The Association Between Hirschsprung Disease And RET,NRG1,NRG3 Gene Polymorphisms In Chinese Population

PART ONE Risk of GWAS-identified genetic variants for HSCR in a Chinese population:a multiple interaction analysisObjectiveIn this study,we selected 2 single nucleiotides polymorphisms(SNPs)at RET gene,and 3 SNPs at NRG1 and NRG3 gene as HSCR risk associated markers,respectively.Accordingly,we designed this case-controlled study to investigate the relevance between these eight SNPs the haplyotypes which consist of these SNPs,and the pathogenesis of HSCR,and we explored the SNP-SNP interactions with an aim to evaluate the disease risk level.As a result,we will try to find out if Our results support the genetic variation of RET and NRG1 being associated with susceptibility to HSCR in the Chinese population.In addition,it is the very first and important step for the molecular mechanism research of the HSCR in the future in the Chinese population.Material and Methods1.Blood sample collection and DNAexaction:The diagnosis of HSCR was based on the criteria of the Fourth International Symposium on Hirschsprung Disease and Related Neurocristopathies and the histological examination of either biopsy tissues or pathology specimens obtained during operations.Blood samples were obtained from healthy individuals as controls who were randomly recruited from the general population in the Wuhan region of Hubei Province.The diagnosis of HSCR was based on the histological examination of either biopsy tissues or pathology specimens obtained during operations.This research project has been approved by the Ethical Committee of the Medical Association of Tongji Medical College,Huazhong University of Science and Technology.Informed consent was obtained from all participants or legal guardians.Genomic DNA was extracted from the white blood cells using a DNA blood midi kit according to the manufacturer's protocols,and the samples were maintained at-80? until use.2.SNP selection and genotyping:Through the(https://www.ncbi.nlm.nih.gov/snp/)SNP database and literature resources,we selected target SNPs.A hospital based case-control study was cinducted.We selected eight SNPs to assess the risk association with HSCR:rs2435357 and rs2506030 at RET;rs2439302,rs7835688 and rs16879552 at RET;and rs 10748842,rs 10883866 and rs6584400 at RET.The genetic polymorphisms were genotyped using the TaqMan SNP Genotyping Assay on a 7900HT Fast Real-Time PCR System.3.Statistical analysis between RET,NRG1 and NRG3 genes and HSCR:The Hardy-Weinberg equilibrium of the SNPs was assessed with a goodness of fit Chi-squared test in the control subjects.To analyze the frequency of alleles and genotypes in all samples,the Chi-square test was used.The ORs and 95%CIs were determined for the association between disease status and genotype using a Chi-squared test,and two-tailed P-values less than 0.05 were considered significant.To avoid the assumptions of genetic models,dominant,recessive and additive models for the three SNPs in association with HSCR were analyzed.The wild type was considered as a reference.The genetic comparisons included a homozygous model,heterozygous model,dominant model and recessive model.The combined effects of SNPs between NRG1(rs2439302)and RET(rs2506030 and rs2435357)on HSCR risk were assessed.ORs and the frequency of the SNP alleles were calculated for disease markers.We used Haploview 4.2 for tests of LD determined by the r2 value.For SNP-SNP interactions,we used a multiple Logistic regression model to estimate the multiplicative interaction effect of the SNPs,For each SNP,the genotypes were coded as 0,1,or 2 indicating the number of HSCR risk alleles in the individual.The unweighted cumulative genetic risk scores of an individual is the total count of disease alleles from the SNPs obtained by adding coded genotypes(scors range:0-6).P-values less than 0.05 were considered significant of all these tests.Results1.The characteristics of 362 HSCR patients and 1,448 healthy subjects.There were 275(75.97%)male and 87(24.03%)female HSCR subjects,while 1,100(75.97%)male subjects and 348(24.03%)female subjects were included in the healthy control group.The average age was 1.01(±0.88)years and 1.18(±0.79)years for the case and control groups.The cases and controls were well matched on the distribution of gender and age(P=1.00 and 0.839,respectively).2.In this case-control study,among the eight GWAS identified genetic markers,four polymorphisms,rs2506030 and rs2435357 in inhancer area at RET,and rs2439302,rs7835688 in intron 1 at NRG1 showed a positive correlation with HSCR susceptibility,with an OR greater than 1.0(P<0.05).However,no genetic correlation was found between HSCR and the three SNPs at RET(rs10748842,rs10883866 and rs6584400)or the SNP at RET(rs 16879552)when comparing the risk allele with the non-risk allele.In addition,no relevancy was observed in the heterozygote or the homozygote variant.3.We calculated the linkage disequilibrium(LD)for the cases and controls.For the rs2435357 and rs2506030 SNPs,the r2 of these two SNPs is 0.041,which suggest they are in low association.The two variants,rs7835688 and rs2439302 at the NRG1 locus,the r2 of these two SNPs is 0.95.The result indicated that rs7835688 and rs2439302 are strongly associated.Therefore,we report rs2439302 as representative of the association.Genotype distributions were in Hardy-Weinberg equilibrium for the polymorphisms in both the HSCR cases and the controls(Chi-squared test,P>0.05),and the statistical test power was>95.0%.4.We further analyzed the combination of these two genes.Compared with other genotypes,the patients carrying homozygous risk alleles of rs2439302-GG and rs2506030-GG or rs2439302-GG and rs2435357-TT had the highest HSCR risk with OR=25.69(95%CI=10.11-65.29,P=9.12E-12)and 25.57(95%CI=10.88-60.07,P=1.02E-13),respectively.The patients carrying homozygous risk alleles also had the highest HSCR risk with an OR=56.53(95%CI=12.16-262.83,P=4.50E-07).Also,we discovered the similar results that the patients carrying two(rs7835688-CC and rs2506030-GG,rs7835688-CC and rs2435357-TT)or three(rs7835688-CC,rs2506030-GG and rs2435357-TT)homozygous risk alleles with an OR=24.13(95%CI=9.54-61.05,P=1.80E-11),OR=24.10(95%CI=9.82-59.13,P=3.68E-12),OR=54.83(95%CI=10.94-274.74,P=1.12E-06),respectively.Conclusion1.Our results support the genetic variation of RET and RET being associated with susceptibility to HSCR in the Chinese population.2.This is the first time to study the association between polymorphisms in RET(rs2435357 and rs2506030)and NRG1(rs2439302 and rs7835688)genes with HSCR in Chinese population.3.Cumulative effect was observed in the study population,and with increasing numbers of risk alleles,these genetic interactions significantly enhance the risk of HSCR.The patients carrying homozygous risk alleles also had the highest HSCR risk.4.Our findings emphasized that cumulative genetic risk varied among the genetic variants of rs2506030,rs2439302,rs2435357and rs7835688.However,it is not very clear that the molecular pathogenesis associated with HSCR,and therefore,more sophisticated biological analyses are necessary to explain the complex interactions between these genes.PART TWO Single nucleotide polymorphisms in the RET and NRG1 genes affected the expression of themObjectiveTo explore whether any of the four genotypes in rs2435357,rs2506030,rs2439302 and rs7835688 were associated with the expression of the RET and NRG1 genes.Material and Methods1.In the part one,the two SNPs,rs2439302 and rs7835688,are in high allelic identity(LD r2 =0.9).If one variant shows significant association with HSCR,the other variant,by the phenomenon of LD,is expected to also show disease association.Therefore,we report rs2439302 as representative of the association.To explore whether any of the three genotypes in rs2435357,rs2506030 and rs2439302 were associated with the expression of the RET and NRG1 genes,we performed western blotting and real-time quantitative reverse transcription PCR(RT-qPCR)on ganglionic colon tissues obtained from ten HSCR patients.2.Immunohistochemical staining:Colon tissue of ganglion segment of HSCR cases embedded inparaffin were cut with 5 ?m thick sections and immunohistochemical stained with RET and NRG1.The expression of RET and NRG1 level were examined.ResultsCompared with rs2435357-CC or rs2506030-AA,the relative expression of RET mRNA,protein and immunohistochemical staining decreased in rs2435357-CT and rs2435357-TT or rs2506030-AG and rs2506030-GG.Image analysis of western blots demonstrated a significant decrease of RET protein expression(P<0.05).RT-qPCR revealed that RET was down-regulated in HSCR gut tissues.The relative expression of RET was lower in heterozygous and homozygous carriers of the HSCR risk allele than that in homozygous wild type subjects(P<0.05).For rs2439302,the highest NRG1 mRNA,protein expression and immunohistochemical staining were found for those individuals homozygous for the G risk allele and the lowest for those homozygous for the CC genotype(P<0.05).ConclusionThe polymorphisms rs2506030 and rs2439302 in RET,and rs7835688 and rs2435357 in NRG1 were functional gene variations in HSCR.It can affect the the transcription of mRNA and expression of protein products.