Diagnosis of multiple endocrine neoplasia [MEN] 2A, 2B and familial medullary thyroid cancer [FMTC] by multiplex PCR and heteroduplex analyses of RET proto-oncogene mutations.

Multiple endocrine neoplasia type 2 [MEN 2] is an autosomal dominant cancer syndrome with two subtypes, 2A and 2B. MEN 2A and medullary thyroid cancer [MTC] are caused by > 25 different point mutations in exons 10, 11, and 13 of the RET proto-oncogene, whereas MEN 2B is caused by a single exon 16-point mutation. Various molecular methods have been used to identify the different mutations, including DNA sequencing, restriction enzymatic analyses, chemical cleavage mismatch, Single Stranded Conformational Polymorphism [SSCP], and Denaturing Gradient Gel Electrophoresis [DGGE]. These techniques, although useful and accurate, are labor intensive and some involve the use of radioactivity. We have developed a multiplex PCR assay simultaneously to amplify exons 10, 11, and 13 of the RET proto-oncogene. The multiplex PCR product is then analyzed on a modified Mutation Detection Enhancement [MDE] matrix for heteroduplex identification and visualized with ethidium bromide. Distinct heteroduplexes were detected for each known RET proto-oncogene mutation available in our laboratory (nine in exon 10, five in exon 11, one in exon 13, and the single exon 16 mutation). Presymptomatic DNA diagnosis of MEN 2 is essential since pentagastrin-stimulated calcitonin studies can occasionally produce false positive results and lead to unnecessary thyroidectomies. Prophylactic thyroidectomy is recommended by age 5 or 6 once a mutation is identified in a patient, since penetrance is very high. MDE heteroduplex detection provides a quick, efficient, and inexpensive method of screening for RET mutations in MTC patients with unknown mutations, or for presymptomatic diagnosis in individuals at risk for inheriting a known RET mutation. Confirmation of the specific mutation can be achieved by restriction enzymatic digestion (if feasible) or by DNA sequencing.