| Background: Hirschsprung's disease (HD) is a common congenital malformation affecting 1 in 5000 live births, which also named congenital aganglionsis and occupies the second in the congenital intestinal malformation. The proportion of cases between male and female is 4:1.The main symptom is complete or incomplete neonatal obstruction. This disease severely effects children's health, even endanger lives. As a complex disease, HD has been ascribed to the absence in the terminal hindgut of ganglion cells from the neural crest, which causes the abnormal contraction of involved intestines, and then the proximal end of the sick colon appears compensatory dilated thickness and forms megacolon. Recently, the incidence of HD increased and scholars around world paid more attention on it. HD is a disease with complex pathogenesis. Though the pathologic base of HD is lack of ganglion cells of affected gut, the causation of the lack of ganglion cells is still ambiguous. Now, surgery is the only effective treatment, patients have to suffer great pain, so how to clarify the pathogenesis ulteriorly, reduce the incidence of HD and seek after effective intraumatic treatment become the most important problem at present.Most HD cases are sporadic, and the percent of familial cases is about 3.6-7.8%. Now,HD is thought to be multifactorial in origin, including genetic and microenviromental influences. Genetic factors are thought to be the most important cause of development of HD. Up to the present, upon 10 genes and 4 regions have been regarded as the major candidate genes and regions ofHSCR, such as RET > EDNRB> EDN3> GDNF. SOX10> ECE-K Neurturin, 9q31 and so on. Among these, two signal pathway, RET/GDNF and EDNRB/EDN3/ECE-1, were studied widely.Now, RET gene is regarded as the most important causal gene of HD. RET gene codes a pattern of receptor tyrosine kinase, which is transmember polymer including 1114 amino acid. The main function of this receptor is to transfer cellular development and differentiation signals. According to the data, the mutations of RET account for about 40-50% familial cases and 15-20% sporadic cases.Hitherto, about 90 RET mutations have been reported in the patients with HSCR. These mutations scatter along the entire RET proto-oncogene and most are point mutations. Recently, some specific single nucleotide polymorphism (SNP) loci were identified to associate with HD, such as A45A on exoi^ L769L on exonl3. In 2005, Chakravarti et al found that 6 SNPs existed in intronl of RET gene were associated with HD obviously, especially RET+3 locus. RET+3 locus lies within an enhancer-like multi-species conserved sequence (MCS+9.7), and it is very important to study the function of this locus. To this day, the study of mutation of RET gene in HD patients has been very roundly in land, but the study about the association between HD and the SNP loci within RET gene is still unseen.The endothelin-B receptor gene (EDNRB) gene is second important causal gene of HD, which encoding a G-protein-coupled heptahelical receptor protein. EDNRB was mapped to chromosome 13q22 and the length of which is 24kb, comprising 7 exons and 6 introns. It has been proved through experiment that the signal pathway mediated by EDNRB and its Hand, and targeted disruption of the mouse EDNRB gene resulted inaganglionic megacolon and spotted coat colour, a phenotype similar to that of human HD. As for EDN3 gene mapped on chromosome 20 codes the most important Hand of EDNRB, endothelin-3. If the mutations occur in the coding sequence of EDN3 gene, the function of protein will be changed or the functional protein can't be formed correctly and the signaling pathway mediated by EDNRB/EDN3 also will be affected. Till to now, there have been 17 mutation patterns of EDNRB gene have been detected, the incidence is 5-10%, while the mutation of EDN3 gene is less than EDNRB gene. Now, mutation study of EDNRB and EDN3 gene in Chinese patients with HD is still few.Objective: (1) To investigate the pattern and incidence of mutation of EDNRB and EDN3 genes in Chinese with sporadic HD, to elucidate the genetic mechanism of Chinese HD patient at the molecular level and to illuminate the relationship between EDNRB/EDN3 genes and Chinese sporadic HD. (2) To study the traits of RET+3 > rs2506004> rs2506005 locus in Chinese patients with sporadic HD and to elucidate the role of SNPS in development of Chinese sporadic HD and seek the haplotypes associated with HD.Methods: Genomic DNA was extracted from blood of sporadic HD patients and normal control. (1) Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was used to analysis all seven exons of EDNRB gene and two exons of EDN3 gene, and the samples with abnormal single strand showed in PCR-SSCP were detected by DNA direct-sequencing to identify the mutation pattern of abnormal sample. (2) PCR-sequencing was used to analysis the genotype of RET+3 ?. rs250600^ rs2506005 locus in HD patients and normal controls, the allele frequency^ linkage disequilibrium and haplotype frequency were analysis by SHEsis software.l20:-UResult: Two variants, L831L in exon 4 and V553M in exon 2 were found in EDNRB gene in seventy-five patients with sporadic HD. L831L which has been reported to be single nucleotide polymorphism with 0.5 genotype frequency was detected in 6 cases. While themissense mutation V553M was detected in 2 cases, which lies on the third transmembrance domain of EDNRB protein. V553M has not been reported in Western population, so perhaps this is a novel mutation specially existed in Chinese population, the mutation incidence is 2/75 (2.8%). And no genetic variations were observed in EDN3 gene.RET+3, rs2506005 and rs2506004 were genotyped in 99 Chinese patients with isolated HSCR and 132 ethnically matched controls. Our data show that the main genotype of RET+3, rs2506005 and rs2506004 in HSCR is TT> CC> AA, respectively, allele frequency of T> C, A is 72%. On the other hand, The main genotype of RET+3, rs2506005 and rs2506004 in control is TC, CT> AC, respectively, allele frequency of T> C, A is 45%. And these three loci is complete linkage disequilibrium, only construct two haplotypes TCC and CTA. CTA is strongly associated with HD (Fisher's Exact P = 1.81 X10"8;OR=3.043, 95% CI 2.054-4.507). The transmission frequency of RET+3: T allele to affected sons and daughters led to a 5.7-fold and 2.1-fold increase in susceptibility in males and in females, respectively.Conclusion: (1) EDNRB gene is the important candidate gene of HD with low mutation incidence (<5%), while the relationship between EDM3 and HD should be investigated more. (2) Haplotype CTA(rs2506005: C;RET+3: T;rs2506004: A) is associated with HD and its effect is sex-dependent. It shows that some special SNPs of HD candidate genes can increase the risk for Chinese population suffering from HD.
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