Interaction of RET proto‐oncogene codon 609 germline mutations with RET haplotypes characterized by c.135G>A alleles modifying MEN 2A or HSCR phenotypes

Germline mutations of the RET proto-oncogene have been identified in patients with the inherited neoplastic syndrome ‘‘multiple endocrine neoplasia type 2’’ (MEN 2, [MIM no. 171400]), which comprises themedullary thyroid carcinoma as an obligatory feature, and with Hirschsprung disease (HSCR [MIM no. 142623]), a developmental disorder of the enteric nervous system characterized by the absence of intramural ganglia in the hindgut. Highly penetrant germline missense mutations of RET proto-oncogene have been detected in each one of six cysteine codons within exon 10 (codons 609, 611, 618, and 620) and exon 11 (codons 630 and 634) in about 95% of MEN 2A and in up to 85% of familial medullary thyroid carcinoma (FMTC) families [Donis-Keller et al., 1993; Mulligan et al., 1993; Eng andMulligan, 1997]. In contrast, between 10 and 40% of the patients with HSCR harbor germline mutations of the RET proto-oncogene, which are primarily point mutations scattered throughout the extracellular domain and within the intracellular tyrosine kinase domain of RET [Edery et al., 1994; Romeo et al., 1994; Angrist et al., 1995; Attie et al., 1995; Seri et al., 1997]. The phenotypic association of MEN 2A/FMTC and HSCR was previously reported in several kindreds. With one exception, this combined phenotype segregates in the involved families with a single RET haplotype harboring a cysteine germline mutation of the codons 609, 618, or 620 [Mulligan et al., 1994]. Since all of these RET germline mutations are described in various families in association withMEN 2A/FMTC alone, and assuming a genetically determined HSCR phenotype, additional genetic alterations resulting in a combination of the MEN 2 and HSCR phenotype can be hypothesized. The present study describes two unrelated kindreds with codon 609 RET mutations associated with different phenotypes and different alleles at an intragenic single nucleotide polymorphic site in exon 2. We analyzed the RET proto-oncogene in two families both harboring a codon 609 RET germline mutation. The only affected individual of the first family (Fig. 1a) has a de novo mutation in association with HSCR, without any of the expected MEN2 features. In the second family, the mutation segregates with the more characteristic MEN2A phenotype (Fig. 1b). Genomic DNA was extracted from leukocytes from peripheral venous blood samples by standard protocols. The amplification and sequencing of all 21 RET exons was performed using primers and reaction conditions described previously [Ceccherini et al., 1994;Mulligan et al., 1994;Fitze et al., 2002]. Furthermore, we determined the genotypes of seven polymorphisms of the coding region of the RET proto-oncogene in all family members [Ceccherini et al., 1994; Fitze et al., 1999]. Direct DNA sequencing of all 21 coding exons of the RET proto-oncogene, including the adjacent intronic boundaries, detected a heterozygous germline mutation of codon 609 (TGC! AGC) in the HSCR affected patient (II-1, Fig. 1a) of the first family. This mutation results in the substitution of a cysteine amino acid residue by serine. In this patient, an additional germline mutation was not found and all clinically unaffected family members were excluded as gene carriers. In the index case, the homozygous c.135A genotype, the heterozygous c.2307T/G genotype, and for all others (c.375C, c.1296G, c.2071G, c.2508C, c.2712C) the homozygous wildtype genotypes were found in the analysis of the coding region polymorphisms. Hence, the C609S mutation was located on a c.135A allele. The genotypes in this family are shown in Figure 1a. In contrast, the index case of the second family (II-2, Fig. 1b) also harbored a heterozygous germline mutation of the codon 609 (TGC! GGC), resulting in a substitution of cysteine by glycine. In this family theC609Gmutation segregatedwith the MEN 2A phenotype. Six of seven identified gene carriers underwent total thyroidectomy, and five were diagnosed with MTC. Only a 5-year-old boy (IV-4) showed a normal thyroid gland histology after prophylactic thyroidectomy. Two of the gene carriers (II-2, II-4) also had a pheochromocytoma. Moreover, the polymorphisms, particularly c.135G>A and c.2307T/G, showed the homozygous wild-type genotype (c.135G, c.375C, c.2071G, c.2307T, c.2508C, c.2712C), with exception of c.1296G>A polymorphism, which was heterozygous in five of seven C609G gene carriers (Fig. 1b). Hence, the C609G mutation was located on a c.135G allele. Whereas the family harboring the C609G (TGC! GGC) mutation showed a typical MEN 2A syndrome without clinical signs ofHSCRor chronic constipation over three generations, a novel de novo C609S (TGC! AGC)mutation in one individual was associated with isolated short-segment HSCR. A prophylactic thyroidectomy at age 30 revealed neitherMTCnor C-cell hyperplasia. Isolated HSCR in association with a common MEN 2 cysteine codon mutation involving either codon 609 (three cases) or codon620 (one case) is relatively rare [Mulligan etal. , 1994;Angrist et al., 1995;Hofstra, 1998]. Yet, coexistence of MEN 2 and HSCR phenotypes segregating with a specific RET allele harboring RET mutations of codons 609, 618, and 620 have been shown in 21 reported families [Chretien et al., 1994;Mulligan et al., 1994;Borst et al., 1995;Blanket al., 1996; Caron et al., 1996; Peretz et al., 1997; Seri et al., 1997; Borrego et al., 1998; Decker et al., 1998; Fitze et al., 2002; Pasini et al., 2002]. Because of the contrasting character of RETmutations in theMEN2 or theHSCRphenotype (gain of function inMEN 2-associated RET mutations versus loss-of-function in HSCRassociated RET mutations), it may be assumed that the combined HSCR/MEN 2 phenotype derives from two different RET germline mutations. However, second RET mutations *Correspondence to: Dr. Guido Fitze, M.D., Department of Pediatric Surgery, University of Technology Dresden, Fetscherstrasse 74, D-01307 Dresden, Germany. E-mail: Guido.Fitze@mailbox.tu-dresden.de