Research Of Polymorphisms Of Endothelin Receptor B And Expression Of SOX10 In The Pathogenesis Of Hirschsprung Disease

Part I Screening for Polymorphisms and Mutations of EDNRB gene in Patients with sporadic Hirschsprung diseaseObjective: To screen for single nucleotide polymorphisms and mutations of endothelin receptor B gene in Chinese patients of Han ethnicity with Hirschsprung disease and probe the correlation of EDNRB gene in the etiology of Hirschsprung disease.Methods: To adopt a case-control study and peripheral blood samples from 104 patients diagnosed with sporadic HD and 120 normal children as controls were collected and DNA was extracted from blood samples by salt fractionation. All exons and intron/exon boundaries of EDNRB gene were amplified by polymerase chain reaction and PCR products were screened for mutations and polymorphisms by single strand conformation polymorphisms.Results: Mutations and polymorphic sites were identified in exon 1, 3, 4, 6 and 7 of susceptibility gene EDNRB in patients with sporadic HD. Mutations were detected especially in patients with SSA. No changes were found in exon 2 and exon 5..Conclusions: EDNRB gene may play an important role in the pathogenesis of Han ethnic Chinese patients with sporadic HD, especially for short-segment HD. Polymorphisms and mutations may affect receptor functions.Part II Analysis of Genotype and Allele distribution and Mutation status of EDNRB gene in Patients with sporadic Hirschsprung diseaseObjective: To study the characteristics of mutations and polymorphisms of susceptibility gene EDNRB in Chinese patients of Han ethnicity with Hirschsprung disease and analyze the relationship between nucleotide change and HD.Methods: Peripheral blood samples from 104 patients diagnosed with sporadic HD and 120 normal children as controls were collected and DNA was extracted from blood samples by salt fractionation. All exons and intron/exon boundaries of EDNRB gene were amplified by polymerase chain reaction and PCR products were screened for mutations and polymorphisms by single strand conformation polymorphisms. Using direct DNA sequencing to detect the samples with abnormal mobility bands and nucleotide sequence alteration was clarified. The differences of allele frequencies and genotype distribution in polymorphic sites were further analyzed between case group and control group.Results: Mutations of EDNRB gene in sporadic HD were detected in seven patients and the mutation rate was 6.7%(7/104), four types of point mutations, including two novel mutations(c711 C>A in exon 3(A237A, 2 cases), c1157 T>G in exon 6(L386R, 2 cases)) and two previously described mutations(c723 T>C in exon 3(D241D, 2 cases), cl278 T>C in exon 7(N426N, 1 case)). Six mutations occurred in short-segment aganglionosis and all patients with mutations are in heterozygous status. Five single nucleotide polymorphisms(SNPs) sites in exons 1, 4, and 6 of EDNRB in the sHD patients and controls were detected. The differences between patients and controls in allele and genotype distribution of the four EDNRB polymorphic sites were statistically significant(P<0.05), including c311 A>T(N104I) in exon 1, c831 G>A(L277L) in exon 4, cl 148 C>A(P383Q) in exon 6 and cl 170 C >A(S390R) in exon 6. Patients with homozygotes of the C allele of c1148 C>A had a 8.2 fold elevated risk for HD(95%CI 3.1～21.7, P<0.001).Conclusions: Mutations of EDNRB gene occurred in Han ethnic Chinese patients with HD, especially in patients with SSA. Furthermore, all patients with mutations were in heterozygous status. There was heterogeneity between mutations and phenotype. Five SNPs were detected in patients and controls and the allele and genotype distributions in four SNPs of EDNRB were statistically different between patients and controls, which showed a significant correlation with HD. Genetic characteristics of EDNRB in sHD patients showed differences among different ethnicities.Part III Study of Hereditary relationship and Susceptibility in Polymorphisms and Mutations of EDNRB gene between Patients with sporadic Hirschsprung disease and their ParentsObjective: To study the hereditary characteristics of polymorphisms and mutations of EDNRB gene in sHD between parental and filial generation, and evaluate the relationship between haplotype and risk for HD.Methods: Peripheral blood samples from 104 patients diagnosed with sporadic HD, 84 parents of 42 patients and 120 normal children as controls were collected. Using PCR-SSCP and direct DNA sequencing to detect mutations and polymorphic sites in EDNRB gene. The differences of allele frequencies and genotype distribution in polymorphic sites were further analyzed between case group, parents group, and control group. Allele frequencies of SNPs in forty-two sHD trios were analyzed by transmission disequilibrium test(TDT), and the association between phenotype of HD and SNPs was analyzed. The haplotype was used to evaluate risk for sHD.Results: The mutation rate of EDNRB gene in sHD was 6.7% (7/104). Three cases occurred germline mutation and two of them inherited from unaffected mother. TDT indicated a nonrandom association of transmitting specific alleles of the twopolymorphic sites to patients, the T allele(c311 A>T) and C allele(c1148 C>A) were preferentially transmitted to filial generation. Allele frequencies distribution was statistically different in 4 SNPs sites between SSA patients and controls, only 1 SNPs site between LSA patients and controls, in 2 SNPs sites(c831 G>A , c1170 C>A) between SSA and LSA patients. Linkage disequilibrium was confirmed in two SNPs sites of c311 A>T and c1148 C>A. The frequency of haplotype of 311A-1148A was much lower in cases group than in controls(2.9% vs 16.7%, P<0.01), conversely in haplotype of 311T-1148C(18.8 % vs 7.9%, P <0.01). Carriers of without 311A-1148A haplotype had a 8.2 fold elevated risk for sHD(OR = 8.153, 95%CI: 3.057～21.741, P<0.001).Conclusions: Germline EDNRB mutation was the major mutation style in Han ethnic Chinese sHD. Five polymorphic sites were detected and SNPs sites of c311 A>T and c1148 C>A showed transmission disequilibrium. Patients with SSA phenotype had a more significant association with EDNRB gene than LSA. Carriers of 311A-1148A haplotype might be protective against sHD, Carriers of without 311A-1148A haplotype had a 8.2 fold elevated risk for sHD(OR = 8.153, 95%CI: 3.057～21.741, P <0.001).Part IV Research of the association between SOX10 candidate gene and sporadic Hirschsprung diseaseObjective: To study the expression of SOX10 gene in the tissue of different bowel segments of patients with sporadic HD and probe for the effects of SOX10 gene in the pathogenesis of sHD.Methods: Surgical specimens were collected from 55 patients with sporadic HD and 24 patients without sHD as controls. The expression level of SOX10 mRNA in different bowel segments(constrictive segment, transitional segment, normal segment)of patients with sHD and control tissues was examined by semiquantitative reverse transcription-polymerase chain reaction, The association of SOX10 and clinicalpathological parameters was further analyzed.Results: SOX10 mRNA was expressed in both differently pathological bowel segments and normal controls. The expression level of SOX10 in constructive bowel segment was (0.766±0.038), which was significantly lower than that in other bowel segments and normal controls(P<0.01). The expression showed no statistical difference between transitional segment(0.834±0.034), normal segment(0.829±0.030) and normal controls (0.824±0.021) (P>0.05). The SOX10 level of constructive segment in SSA was significantly high than that in LSA((0.775±0.037) vs (0.752±0.036),P<0.05).Conclusions: Abnormal expression of SOX10 gene had a close correlation with the etiology of sHD, especially with long-segment HD. SOX10 may be normally required postnatally in the functional maintenance of the enteric nervous system.