined using fluorescein (FITC)-and digoxin-modified primers in the mLAMP process. In the presence of biotin- and FITC-/digoxin-modified primers, the mLAMP yielded numerous biotin- and FITC-/digoxin-attached duplex products, which were detected by LFB through biotin/streptavidin interaction (biotin on the duplex and streptavidin on the gold nanoparticle) and immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the LFB test line). The accumulation of gold nanoparticles generated a characteristic red line, enabling visual and multiplex detection of target pathogens without instrumentation. The limit of detection (LoD), analytical specificity and feasibility of LAMP-LFB technique were successfully examined in pure culture and blood samples. The entire procedure, including specimen (blood samples) processing (30 min), isothermal reaction (40 min) and result reporting (within 2 min), could be completed within 75 min. Thus, this assay offers a simple, rapid, sensitive and specific test for multiplex detection of E. faecalis and S. aureus strains. Furthermore, the LAMP-LFB strategy is a universal technique, which can be extended to detect various target sequences by re-designing the specific LAMP primers. PMID:28239371

Switchable in-line monitor for multi-dimensional multiplexed photonic integrated circuit.

PubMed

Chen, Guanyu; Yu, Yu; Ye, Mengyuan; Zhang, Xinliang

2016-06-27

A flexible monitor suitable for the discrimination of on-chip transmitted mode division multiplexed (MDM) and wavelength division multiplexed (WDM) signals is proposed and fabricated. By selectively extracting part of the incoming signals through the tunable wavelength and mode dependent drop filter, the in-line and switchable monitor can discriminate the wavelength, mode and power information of the transmitted signals. Being different from a conventional mode and wavelength demultiplexer, the monitor is specifically designed to ensure a flexible in-line monitoring. For demonstration, three mode and three wavelength multiplexed signals are successfully processed. Assisted by the integrated photodetectors (PDs), both the measured photo currents and eye diagrams validate the performance of the proposed device. The bit error ratio (BER) measurement results show less than 0.4 dB power penalty between different modes and ~2 dB power penalty for single wavelength and WDM cases under 10^-9 BER level.

Microsphere-Based Multiplex Analysis of DNA Methylation in Acute Myeloid Leukemia

PubMed Central

Wertheim, Gerald B.W.; Smith, Catherine; Figueroa, Maria E.; Kalos, Michael; Bagg, Adam; Carroll, Martin; Master, Stephen R.

2015-01-01

Aberrant regulation of DNA methylation is characteristic of cancer cells and clearly influences phenotypes of various malignancies. Despite clear correlations between DNA methylation and patient outcome, tests that directly measure multiple-locus DNA methylation are typically expensive and technically challenging. Previous studies have demonstrated that the prognosis of patients with acute myeloid leukemia can be predicted by the DNA methylation pattern of 18 loci. We have developed a novel strategy, termed microsphere HpaII tiny fragment enrichment by ligation-mediated PCR (MELP), to simultaneously analyze the DNA methylation pattern at these loci using methylation-specific DNA digestion, fluorescently labeled microspheres, and branched DNA hybridization. The method uses techniques that are inexpensive and easily performed in a molecular laboratory. MELP accurately reflects the methylation levels at each locus analyzed and segregates patients with acute myeloid leukemia into prognostic subgroups. Our results demonstrate the usefulness of MELP as a platform for simultaneous evaluation of DNA methylation of multiple loci. PMID:24373919

High-throughput multiplex HLA-typing by ligase detection reaction (LDR) and universal array (UA) approach.

PubMed

Consolandi, Clarissa

2009-01-01

One major goal of genetic research is to understand the role of genetic variation in living systems. In humans, by far the most common type of such variation involves differences in single DNA nucleotides, and is thus termed single nucleotide polymorphism (SNP). The need for improvement in throughput and reliability of traditional techniques makes it necessary to develop new technologies. Thus the past few years have witnessed an extraordinary surge of interest in DNA microarray technology. This new technology offers the first great hope for providing a systematic way to explore the genome. It permits a very rapid analysis of thousands genes for the purpose of gene discovery, sequencing, mapping, expression, and polymorphism detection. We generated a series of analytical tools to address the manufacturing, detection and data analysis components of a microarray experiment. In particular, we set up a universal array approach in combination with a PCR-LDR (polymerase chain reaction-ligation detection reaction) strategy for allele identification in the HLA gene.

Automated cell-type classification in intact tissues by single-cell molecular profiling

PubMed Central

2018-01-01

A major challenge in biology is identifying distinct cell classes and mapping their interactions in vivo. Tissue-dissociative technologies enable deep single cell molecular profiling but do not provide spatial information. We developed a proximity ligation in situ hybridization technology (PLISH) with exceptional signal strength, specificity, and sensitivity in tissue. Multiplexed data sets can be acquired using barcoded probes and rapid label-image-erase cycles, with automated calculation of single cell profiles, enabling clustering and anatomical re-mapping of cells. We apply PLISH to expression profile ~2900 cells in intact mouse lung, which identifies and localizes known cell types, including rare ones. Unsupervised classification of the cells indicates differential expression of ‘housekeeping’ genes between cell types, and re-mapping of two sub-classes of Club cells highlights their segregated spatial domains in terminal airways. By enabling single cell profiling of various RNA species in situ, PLISH can impact many areas of basic and medical research. PMID:29319504

Costs of genetic testing: Supporting Brazilian Public Policies for the incorporating of molecular diagnostic technologies

PubMed Central

Schlatter, Rosane Paixão; Matte, Ursula; Polanczyk, Carisi Anne; Koehler-Santos, Patrícia; Ashton-Prolla, Patricia

2015-01-01

This study identifies and describes the operating costs associated with the molecular diagnosis of diseases, such as hereditary cancer. To approximate the costs associated with these tests, data informed by Standard Operating Procedures for various techniques was collected from hospital software and a survey of market prices. Costs were established for four scenarios of capacity utilization to represent the possibility of suboptimal use in research laboratories. Cost description was based on a single site. The results show that only one technique was not impacted by rising costs due to underutilized capacity. Several common techniques were considerably more expensive at 30% capacity, including polymerase chain reaction (180%), microsatellite instability analysis (181%), gene rearrangement analysis by multiplex ligation probe amplification (412%), non-labeled sequencing (173%), and quantitation of nucleic acids (169%). These findings should be relevant for the definition of public policies and suggest that investment of public funds in the establishment of centralized diagnostic research centers would reduce costs to the Public Health System. PMID:26500437

Chaotic, informational and synchronous behaviour of multiplex networks

NASA Astrophysics Data System (ADS)

Baptista, M. S.; Szmoski, R. M.; Pereira, R. F.; Pinto, S. E. De Souza

2016-03-01

The understanding of the relationship between topology and behaviour in interconnected networks would allow to charac- terise and predict behaviour in many real complex networks since both are usually not simultaneously known. Most previous studies have focused on the relationship between topology and synchronisation. In this work, we provide analytical formulas that shows how topology drives complex behaviour: chaos, information, and weak or strong synchronisation; in multiplex net- works with constant Jacobian. We also study this relationship numerically in multiplex networks of Hindmarsh-Rose neurons. Whereas behaviour in the analytically tractable network is a direct but not trivial consequence of the spectra of eigenvalues of the Laplacian matrix, where behaviour may strongly depend on the break of symmetry in the topology of interconnections, in Hindmarsh-Rose neural networks the nonlinear nature of the chemical synapses breaks the elegant mathematical connec- tion between the spectra of eigenvalues of the Laplacian matrix and the behaviour of the network, creating networks whose behaviour strongly depends on the nature (chemical or electrical) of the inter synapses.

Multiplex detection of pathogen biomarkers in human blood, serum, and saliva using silicon photonic microring resonators

NASA Astrophysics Data System (ADS)

Estrada, I. A.; Burlingame, R. W.; Wang, A. P.; Chawla, K.; Grove, T.; Wang, J.; Southern, S. O.; Iqbal, M.; Gunn, L. C.; Gleeson, M. A.

2015-05-01

Genalyte has developed a multiplex silicon photonic chip diagnostics platform (MaverickTM) for rapid detection of up to 32 biological analytes from a drop of sample in just 10 to 20 minutes. The chips are manufactured with waveguides adjacent to ring resonators, and probed with a continuously variable wavelength laser. A shift in the resonant wavelength as mass binds above the ring resonators is measured and is directly proportional to the amount of bound macromolecules. We present here the ability to multiplex the detection of hemorrhagic fever antigens in whole blood, serum, and saliva in a 16 minute assay. Our proof of concept testing of a multiplex antigencapture chip has the ability to detect Zaire Ebola (ZEBOV) recombinant soluble glycoprotein (rsGP), Marburg virus (MARV) Angola recombinant glycoprotein (rGP) and dengue nonstructural protein I (NS1). In parallel, detection of 2 malaria antigens has proven successful, but has yet to be incorporated into multiplex with the others. Each assay performs with sensitivity ranging from 1.6 ng/ml to 39 ng/ml depending on the antigen detected, and with minimal cross-reactivity.

An Improved Model Predictive Current Controller of Switched Reluctance Machines Using Time-Multiplexed Current Sensor

PubMed Central

Li, Bingchu; Ling, Xiao; Huang, Yixiang; Gong, Liang; Liu, Chengliang

2017-01-01

This paper presents a fixed-switching-frequency model predictive current controller using multiplexed current sensor for switched reluctance machine (SRM) drives. The converter was modified to distinguish currents from simultaneously excited phases during the sampling period. The only current sensor installed in the converter was time division multiplexing for phase current sampling. During the commutation stage, the control steps of adjacent phases were shifted so that sampling time was staggered. The maximum and minimum duty ratio of pulse width modulation (PWM) was limited to keep enough sampling time for analog-to-digital (A/D) conversion. Current sensor multiplexing was realized without complex adjustment of either driver circuit nor control algorithms, while it helps to reduce the cost and errors introduced in current sampling due to inconsistency between sensors. The proposed controller is validated by both simulation and experimental results with a 1.5 kW three-phase 12/8 SRM. Satisfied current sampling is received with little difference compared with independent phase current sensors for each phase. The proposed controller tracks the reference current profile as accurately as the model predictive current controller with independent phase current sensors, while having minor tracking errors compared with a hysteresis current controller. PMID:28513554

Chemical and Biological Tools for the Preparation of Modified Histone Proteins

PubMed Central

Howard, Cecil J.; Yu, Ruixuan R.; Gardner, Miranda L.; Shimko, John C.; Ottesen, Jennifer J.

2016-01-01

Eukaryotic chromatin is a complex and dynamic system in which the DNA double helix is organized and protected by interactions with histone proteins. This system is regulated through, a large network of dynamic post-translational modifications (PTMs) exists to ensure proper gene transcription, DNA repair, and other processes involving DNA. Homogenous protein samples with precisely characterized modification sites are necessary to better understand the functions of modified histone proteins. Here, we discuss sets of chemical and biological tools that have been developed for the preparation of modified histones, with a focus on the appropriate choice of tool for a given target. We start with genetic approaches for the creation of modified histones, including the incorporation of genetic mimics of histone modifications, chemical installation of modification analogs, and the use of the expanded genetic code to incorporate modified amino acids. Additionally, we will cover the chemical ligation techniques that have been invaluable in the generation of complex modified histones that are indistinguishable from the natural counterparts. Finally, we will end with a prospectus on future directions of synthetic chromatin in living systems. PMID:25863817

A temperature-tolerant multiplex elements and genes screening system for genetically modified organisms based on dual priming oligonucleotide primers and capillary electrophoresis.

PubMed

Fu, Wei; Wei, Shuang; Wang, Chenguang; Du, Zhixin; Zhu, Pengyu; Wu, Xiyang; Wu, Gang; Zhu, Shuifang

2017-08-15

High throughput screening systems are the preferred solution to meet the urgent requirement of increasing number of genetically modified organisms (GMOs). In this study, we have successfully developed a multiplex GMO element screening system with dual priming oligonucleotide (DPO) primers. This system can detect the cauliflower mosaic virus 35S (CaMV 35S), terminator of nopaline synthase gene (NOS), figwort mosaic virus 35S (FMV 35S) promoter, neomycin phosphotransferaseII (NPTII), Bt Cry 1Ab, phosphinothricin acetyltransferase genes (bar) and Streptomyces viridochromogenes (pat) simultaneously, which covers more than 90% of all authorized GMO species worldwide. This system exhibits a high tolerance to annealing temperatures, high specificity and a limit of detection equal to conventional PCR. A total of 214 samples from markets, national entry-exit agencies, the Institute for Reference Materials and Measurement (IRMM) and the American Oil Chemists' Society (AOCS) were also tested for applicability. This screening system is therefore suitable for GMO screening. Copyright © 2017 Elsevier Ltd. All rights reserved.

Femoral vein injury managed by in situ saphenous vein bypass : a case report.

PubMed

Coppin, Th; Kuhnle, M

2014-01-01

Injured veins of the lower limbs may cause massive haemorrhage requiring early control. Operative management of injured veins remains a controversial topic and reconstruction or ligation depends on venous and adjacent tissue damage. Nevertheless, venous reconstruction seems to reduce the complications of venous ligation. The case of a 33 year old women with a venous wound to the right groin is presented. Surgical management consisted of controlling the bleeding and venous revascularisation with an in situ saphenous vein bypass to substitute the injured femoral vein. The patient had an uneventful postoperative period without any complications. The case demonstrates this method of vascular venous repair is the preferable one. Copyright© Acta Chirurgica Belgica.

Development of a multiplex DNA-based traceability tool for crop plant materials.

PubMed

Voorhuijzen, Marleen M; van Dijk, Jeroen P; Prins, Theo W; Van Hoef, A M Angeline; Seyfarth, Ralf; Kok, Esther J

2012-01-01

The authenticity of food is of increasing importance for producers, retailers and consumers. All groups benefit from the correct labelling of the contents of food products. Producers and retailers want to guarantee the origin of their products and check for adulteration with cheaper or inferior ingredients. Consumers are also more demanding about the origin of their food for various socioeconomic reasons. In contrast to this increasing demand, correct labelling has become much more complex because of global transportation networks of raw materials and processed food products. Within the European integrated research project 'Tracing the origin of food' (TRACE), a DNA-based multiplex detection tool was developed-the padlock probe ligation and microarray detection (PPLMD) tool. In this paper, this method is extended to a 15-plex traceability tool with a focus on products of commercial importance such as the emmer wheat Farro della Garfagnana (FdG) and Basmati rice. The specificity of 14 plant-related padlock probes was determined and initially validated in mixtures comprising seven or nine plant species/varieties. One nucleotide difference in target sequence was sufficient for the distinction between the presence or absence of a specific target. At least 5% FdG or Basmati rice was detected in mixtures with cheaper bread wheat or non-fragrant rice, respectively. The results suggested that even lower levels of (un-)intentional adulteration could be detected. PPLMD has been shown to be a useful tool for the detection of fraudulent/intentional admixtures in premium foods and is ready for the monitoring of correct labelling of premium foods worldwide.

Genetic engineering and chemical conjugation of potato virus X.

PubMed

Lee, Karin L; Uhde-Holzem, Kerstin; Fischer, Rainer; Commandeur, Ulrich; Steinmetz, Nicole F

2014-01-01

Here we report the genetic engineering and chemical modification of potato virus X (PVX) for the presentation of various peptides, proteins, and fluorescent dyes, or other chemical modifiers. Three different ways of genetic engineering are described and by these means, peptides are successfully expressed not only when the foot and mouth disease virus (FMDV) 2A sequence or a flexible glycine-serine linker is included, but also when the peptide is fused directly to the PVX coat protein. When larger proteins or unfavorable peptide sequences are presented, a partial fusion via the FMDV 2A sequence is preferable. When these PVX chimeras retain the ability to assemble into viral particles and are thus able to infect plants systemically, they can be utilized to inoculate susceptible plants for isolation of sufficient amounts of virus particles for subsequent chemical modification. Chemical modification is required for the display of nonbiological ligands such as fluorophores, polymers, and small drug compounds. We present three methods of chemical bioconjugation. For direct conjugation of small chemical modifiers to solvent exposed lysines, N-hydroxysuccinimide chemistry can be applied. Bio-orthogonal reactions such as copper-catalyzed azide-alkyne cycloaddition or hydrazone ligation are alternatives to achieve more efficient conjugation (e.g., when working with high molecular weight or insoluble ligands). Furthermore, hydrazone ligation offers an attractive route for the introduction of pH-cleavable cargos (e.g., therapeutic molecules).

[An attempt to identify 22q11.2 microdeletions in samples of the Hungarian schizophrenia DNA bank by multiplex ligation-based probe amplification (MLPA): literature review, methodology and results].

PubMed

Klein, Izabella; Szocs, Katalin; Vincze, Katalin; Benkovits, Judit; Somogyi, Szilvia; Herman, Levente; Rethelyi, Janos M

2016-12-01

Schizophrenia is a severe debilitating psychiatric disorder, with a typical onset in adolescence or early adulthood. This condition is characterized by heterogeneous symptoms (hallucinations, delusions, disorganized behaviour, affective flattening, and social isolation) and a life-time prevalence of 0.5-1.2%. In spite of the efforts to uncover the etiology of the disorder, its pathogenesis and neurobiological background are poorly understood. Given the high heritability in schizophrenia, genetic research remains an important area of focus. Besides the common variations of low penetrance - single nucleotid polymorphisms (SNPs) -, rare variants, mainly copy number variations (CNVs) play a role in the genetic architecture of the disorder. The most frequent CNV associated with schizophrenia is the hemizygous deletion of the 22q11.2 region. According to previous research this genetic variant occurs in 1% of the patients and conversely, 25% of the carriers of the 22q11.2 microdeletion will develop schizophrenia. The 22q11.2 deletion syndrome (22Q11DS, velocardiofacial (VCFS) syndrome, DiGeorge-syndrome) is usually a childhood diagnosis. Its prevalence is 1:2000-4000 considering all births. Patients can demonstrate heart developmental disorders, craniofacial (elongated face, hypertelorism), immunological (thymus-hypoplasia), endocrinological (hypocalcaemia) abnormalities, and neurodevelopmental alterations, but only a proportion will have these abnormalities due to incomplete penetrance. The variable symptoms complicate the recognition of the syndrome in the day to day medical practice. 25% of the known 22Q11DS patients develop schizophrenia but the risk of neuropsychiatric problems, like autism, ADHD and childhood conduct disorder is also increased, while early onset Parkinson's disease in also more frequent in adults. The schizophrenia phenotype is not distinguishable at the moment in patients with or without the 22q11 deletion. But emerging evidence suggests that early onset Parkinson's disease is more frequent in 22Q11DS and the effects of clozapine treatment could be different in schizophrenia with 22Q11DS. The question arises what is the incidence rate of the 22q11.2 microdeletion among our Hungarian DNA samples with schizophrenia. To answer the question, we utilized a new method used in routine genetic diagnostics, multiplex ligation-based probe amplification (MLPA). Although we genotyped the DNA of 315 Hungarian schizophrenia patients, we found no 22Q11DS in this cohort. The findings are discussed in terms of basic research and their translation into everyday clinical practice.

Tetraspanin CD9 participates in dysmegakaryopoiesis and stromal interactions in primary myelofibrosis.

PubMed

Desterke, Christophe; Martinaud, Christophe; Guerton, Bernadette; Pieri, Lisa; Bogani, Costanza; Clay, Denis; Torossian, Frederic; Lataillade, Jean-Jacques; Hasselbach, Hans C; Gisslinger, Heinz; Demory, Jean-Loup; Dupriez, Brigitte; Boucheix, Claude; Rubinstein, Eric; Amsellem, Sophie; Vannucchi, Alessandro M; Le Bousse-Kerdilès, Marie-Caroline

2015-06-01

Primary myelofibrosis is characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in the bone marrow and spleen. The expression of CD9, a tetraspanin known to participate in megakaryopoiesis, platelet formation, cell migration and interaction with stroma, is deregulated in patients with primary myelofibrosis and is correlated with stage of myelofibrosis. We investigated whether CD9 participates in the dysmegakaryopoiesis observed in patients and whether it is involved in the altered interplay between megakaryocytes and stromal cells. We found that CD9 expression was modulated during megakaryocyte differentiation in primary myelofibrosis and that cell surface CD9 engagement by antibody ligation improved the dysmegakaryopoiesis by restoring the balance of MAPK and PI3K signaling. When co-cultured on bone marrow mesenchymal stromal cells from patients, megakaryocytes from patients with primary myelofibrosis displayed modified behaviors in terms of adhesion, cell survival and proliferation as compared to megakaryocytes from healthy donors. These modifications were reversed after antibody ligation of cell surface CD9, suggesting the participation of CD9 in the abnormal interplay between primary myelofibrosis megakaryocytes and stroma. Furthermore, silencing of CD9 reduced CXCL12 and CXCR4 expression in primary myelofibrosis megakaryocytes as well as their CXCL12-dependent migration. Collectively, our results indicate that CD9 plays a role in the dysmegakaryopoiesis that occurs in primary myelofibrosis and affects interactions between megakaryocytes and bone marrow stromal cells. These results strengthen the "bad seed in bad soil" hypothesis that we have previously proposed, in which alterations of reciprocal interactions between hematopoietic and stromal cells participate in the pathogenesis of primary myelofibrosis. Copyright© Ferrata Storti Foundation.

Stable coordination of the inhibitory Ca2+ ion at MIDAS in integrin CD11b/CD18 by an antibody-derived ligand aspartate: Implications for integrin regulation and structure-based drug design

PubMed Central

Mahalingam, Bhuvaneshwari; Ajroud, Kaouther; Alonso, Jose Luis; Anand, Saurabh; Adair, Brian; Horenstein, Alberto L; Malavasi, Fabio; Xiong, Jian-Ping; Arnaout, M. Amin

2011-01-01

A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg2+ ion hexacoordinated at the metal-ion-dependent-adhesion site (MIDAS) in the integrin A-domain. This interaction stabilizes the A-domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A-domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking monoclonal antibody (mAb) 107 binds MIDAS of integrin CD11b/CD18 A-domain (CD11bA), but in contrast, it favors the inhibitory Ca2+ ion over Mg2+ at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of Ca2+ at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca2+. Binding of Fab 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that denticity of the ligand Asp/Glu can modify divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca2+ ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists. PMID:22095715

Efficacy of carvedilol versus propranolol versus variceal band ligation for primary prevention of variceal bleeding.

PubMed

Abd ElRahim, Ayman Yosry; Fouad, Rabab; Khairy, Marwa; Elsharkawy, Aisha; Fathalah, Waleed; Khatamish, Haytham; Khorshid, Omayma; Moussa, Mona; Seyam, Moataz

2018-01-01

Band ligation and propranolol are the current therapies for primary prevention of variceal bleeding. Carvedilol is a rising nonselective beta-blocker used for reducing portal pressure with favorable outcome. The aim of this study to assess the efficacy of carvedilol, propranolol, and band ligation for primary prevention of variceal bleeding based on the effect of each regimen on progression of Child score and portal hypertensive gastropathy after 1 year. The study included 264 cirrhotic patients with medium/large-sized varices who were candidates for primary prophylaxis of variceal bleeding. Patients were randomly divided into three groups: group I: band ligation; group II: propranolol; group III: carvedilol. Group I showed higher success rate of 75 %, followed by group III with 70.2 % and group II with 65.2 %. Risk of bleeding was comparable between the three groups, with group II carrying the highest rate of complications (34.7 %) followed by group III (14.2 %) and finally group I (5.7 %). After 1 year of follow-up, Child score did not improve in any of the studied groups, while portal hypertensive gastropathy significantly increased in group I but decreased in groups II and III. Band ligation is the best treatment option for primary prevention of variceal bleeding with minimal complications. Carvedilol is a good pharmaceutical alternative medicine to propranolol with lesser side-effects. Progress of liver disease as represented by Child score is not affected by any of the primary variceal prophylactic regimens, although medical treatment reduces portal hypertensive gastropathy. Choice of treatment depends on patient will, compliance with treatment, and endoscopist competence.

Taurine zinc solid dispersions enhance bile-incubated L02 cell viability and improve liver function by inhibiting ERK2 and JNK phosphorylation during cholestasis.

PubMed

Wang, Yu; Mei, Xueting; Yuan, Jingquan; Lai, Xiaofang; Xu, Donghui

2016-07-29

Dietary intakes of taurine and zinc are associated with decreased risk of liver disease. In this study, solid dispersions (SDs) of a taurine zinc complex on hepatic injury were examined in vitro using the immortalized human hepatocyte cell line L02 and in a rat model of bile duct ligation. Sham-operated and bile duct ligated Sprague-Dawley rats were treated with the vehicle alone or taurine zinc (40, 80, 160mg/kg) for 17days. Bile duct ligation significantly increased blood lipid levels, and promoted hepatocyte apoptosis, inflammation and compensatory biliary proliferation. In vitro, incubation with bile significantly reduced L02 cell viability; this effect was significantly attenuated by pretreatment with SP600125 (a JNK inhibitor) and enhanced when co-incubated with taurine zinc SDs. In vivo, administration of taurine zinc SDs decreased serum alanine aminotransferase and aspartate aminotransferase activities in a dose-dependent manner and attenuated the increases in serum total bilirubin, total cholesterol and low density lipoprotein cholesterol levels after bile duct ligation. Additionally, taurine zinc SDs downregulated the expression of interleukin-1β and inhibited the phosphorylation of Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase2 (ERK2) in the liver after bile duct ligation. Moreover, taurine zinc SDs had more potent blood lipid regulatory and anti-apoptotic effects than the physical mixture of taurine and zinc acetate. Therefore, we speculate that taurine zinc SDs protect liver function at least in part via a mechanism linked to reduce phosphorylation of JNK and ERK2, which suppresses inflammation, apoptosis and cholangiocyte proliferation during cholestasis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Thrombospondin-1 expression may be implicated in liver atrophic mechanism due to obstructed portal venous flow.

PubMed

Hayashi, Hiromitsu; Kuroki, Hideyuki; Higashi, Takaaki; Takeyama, Hideaki; Yokoyama, Naomi; Okabe, Hirohisa; Nitta, Hidetoshi; Beppu, Toru; Takamori, Hiroshi; Baba, Hideo

2017-07-01

Liver is an amazing organ that can undergo regenerative and atrophic changes inversely, depending on blood flow conditions. Although the regenerative mechanism has been extensively studied, the atrophic mechanism remains to be elucidated. To assess the molecular mechanism of liver atrophy due to reduced portal blood flow, we analyzed the gene expressions between atrophic and hypertrophic livers induced by portal vein embolization in three human liver tissues using microarray analyses. Thrombospondin (TSP)-1 is an extracellular protein and a negative regulator of liver regeneration through its activation of the transforming growth factor-β/Smad signaling pathway. TSP-1 was extracted as the most upregulated gene in atrophic liver compared to hypertrophic liver due to portal flow obstruction in human. Liver atrophic and hypertrophic changes were confirmed by HE and proliferating cell nuclear antigen staining and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling. In an in vivo model with portal ligation, TSP-1 and phosphorylated Smad2 expression were continuously induced at 6 h and thereafter in the portal ligated liver, whereas the induction was transient at 6 h in the portal non-ligated liver. Indeed, while cell proliferation represented by proliferating cell nuclear antigen expression at 48 h was induced in the portal ligated liver, the sinusoidal dilatation and hepatocyte cell death with terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling was detectable at 48 h in the portal ligated liver. Obstructed portal flow induces persistent TSP-1 expression and transforming growth factor-β/Smad signal activation in atrophic liver. Thrombospondin-1 may be implicated in the liver atrophic change due to obstructed portal flow as a pro-atrophic factor. © 2016 The Japan Society of Hepatology.

Dynamic article: surgical anatomical planes for complete mesocolic excision and applied vascular anatomy of the right colon.

PubMed

Açar, Halil İbrahim; Cömert, Ayhan; Avşar, Abdullah; Çelik, Safa; Kuzu, Mehmet Ayhan

2014-10-01

Lower local recurrence rates and better overall survival are associated with complete mesocolic excision with central vascular ligation for treatment of colon cancer. To accomplish this, surgeons need to pay special attention to the surgical anatomical planes and vascular anatomy of the colon. However, surgical education in this area has been neglected. The aim of this study is to define the correct surgical anatomical planes for complete mesocolic excision with central vascular ligation and to demonstrate the correct dissection technique for protecting anatomical structures. Macroscopic and microscopic surgical dissections were performed on 12 cadavers in the anatomy laboratory and on autopsy specimens. The dissections were recorded as video clips. Dissections were performed in accordance with the complete mesocolic excision technique on 10 male and 2 female cadavers. Vascular structures, autonomic nerves, and related fascias were shown. Within each step of the surgical procedure, important anatomical structures were displayed on still images captured from videos by animations. Three crucial steps for complete mesocolic excision with central vascular ligation are demonstrated on the cadavers: 1) full mobilization of the superior mesenteric root following the embryological planes between the visceral and the parietal fascias; 2) mobilization of the mesocolon from the duodenum and the pancreas and identification of vascular structures, especially the veins around the pancreas; and 3) central vascular ligation of the colonic vessels at their origin, taking into account the vascular variations within the mesocolonic vessels and the autonomic nerves around the superior mesenteric artery. The limitation of this study was the number of the cadavers used. Successful complete mesocolic excision with central vascular ligation depends on an accurate knowledge of the surgical anatomical planes and the vascular anatomy of the colon.

Development of authentication code for multi-access optical code division multiplexing based quantum key distribution

NASA Astrophysics Data System (ADS)

Taiwo, Ambali; Alnassar, Ghusoon; Bakar, M. H. Abu; Khir, M. F. Abdul; Mahdi, Mohd Adzir; Mokhtar, M.

2018-05-01

One-weight authentication code for multi-user quantum key distribution (QKD) is proposed. The code is developed for Optical Code Division Multiplexing (OCDMA) based QKD network. A unique address assigned to individual user, coupled with degrading probability of predicting the source of the qubit transmitted in the channel offer excellent secure mechanism against any form of channel attack on OCDMA based QKD network. Flexibility in design as well as ease of modifying the number of users are equally exceptional quality presented by the code in contrast to Optical Orthogonal Code (OOC) earlier implemented for the same purpose. The code was successfully applied to eight simultaneous users at effective key rate of 32 bps over 27 km transmission distance.

Detection of Shiga-Like Toxin (stx1 and stx2), Intimin (eaeA), and Enterohemorrhagic Escherichia coli (EHEC) Hemolysin (EHEC hlyA) Genes in Animal Feces by Multiplex PCR

PubMed Central

Fagan, Peter K.; Hornitzky, Michael A.; Bettelheim, Karl A.; Djordjevic, Steven P.

1999-01-01

A multiplex PCR was developed for the rapid detection of genes encoding Shiga toxins 1 and 2 (stx1 and stx2), intimin (eaeA), and enterohemolysin A (hlyA) in 444 fecal samples derived from healthy and clinically affected cattle, sheep, pigs, and goats. The method involved non-solvent-based extraction of nucleic acid from an aliquot of an overnight culture of feces in EC (modified) broth. The detection limit of the assay for both fecal samples and pure cultures was between 18 and 37 genome equivalents. stx1 and hlyA were the most commonly encountered virulence factors. PMID:9925634

Unsteady Flow Field Measurements Using LDV (Laser Doppler Velocimetry).

DTIC Science & Technology

1987-12-01

data and digitized velocity data F.-o- the LDV signal processors were channeled to a 3D -LDV Computer ,nterface (CI). The CI, multiplexing the inputs...beam al _:gnm.ent Fr L.Zr’.s3 , - steering wedges, within the Bragg ’ell modules serves l: bring the beams back t: parallel. A 7i:r~s:: pe -b:t .e, pla...INITIALS DESCRIPTION C 07/26/83 TML Adapted from DAPNT C 12/12,/85 CLH Modified to print results in either Coctal or integer. C 02/25/87 GBG Modified

Effect of ligation method on maxillary arch force/moment systems for a simulated lingual incisor malalignment.

PubMed

Seru, Surbhi; Romanyk, Dan L; Toogood, Roger W; Carey, Jason P; Major, Paul W

2014-01-01

The objectives of this study were to determine whether there is a difference in the magnitude of forces and moments produced by elastic ligation when compared to passive ligation, and whether these forces and moments propagate differently along the arch for the two ligation types. A lingual incisor malalignment was used in this study. The Orthodontic Simulator (OSIM) was used to quantify the three-dimensional forces and moments applied on the teeth given a lingually displaced incisor. A repeated measures MANOVA was performed to statistically analyze the data. The interaction factor illustrated convincing evidence that there is a difference in maximum force and moment values for all outcome variables between ligation types considering all tooth positions along the arch. The mean differences for FX and FY between ligation types were found to be clinically significant, with values for elastic ligation consistently higher than passive ligation. It was found that the maximum forces and moments produced by elastic ligation are greater than those produced by passive ligation and that the magnitude of this difference for the mesiodistal and buccolingual forces is clinically relevant. Additionally, it was determined that elastic ligation causes forces and moments to propagate further along the arch than passive ligation for all outcome variables.

Incidence of white spot lesions among patients treated with self- and conventional ligation systems.

PubMed

Akin, Mehmet; Tezcan, Mucella; Ileri, Zehra; Ayhan, Faruk

2015-07-01

The aim of this study was to investigate the incidence of white spot lesions (WSLs) and its relationship with various patient and treatment variables, in patients treated with self-ligation and conventional ligation orthodontic bracket systems. Two-hundred randomly selected patient records (136 female, 64 male) for self-ligation and (108 female, 92 male) for conventional ligation groups were examined to determine WSL development. In the self-ligation group, Damon 3MX (Ormco, Glendora, Calif) brackets had been used, and in the conventional ligation group, Equilibrium 2 (Dentaurum, Phorzeim, Germany) had been used. Labial surfaces of 24 teeth in the pre- and post-treatment photographic records were scored using the WSL index. The prevalence of patients who developed at least 1 WSL before treatment was 19%, whereas after treatment, it was 49% in the self-ligation and 54% in the conventional ligation groups. Before treatment, the patients had only mild WSL, but after treatment, severe WSL and cavitation were observed in both groups. Bracket type, age, and hygiene care were significantly associated with new WSL development (P = 0.008, P = 0.004, P = 0.013, respectively). Bracket type and more importantly, the hygiene care therapy provided appeared to influence the development of new WSLs. Ligation can promote plaque accumulation and thereby new WSL development in conventional bracket systems. This article investigates the incidence of WSLs in patients treated with self-ligation and conventional ligation. The present study showed that incidence of WSL less in the self-ligation than in the conventional ligation but hygiene care was mostly important factor in developed WSL.

[Comparative genomic hybridisation as a first option in genetic diagnosis: 1,000 cases and a cost-benefit analysis].

PubMed

Castells-Sarret, Neus; Cueto-González, Anna M; Borregan, Mar; López-Grondona, Fermina; Miró, Rosa; Tizzano, Eduardo; Plaja, Alberto

2017-09-25

Conventional cytogenetics diagnoses 3-5% of patients with unexplained developmental delay/intellectual disability and/or multiple congenital anomalies. The Multiplex Ligation-dependent Probe Amplification increases diagnostic rates from between 2.4 to 5.8%. Currently the comparative genomic hybridisation array or aCGH is the highest performing diagnostic tool in patients with developmental delay/intellectual disability, congenital anomalies and autism spectrum disorders. Our aim is to evaluate the efficiency of the use of aCGH as first-line test in these and other indications (epilepsy, short stature). A total of 1000 patients referred due to one or more of the abovementioned disorders were analysed by aCGH. Pathogenic genomic imbalances were detected in 14% of the cases, with a variable distribution of diagnosis according to the phenotypes: 18.9% of patients with developmental delay/intellectual disability; 13.7% of multiple congenital anomalies, 9.76% of psychiatric pathologies, 7.02% of patients with epilepsy, and 13.3% of patients with short stature. Within the multiple congenital anomalies, central nervous system abnormalities and congenital heart diseases accounted for 14.9% and 10.6% of diagnoses, respectively. Among the psychiatric disorders, patients with autism spectrum disorders accounted for 8.9% of the diagnoses. Our results demonstrate the effectiveness and efficiency of the use of aCGH as the first line test in genetic diagnosis of patients suspected of genomic imbalances, supporting its inclusion within the National Health System. Copyright © 2017. Publicado por Elsevier España, S.L.U.

MECP2 gene study in a large cohort: testing of 240 female patients and 861 healthy controls (519 females and 342 males).

PubMed

Maortua, Hiart; Martínez-Bouzas, Cristina; García-Ribes, Ainhoa; Martínez, María-Jesus; Guillen, Encarna; Domingo, María-Rosario; Calvo, María-Teresa; Guitart, Miriam; Gabau, Elisabeth; Botella, María-Pilar; Gener, Blanca; Rubio, Izaskun; López-Aríztegui, María-Asunción; Tejada, María-Isabel

2013-09-01

The MECP2 gene located on Xq28 is one of the most important genes contributing to the spectrum of neurodevelopmental disorders. Therefore, we present our experience in the molecular study of this gene. MECP2 was thoroughly tested for the presence of mutations (sequencing of four exons and rearrangements) in 120 female patients: 28 with classic Rett syndrome, five with atypical Rett syndrome, and 87 with heterogeneous phenotypes with some Rett-like features. Another 120 female patients with intellectual disability of unknown origin were also studied, but in these cases we only tested exons 3 and 4. Finally, 861 healthy controls (519 females and 342 males) were also studied for exon 3 and 4. Eighteen different pathological mutations were found, five of them previously undescribed, and four large deletions detected by multiplex ligation-dependent probe amplification. All were de novo mutations not present in the parents. In conclusion, i) MECP2 is one of the most important genes in the diagnosis of genetic intellectual disability in females; ii) MECP2 must be studied not only in patients with classical/atypical Rett syndrome but also in patients with other phenotypes related to Rett syndrome; and iii) for the new variants, it is important to perform complementary studies, including the analysis of large populations of healthy individuals and the use of in silico programs. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Validation and Implementation of BRCA1/2 Variant Screening in Ovarian Tumor Tissue.

PubMed

de Jonge, Marthe M; Ruano, Dina; van Eijk, Ronald; van der Stoep, Nienke; Nielsen, Maartje; Wijnen, Juul T; Ter Haar, Natalja T; Baalbergen, Astrid; Bos, Monique E M M; Kagie, Marjolein J; Vreeswijk, Maaike P G; Gaarenstroom, Katja N; Kroep, Judith R; Smit, Vincent T H B M; Bosse, Tjalling; van Wezel, Tom; van Asperen, Christi J

2018-06-21

BRCA1/2 variant analysis in tumor tissue could streamline the referral of patients with epithelial ovarian, fallopian tube, or primary peritoneal cancer to genetic counselors and select patients who benefit most from targeted treatment. We investigated the sensitivity of BRCA1/2 variant analysis in formalin-fixed, paraffin-embedded tumor tissue using a combination of next-generation sequencing and copy number variant multiplex ligation-dependent probe amplification. After optimization using a training cohort of known BRCA1/2 mutation carriers, validation was performed in a prospective cohort (Clinical implementation Of BRCA1/2 screening in ovarian tumor tissue: COBRA-cohort) in which screening of BRCA1/2 tumor DNA and leukocyte germline DNA was performed in parallel. BRCA1 promoter hypermethylation and pedigree analysis were also performed. In the training cohort 45 of 46 germline BRCA1/2 variants were detected (sensitivity 98%). In the COBRA cohort (n=62), all six germline variants were identified (sensitivity 100%), together with five somatic BRCA1/2 variants and eight cases with BRCA1 promoter hypermethylation. In four BRCA1/2 variant-negative patients, surveillance or prophylactic management options were offered based on positive family histories. We conclude that BRCA1/2 formalin-fixed, paraffin-embedded tumor tissue analysis reliably detects BRCA1/2 variants. When taking family history of BRCA1/2 variant-negative patients into account, tumor BRCA1/2 variant screening allows more efficient selection of epithelial ovarian cancer patients for genetic counselling and simultaneously selects patients who benefit most from targeted treatment. Copyright © 2018. Published by Elsevier Inc.

Molecular diagnosis of α-thalassemia in a multiethnic population.

PubMed

Gilad, Oded; Shemer, Orna Steinberg; Dgany, Orly; Krasnov, Tanya; Nevo, Michal; Noy-Lotan, Sharon; Rabinowicz, Ron; Amitai, Nofar; Ben-Dor, Shifra; Yaniv, Isaac; Yacobovich, Joanne; Tamary, Hannah

2017-06-01

α-Thalassemia, one of the most common genetic diseases, is caused by deletions or point mutations affecting one to four α-globin genes. Molecular diagnosis is important to prevent the most severe forms of the disease. However, the diagnosis of α-thalassemia is complex due to a high variability of the genetic defects involved, with over 250 described mutations. We summarize herein the findings of genetic analyses of DNA samples referred to our laboratory for the molecular diagnosis of α-thalassemia, along with a detailed clinical description. We utilized a diagnostic algorithm including Gap-PCR, to detect known deletions, followed by sequencing of the α-globin gene, to identify known and novel point mutations, and multiplex ligation-dependent probe amplification (MLPA) for the diagnosis of rare or novel deletions. α-Thalassemia was diagnosed in 662 of 975 samples referred to our laboratory. Most commonly found were deletions (75.3%, including two novel deletions previously described by us); point mutations comprised 25.4% of the cases, including five novel mutations. Our population included mostly Jews (of Ashkenazi and Sephardic origin) and Muslim Arabs, who presented with a higher rate of point mutations and hemoglobin H disease. Overall, we detected 53 different genotype combinations causing a spectrum of clinical phenotypes, from asymptomatic to severe anemia. Our work constitutes the largest group of patients with α-thalassemia originating in the Mediterranean whose clinical characteristics and molecular basis have been determined. We suggest a diagnostic algorithm that leads to an accurate molecular diagnosis in multiethnic populations. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Clinical and Molecular Heterogeneity in Brazilian Patients with Sotos Syndrome

PubMed Central

Vieira, Gustavo H.; Cook, Melissa M.; Ferreira De Lima, Renata L.; Frigério Domingues, Carlos E.; de Carvalho, Daniel R.; Soares de Paiva, Isaias; Moretti-Ferreira, Danilo; Srivastava, Anand K.

2015-01-01

Sotos syndrome (SoS) is a multiple anomaly, congenital disorder characterized by overgrowth, macrocephaly, distinctive facial features and variable degree of intellectual disability. Haploinsufficiency of the NSD1 gene at 5q35.3, arising from 5q35 microdeletions, point mutations, and partial gene deletions, accounts for a majority of patients with SoS. Recently, mutations and possible pathogenetic rare CNVs, both affecting a few candidate genes for overgrowth, have been reported in patients with Sotos-like overgrowth features. To estimate the frequency of NSD1 defects in the Brazilian SoS population and possibly reveal other genes implicated in the etiopathogenesis of this syndrome, we collected a cohort of 21 Brazilian patients, who fulfilled the diagnostic criteria for SoS, and analyzed the NSD1 and PTEN genes by means of multiplex ligation-dependent probe amplification and mutational screening analyses. We identified a classical NSD1 microdeletion, a novel missense mutation (p.C1593W), and 2 previously reported truncating mutations: p.R1984X and p.V1760Gfs*2. In addition, we identified a novel de novo PTEN gene mutation (p.D312Rfs*2) in a patient with a less severe presentation of SoS phenotype, which did not include pre- and postnatal overgrowth. For the first time, our study implies PTEN in the pathogenesis of SoS and further emphasizes the existence of ethno-geographical differences in NSD1 molecular alterations between patients with SoS from Europe/North America (70-93%) and those from South America (10-19%). PMID:25852445

Investigation of major genetic alterations in neuroblastoma.

PubMed

Costa, Régis Afonso; Seuánez, Héctor N

2018-06-01

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. This malignancy shows a wide spectrum of clinical outcome and its prognosis is conditioned by manifold biological and genetic factors. We investigated the tumor genetic profile and clinical data of 29 patients with NB by multiplex ligation-dependent probe amplification (MLPA) to assess therapeutic risk. In 18 of these tumors, MYCN status was assessed by fluorescence in situ hybridization (FISH). Copy number variation was also determined for confirming MLPA findings in two 6p loci. We found 2p, 7q and 17q gains, and 1p and 11q losses as the most frequent chromosome alterations in this cohort. FISH confirmed all cases of MYCN amplification detected by MLPA. In view of unexpected 6p imbalance, copy number variation of two 6p loci was assessed for validating MLPA findings. Based on clinical data and genetic profiles, patients were stratified in pretreatment risk groups according to international consensus. MLPA proved to be effective for detecting multiple genetic alterations in all chromosome regions as requested by the International Neuroblastoma Risk Group (INRG) for therapeutic stratification. Moreover, this technique proved to be cost effective, reliable, only requiring standard PCR equipment, and attractive for routine analysis. However, the observed 6p imbalances made PKHD1 and DCDC2 inadequate for control loci. This must be considered when designing commercial MLPA kits for NB. Finally, four patients showed a normal MLPA profile, suggesting that NB might have a more complex genetic pattern than the one assessed by presently available MLPA kits.

Spectrum of mutations in homozygous familial hypercholesterolemia in India, with four novel mutations.

PubMed

Setia, Nitika; Saxena, Renu; Arora, Anjali; Verma, Ishwar C

2016-12-01

Homozygous familial hypercholesterolemia (FH) is a rare but serious, inherited disorder of lipid metabolism characterized by very high total and LDL cholesterol levels from birth. It presents as cutaneous and tendon xanthomas since childhood, with or without cardiac involvement. FH is commonly caused by mutations in three genes, i.e. LDL receptor (LDLR), apolipoprotein B (ApoB) and PCSK9. We aimed to determine the spectrum of mutations in cases of homozygous FH in Asian Indians and evaluate if there was any similarity to the mutations observed in Caucasians. Sixteen homozygous FH subjects from eleven families were analyzed for mutations by Sanger sequencing. Large rearrangements in LDLR gene were evaluated by multiplex ligation probe dependent amplification (MLPA) technique. Ten mutations were observed in LDLR gene, of which four mutations were novel. No mutation was detected in ApoB gene and common PCSK9 mutation (p.D374Y). Fourteen cases had homozygous mutations; one had compound heterozygous mutation, while no mutation was detected in one clinically homozygous case. We report an interesting "Triple hit" case with features of homozygous FH. The spectrum of mutations in the Asian Indian population is quite heterogeneous. Of the mutations identified, 40% were novel. No mutation was observed in exons 3, 9 and 14 of LDLR gene, which are considered to be hot spots in studies done on Asian Indians in South Africa. Early detection followed by aggressive therapy, and cascade screening of extended families has been initiated to reduce the morbidity and mortality in these patients. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Aetiological bases of 46,XY disorders of sex development in the Hong Kong Chinese population.

PubMed

Chan, Angel O K; But, W M; Lee, C Y; Lam, Y Y; Ng, K L; Loung, P Y; Lam, Aimen; Cheng, C W; Shek, C C; Wong, W S; Wong, K F; Wong, M Y; Tse, W Y

2015-12-01

Disorders of sex development are due to congenital defects in chromosomal, gonadal, or anatomical sex development. The objective of this study was to determine the aetiology of this group of disorders in the Hong Kong Chinese population. Five public hospitals in Hong Kong. Patients with 46,XY disorders of sex development under the care of paediatric endocrinologists between July 2009 and June 2011. Measurement of serum gonadotropins, adrenal and testicular hormones, and urinary steroid profiling. Mutational analysis of genes involved in sexual differentiation by direct DNA sequencing and multiplex ligation-dependent probe amplification. Overall, 64 patients were recruited for the study. Their age at presentation ranged from birth to 17 years. The majority presented with ambiguous external genitalia including micropenis and severe hypospadias. A few presented with delayed puberty and primary amenorrhea. Baseline and post-human chorionic gonadotropin-stimulated testosterone and dihydrotestosterone levels were not discriminatory in patients with or without AR gene mutations. Of the patients, 22 had a confirmed genetic disease, with 11 having 5α-reductase 2 deficiency, seven with androgen insensitivity syndrome, one each with cholesterol side-chain cleavage enzyme deficiency, Frasier syndrome, NR5A1-related sex reversal, and persistent Müllerian duct syndrome. Our findings suggest that 5α-reductase 2 deficiency and androgen insensitivity syndrome are possibly the two most common causes of 46,XY disorders of sex development in the Hong Kong Chinese population. Since hormonal findings can be unreliable, mutational analysis of the SRD5A2 and AR genes should be considered the first-line tests for these patients.

Clinical and molecular evaluation of SHOX/PAR1 duplications in Leri-Weill dyschondrosteosis (LWD) and idiopathic short stature (ISS).

PubMed

Benito-Sanz, S; Barroso, E; Heine-Suñer, D; Hisado-Oliva, A; Romanelli, V; Rosell, J; Aragones, A; Caimari, M; Argente, J; Ross, J L; Zinn, A R; Gracia, R; Lapunzina, P; Campos-Barros, A; Heath, K E

2011-02-01

Léri-Weill dyschondrosteosis (LWD) is a skeletal dysplasia characterized by disproportionate short stature and the Madelung deformity of the forearm. SHOX mutations and pseudoautosomal region 1 deletions encompassing SHOX or its enhancers have been identified in approximately 60% of LWD and approximately 15% of idiopathic short stature (ISS) individuals. Recently SHOX duplications have been described in LWD/ISS but also in individuals with other clinical manifestations, thus questioning their pathogenicity. The objective of the study was to investigate the pathogenicity of SHOX duplications in LWD and ISS. Multiplex ligation-dependent probe amplification is routinely used in our unit to analyze for SHOX/pseudoautosomal region 1 copy number changes in LWD/ISS referrals. Quantitative PCR, microsatellite marker, and fluorescence in situ hybridization analysis were undertaken to confirm all identified duplications. During the routine analysis of 122 LWD and 613 ISS referrals, a total of four complete and 10 partial SHOX duplications or multiple copy number (n > 3) as well as one duplication of the SHOX 5' flanking region were identified in nine LWD and six ISS cases. Partial SHOX duplications appeared to have a more deleterious effect on skeletal dysplasia and height gain than complete SHOX duplications. Importantly, no increase in SHOX copy number was identified in 340 individuals with normal stature or 104 overgrowth referrals. MLPA analysis of SHOX/PAR1 led to the identification of partial and complete SHOX duplications or multiple copies associated with LWD or ISS, suggesting that they may represent an additional class of mutations implicated in the molecular etiology of these clinical entities.

Genes commonly deleted in childhood B-cell precursor acute lymphoblastic leukemia: association with cytogenetics and clinical features

PubMed Central

Schwab, Claire J.; Chilton, Lucy; Morrison, Heather; Jones, Lisa; Al-Shehhi, Halima; Erhorn, Amy; Russell, Lisa J.; Moorman, Anthony V.; Harrison, Christine J.

2013-01-01

In childhood B-cell precursor acute lymphoblastic leukemia, cytogenetics is important in diagnosis and as an indicator of response to therapy, thus playing a key role in risk stratification of patients for treatment. Little is known of the relationship between different cytogenetic subtypes in B-cell precursor acute lymphoblastic leukemia and the recently reported copy number abnormalities affecting significant leukemia associated genes. In a consecutive series of 1427 childhood B-cell precursor acute lymphoblastic leukemia patients, we have determined the incidence and type of copy number abnormalities using multiplex ligation-dependent probe amplification. We have shown strong links between certain deletions and cytogenetic subtypes, including the novel association between RB1 deletions and intrachromosomal amplification of chromosome 21. In this study, we characterized the different copy number abnormalities and show heterogeneity of PAX5 and IKZF1 deletions and the recurrent nature of RB1 deletions. Whole gene losses are often indicative of larger deletions, visible by conventional cytogenetics. An increased number of copy number abnormalities is associated with NCI high risk, specifically deletions of IKZF1 and CDKN2A/B, which occur more frequently among these patients. IKZF1 deletions and rearrangements of CRLF2 among patients with undefined karyotypes may point to the poor risk BCR-ABL1-like group. In conclusion, this study has demonstrated in a large representative cohort of children with B-cell precursor acute lymphoblastic leukemia that the pattern of copy number abnormalities is highly variable according to the primary genetic abnormality. PMID:23508010

Contribution of Rare Copy Number Variants to Isolated Human Malformations

PubMed Central

Serra-Juhé, Clara; Rodríguez-Santiago, Benjamín; Cuscó, Ivon; Vendrell, Teresa; Camats, Núria; Torán, Núria; Pérez-Jurado, Luis A.

2012-01-01

Background Congenital malformations are present in approximately 2–3% of liveborn babies and 20% of stillborn fetuses. The mechanisms underlying the majority of sporadic and isolated congenital malformations are poorly understood, although it is hypothesized that the accumulation of rare genetic, genomic and epigenetic variants converge to deregulate developmental networks. Methodology/Principal Findings We selected samples from 95 fetuses with congenital malformations not ascribed to a specific syndrome (68 with isolated malformations, 27 with multiple malformations). Karyotyping and Multiplex Ligation-dependent Probe Amplification (MLPA) discarded recurrent genomic and cytogenetic rearrangements. DNA extracted from the affected tissue (46%) or from lung or liver (54%) was analyzed by molecular karyotyping. Validations and inheritance were obtained by MLPA. We identified 22 rare copy number variants (CNV) [>100 kb, either absent (n = 7) or very uncommon (n = 15, <1/2,000) in the control population] in 20/95 fetuses with congenital malformations (21%), including 11 deletions and 11 duplications. One of the 9 tested rearrangements was de novo while the remaining were inherited from a healthy parent. The highest frequency was observed in fetuses with heart hypoplasia (8/17, 62.5%), with two events previously related with the phenotype. Double events hitting candidate genes were detected in two samples with brain malformations. Globally, the burden of deletions was significantly higher in fetuses with malformations compared to controls. Conclusions/Significance Our data reveal a significant contribution of rare deletion-type CNV, mostly inherited but also de novo, to human congenital malformations, especially heart hypoplasia, and reinforce the hypothesis of a multifactorial etiology in most cases. PMID:23056206

Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array

PubMed Central

Huang, Huan; Li, Shuo; Sun, Lizhou; Zhou, Guohua

2015-01-01

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed “multiplex ligation-dependent probe amplification–digital amplification coupled with hydrogel bead-array” (MLPA–DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA–DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA–DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC. PMID:25880764

Novel glucokinase mutations in patients with monogenic diabetes - clinical outline of GCK-MD and potential for founder effect in Slavic population.

PubMed

Borowiec, M; Antosik, K; Fendler, W; Deja, G; Jarosz-Chobot, P; Mysliwiec, M; Zmyslowska, A; Malecki, M; Szadkowska, A; Mlynarski, W

2012-03-01

Glucokinase (GCK) gene mutations are the causative factor of GCK-MD (monogenic diabetes) characterized by a mild clinical phenotype and potential for insulin withdrawal. This study presents the results of a nationwide genetic screening for GCK-MD performed in Poland. A group of 194 patients with clinical suspicion of GCK-MD and 17 patients with neonatal diabetes were subjected to GCK sequencing. Patients negative for GCK mutations were subjected to multiplex ligation-dependent probe amplification (MLPA) to detect deletions or insertions. A total of 44 GCK heterozygous mutations were found in 68 probands (35%). Among those, 20 mutations were novel ones: A282fs, D198V, E158X, G246V, G249R, I348N, L165V, L315Q, M115I, N254S, P284fs, Q338P, R377L, R43C, R46S, S212fs, S212P, T255N, V406A and Y214D. No abnormalities were detected in MLPA analysis. Homozygous D278E mutation was found in one patient with neonatal diabetes. The most frequently observed combinations of symptoms typical for GCK-MD were mild diabetes and/or fasting hyperglycaemia (98.3%), positive C-peptide at diagnosis (76%) and dominant mode of inheritance (59%). This study outlines numerous novel mutations of the GCK gene present in white Caucasians of Slavic origin. Thorough clinical assessment of known factors associated with GCK-MD may facilitate patient selection. © 2011 John Wiley & Sons A/S.

Assessment of chromosomal imbalances in CIMP-high and CIMP-low/CIMP-0 colorectal cancers.

PubMed

Kozlowska, Joanna; Karpinski, Pawel; Szmida, Elzbieta; Laczmanska, Izabela; Misiak, Blazej; Ramsey, David; Bebenek, Marek; Kielan, Wojciech; Pesz, Karolina A; Sasiadek, Maria M

2012-08-01

Data presented in a number of recent studies have revealed a negative correlation between CpG island methylator phenotype (CIMP) and chromosomal instability (CIN) measured by a loss of heterozygosity (LOH) of selected loci, suggesting that CIN and CIMP represent two independent mechanisms in sporadic colorectal cancer (CRC) carcinogenesis. However, CIN is a heterogeneous phenomenon, which may be studied not only by employing LOH analysis but also by observing chromosomal imbalances (gains and deletions). The current study aimed to investigate the relationship between CIMP and chromosomal gains and deletions (assessed by comparative genomic hybridization) in a group of 20 CIMP-high and 79 CIMP-low/CIMP-0 CRCs. Our results revealed that the mean numbers of gains and of total chromosomal imbalances were significantly greater (p = 0.004 and p = 0.007, respectively) in the CIMP-low/CIMP-0 group compared to the CIMP-high group, while no significant difference was observed between the mean numbers of losses (p = 0.056). The analysis of copy number changes of 41 cancer-related genes by multiplex ligation-dependent probe amplification showed that CRK gene was exclusively deleted in CIMP-low/CIMP-0 tumors (p = 0.02). Given that chromosomal losses play an important role in tumor suppressor inactivation and chromosomal gains, in the activation of proto-oncogenes, we hypothesize that tumor suppressor inactivation plays similar roles in both CIMP-high and CIMP-low/CIMP-0 CRCs, while the predominance of chromosomal gains in CIMP-low/CIMP-0 tumors may suggest that the activation of proto-oncogenes is the underlying mechanism of CIMP-low/CIMP-0 CRC progression.

Assessment of HER2 status in breast cancer biopsies is not affected by accelerated tissue processing.

PubMed

Bulte, Joris P; Halilovic, Altuna; Kalkman, Shona; van Cleef, Patricia H J; van Diest, Paul J; Strobbe, Luc J A; de Wilt, Johannes H W; Bult, Peter

2018-03-01

To establish whether core needle biopsy (CNB) specimens processed with an accelerated processing method with short fixation time can be used to determine accurately the human epidermal growth factor receptor 2 (HER2) status of breast cancer. A consecutive case-series from two high-volume breast clinics was created. We compared routine HER2 immunohistochemistry (IHC) assessment between accelerated processing CNB specimens and routinely processed postoperative excision specimens. Additional amplification-based testing was performed in cases with equivocal results. The formalin fixation time was less than 2 h and between 6 and 72 h, respectively. Fluorescence in-situ hybridisation and multiplex ligation-dependent probe amplification were used for amplification testing. One hundred and forty-four cases were included, 15 of which were HER2-positive on the routinely processed excision specimens. On the CNB specimens, 44 were equivocal on IHC and required an amplification-based test. Correlation between the CNB specimens and the corresponding excision specimens was high for final HER2 status, with an accuracy of 97% and a kappa of 0.85. HER2 status can be determined reliably on CNB specimens with accelerated processing time using standard clinical testing methods. Using this accelerated technology the minimum 6 h of formalin fixation, which current guidelines consider necessary, can be decreased safely. This allows for a complete and expedited histology-based diagnosis of breast lesions in the setting of a one-stop-shop, same-day breast clinic. © 2018 The Authors. Histopathology Published by John Wiley & Sons Ltd.

Microduplications at 22q11.21 are associated with non-syndromic classic bladder exstrophy.

PubMed

Draaken, Markus; Reutter, Heiko; Schramm, Charlotte; Bartels, Enrika; Boemers, Thomas M; Ebert, Anne-Karoline; Rösch, Wolfgang; Schröder, Annette; Stein, Raimund; Moebus, Susanne; Stienen, Dietlinde; Hoffmann, Per; Nöthen, Markus M; Ludwig, Michael

2010-01-01

The exstrophy-epispadias complex (EEC) comprises a spectrum of urogenital anomalies in which part or all of the distal urinary tract fails to close. The present study aimed to identify microaberrations characterized by loss or gain of genomic material that contribute to the EEC at a genome-wide level. Molecular karyotyping, utilizing 549,839 single nucleotide polymorphisms (SNPs) with an average spacing of 5.7 kilobases, was performed to screen an initial cohort of 16 patients with non-syndromic EEC. A de novo microduplication involving chromosomal region 22q11.21 was identified in one patient with classic exstrophy of the bladder (CBE). Subsequent multiplex ligation-dependent probe amplification (MLPA) analysis was performed with an MLPA 22q11 kit in a further 50 non-syndromic EEC cases. We identified one CBE patient with an overlapping 22q11.21 duplication in whom the duplication had been transmitted from the unaffected mother. Chromosomal region 22q11 is well known for its susceptibility to genomic rearrangements, and these are associated with various syndromes including the velo-cardio-facial/DiGeorge syndrome (VCFS/DGS), the der(22) syndrome, and the cat-eye syndrome. Duplications in this region result in a wide and variable spectrum of clinical presentations that include features of the VCFS/DGS, while some carriers present with a completely normal phenotype. Our findings extend the phenotypic spectrum of the 22q11.2 duplication syndrome, and indicate that this aberration predisposes to CBE with incomplete penetrance. Copyright 2009 Elsevier Masson SAS. All rights reserved.

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

PubMed

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

2017-01-01

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