Whole genome sequencing reveals epistasis effects within RET for Hirschsprung disease.

Common variants in RET and NRG1 have been associated with Hirschsprung disease (HSCR), a congenital disorder characterised by incomplete innervation of distal gut, in East Asian (EA) populations. However, the allelic effects so far identified do not fully explain its heritability, suggesting the presence of epistasis, where effect of one genetic variant differs depending on other (modifier) variants. Few instances of epistasis have been documented in complex diseases due to modelling complexity and data challenges. We proposed four epistasis models to comprehensively capture epistasis for HSCR between and within RET and NRG1 loci using whole genome sequencing (WGS) data in EA samples. 65 variants within the Topologically Associating Domain (TAD) of RET demonstrated significant epistasis with the lead enhancer variant (RET+3; rs2435357). These epistatic variants formed two linkage disequilibrium (LD) clusters represented by rs2506026 and rs2506028 that differed in minor allele frequency and the best-supported epistatic model. Intriguingly, rs2506028 is in high LD with one cis-regulatory variant (rs2506030) highlighted previously, suggesting that detected epistasis might be mediated through synergistic effects on transcription regulation of RET. Our findings demonstrated the advantages of WGS data for detecting epistasis, and support the presence of interactive effects of regulatory variants in RET for HSCR.

Whole-genome analysis of noncoding genetic variations identifies multiscale regulatory element perturbations associated with Hirschsprung disease.

It is widely recognized that noncoding genetic variants play important roles in many human diseases, but there are multiple challenges that hinder the identification of functional disease-associated noncoding variants. The number of noncoding variants can be many times that of coding variants; many of them are not functional but in linkage disequilibrium with the functional ones; different variants can have epistatic effects; different variants can affect the same genes or pathways in different individuals; and some variants are related to each other not by affecting the same gene but by affecting the binding of the same upstream regulator. To overcome these difficulties, we propose a novel analysis framework that considers convergent impacts of different genetic variants on protein binding, which provides multiscale information about disease-associated perturbations of regulatory elements, genes, and pathways. Applying it to our whole-genome sequencing data of 918 short-segment Hirschsprung disease patients and matched controls, we identify various novel genes not detected by standard single-variant and region-based tests, functionally centering on neural crest migration and development. Our framework also identifies upstream regulators whose binding is influenced by the noncoding variants. Using human neural crest cells, we confirm cell stage-specific regulatory roles of three top novel regulatory elements on our list, respectively in the RET, RASGEF1A, and PIK3C2B loci. In the PIK3C2B regulatory element, we further show that a noncoding variant found only in the patients affects the binding of the gliogenesis regulator NFIA, with a corresponding up-regulation of multiple genes in the same topologically associating domain.

Cancer gene mutations in congenital pulmonary airway malformation patients.

BACKGROUND: Newborns affected with congenital pulmonary airway malformations (CPAMs) may present with severe respiratory distress or remain asymptomatic. While surgical resection is the definitive treatment for symptomatic CPAMs, prophylactic elective surgery may be recommended for asymptomatic CPAMs owing to the risk of tumour development. However, the implementation of prophylactic surgery is quite controversial on the grounds that more evidence linking CPAMs and cancer is needed. The large gap in knowledge of CPAM pathogenesis results in uncertainties and controversies in disease management. As developmental genes control postnatal cell growth and contribute to cancer development, we hypothesised that CPAMs may be underlain by germline mutations in genes governing airways development. METHODS: Sequencing of the exome of 19 patients and their unaffected parents. RESULTS: A more than expected number of mutations in cancer genes (false discovery rate q-value <5.01×10-5) was observed. The co-occurrence, in the same patient, of damaging variants in genes encoding interacting proteins is intriguing, the most striking being thyroglobulin (TG) and its receptor, megalin (LRP2). Both genes are highly relevant in lung development and cancer. CONCLUSIONS: The overall excess of mutations in cancer genes may account for the reported association of CPAMs with carcinomas and provide some evidence to argue for prophylactic surgery by some surgeons.

Identification of Genes Associated With Hirschsprung Disease, Based on Whole-Genome Sequence Analysis, and Potential Effects on Enteric Nervous System Development.

BACKGROUND & AIMS: Hirschsprung disease, or congenital aganglionosis, is believed to be oligogenic-that is, caused by multiple genetic factors. We performed whole-genome sequence analyses of patients with Hirschsprung disease to identify genetic factors that contribute to disease development and analyzed the functional effects of these variants. METHODS: We performed whole-genome sequence analyses of 443 patients with short-segment disease, recruited from hospitals in China and Vietnam, and 493 ethnically matched individuals without Hirschsprung disease (controls). We performed genome-wide association analyses and gene-based rare-variant burden tests to identify rare and common disease-associated variants and study their interactions. We obtained induced pluripotent stem cell (iPSC) lines from 4 patients with Hirschsprung disease and 2 control individuals, and we used these to generate enteric neural crest cells for transcriptomic analyses. We assessed the neuronal lineage differentiation capability of iPSC-derived enteric neural crest cells using an in vitro differentiation assay. RESULTS: We identified 4 susceptibility loci, including 1 in the phospholipase D1 gene (PLD1) (P = 7.4 × 10-7). The patients had a significant excess of rare protein-altering variants in genes previously associated with Hirschsprung disease and in the ß-secretase 2 gene (BACE2) (P = 2.9 × 10-6). The epistatic effects of common and rare variants across these loci provided a sensitized background that increased risk for the disease. In studies of the iPSCs, we observed common and distinct pathways associated with variants in RET that affect risk. In functional assays, we found variants in BACE2 to protect enteric neurons from apoptosis. We propose that alterations in BACE1 signaling via amyloid ß precursor protein and BACE2 contribute to pathogenesis of Hirschsprung disease. CONCLUSIONS: In whole-genome sequence analyses of patients with Hirschsprung disease, we identified rare and common variants associated with disease risk. Using iPSC cells, we discovered some functional effects of these variants.

Uncovering the genetic lesions underlying the most severe form of Hirschsprung disease by whole-genome sequencing.

Hirschsprung disease (HSCR) is a complex birth defect characterized by the lack of ganglion cells along a variable length of the distal intestine. A large proportion of HSCR patients remain genetically unexplained. We applied whole-genome sequencing (WGS) on 9 trios where the probands are sporadically affected with the most severe form of the disorder and harbor no coding sequence variants affecting the function of known HSCR genes. We found de novo protein-altering variants in three intolerant to change genes-CCT2, VASH1, and CYP26A1-for which a plausible link with the enteric nervous system (ENS) exists. De novo single-nucleotide and indel variants were present in introns and non-coding neighboring regions of ENS-related genes, including NRG1 and ERBB4. Joint analysis with those inherited rare variants found under recessive and/or digenic models revealed both patient-unique and shared genetic features where rare variants were found to be enriched in the extracellular matrix-receptor (ECM-receptor) pathway (p = 3.4 × 10-11). Delineation of the genetic profile of each patient might help finding common grounds that could lead to the discovery of shared molecules that could be used as drug targets for the currently ongoing cell therapy effort which aims at providing an alternative to the surgical treatment.

Genetic study of congenital bile-duct dilatation identifies de novo and inherited variants in functionally related genes.

BACKGROUND: Congenital dilatation of the bile-duct (CDD) is a rare, mostly sporadic, disorder that results in bile retention with severe associated complications. CDD affects mainly Asians. To our knowledge, no genetic study has ever been conducted. METHODS: We aim to identify genetic risk factors by a "trio-based" exome-sequencing approach, whereby 31 CDD probands and their unaffected parents were exome-sequenced. Seven-hundred controls from the local population were used to detect gene-sets significantly enriched with rare variants in CDD patients. RESULTS: Twenty-one predicted damaging de novo variants (DNVs; 4 protein truncating and 17 missense) were identified in several evolutionarily constrained genes (p < 0.01). Six genes carrying DNVs were associated with human developmental disorders involving epithelial, connective or bone morphologies (PXDN, RTEL1, ANKRD11, MAP2K1, CYLD, ACAN) and four linked with cholangio- and hepatocellular carcinomas (PIK3CA, TLN1 CYLD, MAP2K1). Importantly, CDD patients have an excess of DNVs in cancer-related genes (p < 0.025). Thirteen genes were recurrently mutated at different sites, forming compound heterozygotes or functionally related complexes within patients. CONCLUSIONS: Our data supports a strong genetic basis for CDD and show that CDD is not only genetically heterogeneous but also non-monogenic, requiring mutations in more than one genes for the disease to develop. The data is consistent with the rarity and sporadic presentation of CDD.

Sacral agenesis: a pilot whole exome sequencing and copy number study.

BACKGROUND: Caudal regression syndrome (CRS) or sacral agenesis is a rare congenital disorder characterized by a constellation of congenital caudal anomalies affecting the caudal spine and spinal cord, the hindgut, the urogenital system, and the lower limbs. CRS is a complex condition, attributed to an abnormal development of the caudal mesoderm, likely caused by the effect of interacting genetic and environmental factors. A well-known risk factor is maternal type 1 diabetes. METHOD: Whole exome sequencing and copy number variation (CNV) analyses were conducted on 4 Caucasian trios to identify de novo and inherited rare mutations. RESULTS: In this pilot study, exome sequencing and copy number variation (CNV) analyses implicate a number of candidate genes, including SPTBN5, MORN1, ZNF330, CLTCL1 and PDZD2. De novo mutations were found in SPTBN5, MORN1 and ZNF330 and inherited predicted damaging mutations in PDZD2 (homozygous) and CLTCL1 (compound heterozygous). Importantly, predicted damaging mutations in PTEN (heterozygous), in its direct regulator GLTSCR2 (compound heterozygous) and in VANGL1 (heterozygous) were identified. These genes had previously been linked with the CRS phenotype. Two CNV deletions, one de novo (chr3q13.13) and one homozygous (chr8p23.2), were detected in one of our CRS patients. These deletions overlapped with CNVs previously reported in patients with similar phenotype. CONCLUSION: Despite the genetic diversity and the complexity of the phenotype, this pilot study identified genetic features common across CRS patients.

Trans-ethnic meta-analysis of genome-wide association studies for Hirschsprung disease.

Hirschsprung disease (HSCR) is the most common cause of neonatal intestinal obstruction. It is characterized by the absence of ganglia in the nerve plexuses of the lower gastrointestinal tract. So far, three common disease-susceptibility variants at the RET, SEMA3 and NRG1 loci have been detected through genome-wide association studies (GWAS) in Europeans and Asians to understand its genetic etiologies. Here we present a trans-ethnic meta-analysis of 507 HSCR cases and 1191 controls, combining all published GWAS results on HSCR to fine-map these loci and narrow down the putatively causal variants to 99% credible sets. We also demonstrate that the effects of RET and NRG1 are universal across European and Asian ancestries. In contrast, we detected a European-specific association of a low-frequency variant, rs80227144, in SEMA3 [odds ratio (OR) = 5.2, P = 4.7 × 10-10]. Conditional analyses on the lead SNPs revealed a secondary association signal, corresponding to an Asian-specific, low-frequency missense variant encoding RET p.Asp489Asn (rs9282834, conditional OR = 20.3, conditional P = 4.1 × 10-14). When in trans with the RET intron 1 enhancer risk allele, rs9282834 increases the risk of HSCR from 1.1 to 26.7. Overall, our study provides further insights into the genetic architecture of HSCR and has profound implications for future study designs.

Genome-wide search followed by replication reveals genetic interaction of CD80 and ALOX5AP associated with systemic lupus erythematosus in Asian populations.

OBJECTIVES: Genetic interaction has been considered as a hallmark of the genetic architecture of systemic lupus erythematosus (SLE). Based on two independent genome-wide association studies (GWAS) on Chinese populations, we performed a genome-wide search for genetic interactions contributing to SLE susceptibility. METHODS: The study involved a total of 1 659 cases and 3 398 controls in the discovery stage and 2 612 cases and 3 441 controls in three cohorts for replication. Logistic regression and multifactor dimensionality reduction were used to search for genetic interaction. RESULTS: Interaction of CD80 (rs2222631) and ALOX5AP (rs12876893) was found to be significantly associated with SLE (OR_int=1.16, P_int_all=7.7E-04 at false discovery rate<0.05). Single nuclear polymorphism rs2222631 was found associated with SLE with genome-wide significance (P_all=4.5E-08, OR=0.86) and is independent of rs6804441 in CD80, whose association was reported previously. Significant correlation was observed between expression of these two genes in healthy controls and SLE cases, together with differential expression of these genes between cases and controls, observed from individuals from the Hong Kong cohort. Genetic interactions between BLK (rs13277113) and DDX6 (rs4639966), and between TNFSF4 (rs844648) and PXK (rs6445975) were also observed in both GWAS data sets. CONCLUSIONS: Our study represents the first genome-wide evaluation of epistasis interactions on SLE and the findings suggest interactions and independent variants may help partially explain missing heritability for complex diseases.

Chinese family with diffuse oesophageal leiomyomatosis: a new COL4A5/COL4A6 deletion and a case of gonosomal mosaicism.

BACKGROUND: Diffuse oesophageal leiomyomatosis (DOL) is a rare disorder characterized by tumorous overgrowth of the muscular wall of the oesophagus. DOL is present in 5 % of Alport syndrome (AS) patients. AS is a rare hereditary disease that involves varying degrees of hearing impairment, ocular changes and progressive glomerulonephritis leading to renal failure. In DOL-AS patients, the genetic defect consists of a deletion involving the COL4A5 and COL4A6 genes on the X chromosome. CASE PRESENTATION: We report a two-generation family (4 individuals; parents and two children, one male and one female) with two members (mother and son) affected with oesophageal leiomyomatosis. Signs of potential renal failure, which characterizes AS, were only apparent in the index patient (son) 2 years and three months after the initial diagnosis of DOL. Blood DNA from the four family members were submitted to exome sequencing and array genotyping to perform a genome wide screening for disease causal single nucleotide (SN) and copy number (CN) variations. Analyses revealed a new 40kb deletion encompassing from intron 2 of COL4A5 to intron 1 of COL4A6 at Xq22.3. The breakpoints were also identified. Possible confounding pathogenic exonic variants in genes known to be involved in other extracellular matrices disorders were also shared by the two affected individuals. Meticulous analysis of the maternal DNA revealed a case of gonosomal mosaicism. CONCLUSIONS: This is the first report of gonadosomal mosaicism associated to DOL-AS.

Depletion of the IKBKAP ortholog in zebrafish leads to hirschsprung disease-like phenotype.

AIM: To investigate the role of IKBKAP (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein) in the development of enteric nervous system (ENS) and Hirschsprung disease (HSCR). METHODS: In this study, we injected a morpholino that blocked the translation of ikbkap protein to 1-cell stage zebrafish embryos. The phenotype in the ENS was analysed by antibody staining of the pan-neuronal marker HuC/D followed by enteric neuron counting. The mean numbers of enteric neurons were compared between the morphant and the control. We also studied the expressions of ret and phox2bb, which are involved in ENS development, in the ikbkap morpholino injected embryos by quantitative reverse transcriptase polymerase chain reaction and compared them with the control. RESULTS: We observed aganglionosis (χ2, P<0.01) and a reduced number of enteric neurons (38.8±9.9 vs 50.2±17.3, P<0.05) in the zebrafish embryos injected with ikbkap translation-blocking morpholino (morphant) when compared with the control embryos. Specificity of the morpholino was confirmed by similar results obtained using a second non-overlapping morpholino that blocked the translation of ikbkap. We further studied the morphant by analysing the expression levels of genes involved in ENS development such as ret, phox2bb and sox10, and found that phox2bb, the ortholog of human PHOX2B, was significantly down-regulated (0.51±0.15 vs 1.00±0, P<0.05). Although we also observed a reduction in the expression of ret, the difference was not significant. CONCLUSION: Loss of IKBKAP contributed to HSCR as demonstrated by functional analysis in zebrafish embryos.

HOXB5 binds to multi-species conserved sequence (MCS+9.7) of RET gene and regulates RET expression.

RET gene is crucial for the development of enteric nervous system, and dys-regulation of RET expression causes Hirschsprung disease. HOXB5 regulates RET transcription, and perturbations in transcriptional regulation by HOXB5 caused reduced RET expression and defective enteric nervous system development in mice. The mechanisms by which HOXB5 regulate RET transcription are unclear. Thus, unraveling the regulatory mechanisms of HOXB5 on RET transcription could lead to a better understanding of the etiology of Hirschsprung disease. In this study, we identified and confirmed HOXB5 binding to the multi-species conserved sequence (MCS+9.7) in the first intron of the RET gene. We developed a RET mini-gene reporter system, and showed that MCS+9.7 enhanced HOXB5 trans-activation from RET promoter in human neuroblastoma SK-N-SH cells and in chick embryos. The deletion of HOXB5 binding site interfered with HOXB5 trans-activation. Furthermore, transfection of HOXB5 induced endogenous RET transcription, enhanced the co-precipitation of TATA-box binding protein with the transcription start site of RET, and induced histone H3K4 trimethylation in chromatin regions upstream and downstream of RET transcription start site. In conclusion, (i) HOXB5 physically interacted with MCS+9.7 and enhanced RET transcription, (ii) HOXB5 altered chromatin conformation and histone modification of RET locus, which could facilitate the formation of transcription complex, and enhance RET transcription, (iii) expression of RET was mediated by a complex regulatory network of transcription factors functioning in a synergistic, additive and/or independent manners. Hence, dys-regulation of RET expression by HOXB5 could result in insufficient RET expression and Hirschsprung disease.

Three SNPs in chromosome 11q23.3 are independently associated with systemic lupus erythematosus in Asians.

Systemic lupus erythematosus (SLE) has a complex etiology and is affected by both genetic and environmental factors. Although more than 40 loci have shown robust association with SLE, the details of these loci, such as the independent contributors and the genes involved, are still unclear. In this study, we performed meta-analysis of two existing genome-wide association studies (GWASs) on Chinese Han populations from Hong Kong and Anhui, China, and followed the findings by further replication on three additional Chinese and Thailand cohorts with a total of 4254 cases and 6262 controls matched geographically and ethnically. We discovered multiple susceptibility variants for SLE in the 11q23.3 region, including variants in/near PHLDB1 (rs11603023, P(_combined) = 1.25E-08, OR = 1.20), DDX6 (rs638893, P(_combined) = 5.19E-07, OR = 1.22) and CXCR5 (rs10892301, P(_combined) = 2.51E-08, OR = 0.85). Genetic contributions from the newly identified variants were all independent of SNP rs4639966, whose association was reported from the previous GWAS. In addition, the three newly identified variants all showed independent association with the disease through modeling by both stepwise and conditional logistic regression. The presence of multiple independent variants in this region emphasizes its role in SLE susceptibility, and also hints the possibility that distinct biological mechanisms might be involved in the disease involving this genomic region.

Gene network analysis of candidate loci for human anorectal malformations.

Anorectal malformations (ARMs) are birth defects that require surgery and carry significant chronic morbidity. Our earlier genome-wide copy number variation (CNV) study had provided a wealth of candidate loci. To find out whether these candidate loci are related to important developmental pathways, we have performed an extensive literature search coupled with the currently available bioinformatics tools. This has allowed us to assign both genic and non-genic CNVs to interrelated pathways known to govern the development of the anorectal region. We have linked 11 candidate genes to the WNT signalling pathway and 17 genes to the cytoskeletal network. Interestingly, candidate genes with similar functions are disrupted by the same type of CNV. The gene network we discovered provides evidence that rare mutations in different interrelated genes may lead to similar phenotypes, accounting for genetic heterogeneity in ARMs. Classification of patients according to the affected pathway and lesion type should eventually improve the diagnosis and the identification of common genes/molecules as therapeutic targets.

RET and NRG1 interplay in Hirschsprung disease.

Hirschsprung disease (HSCR, aganglionic megacolon) is a complex genetic disorder of the enteric nervous system (ENS) characterized by the absence of enteric neurons along a variable length of the intestine. While rare variants (RVs) in the coding sequence (CDS) of several genes involved in ENS development lead to disease, the association of common variants (CVs) with HSCR has only been reported for RET (the major HSCR gene) and NRG1. Importantly, RVs in the CDS of these two genes are also associated with the disorder. To assess independent and joint effects between the different types of RET and NRG1 variants identified in HSCR patients, we used 254 Chinese sporadic HSCR patients and 143 ethnically matched controls for whom the RET and/or NRG1 variants genotypes (rare and common) were available. Four genetic risk factors were defined and interaction effects were modeled using conditional logistic regression analyses and pair-wise Kendall correlations. Our analysis revealed a joint effect of RET CVs with RET RVs, NRG1 CVs or NRG1 RVs. To assess whether the genetic interaction translated into functional interaction, mouse neural crest cells (NCCs; enteric neuron precursors) isolated from embryonic guts were treated with NRG1 (ErbB2 ligand) or/and GDNF (Ret ligand) and monitored during the subsequent neural differentiation process. Nrg1 inhibited the Gdnf-induced neuronal differentiation and Gdnf negatively regulated Nrg1-signaling by down-regulating the expression of its receptor, ErbB2. This preliminary data suggest that the balance neurogenesis/gliogenesis is critical for ENS development.

Screening of the RET gene of Vietnamese Hirschsprung patients identifies 2 novel missense mutations.

BACKGROUND/PURPOSE: Hirschsprung disease (HSCR; megacolon, congenital aganglionosis) is a congenital disorder characterized by the absence of ganglion cells along variable segments of the gut. Both rare (RV) and common variants of the RET gene are associated with HSCR. The aim of this study is to assess, for the first time, the variation in the RET gene of Vietnamese HSCR patients. METHODS: We used Sanger sequencing to screen the coding sequence of the RET gene of 97 Vietnamese HSCR patients of Southern Chinese ancestry. The healthy population consisted of 250 Southern Chinese individuals with no diagnosis of HSCR. RESULTS: We detected 8 heterozygous RVs distributed among 13 patients (13.40%) and that were not present in healthy individuals. Among those variants, there were 2 novel and deleterious (R133C [c.397 C>T]; R144C [c.430 C>T]) missense amino acid substitutions, 2 novel silent variants (P667P [c.2001 A>T]; Y809Y [c.2427 C>T]), and 4 previously described missense substitutions (R114H [c.341 G>A]; V292M [c.874 G>A]; G533S [c.1597 G>A]; R982C [c.2944 C>T]). As expected, the common RET coding sequence variants rs1800858 (A45A [c.135 G>A]) and rs1800861 (L769L [c.2307 T>G]) were highly associated with the disease. CONCLUSIONS: The identification of novel deleterious variants together with the fact RET RVs are virtually unique to HSCR patients indicates that the RET gene is a target for mutations among Hirschsprung patients of any population.

Functional analyses of RET mutations in Chinese Hirschsprung disease patients.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital disease characterized by the absence of ganglion cells in various length of distal digestive tract. The rearranged during transfection gene (RET) is considered the major gene in HSCR. Although an increasing number of HSCR-associated RET coding sequence (CDS) mutations have been identified in recent years, not many have been investigated for functional consequence on the RET protein. METHODS AND RESULTS: We examined the functional implications of the de novo RET-CDS mutations V145G, Y483X, V636fsX1, and F961L that we first identified in sporadic Chinese patients with HSCR. The V145G disrupted RET glycosylation and F961L RET phosphorylation. Presumably, the truncation mutations would affect the translocation or the anchoring of the RET protein onto the cellular membrane. CONCLUSION: The study of RET-CDS mutations that appear de novo is essential not only for understanding the mechanistic of the disease but also for penetrance and recurrence risk estimations, being the ultimate goal for the improvement in disease management and counseling.

RET mutational spectrum in Hirschsprung disease: evaluation of 601 Chinese patients.

Rare (RVs) and common variants of the RET gene contribute to Hirschsprung disease (HSCR; congenital aganglionosis). While RET common variants are strongly associated with the commonest manifestation of the disease (males; short-segment aganglionosis; sporadic), rare coding sequence (CDS) variants are more frequently found in the lesser common and more severe forms of the disease (females; long/total colonic aganglionosis; familial).Here we present the screening for RVs in the RET CDS and intron/exon boundaries of 601 Chinese HSCR patients, the largest number of patients ever reported. We identified 61 different heterozygous RVs (50 novel) distributed among 100 patients (16.64%). Those include 14 silent, 29 missense, 5 nonsense, 4 frame-shifts, and one in-frame amino-acid deletion in the CDS, two splice-site deletions, 4 nucleotide substitutions and a 22-bp deletion in the intron/exon boundaries and 1 single-nucleotide substitution in the 5' untranslated region. Exonic variants were mainly clustered in RET the extracellular domain. RET RVs were more frequent among patients with the most severe phenotype (24% vs. 15% in short-HSCR). Phasing RVs with the RET HSCR-associated haplotype suggests that RVs do not underlie the undisputable association of RET common variants with HSCR. None of the variants were found in 250 Chinese controls.

Haplotype analysis reveals a possible founder effect of RET mutation R114H for Hirschsprung's disease in the Chinese population.

BACKGROUND: Hirschsprung'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. The RET gene is the major gene implicated in this gastrointestinal disease. A highly recurrent mutation in RET (RET(R114H)) has recently been identified in approximately 6-7% of the Chinese HSCR patients which, to date, has not been found in Caucasian patients or controls nor in Chinese controls. Due to the high frequency of RET(R114H) in this population, we sought to investigate whether this mutation may be a founder HSCR mutation in the Chinese population. METHODOLOGY AND PRINCIPAL FINDINGS: To test whether all RET(R114) were originated from a single mutational event, we predicted the approximate age of RET(R114H) by applying a Bayesian method to RET SNPs genotyped in 430 Chinese HSCR patients (of whom 25 individuals had the mutation) to be between 4-23 generations old depending on growth rate. We reasoned that if RET(R114H) was a founder mutation then those with the mutation would share a haplotype on which the mutation resides. Including SNPs spanning 509.31 kb across RET from a recently obtained 500 K genome-wide dataset for a subset of 181 patients (14 RET(R114H) patients), we applied haplotype estimation methods to determine whether there were any segments shared between patients with RET(R114H) that are not present in those without the mutation or controls. Analysis yielded a 250.2 kb (51 SNP) shared segment over the RET gene (and downstream) in only those patients with the mutation with no similar segments found among other patients. CONCLUSIONS: This suggests that RET(R114H) is a founder mutation for HSCR in the Chinese population.

Lack of association between nNOS -84G>A polymorphism and risk of infantile hypertrophic pyloric stenosis in a Chinese population.

BACKGROUND: Infantile hypertrophic pyloric stenosis (IHPS) is one of the most common gastrointestinal obstructions in the infancy requiring surgery. Reduced expression of neuronal nitric oxide synthase (nNOS), which plays an important role in the regulation of the human pyloric muscle, is thought to underlie IHPS. The role of nNOS in IHPS has been supported by the genetic association of a functional regulatory nNOS polymorphism (-84G>A) with IHPS in whites. We reasoned that the corroboration of this association in a population of different ethnic origin would prompt follow-up studies and further investigation of the IHPS pathology at molecular level. Thus, we attempted to reproduce the original findings in a Chinese population of comparable size in what would be the first genetic study on IHPS conducted in Chinese. METHODS: nNOS -84G>A genotypes were analyzed in 56 patients and 86 controls by polymerase chain reaction and DNA sequencing. Logistic regression was used to compute odds ratios. RESULTS: Our study could not corroborate the association previously reported. Although the frequency of the IHPS-associated allele (-84A) in controls (0.205) was similar to that reported for white controls, there was a dramatic difference in -84A frequencies between white and Chinese patients (0.198). Similarly, there was no difference in the nNOS -84G>A genotype distribution between patients and controls, even when the GA and AA genotypes were combined to compare GG genotype (odds ratio, 1.01; 95% confidence interval, 0.47-2.19). CONCLUSIONS: Failure to replicate the initial finding does not detract from its validity, because genetic effects may differ across populations. Differences across populations in linkage disequilibrium and/or allele frequencies may contribute to this lack of replication. The role nNOS in IHPS awaits further investigation.