DIAGNOSIS OF MULTIPLE ENDOCRINE NEOPLASIA [MEN] 2A, 2B AND FAMILIAL MEDULLARY-THYROID CANCER [FMTC] BY MULTIPLEX PCR AND HETERODUPLEX ANALYSES OF RET PROTOONCOGENE MUTATIONS

Multiple endocrine neoplasia type 2 [MEN 2] is an autosomal dominant c ancer syndrome with two subtypes, 2A and 2B. MEN 2A and medullary thyr oid 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 u sed to identify the different mutations, including DNA sequencing, res triction enzymatic analyses, chemical cleavage mismatch, Single Strand ed Conformational Polymorphism [SSCP], and Denaturing Gradient Gel Ele ctrophoresis [DGGE]. These techniques, although useful and accurate, a re 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. Dis tinct heteroduplexes were detected for each known RET proto-oncogene m utation 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 calciton in studies can occasionally produce false positive results and lead to unnecessary thyroidectomies. Prophylactic thyroidectomy is recommende d by age 5 or 6 once a mutation is identified in a patient, since pene trance is very high. MDE heteroduplex detection provides a quick, effi cient, and inexpensive method of screening for RET mutations in MTC pa tients with unknown mutations, or for presymptomatic diagnosis in indi viduals at risk for inheriting a known RET mutation. Confirmation of t he specific mutation can be achieved by restriction enzymatic digestio n (if feasible) or by DNA sequencing. (C) 1996 Wiley-Liss, Inc.