In this article, we summarize our recent findings on rearranged during transfection (RE7) mutations in a series of 46 sporadic as well as multiple endocrine neoplasia (MEN) type 2- associated tumors and present results of our family screening efforts to identify MEN 2 and MEN 1 gene carriers. A nonisotopic polymerase chain reaction-based single-strand conformation polymorphism (PCR-SSCP) analysis and heteroduplex gel electrophoresis method was used to screen DNA extracted from archival specimens of 22 patients with MEN 2-associated and 24 patients with sporadic tumors for mutations in RETexons 1O, 11, 13, and 16. Point mutations were identified by nonisotopic cycle sequencing of PCR products using an automated DNA sequencer. We found six different missense germ line mutations at cysteine residues encoded by exons 10 and 11 in all patients with MEN 2A or familial medullary thyroid carcinoma (FMTC). The frequency of mutations at codon 634 was higher in patients with MEN 2A than with FMTC and a (63)Cys - Arg mutation was associated with parathyroid disease. A germline Met -* Thr point mutation at codon 918 of the RETtyrosine kinase domain encoded by exon 16 was identified in all MEN 2B patients. Nonpredicted inheritable medullary thyroid carcinomas (MTCs) were detected in two patients and a mosaic postzygotic mutation was found in one additional patient. Tumor-specific (somatic) Met - Thr point mutations at codon 918 were identified in 5 of 13 sporadic MTCs and 2 of 8 sporadic pheochromocytomas (PCCs). The remaining sporadic tumors lacked mutations in all four RET exons tested. In exon 13, a nucleic acid polymorphism (CTT/CTG; Leu) at codon 769 was identified, which is present in approx 40% of the examined population. Our study demonstrates that the molecular methods used are not only suitable to identify asymptomatic individuals at risk for MEN 2A, FMTC, and MEN 2B, but also to distinguish sporadic from inherited tumors using archival tissue specimens; and that more tumors than clinically expected are inheritable, indicating the need for genetic analysis of all MTC and PCC patients.

Background. The distinction of sporadic from inherited medullary thyroid carcinomas (MTCs) is of clinical importance because of the differences in prognosis, and the need for family screening for genetic counseling required in the latter. Germline mutations in the RET protooncogene are associated with multiple endocrine neoplasia (MEN) type 2A, familial medullary thyroid carcinoma (FMTC), and MEN type 2B. Somatic point mutations in the same gene have been identified in a subset of sporadically occurring medullary thyroid carcinomas. Methods. A nonisotopic polymerase chain reaction‐(PCR) based single strand conformation polymorphism (SSCP) analysis and heteroduplex gel electrophoresis method was used to screen DNA extracted from 32 form‐aldehyde fixed and paraffin embedded MTC specimens and normal tissue or blood of the same patient for point mutations in RET exons 10, 11, and 16. Point mutations were identified by nonisotopic cycle sequencing of PCR‐products using an automated DNA‐sequencer. Results were compared with the disease phenotype, clinical findings, and follow‐up. Results. Six different missense germline mutations were identified at cysteine residues 618, 630, and 634 of the cysteine‐rich extracellular RET domain encoded by exons 10 and 11 in all patients with FMTC and MEN 2A. The frequency of mutations at codon 634 was higher in patients with MEN 2A than with FMTC and a 634 Cys → Arg mutation was associated with parathyroid disease in three patients. A germline Met → Thr point mutation at codon 918 of the RET tyrosine kinase domain was identified in all three patients with MEN 2B. Two patients with clinically sporadic MTCs and negative family history exhibited a RET germline mutation at codon 634, indicating the presence of an nonpredicted inherited MTC. Further‐more, one patient had a 618 Cys → Ser mutation in the tumor and nontumorous thyroid DNA but not in blood DNA, indicating a mosaic mutation affecting thyroid tissue but not blood cells. Tumor specific (somatic) Met → Thr point mutations at codon 918 were identified in 5 of 13 sporadic MTCs. The remaining eight sporadic MTCs lacked mutations in all three RET exons tested. Conclusions. This study demonstrates that (1) the molecular methods are not only suitable to identify asymptomatic individuals at risk for MEN 2A, FMTC, and MEN 2B but also to distinguish heritable from non‐heritable MTCs using archival tissue specimens, and (2) that more MTCs than clinically expected are heritable, indicating the need for genetic analysis of all patients with MTC. Cancer 1995; 76:479–89.