Genome-wide copy number variation study in anorectal malformations

Anorectal malformations (ARMs, congenital obstruction of the anal opening) are among the most common birth defects requiring surgical treatment (2-5/10 000 live-births) and carry significant chronic morbidity. ARMs present either as isolated or as part of the phenotypic spectrum of some chromosomal abnormalities or monogenic syndromes. The etiology is unknown. To assess the genetic contribution to ARMs, we investigated single-nucleotide polymorphisms and copy number variations (CNVs) at genome-wide scale. A total of 363 Han Chinese sporadic ARM patients and 4006 Han Chinese controls were included. Overall, we detected a 1.3-fold significant excess of rare CNVs in patients. Stratification of patients by presence/absence of other congenital anomalies showed that while syndromic ARM patients carried significantly longer rare duplications than controls (P = 0.049), non-syndromic patients were enriched with both rare deletions and duplications when compared with controls (P = 0.00031). Twelve chromosomal aberrations and 114 rare CNVs were observed in patients but not in 868 controls nor 11 943 healthy individuals from the Database of Genomic Variants. Importantly, these aberrations were observed in isolated ARM patients. Gene-based analysis revealed 79 genes interfered by CNVs in patients only. In particular, we identified a de novo DKK4 duplication. DKK4 is a member of the WNT signaling pathway which is involved in the development of the anorectal region. In mice, Wnt disruption results in ARMs. Our data suggest a role for rare CNVs not only in syndromic but also in isolated ARM patients and provide a list of plausible candidate genes for the disorder.postprin

State-of-the-art microscopy to understand islets of Langerhans:what to expect next?

The discovery of Langerhans and microscopic description of islets in the pancreas were crucial steps in the discovery of insulin. Over the past 150 years, many discoveries in islet biology and type 1 diabetes have been made using powerful microscopic techniques. In the past decade, combination of new probes, animal and tissue models, application of new biosensors and automation of light and electron microscopic methods and other (sub)cellular imaging modalities have proven their potential in understanding the beta cell under (patho)physiological conditions. The imaging evolution, from fluorescent jellyfish to real-time intravital functional imaging, the revolution in automation and data handling and the increased resolving power of analytical imaging techniques are now converging. Here, we review innovative approaches that address islet biology from new angles by studying cells and molecules at high spatiotemporal resolution and in live models. Broad implementation of these cellular imaging techniques will shed new light on cause/consequence of (mal)function in islets of Langerhans in the years to come

Genome-wide profile of copy number variants for Hirschsprung disease

965/W/Poster Board: no. 623Hirschsprung disease (HSCR, aganglionic megacolon) is a developmental disorder characterised by the absence of the enteric ganglia along the intestinal tract. In addition to the major implicating gene RET, common polymorphisms, rare mutations and chromosomal abnormalities have been found to be associated with HSCR. The most common associated chromosomal abnormality is trisomy 21 (Down syndrome). Other chromosomal aberrations including 13q chromosomal deletions were also reported, which pinpointed the region for the discovery of disesase susceptibility loci, such as EDNRB. To characterize the copy number variants for HSCR, we analyzed 139 HSCR patients and 333 controls previously genotyped by Affymetrix 500k SNP arrays using Birdsuite. For HSCR patients, we identified 92 duplications and 60 deletions of at least 100kb. The total burden for rare and singleton CNVs increased for cases when compared to controls, both for the number of CNVs per individual (p = 0.0008) and the number of overlapping genes (p < 0.0004). In particular, long singleton duplications and deletions were identified for HSCR patients. Focusing on highly confident structural variations revealed three duplications ranging from 1.8 to 10Mb, spanning 8p21, 15q11-13 and 16p12. Two nonoverlapping deletions (14.9 & 29.7Mb) with both breakpoints residing in 11q23 were also shown. As previously reported, a 16Mb deletion overlapping with EDNRB was found on one HSCR patient. Together with other shorter variants, this finding could account for a sustainable proportion of susceptibilty to HSCR.The 59th Annual Meeting of the American Society of Human Genetics (ASHG), Honolulu, HI., 20-24 October 2009

Unraveling the genetic basis of nasopharyngeal carcinoma using next-generation sequencing approaches

Section 2 - Poster 1: no. 1921BACKGROUND: Nasopharyngeal carcinoma (NPC) is an epithelial malignancy that originates in the nasopharynx. This cancer is highly prevalent in Asia, especially southern China. Familiar genetic predisposition is observed in 5-10% of NPC patients. It is well-accepted that host genetics, EBV infection, and environmental factors together contribute to NPC development. PURPOSE: The genetic basis of NPC has not been fully elucidated. Therefore, we aim to use next-generation sequencing approaches to identify genes or pathways associated with NPC risk and to understand the genetic basis of NPC. METHODS: In the discovery cohort, 67 family history-positive (FH+) NPC cases from 56 families, 39 early-age onset cases, and 51 sporadic cases were subjected to whole-exome sequencing (WES). WES data of additional 415 non-cancer controls were obtained from non-cancer studies. Two strategies, mixed model association analysis and systematic filtering methods, were used for the analysis. The LightSNiP assay, which detects variants by melting curve analysis, and targeted sequencing approach were applied in an independent validation cohort. RESULTS: Association analysis identified a 3’ UTR variant in G protein-coupled receptor 114 (GPR114) with genome-wide significance (p-value 1.1x10-8). The risk allele was observed in 10 cases from 5 FH+ families and 1 sporadic case, but was not found in controls and public databases. In the validation study, LightSNiP assay identified the variant in 4 out of 210 FH+ cases, 25 out of 1725 sporadic cases, and 11 out of 1849 controls. The odds ratios are 3.1 and 2.35 for FH+ and sporadic cases, respectively. All the variants identified were validated by Sanger sequencing. GPR114 is expressed in both NPC and normal nasopharyngeal tissues. Further functional studies are now underway for GPR114. Using the systematic filtering analysis strategy, we identified the genes with rare damaging variants (minor allele frequency≤0.01) in at least four FH+ families and excluded the genes which are likely to be false positive signals in WES or not expressed in NPC or normal nasopharyngeal tissues. In total 455 genes were identified. Gene ontology analysis identified enriched terms including chromatin modification (FDR=0.02) and extracellular matrix (FDR=0.006). Targeted sequencing of candidate genes in about 300 additional FH+ cases and 300 age- and gender-matched controls is underway. CONCLUSIONS: In our study, a GPR114 3’ UTR variant was found to be associated with increased risk of NPC in both familiar and sporadic cases. We also identified rare damaging variants enriched in chromatin modification and extracellular matrix. Our study provides an enhanced road map for comprehensive understanding of the genetic basis of NPC.link_to_OA_fulltex

Identification of rare variants in the NRG1 gene of Hirschsprung's patients

725/W/Poster Board #383Hirschsprung’s disease (HSCR, aganglionic megacolon), is a congenital disorder characterized by the absence of enteric ganglia in variable portions of the distal intestine. HSCR has a complex pattern of inheritance and presents mainly sporadically. Besides the major HSCR gene, RET, and other implicating genes (e.g. EDNRB), there is evidence that other loci contribute to HSCR. Recently, through a genome-wide association study, we identified NRG1 as a new HSCR contributing locus. Several lines of evidence indicate that in addition to common variants/SNPs, rare variants may contribute substantially to the multifactorial inheritance of complex diseases, and that the genes with disease-associated SNPs are to be considered candidates for the search of deleterious rare variants. We hypothesized that rare NRG1 variants contributing to HSCR may exist. To identify these variants, we have sequenced the 17 exons (including exon/intron boundaries) of NRG1 of 386 HSCR patients and 100 controls on an ABI 3730xl DNA Analyzer. We have identified 7 novel rare variants in 7 patients that cause 4 non-synonymous amino-acid changes, a truncation of the protein, a disruption of the splice site and an alteration of the conserved region intronic region in the exon boundary. The non-synonymous amino-acid substitutions affect highly conserved residues and have not been found in the controls. The four patients bearing NRG1 rare variants have no diseaseassociated variants in RET or any other HSCR-associated gene. The finding of rare NRG1 variants with an obvious functional effect on the NRG1 protein, suggest that NRG1 is one of the several molecules contributing to the pathology of HSCR. More controls are currently being sequenced.The 59th Annual Meeting of the American Society of Human Genetics (ASHG), Honolulu, HI., 20-24 October 2009

A RET founder mutation in Chinese hirschsprung's patients

768/W/Poster Board: no. 426Hirschsprung’s disease (HSCR) is a congenital disorder associated with the lack of intramural ganglion cells in the myenteric and sub-mucosal plexuses along varying segments of the gastrointestinal tract. Rearranged during transfection (RET) gene is implicated in HSCR and is the major gene of this gastrointestinal disease. To date, over 200 low frequency recurrent RET coding sequence (CDS) mutations have been identified in HSCR patients. However, a highly recurrent RET(R114H) mutation has been identified in 10% of the Chinese HSCR patients which has never been found in Caucasians patients or controls nor in 400 Chinese controls. The high frequency of RET(R114H) in our population together with the fact that it is not a “de novo” mutation in the context of the most HSCR-associated RET-haplotype, suggests that it may be a founder HSCR mutation in the Chinese population. Initial investigation involved applying a Bayesian method to 21 single nucleotide polymorphisms (SNPs; across a 62kb region of RET) genotyped in 421 Chinese HSCR patients (of which 24 individuals had the mutation) to predict the approximate age of RET(R114H). The approach allowed the inference of the mutation age based on the observed linkage disequilibrium (LD) at multiple SNPs which predicted the mutation to be between 12 and 13 generations old. Including SNPs from a recently obtained genome-wide 500K dataset for 181 of the above mentioned patients (which now only included 14 patients who had the RET(R114H) mutation), we applied haplotype estimation methods to determine whether there were any segments shared between patients with the RET(R114H) compared to those without the mutation and controls. Data consisted a total of 92 SNPs spanning a 510kb region over the RET gene. Analysis yielded a 256kb (76 SNP) shared segment over the RET gene (and downstream) in only those patients with the mutation with no similar segments found among other patients or controls. This suggests that RET(R114H) is a possible founder effect for Hirschsprung’s disease in the Chinese population.The 59th Annual Meeting of the American Society of Human Genetics (ASHG), Honolulu, HI., 20-24 October 2009

Quantifying epistasis between two sets of signaling pathway genes by canonical correlation analysis

1852/T/Poster Board: no. 401This paper focuses on three aspects related to the conceptualization and application of canonical correlation analysis (CCA) as a statistical model in genetic study: 1) partial canonical correlation analysis to control the effect of population stratification, 2) epistasis analysis for case control study and 3) backward or stepwise canonical analysis to provide more insight into the dynamics of the complex genetic network. We applied these methods to simulated data and to a genetic pathway study of Hirschsprung disease. The results suggest the DLL3 gene from the NOTCH pathways interacted with the PTCH1 gene from the SHH pathway. The first canonical correlation is 0.444 (p-value from the Wilks Lamda test is 8.40E-06) among the cases and 0.157 (p-value 0.236) among the control. The difference between the correlations is significant (p-value 0.00011), after Fisher z transformation.The 59th Annual Meeting of the American Society of Human Genetics (ASHG), Honolulu, HI., 20-24 October 2009

Genomi-wide association study on anorectal malformations in the Chinese population

Poster Presentation: abstract 2942/TAnorectal malformations (ARM) represent a complex group of congenital diseases characterized by the obstruction of anal opening. Occurring in 1 out of every 4,000 to 5,000 individuals, ARMs are one of the most common pediatric surgical problems. The spectrum of ARMs ranges from anal stenosis to anal atresia/imperforated anus with/without fistula to persistent cloaca. The etiology of ARMs remains unknown, although there is strong evidence for a genetic component. This is indicated by the very early developmental disruption, its recurrence in families and the high concordance of its existence with some abnormalities. Though several candidate genes e.g. endothelin-β receptor (EDNRB) and sonic hedgehog (Shh) were proposed for their potential roles in the development of ARMs, the causes of ARMs still remain unknown. To explore the genetic contribution to the pathogenesis of ARM, we performed systematic analyses of genetic markers, in 176 Chinese patients and 2986 ethnically matched individuals as controls. The genome-wide association study (GWAS) was performed using the Illumina Human 610-Quad BeadChips with 488, 394 SNPs successfully genotyped. An association peak encompassing two gene members of the solute carrier (SLC) family was observed, with p-value= (odds ratio 1.94, 95% CI: 1.54-2.45 for allele C) for the most associated SNP (rs11045422). To confirm the observed association, those statistically significant SNPs will be genotyped in an independent set of cases and controls. Bioinformatics and experimental analysis will be used to study the biological relevance of SLC genes in ARMs.link_to_OA_fulltextThe 60th Annual Meeting of the American Society of Human Genetics (ASHG 2010), Washington D.C., 2-6 November 2010

Genome-wide copy number analysis uncovers a new HSCR gene: NRG3

The ASHG 61st Annual Meeting will be subsumed in the 12th ICHG MeetingPoster Session Title: Complex Traits: Theory and Methods: no. 415WHirschsprung disease (HSCR) is a congenital disorder characterized by aganglionosis of the distal intestine. To assess the contribution of Copy Number Variants (CNVs) to HSCR, we analysed the data generated from our previous genome-wide association study on HSCR patients whereby we identified NRG1 as a new HSCR susceptibility locus. Analysis of 129 Chinese patients and 331 ethnically matched controls showed that HSCR patients have a greater burden of rare CNVs (p=1.50x10-5), particularly for those encompassing genes (p=5.00x10-6). We identified 246 rare-genic CNVs exclusive to patients. Among those, we detected a NRG3 deletion (p=1.64x10-3). Subsequent NRG3 follow-up (96 additional patients and 105 controls) revealed 9 deletions and 2 de novo duplications among patients and two deletions among controls (p=1.67x10-6). Importantly, NRG3 is a paralog of NRG1. Stratification of patients by presence/absence of HSCR-associated syndromes showed that while syndromic-HSCR patients carried significantly longer CNVs than the non-syndromic or controls (p=1.50x10-5), non-syndromic patients were enriched in CNV number when compared to controls (p=4.00x10-6) or the syndromic counterpart. Our results suggest a role for NRG3 in HSCR etiology and provide insights into the relative contribution of structural variants in both syndromic and non-syndromic HSCR. This would be the first genome-wide catalog of copy number variants identified in HSCR.link_to_OA_fulltex

Fine mapping of Hirschsprung’s disease loci in 9q31

721/W/Poster Board #379Hirschsprung’s disease (HSCR) is a congenital disorder in which there is an absence of ganglion cells in variable portions of the lower digestive tract, according to which patients are classified. The RET gene is the largest risk factor in HSCR, although reduced penetrance of RET mutations, absence of RET mutations in some patients, and variable expression of the HSCR phenotype indicate that more than one gene is involved. A RET-dependent modifier which segregates in families harboring no or hypomorphic RET mutations was mapped to 9q31. Fine mapping of the region performed on 142 Dutch trios by genotyping 370 tag-SNPs spanning approximately 7 Mb (from 108.5-115.5 Mb) of 9q31 on an Illumina GoldenGate platform identified two different 9q31 HSCR-associated regions in which genes with biological plausibility lie. Since evaluation of an association in a population of different origin from that of the initial finding increases the association confidence and, since linkage disequilibrium (LD) differences across populations can be used to narrow the regions of interest, we genotyped 181 Chinese HSCR patients and 179 controls for 38 tag-SNPs chosen from the CHB population spanning the 9q31 regions of interest. In addition, we made use of genotype data for the 9q31 region obtained from a genome-wide association study recently conducted in Chinese HSCR patients. Only one of the two 9q31 HSCR-associated regions identified in the Dutch population was associated in Chinese (p = 0.021), although the most associated SNPs within the region differed, probably due to differences in LD and/or different genotyping densities. Importantly, the associated region encompasses IKBKAP and CTNNAL1, which have been linked to neurodevelopmental disorders.The 59th Annual Meeting of the American Society of Human Genetics (ASHG), Honolulu, HI., 20-24 October 2009