Elsevier

The Lancet

Volume 359, Issue 9313, 6 April 2002, Pages 1200-1205
The Lancet

Mechanisms of Disease
Association between c135G/A genotype and RET proto-oncogene germline mutations and phenotype of Hirschsprung's disease

https://doi.org/10.1016/S0140-6736(02)08218-1Get rights and content

Summary

Background

Several genes, including the major susceptibility gene RET, have roles in development of Hirschsprung's disease. Results of genetic-linkage analysis of patients with familial disease with both long-segment and short-segment phenotypes have shown close linkage with the RET locus. We aimed to investigate whether both RET mutations and polymorphisms contribute to phenotype of Hirschsprung's disease.

Methods

We looked at the coding region of all 21 exons of the RET proto-oncogene, including the flanking intronic sequences, by direct DNA sequencing in 76 caucasians from Germany with Hirschsprung's disease.

Findings

20 different mutations were detected in 18 patients. Mutations were under-represented in patients with a homozygous RET c135A/A genotype in association with short-segment phenotype. Short-segment phenotype also arose if the RET mutation was on the c135A allele; conversely, a RET germline mutation on the c135G allele resulted in long-segment phenotype, particularly in heterozygous c135G/A patients.

Interpretation

These observations lend support to the idea that both RET alleles have a role in pathogenesis of Hirschsprung's disease, in a dose-dependent fashion. We also showed that the c135G/A polymorphism modifies the phenotype by a within-gene interaction between the c135A variant and a mutation.

Introduction

Hirschsprung's disease is a dysgenetic neurocristopathy1 that is characterised histopathologically by congenital absence of the intramural ganglia of the plexus myentericus and submucosus in the hindgut. This disorder arises in one in 5000 livebirths, predominantly in males.2 Functional intestinal stenosis develops in Hirschsprung's disease, with consecutive dilatation and hypertrophy of the upper intestine attributable to an aganglionic colonic segment of variable lengths. Complex familial segregation data for people with Hirschsprung's disease showed that, in cases of aganglionosis beyond the sigmoid colon, the mode of inheritance was most probably autosomal dominant with incomplete penetrance.2 In all other cases, the pattern of inheritance was probably either multifactorial or caused by a recessive gene with very low penetrance, without predominance of either one.2

Molecular-genetic analysis has identified several genes that have a role in development of Hirschsprung's disease; the major susceptibility gene for this disorder is the RET proto-oncogene. This gene, which encodes a receptor tyrosine kinase, is expressed in tissues derived from the neural crest and neoplasms.

Missense germline mutations affecting hot spots in exons 10, 11, and 13–16 of the RET proto-oncogene have been reported in autosomal dominant inherited multiple endocrine neoplasia type 2 syndromes. These mutations result in ligand-independent gain in function of the RET-signalling pathway.3, 4, 5 By contrast, RET mutations seen in people with Hirschsprung's disease result in either truncation of RET protein or possible functional inactivation of the molecule. Although loss of one allele in some patients with Hirschsprung's disease suggests haploinsufficiency,6 retention of one wild-type allele in those with a possible inactivating RET mutation seems to suggest dominant negative action of the mutated RET allele.7

Genes coding for functional ligands of the RET-receptor complex, such as the glial-cell-line-derived neurotrophic factor (GDNF), neurturin (NTN), artemin (ARTN), persephin (PSPN), and corresponding members of the GDNF-family receptor α genes (GFRα-1–4), have also been suggested to be putative susceptibility genes associated with Hirschsprung's disease. Rare heterozygous germline mutations of GDNF and NTN have been detected in patients with this disorder, often in combination with RET mutations, lending support to the idea of synergistic heterozygosity.8, 9, 10, 11, 12

Patients with Shah-Waardenburg syndrome with megacolon have a homozygous founder mutation in the G-protein-coupled endothelin B receptor gene (EDNRB),13 whereas heterozygous mutations of EDNRB and EDN3 have been identified in individuals with isolated Hirschsprung's disease.14 Heterozygous mutations of SOX10 have been described in patients with megacolon in Shah-Waardenburg syndrome.15 Furthermore, SIP1 mutations have been reported in patients with megacolon in combination with mental retardation and facial dysmorphy.16

Nevertheless, genetic-linkage analysis of people with familial Hirschsprung's disease with both long-segment and short-segment phenotype has shown tight linkage and association with the RET proto-oncogene locus.17, 18 Seri and colleagues19 recorded RET germline mutations in 75% of patients with a long aganglionic segment, and Attie and co-workers20 recorded mutations in up to 50% of people with familial Hirschsprung's disease. Svensson and colleagues21 did a population-based study and showed a low frequency of RET proto-oncogene mutations (7·2%) in Swedish patients with this disorder.

Simultaneous and independent findings by us and others have shown a strong association between sporadic phenotype of Hirschsprung's disease and specific alleles of c135G/A and c2307T/G polymorphisms in the RET proto-oncogene. The c135A and c2307G alleles represent RET variants that potentially cause or modify this disorder with a low penetrance.22, 23 Moreover, Borrego and colleagues24 suggest that genotypes comprising specific pairs of RET haplotypes predispose to Hirschsprung's disease.

We postulated that in people with Hirschsprung's disease, both RET mutations and polymorphisms contribute to phenotype. Thus, we aimed to investigate the coding region of all 21 exons of the RET proto-oncogene, including the flanking intronic sequences. To investigate our hypothesis, we adhered to the following hierarchical procedure. First, we compared the number of mutations in patients with Hirschsprung's disease with long-segment phenotype with the number of mutations in those with short-segment phenotype. Second, we investigated c135G/A variants in these groups, since c135A RET variant has shown the strongest association with disease phenotype compared with other characterised RET variants (76·3% c135G alleles in 156 controls vs 26·6% in 62 patients).23 Third, we established haplotypes comprising the c135 RET locus and identified RET mutations, thus allowing analysis of the association between disease phenotype and c135G/A polymorphism on chromosomes that also carry a RET germline mutation.

Section snippets

Patients

We studied 76 caucasians with Hirschsprung's disease from Germany. Men were 3·75 times more likely to be affected than women. Only five (6·6%) patients had a family history for the disease. In addition to this disease, two patients had Down's syndrome and another had concomitant Fallot's tetralogy. Patients with a RET germline mutation in one of the codons associated with multiple endocrine neoplasia type 2 syndromes underwent prophylactic total thyroidectomy.

All patients fulfilled histological

Results

Sequence analysis of the RET proto-oncogene in 76 patients with Hirschsprung's disease revealed 20 mutations in 18 patients (24%), including one nonsense (5%), 13 missense (65%), two splice-site (10%), and four silent (20%; panel 2). Although five mutations (in patients 2, 4, 5, 9, and 12) have been reported, 19, 25, 26, 28, 29, 30, 31 none of the remaining 15 has been detected in the 50 randomly selected healthy individuals. Two patients had two mutations each—one missense and one silent

Discussion

Hirschsprung's disease has been shown to be associated with multiple genes, suggesting genetic heterogeneity. Mutations in any one of several genes are sufficient for genotype expression of this disorder. Furthermore, Hirschsprung's disease is a complex multigenetic disorder, in which the cumulative effect of mutations in multiple genes contributes to phenotype in one individual.18 Although molecular-genetic analysis has identified several genes involved in the cause of Hirschsprung's disease,

GLOSSARY

dysgenetic neurocristopathy
Congenital malformation resulting from derangement of neural-crest migration, colonisation, or cytodifferentiation.
haploinsufficiency
Arises when the normal phenotype requires the protein product of both alleles, and reduction of 50% of gene function results in an abnormal phenotype.
haplotype
The particular combination of alleles seen at linked loci of some chromosome.
linkage disequilibrium
The situation in which alleles of two loci on a random chromosome do not arise

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