Association And Whole Genome Sequencing Based Study On Risk Locus For Hirschsprung Disease

Chapter 1: Case-control and Meta-Analysis based study for NRG1 variants rs7835688 and rs16879552 on Hirschsprung disease susceptibilityBackground:Hirschsprung Disease(HD)is the most common malformation of the enteric nervous system,which has an association with single nucleotide polymorphisms(SNPs).NRG1 was first identified as a susceptibility locus for HD by genome-wide association study in Chinese.Within the NRG1 region,SNPs rs7835688(G > C)and rs16879552(T > C)showed the strongest overall associations with HD.Up to now,several case control studies have been performed to explore the relationship between rs7835688 and rs16879552 variants and HD risk,but the results are still controversial due to the inconsistency among these studies.In that case,we performed this case-control and Meta-Analysis based study to provide the evidence for the implication of NRG1 rs7835688 and rs16879552 polymorphisms in the HD susceptibility.Methods:(1)A hospital-based case-control and fine mapping study was conducted to explore the association between NRG1 locus rs7835688 and rs16879552 and HD susceptibility.(2)After the case-control study,a meta-analysis was performed by combining the fine mapping data with the results from other relevant,previously published case-control studies to further investigate such associations.(3)Subgroup analysis were performed by ethnicity(Asian or Caucasian)to explore the association between rs7835688 and rs16879552 and HD risk.(4)According to the extent of the affected bowel,we divided the HD into two types: short-HD and long-HD/TCA,then stratified analysis was conducted based on the type of HD.Results:(1)Our case-control study indicated that rs7835688 was significantly associated with increased risk of HD in the Chinese Han population under any genetic models.However,rs16879552 was not found to be associated with HD.(2)In total,8 articles for rs7835688 and 9 articles for rs16879552 were included in the final analysis.For rs7835688,5 studies were satisfied with the Hardy-Weinberg equilibrium(HWE).As for rs16879552,4 studies were satisfied with the HWE except one.For the rest 4 studies,the authors pointed out that the genotype distribution in the controls for rs7835688 and rs16879552 didn't violate HWE,but the exact data was not given.Of all of the studies included,7 studies involved Asians,2 studies investigated Caucasians.All studies followed a case–control design,2 of them were GWAS;5 studies used population-based controls,and 4 studies used hospital-based controls.(3)The combined results showed a significant association between HD risk and rs7835688 in all genetic models(per-allele model: OR = 1.66,95% CI = 1.35–2.05;P = 1.940E-06).Rs16879552 was significantly associated with HD in per-allele(OR = 1.50,95% CI = 1.27–1.76;P = 1.087E-06),additive and recessive model,except for dominant model.(4)Stratified analysis by ethnicity showed that rs7835688 and rs16879552 were only causative for Asians,but not risk locus for Caucasians.(5)Pooled data based on segment length suggested that patients with rs7835688 had a significantly higher risk for short-segment HD in all genotypes;but rs16879552 was only found to be associated with long-segment HD/total colonic aganglionosis at the allele level.Conclusions: Our study have replicated the positive associations among NRG1 polymorphisms(rs7835688 and rs16879552)and HD susceptibility.The results highlight the importance of genetic variation of NRG1 and provide justification for further investigation of NRG1's role in the pathophysiology of HD.Chapter 2: Identification of new Hirschsprung disease causative gene by whole genome sequencing and functional validation of the geneBackground: As a congenital malformation of the lower gastrointestinal tract,Hirschsprung disease(HD)can be attributed to the migration of the neural crest cells(ENCCs)been disrupted during embryonic development.This disorder leads to an absence of enteric ganglia in the submucosal and myenteric plexuses along a variable length of the gut which producing a functional intestinal obstruction.When ENCCs migrate along the intestine,they are regulated by many transcription factors,cell surface receptors,and neurotrophic signals.Any gene mutations that encode these critical molecules can lead to the aganglionosis that occurs in HD.Genetic studies have identified several causative genes for HD,but they together explain less than 20% of all HD cases.This study aimed to analyze all kinds of genetic variation and identify new HD gene;to explore the expression of target gene in HD tissue and paired normal colon tissues;to clarify the location difference between wild type and mutant type of target gene;and to study the impact of knock-down target gene on proliferation and migration of ENCCs.Methods:(1)DNA was isolated from peripheral blood of 13 trios(affected child and unaffected parents)of HD recruited in our center.After quality control,whole genome sequencing was conducted and various bioinformatics methods were applied to explore all kinds of genomic variation of HD related gene.(2)We searched NCBI GEO Datasets to find out the expression of ALK gene in the human fetuse tissues.(3)Immunohistochemistry,PCR and western-blot were used to examine the location and expression of ALK in HD affected tissues and normal colon samples.(4)In vitro,we transfected the p LKO.1-puro-sh RNA-ALK vector carring validated knoch-down sequencing into ENCCs.The knock-down efficacy was verified by immunofluorescence analysis.Traditional MTT assay and migration assay in an x CELLigence RTCA DP system were conducted to observe the impact of silencing ALK gene on proliferation and migration of ESCCs.Results:(1)Totally there were 2149 mutations were identified in coding regions,of which 1927 mutations were predicted to be pathogenic/likely pathogenic according to ACMG standards and guidelines.In addition,shared mutations screening identified 222 locus.(2)SIFT,Polyphen,Mutation Taster and CADD softwares were employed to predict whether an amino acid substitution change affects the function of the protein.Applying the above strategy,ALK(anaplastic lymphoma kinase)gene was found to be causative gene for HD.One homozygous(c.C1648 T [p.L550F]])and one compound heterozygous(c.C3035 T [p.T1012M] and c.G487 T [p.V163L])ALK variants were detected.(3)Thirty-five human fetal samples from 6 tissues(3-7 replicates per tissue)collected between 10 and 20 weeks gestational time were sequenced using Illumina Tru Seq Stranded Total RNA(from NCBI GEO Datasets).The results demonstrated that the expression of ALK were significantly higher in stomach and intestine than any other organs.(4)Wild type of ALK was mainly expressed in the ganglions and nerve fibers,which located in both membrane and cytoplasm.Compared with normal colon,the expression of ALK was significantly lower in the aganglionic intestine.(5)ALK knockdown strongly decreased the proliferation and migration ability of ENCCsConclusions:(1)By means of whole genome sequencing,we identified all kinds of genetic variation including In Dels,SNV,SV and CNV.ALK is confirmed as a new causative gene for HD.(2)ALK is involved in the biological behavior of ENCCs,and its down regulation will inhibit the proliferation and migration of ENCCs.