Genetic information from discordant sibling pairs points to ESRP2 as a candidate trans-acting regulator of the CF modifier gene SCNN1B

SCNN1B encodes the beta subunit of the epithelial sodium channel ENaC. Previously, we reported an association between SNP markers of SCNN1B gene and disease severity in cystic fibrosis-affected sibling pairs. We hypothesized that factors interacting with the SCNN1B genomic sequence are responsible for intrapair discordance. Concordant and discordant pairs differed at six SCNN1B markers (Praw = 0.0075, Pcorr = 0.0397 corrected for multiple testing). To identify the factors binding to these six SCNN1B SNPs, we performed an electrophoretic mobility shift assay and captured the DNA-protein complexes. Based on protein mass spectrometry data, the epithelial splicing regulatory protein ESRP2 was identified when using SCNN1B-derived probes and the ESRP2-SCNN1B interaction was independently confirmed by coimmunoprecipitation assays. We observed an alternative SCNN1B transcript and demonstrated in 16HBE14o- cells that levels of this transcript are decreased upon ESRP2 silencing by siRNA. Furthermore, we confirmed that mildly and severely affected siblings have different ESPR2 genetic backgrounds and that ESRP2 markers are linked to the response of CF patients' nasal epithelium to amiloride, indicating ENaC involvement (Pbest = 0.0131, Pcorr = 0.068 for multiple testing). Our findings demonstrate that sibling pairs clinically discordant for CF can be used to identify meaningful DNA regulatory elements and interacting factors

Unraveling structural rearrangements of the CFH gene cluster in atypical hemolytic uremic syndrome patients using molecular combing and long-fragment targeted sequencing

Complement factor H (CFH) and its related proteins have an essential role in regulating the alternative pathway of the complement system. Mutations and structural variants (SVs) of the CFH gene cluster, consisting of CFH and its five related genes (CFHR1-5), have been reported in renal pathologies as well as in complex immune diseases like age-related macular degeneration and systemic lupus erythematosus. SV analysis of this cluster is challenging due to its high degree of sequence homology. Following first-line NGS gene panel sequencing, we applied Genomic Vision's Molecular Combing Technology, to detect and visualize SVs within the CFH gene cluster and resolve its structural haplotypes completely. This approach was tested in three patients with atypical hemolytic uremic syndrome (aHUS) and known SVs, and 18 patients with aHUS or complement factor 3 glomerulopathy with unknown CFH gene cluster haplotypes. Three SVs, a CFH/CFHR1 hybrid gene in two patients and a rare heterozygous CFHR4/CFHR1 deletion in trans with the common CFHR3/CFHR1 deletion in a third patient were newly identified. For the latter, the breakpoints were determined using a targeted enrichment approach for long DNA fragments (Samplix Xdrop) in combination with Oxford Nanopore sequencing. Molecular combing in addition to NGS was able to improve the molecular genetic yield in this pilot study. This (cost-)effective approach warrants validation in larger cohorts with CFH/CFHR-associated disease

Rare coding variants in genes encoding GABA(A) receptors in genetic generalised epilepsies : an exome-based case-control study

Background Genetic generalised epilepsy is the most common type of inherited epilepsy. Despite a high concordance rate of 80% in monozygotic twins, the genetic background is still poorly understood. We aimed to investigate the burden of rare genetic variants in genetic generalised epilepsy. Methods For this exome-based case-control study, we used three different genetic generalised epilepsy case cohorts and three independent control cohorts, all of European descent. Cases included in the study were clinically evaluated for genetic generalised epilepsy. Whole-exome sequencing was done for the discovery case cohort, a validation case cohort, and two independent control cohorts. The replication case cohort underwent targeted next-generation sequencing of the 19 known genes encoding subunits of GABA(A) receptors and was compared to the respective GABA(A) receptor variants of a third independent control cohort. Functional investigations were done with automated two-microelectrode voltage clamping in Xenopus laevis oocytes. Findings Statistical comparison of 152 familial index cases with genetic generalised epilepsy in the discovery cohort to 549 ethnically matched controls suggested an enrichment of rare missense (Nonsyn) variants in the ensemble of 19 genes encoding GABA(A) receptors in cases (odds ratio [OR] 2.40 [95% CI 1.41-4.10]; p(Nonsyn)=0.0014, adjusted p(Nonsyn)=0.019). Enrichment for these genes was validated in a whole-exome sequencing cohort of 357 sporadic and familial genetic generalised epilepsy cases and 1485 independent controls (OR 1.46 [95% CI 1.05-2.03]; p(Nonsyn)=0.0081, adjusted p(Nonsyn)=0.016). Comparison of genes encoding GABA(A) receptors in the independent replication cohort of 583 familial and sporadic genetic generalised epilepsy index cases, based on candidate-gene panel sequencing, with a third independent control cohort of 635 controls confirmed the overall enrichment of rare missense variants for 15 GABA(A) receptor genes in cases compared with controls (OR 1.46 [95% CI 1.02-2.08]; p(Nonsyn)=0.013, adjusted p(Nonsyn)=0.027). Functional studies for two selected genes (GABRB2 and GABRA5) showed significant loss-of-function effects with reduced current amplitudes in four of seven tested variants compared with wild-type receptors. Interpretation Functionally relevant variants in genes encoding GABA(A) receptor subunits constitute a significant risk factor for genetic generalised epilepsy. Examination of the role of specific gene groups and pathways can disentangle the complex genetic architecture of genetic generalised epilepsy. Copyright (C) 2018 The Author(s). Published by Elsevier Ltd.Peer reviewe

Mutations in the MEN I gene in sporadic neuroendocrine tumours of gastroenteropancreatic system

Contains fulltext : 24465___.PDF (publisher's version ) (Open Access

Failure to replicate an allelic association between an exon 8 polymorphism of the human alpha1A calcium channel gene and common syndromes of idiopathic generalized epilepsy

The present replication study tested the validity of a previously reported allelic association between a single nucleotide polymorphism in exon 8 (SNP8) of the gene encoding the α1A-calcium channel subunit (CACNA1A) and common subtypes of idiopathic generalized epilepsy (IGE). Pyrosequencing was applied to assess the SNP8 genotypes in 354 unrelated German IGE probands, both parents of 118 IGE probands, and 186 healthy control subjects of German descent. Our population-based association analysis did not provide evidence for an allelic association of SNP8 with either IGE or two phenotypically more homogeneous IGE subtypes, consisting of either 139 probands with juvenile myoclonic epilepsy or 207 probands whose IGE started with typical absence seizures (P>0.72). In addition, the transmission disequilibrium test did not indicate a preferential transmission of SNP8 alleles in 97 informative parent-child transmissions (McNemar χ2=0.093, df=1, P=0.76). Accordingly, we failed to confirm previous evidence that genetic variation of the CACNA1A gene confers susceptibility to common IGE syndromes

New universal primers facilitate PyrosequencingTM

A cost-driving factor in Pyrosequencing is the need for single-stranded PCR products that are usually obtained by biotin-labeling of one primer. We designed new universal primers that allow the introduction of biotin during the specific PCR at either the forward or the reverse primer in a single reaction. When converting five human single nucleotide polymorphism assays from the standard format into the universal format, we obtained pyrograms of similar good quality. Although the universal nonhuman sequences are unlikely to form loop structures, we mostly failed to establish assays without using the proprietary software from Biotage AB. Nevertheless, our universal primer pair adds flexibility to the process of assay design due to optional strand selection and contributes to the reduction of genotyping costs

Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis

Tumorigenesis is characterized by alterations of methylation profiles including loss and gain of 5-methylcytosine. Recently, we identified a single CpG, which seemed to be consistently hypomethylated in pilocytic astrocytomas but not in other gliomas. To evaluate its applicability as a biomarker, we examined its methylation status in a large panel of gliomas (n = 97). Methylation-dependent DNA sequence variation may be considered a kind of single nucleotide polymorphism (methylSNP). MethylSNPs can be easily converted into common SNPs of the C/T type by sodium bisulfite treatment of the DNA and afterwards subjected to conventional SNP typing. We adapted SnaPshot™ and Pyrosequencing™ to determine the methylation of our test CpG in a quantitative manner. The adapted methods, called SNaPmeth and PyroMeth, respectively, gave nearly identical results, however data obtained with PyroMeth showed less scattering. Furthermore, the integrated software for allele frequency determination from Pyrosequencing could be used directly for data analysis while SnaPmeth data had to be exported and processed manually. Although data did not confirm our previous result of a preferential hypomethylation of the tested CpG in pilocytic astrocytomas, we consider quantitative methylSNP analysis by SNaPmeth or PyroMeth a favorable alternative to existing high-throughput methylation assays. It combines single CpG analysis with accurate quantitation and is amenable to high throughput

Genetic variation at the CYP2C locus and its association with torsemide biotransformation

In 97 unselected volunteers and two additional homozygous carriers of CYP2C9(*)3, we investigated the oral clearance of torsemide in relation to 37 polymorphisms at the CYP2C gene locus. Torsemide total oral clearance was linearly associated with the number of CYP2C9(*)3 alleles (geometric mean: 59, 40 and 20 ml/min in carriers of no, one and two alleles) and so were the methyl- and ring-hydroxylation but not the carboxylation clearance. Haplotypes including the CYP2C9(*)3 allele were similarly associated with the clearances but no other variant and no haplotype not including the CYP2C9(*)3 variant. The extended haplotype length (EHL) of the CYP2C9 haplotypes was positively associated with higher activity of the gene product. Torsemide total oral clearance was predictable with r(2)=82.1% using plasma concentrations at 0.5, 1, 2 and 24 h. In conclusion, torsemide's biotransformation strongly depended on the CYP2C9(*)3 variant but no other. Higher clearance CYP2C9 haplotypes appear to be evolutionarily selected

Association of ANKH gene polymorphisms with radiographic hand bone size and geometry in a Chuvasha population

We performed a family-based association study to test the hypothesis that genetic variation at the human orthologue of the mouse progressive ankylosis gene (ANKH) is involved in determining bone size (BS) and bone geometry (BG). The study population comprised 126 nuclear families with 574 adult Chuvashian individuals living in small villages in the Russian Federation. Quantitative bone traits were determined by analyzing plain hand radiographs. Familial correlations for all studied traits revealed a high degree of heritability in this ethnically homogeneous population. Three simple tandem repeat (STR) polymorphisms, one intragenic and two flanking markers, as well as six single nucleotide polymorphisms (SNPs) were tested. The SNPs were detected by re-sequencing experiments and covered ANKH exons with their flanking splice sites and the promoter region. We used three different transmission disequilibrium tests (TDTs) and obtained multiple significant association signals for all investigated bone traits. Alleles of several markers located at different positions of the ANKH locus, including the promoter, consistently revealed the association. The bone traits tested are closely related to bone fragility suggesting a role for ANKH in osteoporosis

New strategies for efficient typing of HLA class-II loci DQB1 and DRB1 by using PyrosequencingTM

The characterization of genetic risk factors for complex diseases located on chromosome-6 frequently requires human leucocyte antigen (HLA) genotyping of large patient cohorts. Currently available methods do not support high-throughput HLA typing beyond the major allele group level. We, thus, developed a high-throughput approach for the HLA-DQB1 and HLA-DRB1 loci that is based on Pyrosequencing™. Pyrosequencing™ offers a higher degree of automation than direct sequencing or oligotyping. Using a dispensation order optimized for the particular HLA locus, rapid group typing and fine resolution can be achieved. We implemented the method for two important HLA loci - DQB1 and DRB1. The HLA-DQB1 typing method comprises the following steps: splitting the potential alleles after a generic polymerase chain reaction (PCR) amplification into groups with a first Pyrosequencing™ reaction and resolving the split allele groups by means of five further Pyrosequencing™ reactions. The HLA-DR gene family is known to be the most polymorphic one in the HLA class-II region because of a large number of DRB1 alleles. Because of this complex nature, HLA-DRB1 typing was performed by means of a combination of sequence-specific PCR typing and Pyrosequencing™. HLA-DQB1 typing and HLA-DRB1 typing were performed successfully by using standard DNA samples with the help of known HLA genotypes and in a blind study by using the samples from the Deutscher Zell Austausch 2002 and 2003. The approach was optimized and was practically tested for genotyping in disease association studies. Our well-elaborated Pyrosequencing™-based protocols offer a new alternative to the existing HLA class-II typing methods and represent a convenient and economic solution, a unique combination of high accuracy with high-sample throughput