The Association Analysis Of RET, EDNRB And PHOX2B Genes With Hirschsprung Disease In The Han Chinese Population

Background and objective:Hirschsprung disease(HSCR,OMIM 142623) represents the main genetic cause of functional intestinal obstruction with an incidence of 1/5000 live births.This developmental disorder is a neurocristopathy and is characterized by the absence of the intestinal ganglion cells of the nerve plexuses in variable lengths of the digestive tract. Symptoms in infants include difficult bowel movements,poor feeding,poor weight gain, and progressive abdominal distention.Males are affected four times more often than females,a difference most prominent in short segment HSCR.Hirschsprung disease is considered a genetic disease,caused by genetic alterations. To date,at least 11 genes have been associated with sporadic or syndromic forms of HSCR.These 'HSCR genes' are generally related to the developmental program of neural crest cells,and include the RET proto-oncogene,glial cell line-derived neurotrophic factor(GDNF),neurturin(NTN),endothelin 3(EDN3),endothelin receptor B(EDNRB),endothelin converting enzyme 1(ECE1),transcriptional factors SOX10 and PHOX2B,Smad interacting protein 1(SIP1),KIAA1279,and TITF1.In this study,we selected 12 single nucleiotides polymorphisms(SNPs) and one microsatellite located in the RET gene and 4 SNPs located in the PHOX2B gene as the gene markers.By using the case-control association analysis,we will investigate the relationship between HSCR and the single SNP and the haplotypes which consist of these SNPs.Meanwhile,mutation analysis of the endothelin family genes was investigated.As a result,we will try to find out if these genes are the susceptibility genes of the HSCR in Han Chinese population.The association study between HSCR and these genes may make it more clearly for the genetic bases of the aetiology of HSCR.In addition,it is the very first and important step for the molecular mechanism research of the HSCR in the future in the Han Chinese population.PartⅠThe association analysis of RET gene with sporadic Hirschsprung disease in the Han Chinese populationSubjects and methods:1.Blood sample collection and DNA extraction:Blood samples were obtained from 125 unrelated cases(96 males,29 females),diagnosed with sporadic HSCR.All patients were histological confirmed with criteria of the Fourth International Symposium on Hirschsprung Disease and Related Neurocristopathies. Control DNAs were obtained from 148 unaffected individuals of Han Chinese.This study was approved by the Ethics Committee of Zhejiang University and all subjects gave informed consent for the genetic analyses.2.SNP selection and genotyping:We selected 12 SNPs(SNP1～SNP12) and one microsatellite(CA)n in the RET through the SNP database(http:// www.ncbi.nlm.nih.gov/snp/).Genotyping was performed by the ligase detection reaction(LDR),which has been demonstrated to be a highly specific and sensitive assay for SNP detection.Fluorescent dye-labeled PCR products were used with an ABI PRISM 377 DNA Sequencer for genotyping,and the data were analyzed by using Genemapper software.3.Statistical analysis:The deviation from Hardy-Weinberg equilibrium(HWE) was examined in controls by the(?)~2 test.Based on the logistic regression method,the case-control association of genotypes in five inheritance models(codominant,dominant,recessive,overdominant,log-additive) was tested for all the 12 single SNPs.Pairwise Linkage Disequilibrium(LD) was calculated for the cases and controls in Han Chinese population.Constructed the haplotype blocks based on the results of LD analyses.The association analysis of the haplotypes with HSCR was similar to that of genotypes of single SNP by logistic regression.The significance of all these tests was 0.05.Results:1.Significant genetic association between three coding region SNPs(SNP8 (rs1800858),SNP9(rs1800860),SNP11(rs1800863)) and HSCR were observed in the experiment(P<0.05).The results indicated that all the non-coding 8 SNPs(SNP1 (rs741763),SNP2(rs2435365),SNP3(rs1864410),SNP4(rs2435364),SNP5(rs2435362), SNP6(rs752975) and SNP7(rs2505535) located beween 5' UTR and exon2,SNP12 (rs2075912) located in intron 19) were significantly associated with HSCR(P< 0.001).We found that a large proportion of our patients were homozygous at the markers that showed the highest association with HSCR,whereas these homozygous genotypes were comparatively lower in controls.The C allele of rs2505535,G allele of rsl800863 and the A allele of rs1800860 were represented as protecting alleles for the Chinese population.Rs2505535 and rs1800860 showed significant difference between S-HSCR and L-HSCR patients(P =0.015,P =0.006).The best inheritance models were obtained according to the smallest AIC(Akaike information) value.For SNP1,SNP4,SNP5 and SNP6,the recessive model were accepted as the best inheritance model;for SNP2,SNP8,SNP11 and SNP12,the log-additive model were accepted as the best inheritance model;for SNP3 and SNP7 the co-dominant model were accepted as the best inheritance model;for SNP9 the dominat model was accepted as the best inheritance model.Unexpectedly,microsatellite(CA)_n were not associated with HSCR. 2.We found strong LD(D'>0.75) between some pairs of the markers in RET gene. Therefore,we constructed two different haplotype blocks consisting of some nearby SNPs which were in strong LD,these two haplotypes were SNP1/SNP2/SNP3/SNP4 /SNP5/SNP6/SNP7/SNP8 and SNP10/SNP11.3.Haplotype-specific association analysis revealed that the two haplotype blocks showed significant association with HSCR(P<0.0001).Conclusion:(1).This is the first time to study the association between RET non-coding mutations with HSCR in Han Chinese population,significant genetic association between non-coding RET SNPs(located in a region 4 kb upstream of the gene through 23 kb of intron 1,and intron 19) and HSCR were observed.The results are consistent with the hypothesis that non-coding mutations in RET play important roles in the development of HSCR.(2).The unknown functional disease variant(s),with a dosage-dependent effect in HSCR,is likely to be located in the 5'region of the RET gene.The study suggests that the RET gene is a major genetic risk factor for HSCR in the Han Chinese population.PartⅡScreening for polymorphisms and mutations of the endothelin signaling pathway genes in the Han Chinese patients with sporadic Hirschsprung diseaseSubjects and methods:1.Blood sample collection and DNA extraction:Blood samples were obtained from 102 unrelated cases,diagnosed with sporadic HSCR.All patients were histological confirmed with criteria of the Fourth International Symposium on Hirschsprung Disease and Related Neurocristopathies.Control DNAs were obtained from 43 unaffected individuals of Han Chinese.The study was approved by the Ethics Committee of Zhejiang University and all subjects gave informed consent for the genetic analyses.2.Mutations and polymorphisms of susceptibility genes EDNRB/EDN3/ECE1 were analyzed by polymerase chain reaction and direct DNA sequencing.3.The differences of allele frequencies and genotype distribution in polymorphic sites were further analyzed between case group and control group.Results:1.Mutations of exon3 of EDN3 gene in HSCR were detected in 3 patients and the mutation rate was 0.029,three types of points mutations(c373 C→T(L125L), c394 C→T(T132X),c406 G→A(G135R)) were found.2.Mutations of EDNRB gene in HSCR were detected in seven patients and the mutation rate was 0.069,three types of point mutations,including two novel mutations(c29G→A(R10H ) in exon 1,3cases; IVS4+16T→C,1 case),one previously described mutation(c553G→A(V185M) in exon2,3 cases).One single nucleotide polymorphisms in exon 4(c831 G→A) were detected and the single SNP showed no significant association with HSCR(P >0.05).3. Statistically significant differences were found for rs212528 and rs212529 and the overdominant model were accepted as the best inheritance model.Conclusion:(1).Mutations of EDN3 and EDNRB gene occurred in Han Chinese patients with HSCR,especially in patients with short segments HSCR.Furthermore,all patients with mutations were heterozygous status.(2).One SNP was detected in exon 4 of the EDNRB gene which showed no significant association with HSCR.The results showed that the endothelin signaling pathway genes may act as secondary susceptibility for HSCR in Han Chinese population.PartⅢThe association analysis of PHOX2B gene with sporadic Hirschsprung disease in Han Chinese populationSubjects and methods:1.Blood sample collection and DNA extraction:Blood samples were obtained from 102 unrelated cases,diagnosed with sporadic HSCR.All patients were histological confirmed with criteria of the Fourth International Symposium on Hirschsprung Disease and Related Neurocristopathies.The control DNAs were obtained from 96 unaffected individuals of Han Chinese.The study was approved by the Ethics Committee of Zhejiang University and all subjects gave informed consent for the genetic analyses.2.SNP selection and genotyping:Polymerase chain reaction amplification and direct sequencing were used to screen PHOX2B coding regions and intron/exon boundaries for mutations and polymorphisms 3.Statistical analysis:The deviation from Hardy-Weinberg equilibrium(HWE) was examined in controls by the(?)~2 test.Based on the logistic regression method,the case-control association of genotypes in five inheritance models(codominant,dominant,recessive, overdominant,log-additive) were tested for all the 4 single SNPs.Pairwise Linkage Disequilibrium(LD) was calculated for the cases and controls in Han Chinese population.Constructed the haplotype blocks based on the results of LD analyses.The association analysis of the haplotypes with HSCR was similar to that of genotypes of single SNP by logistic regression.The significance of all these tests was 0.05.Results:In this study,we genotyped four single nucleotide polymorphisms(SNP1, c.701 A>G(novel);SNP2,IVS2+100 A>G;SNP3,c.1121 A>C;SNP4,c.1124 del15) located within the PHOX2B gene.Statistically significant differences were found for c.701 A>G and IVS2+100 A>G and the log-additive model were accepted as the best inheritance model.SNP2 showed significant difference between S-HSCR and L-HSCR patients(P =0.014).We also showed that the haplotype -A G A N composed of four SNPs exhibited significant association with the disease(P = 0.03),this haplotype was more frequently observed in cases than in controls(OR:2.31,95%CI:1.11-4.82).Conclusion:(1).c.701 A>G and IVS2+100 A>G in PHOX2B gene are significantly associated with HSCR in Han Chinese patients.(2).One single haplotype composed of four markers is associated with HSCR.These results suggest that a common HSCR predisposing variant in the PHOX2B gene might play an important predisposing role in the pathogenesis of HSCR.Our study provides further evidence that the PHOX2B gene is involved in the susceptibility to HSCR in the Han Chinese population.