A founding locus within the RET proto-oncogene may account for a large proportion of apparently sporadic Hirschsprung disease and a subset of cases of sporadic medullary thyroid carcinoma

Abstract

Hirschsprung disease (HSCR) is a common congenital disorder characterized by aganglionosis of the gut. The seemingly unrelated multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant disorder characterized by medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism. Yet, germline mutations in the RET proto-oncogene are associated with both MEN 2 and HSCR. In the former, gain-of-function mutations in a limited set of codons is found, whereas, in the latter, loss-of-function mutations are found. However, germline RET mutation is associated with only 3% of a population-based series of isolated HSCR, and little is known about susceptibility to sporadic MTC. We have found previously that specific haplotypes comprising RET coding single-nucleotide polymorphisms (SNPs) comprising exon 2 SNP A45A were strongly associated with HSCR, whereas haplotypes associated with exon 14 SNP S836S were associated with MTC. In this study, we describe three novel intron 1 SNPs, and, together with the coding SNP haplotypes, the data suggest the presence of distinct ancestral haplotypes for HSCR and sporadic MTC in linkage disequilibrium with a putative founding susceptibility locus/loci. The data are consistent with the presence of a very ancient, low-penetrance founder locus approximately 20-30 kb upstream of SNP A45A, but the failure of the SNPs to span the locus presents challenges in modeling mode of transmission or ancestry. We postulate that this founding locus is germane to both isolated HSCR and MTC but also that different mutations in this locus would predispose to one or the other.

Figure 1

Characterization of novel SNPs in RET intron 1. A, Schematic representation on 3′ end of intron 1 of the RET proto-oncogene. Codon 45 and the 3 IVS1 SNPs are indicated. B, left panel, genotyping of IVS1-126G→T by fluorescent SSCP. Lanes 1, 2, 5, 6, and 11–14, genotype −/−. Lanes 3, 4, and 7–10, genotype +/−. Lanes 15 and 16, genotype +/+. Right panel, genotyping of IVS1-126G→T by differential restriction with NlaIII. Lane 2, a nondigested sample. Lane 3, genotype −/−. Lanes 4 and 5, genotype +/−. Lane 6, genotype +/+. C, FRET analysis of IVS1-1370C→T and IVS1-1463T→C. In each case, amplicons containing the polymorphisms have a melting point (MP) which is >9°C lower (in the case of IVS1-1370C→T) and 3°C higher (in the case of IVS1-1463T→C) than the amplicons with the wild-type sequence. Genotype −/− , +/−, and +/+ are represented in red, blue, and green, respectively.

Figure 2

Excess proportion of ancestral haplotypes among transmitted chromosomes to HSCR patients (see text). The fitted line of expected linkage disequilibrium suggests that the ancestral mutation lies in or near the 5′ region of RET—that is, within intron 1. A schematic of partial RET gene structure on which the positions of the 10 SNPs are placed lies below the plot. Numbers on top of the gene schematic are exon numbers, and the codes below represent the nature and position of the SNPs.

Figure 3

Comparison of the frequencies and distribution of RET IVS1 haplotypes and genotypes between HSCR or patients with MTC and control subjects. χ² (Yates's correction) and P values are denoted below each comparison.