Mutations and polymorphisms of Hirschsprung disease candidate genes in Thai patients

Abstract

Mutation and polymorphism data for Hirschsprung disease (HSCR) varies among ethnic groups. Single nucleotide polymorphisms (SNP) of RET protooncogene (RET) were recently shown to be associated with the disease, and with disease severity, in different populations. In this study, comprehensive analysis of RET, GDNF, EDNRB, ET-3, and SOX-10 genes among sporadic HSCR in Thailand was conducted by standard PCR-SSCP, RFLP, and sequencing methods. Of 41 patients, 30 cases had rectosigmoid disease (RSD) and 11 cases were assigned to the long-segment disease (LSD) group. Four missense mutations of RET, S100M, R231H, T278N, and G533S, were identified in three patients. One novel missense mutation, V111Q, was detected in EDNRB. For ET-3, two novel missense mutations, D166E and C173R, occurred concomitantly in a patient. The incidence of missense mutation was significantly higher in our female HSCR patient than in the male counterpart. Statistical analysis of the SNPs revealed a significant difference between allele distribution of RET L769L in patients in the LSD and RSD groups. The predominant genotype construct of RET A45A/L769L in our HSCR was GG/GG, which is obviously different from results from all previous studies. The GG/GG genotype construct was associated with RSD and with males. The study also detected a variant allele of RET S836S which has never been reported in Asian cohorts.

Association of genetic polymorphisms in the RET-protooncogene and NRG1 with Hirschsprung disease in Thai patients

“…To our knowledge, this is the first study that has used a fluorescence-based SNP detection assay for genotyping RET-protooncogene and NRG1, as previous studies have usually used PCR-direct sequencing, single-stranded conformation polymorphisms and restriction fragment length polymorphisms. 5,13,14,16,20,21 This fluorescence-based technique relies on different properties of two probes that are specific to the alleles of interest, each labeled with different fluorescent markers. It offers several advantages compared with older techniques; not only does it feature high specificity and accuracy, but also not requiring a post-amplification step reduces the time and cost of genotyping.…”

“…5 The haplotype tagging SNPs were selected from the International HapMap project database, based on criteria of r 2 more than 0.8 and minor allele frequency more than 0.05 on individual genotyping.…”

“…7 To complete the fine mapping within the region where rs2506011 belonged to, five captured SNPs of rs2506011 (rs2505540, rs2506021, rs2506020, rs2506010 and rs3123655) were picked. In addition, three SNPs from our previous study 5 (rs1800858, rs1800861, rs1800862) were included for re-validation.…”

“…5 Briefly, the 390 bp amplified fragment was cut using the BstZI enzyme (Promega, Fitchburg, WI, USA) according to the manufacturer's instructions. The digested products were analyzed on 3% agarose gel, in which individuals with GG (major allele homozygosity) showed two bands of 107 and 283 bp, those with GA (heterozygosity) showed three bands of 107, 283 and 390 bp, and those with AA (minor allele homozygosity) showed a single undigested band of 390 bp (not shown).…”

“…4 The incidence of HSCR in Thailand has never been studied, but it is believed to be more prevalent in the southern part of the country. 5 The etiology of HSCR is multifactorial, involving multiple genetic and environmental factors. 6 To date, at least 11 genes have been identified as susceptibility loci in HSCR: RET-protooncogene, GDNF, NTN, EDNRB, EDN3, SOX10, ECE-1, ZFHX1B, PHOX2B, KIAA1279 4,6 and the most recently found, NRG1, identified by a genome-wide association study (GWAS).…”