Mechanisms of DiseaseAssociation between c135G/A genotype and RET proto-oncogene germline mutations and phenotype of Hirschsprung's disease
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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Cited by (95)
Clinical and genetic correlations of familial Hirschsprung's disease
2015, Journal of Pediatric SurgeryCitation Excerpt :Of the possible significant areas of the gene, the RET intronic variations [SNP1 (rs2506004) or SNP2 (rs 2435357)] would appear to be important. One previous report indicated RET haplotypes in 18 out of 76 patients [30] and the presence of the intron 1 polymorphism (rs2435357), was present in 79% of all patients with HSCR, suggesting an increased risk HSCR in homozygotes for this haplotype (Odds ratio > 20) [28,31]. Because this RET variant occurs more frequently (60%) in patients without any RET coding variant than the 14% with a demonstrable gene variant it has been suggested to have a relationship to isolated S-HSCR in males [24].
Genetic mosaicism of a frameshift mutation in the RET gene in a family with Hirschsprung disease
2014, GeneCitation Excerpt :The polymorphisms are found in the coding region (Fitze et al., 1999), in introns or the 3′ untranslated region (Fitze et al., 2003a) as well as the promoter (Emison et al., 2010; Fitze et al., 2003b). In some cases it could be demonstrated that the phenotype of HSCR can be modified by the interaction between a certain polymorphism with a mutation and that this interaction is allele-specific (Fitze et al., 2002). The RET protein is a transmembrane tyrosine kinase that associates with GFRs to form complete receptors for ligands like GDNF (glial cell derived neurotrophic factor) and other GDNF-family ligands (GFLs).
Distribution of RET proto-oncogene variants in children with appendicitis
2022, Molecular Genetics and Genomic MedicineThe Emerging Genetic Landscape of Hirschsprung Disease and Its Potential Clinical Applications
2021, Frontiers in PediatricsNeonatal thrombotic microangiopathy secondary to factor I variant with Hirschsprung disease
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