1 J. Endocrinol. Invest. 36: ???-???, 2013 DOI: 10.3275/8997 ABSTRACT. Background: We previously identified a four- generation family with medullary thyroid cancer (MTC) and a germline p.Y791F RET mutation whose cancer lacked a strong genotype-phenotype correlation. The entire gene coding region of the RET gene should be sequenced when genotype-phenotype discrepancies are observed in patients with multiple endocrine neoplasia type 2 (MEN 2), even if a RET hotspot mutation has been identified. Methods: A new genetic test was performed in the index case of this family with the p.Y791F RET germline mutation. The entire cod- ing region of the RET gene was investigated by direct se- quencing of PCR products. Once a mutation was identified, the target exon was sequenced in all at-risk relatives. Re- sults: An additional p.C634Y germline mutation in the RET gene was identified in the reported family. The double mu- tation occurred in cis and segregated with the phenotype. Through the Brazilian Genetic Screening Program devel- oped at our institution, we additionally report the combi- nation of these two mutations (p.C634Y/p.Y791F) in the RET gene in four other unrelated families. The overall pen- etrance of MTC and pheochromocytoma in patients with the p.C634Y/p.Y791F mutations was 79% and 13%, re- spectively. Conclusion: Our data emphasises that a com- prehensive analysis of the RET gene may reveal multiple germline mutations in MEN 2 patients who exhibit an atyp- ical clinical course of the disease. (J. Endocrinol. Invest. 36: ??-??, 2013) © 2013, Editrice Kurtis INTRODUCTION Multiple endocrine neoplasia type 2 (MEN 2) is an auto- somal dominant-inherited tumour syndrome that can be classified into three clinical phenotypes (reviewed by San- toro et al.) (1). Germline mutations in the RET gene have been identified as the underlying basis of MEN 2 syn- drome. In MEN 2A and familial medullary thyroid carcino- ma (FMTC) families, most of the mutations occur in one of the six cysteine codons within exon 10 and exon 11. Non- cysteine mutations located within exons 5, 8, 10, 11, 13-15 have also been reported (2-5). Almost all individuals with MEN 2B exhibit p.M918T mutation in exon 16 (6). In ad- dition, mutations within exon 14 (p.V804M), in conjunction with a second RET mutation located within exons 13, 14 or 15, and mutations within exon 15 (p.A883F) have also been described in a few cases of MEN 2B (4). For the most prevalent mutations, a striking genotype- phenotype correlation has been observed. Therefore, based on genotype-phenotype correlation, the 7 th Inter- national Workshop on MEN published the first consensus that classified RET mutations into three risk levels (7). Sub- sequently, the American Thyroid Association (ATA) in- corporated the current medullary thyroid carcinoma (MTC) literature with evidence-based medicine to sum- marise the state-of-the-art knowledge into the new MTC management guidelines (4). Since patients with MEN 2A- associated mutations at codon 634 develop MTC at younger ages and exhibit higher rates of pheochromo- cytoma (PHEO) and primary hyperparathyroidism (PHPT), codon 634 mutations were categorised within a separate risk level, and all RET mutations were stratified into four risk levels (A to D). Although the ATA updated the classification system to incorporate all known mutations, the pathogenic roles of recently described rare RET mutations and variants of un- certain significance (VUS) remain less clear. Further, the presence of double mutations in the RET gene necessi- tates better classification and management. Within this field of double RET mutations, we recently faced a new dilemma related to the RET p.Y791F muta- tion. This mutation was initially identified in a patient with apparently sporadic MTC. Because no additional muta- tions were described in exons 10, 11 and 13-16 of RET gene, this RET p.Y791F mutation was considered a new hotspot for MEN 2 syndrome (8). Although this mutation was also associated with susceptibility to apparently spo- radic PHEO and Hirschsprung’s disease (9), the RET Key-words: MEN 2A, RET, p.C634Y, p.Y791F, medullary thyroid carcinoma. Correspondence: J. Cerutti, PhD, Genetic Basis of Thyroid Tumour Laboratory, Rua Pedro de Toledo 669, 11° andar, Universidade Federal de São Paulo, 04039-032, São Paulo, SP, Brazil. E-mail: j.cerutti@unifesp.br Accepted May 9, 2013. First published online May 30, 2013. Comprehensive analysis of RET gene should be performed in patients with multiple endocrine neoplasia type 2 (MEN 2) syndrome and noapparent genotype-phenotype correlation: An appraisal of p.Y791F and p.C634Y RET mutations in five unrelated Brazilian families F.O.F. Valente 1 , M.R. Dias da Silva 1 , C.P. Camacho 1 , I.S. Kunii 1 , A.U. Bastos 2 , C.C.N. da Fonseca 2 , H.P.C. Simião 1 , R. Tamanaha 2 , R.M.B. Maciel 1 , and J.M. Cerutti 1,2 1 Laboratory of Molecular and Translational Endocrinology, Division of Endocrinology, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP; 2 Genetic Basis of Thyroid Tumor Laboratory, Division of Genetics, Department of Morphology and Genetics, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil