The Association Analysis Of RET3'UTR And SOX10Morphisms With Hirschsorung's Disease In The Han Chinese Population

Background and objectiveHirschsprung's disease (HSCR, OMIN142623), also known as intestinal aganglionosis, is a congenital anomaly of the large intestine arising from a developmental abnormality of the enteric nervous system (ENS). The pathology of HSCR is characterized by the absence of ganglion cells in the myenteric and submucosal plexuses of the distal intestine, resulting in absent peristalsis in the affected bowel. This leads to the dilatation and hypertrophy of the proximal large bowel causing the symptoms of megacolon. There is significant racial variation in the incidence of the disease, Asian populations, including Chinese, with the highest incidence2.8/10000, and Caucasians1.5/10000, and lowest in Hispanics1/10000. The male to female ratio is4:1, and the gender imbalance is particularly evident in S-HSCR.Hirschsprung's disease is considered a genetic disease, caused by genetic alterations. To date, at least11genes have been associated with sporadic or syndromic forms of HSCR. These'HSCR genes'are generally related to the development program of neural crest cells, and include the RET proto-oncogene, glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), endothelin3(END3), endothelin receptor B (EDNRB), endothelin coverting enzyme1(ECE1), transcriptional factors SOX10, ZFHX1B (SIP1), PHOX2B, KIAA1279, L1CAM and TITF1.In this study, we selected7single nucleotides polymorphisms (SNPs) located in3'UTR of the RET gene and SNPs in exon of SOX10gene 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. 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 etiology of HSCR.Part I The Association Analysis of3'UTR Polymorphisms in RET Gene with Hirschsprung's Disease in the Han Chinese PopulationSubjects and methods:1. Blood sample collection and DNA extraction:Blood samples were obtained from107unrelated cases, diagnosed with sporadic HSCR. All patients were histological donfirmed with criteria of the Fourth International Symposium on Hirschsprung Disease and Related Neurocristopathies. Control DNAs were obtained from89unaffected 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:We selected7SNPs in the RET through the SNP database (http://www.ncbi.nlm.nih.gov/snp/). Genotyping was performed by PCR amplying the target fragment and then sequencing.3. Statistical analysis:The deviation from Hardy-Weinberg equilibrium (HWE) was examined in controls by the χ2test. 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 the12single 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 was0.05.4. The analysis of polymorphisms affecting secondary structure of m RNA was conducted by using the online mfold web server (http://www.introni.it/rnafold.html) and RNAstructure4.5.Results:1.5SNPs were identified in the RET3'UTR in HSCR cases, rs17028, rs3026782, rs2742240, rs2435355, rs2742241, respectively. The variants alleles of rs76759710and rs3026785are monomorphic SNP in Chinese population, the alleles frequencies of five SNPs in RET3'UTR were significantly different between the cases and controls. The ORs value ranged from0.2834to0.5117. None of these SNPs were significantly different between S-HSCR and L-HSCR patients. The alleles homozygous of these SNPs showed highly negative association with HSCR, and homozygous genotypes had comparatively higher frequency in controls (table2). The ORs for the homozygote genotypes ranged from2.52to7.64, and in heterozygote genotypes from2.23to3.60. This high OR value implied the protective effect against the development of HSCR. Because the frequencies of variant genotype for rs3026782were low, we combined it with heterozygote genotype together to analysis. The heterozygote genotype was also a protective role in HSCR, with the OR of3.975. Then logistic regression was used to carry out association analysis after modeling of the SNPs effects as additive, dominant or recessive. The best inheritance models were decided according to the smallest AIC (Akaike information criterion) value. For rsl7028, rs2435355, the dominant model was accepted as the best inheritance model. As for rs2742240and rs2742241, long-additive was accepted as the best inheritance model. For rs3026782, the overdominant model was accepted as the best inheritance model.2. Pairwise LD between the five SNPs was calculated for cases and controls. We found strong LD (D'>0.75) between some SNPs in the RET3'UTR region, including rsl7028, rs3026782, rs2742240, rs2435355and rs2742241(D'>0.75), rs3026782and rs274220(D'=0.9979), rs2742240and rs2435355(D'=0.7737), rs2435355and rs2742241(D'=0.9995).3. We further estimated frequencies of the haplotypes comprising the seven SNPs. Nine different haplotypes encompassing the seven SNPs were observed in cases and controls. Among these, haplotype7,8and9showed significant association with normal control (p<0.0001). haplotype2,3,4, and5also showed different between cases and controls (P=0.0041,0.013,0.0041and0.001, respectively).4. We analysis the change of Gibbs free energy of these regions including variant alleles by using the mfold software and to assess the possible effect of normal and variant alleles on mRNA secondary structure. A fragment of265bp surrounding rs17028and250bp surrounding rs3026785showed different Gibbs free energy between the normal and variant fragments, i.e.,-73.0kcal/mol to-75.6kcal/mol with normal to variant in rs17028fragment and-34.7kcal/mol to-37.1kcal/mol with normal to variant in rs3026785, which was expected to maintain the stability of RNA secondary structure. However, in the other five variants, the change of Gibbs free energy was decreased in variants, compared with the normal fragments. Conclusion:This is the first time to study the association between polymorphisms in3'UTR of RET gene with HSCR in Han Chinese population, negative genetic association between SNPs and HSCR was observed. These SNPs were protective alleles in HSCR. These SNPs seems to affect the secondary structure of RET mRNA stability, interfere with the mRNA degradation and modulate the expression levels of RET gene.Part II The Association Analysis of the SOX10Polymorphism with Hirschsprung's Disease in the Han Chinese PopulationSubjects and methods:1. Blood sample collection and DNA extraction:Blood samples were obtained from104unrelated cases, diagnosed with sporadic HSCR. All patients were histological donfirmed with criteria of the Fourth International Symposium on Hirschsprung Disease and Related Neurocristopathies. Control DNAs were obtained from96unaffected 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. The polymerase chain reaction amplification and direct sequencing were used to screen and genotype4exons of the SOX10gene for mutations and polymorphisms in104patients with sporadic HSCR and96ethnically matched controls.3. Statistical analysis:The deviation from Hardy-Weinberg equilibrium (HWE) was examined in controls by the χ2test. 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 the12single 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 was0.05. Results:1. We detected4SNPs in the SOX10gene. The first polymorphism identified was a C→>T transition (referred to as SNP1:c.18C>T) in exon2, encoding the sixth Aspartic acid. The second polymorphism identified was an G>T transition (referred to as SNP2:c.122G>T) in exon2, which alters a glycine to valine (GGC→>GTC). The third polymorphism identified was an C>G transition in intron2(referred to as SNP3); the fourth polymorphism identified was an T>C transition (referred to as SNP4:c.927T>C) which was a synonymous mutation in a coding region of309th Histidine.2. Association analysis:The genotype distribution and allele frequencies of4SNPs has no significant difference between the cases and controls. Using logistic regression analysis, we found no SNP sites associated with HSCR in five different genetic models to analyze correlation of a single SNP and HSCR. Conclusion:We were the first to identify SNP1in HSCR in the Chinese population and a novel polymorphism SNP2with HSCR. No SOXIO mutations have been found in Han Chinese with isolated HSCR. This study showed that The SOXIO gene is unlikely to be a major HSCR gene in the Chinese Han population.