CASE REPORT Jonathan Irish, MD, FRCSC, Section Editor Novel tandem germline RET proto-oncogene mutations in a patient with multiple endocrine neoplasia type 2B: Report of a case and a literature review of tandem RET mutations with in silico analysis Kanako–Tanase Nakao, MD, 1 Takeshi Usui, MD, PhD, 2 * Mayumi Ikeda, MD, 1 Yusuke Mori, MD, 3 Tetsuro Yamamoto, MD, 4 Sachiko–Tsukamoto Kawashima, MD, 1 Kazutaka Nanba, MD, 1 Akiko Yuno, MD, 1,5 Tamiko Tamanaha, MD, 1 Tetsuya Tagami, MD, PhD, 1 Mitsuhide Naruse, MD, PhD, 2 Ryo Asato, MD, PhD, 3 Akira Shimatsu, MD, PhD 2 1 Department of Endocrinology and Metabolism, National Hospital Organization, Kyoto Medical Center, Kyoto, Japan, 2 Clinical Research Institute, National Hospital Organization, Kyoto Medical Center, Kyoto, Japan, 3 Department of Head and Neck Surgery, National Hospital Organization, Kyoto Medical Center, Kyoto, Japan, 4 Department of Surgical Pathology, National Hospital Organization, Kyoto Medical Center, Kyoto, Japan, 5 Department of Endocrinology and Metabolism, Kin-i-kyo Chuo Hospital, Sapporo, Japan. Accepted 6 December 2012 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.23241 ABSTRACT: Background. Multiple endocrine neoplasia type 2B (MEN2B) is the rarest and most aggressive form of MEN2. MEN2B cases usually carry either an M918T or A883T mutation of the RET, but to date, there are 3 atypical MEN2B caused by tandem mutations. Methods and Results. A 32-year-old woman with no family history of medullary thyroid carcinoma (MTC) presented with a neck tumor and multiple mucosal nodules. She was diagnosed with MEN2B. Genetic analyses of RET revealed that she had 2 mutations, Q781R and V804M. Subclone and genetic analyses revealed that Q781R was on the paternal allele and V804M was a de novo. In silico analysis of the tandem mutations showed a high prediction score. Conclusions. We describe a novel combination of tandem RET mutations (Q781R/V804M) in a MEN2B-like patient. In silico analysis showed a high prediction score, which was compatible with the clinical phenotype in the present case. V C 2012 Wiley Periodicals, Inc. Head Neck 00: 000–000, 2012 KEY WORDS: RET, MEN2B, tandem mutation, de novo, in silico INTRODUCTION Multiple endocrine neoplasia type 2 (MEN2) is an auto- somal dominant inherited neoplasia syndrome caused by activating mutations in the RET proto-oncogene. 1,2 MEN2 is classified into 3 subtypes: MEN2A, MEN2B, and fami- lial medullary thyroid carcinoma (FMTC). MEN2A, which accounts for 90% to 95% of MEN2 cases, is characterized by the development of medullary thyroid carcinoma (MTC) in more than 90% of RET mutation carriers; depending on the mutation, pheochromocytoma, and primary hyperpara- thyroidism also develop in 0% to 50% and 0% to 20%, respectively, of individuals with MEN2A. 3 FMTC is a clin- ical variant of MEN2A in which MTC is the only manifes- tation. 4 In contrast, MEN2B accounts for 5% to 10% of MEN2 cases 3 and is characterized by MTC (100% of cases), pheochromocytoma (up to 50% of cases), and spe- cific phenotypic features such as mucosal neuromas of the lips, tongue, and conjunctiva (‘mucosal neuromas’), enlarged lips, medullated corneal-nerve fibers, ganglioneur- omatosis of the gastrointestinal tract, and an asthenic ‘Mar- fanoid’ body habitus. 4 More than 95% of patients with MEN2B carry an M918T mutation of RET, and 2% to 3% of patients har- bor an A883F mutation. The frequency of de novo gene mutations is approximately 50% in patients with MEN2B, much higher than the 2% to 9% in MEN2A cases and this, rather than tumor behavior, is considered to be a rea- son for the delayed diagnosis and higher mortality of patients with MEN2B. 4 Although a single mutation of RET, such as M918T or A883T, is enough to cause MEN2 in the majority of cases, double mutations of RET have been reported to cause MEN2 in a limited number of cases. 5–13 Herein, we describe a patient with MEN2B who carries a novel com- bination of double RET germline mutations, V804M and Q781R, on the same allele. To date, 5 other MEN2 cases with RET 804-associated double mutations have been reported. 5–9 To understand the significance of the tandem mutation, we review such cases and perform in silico analysis using the Align-Grantham Variation-Grantham Deviation (Align-GVGD) web-based program, which is proposed to be a valid alternative to in vitro analysis for determining the transforming ability of the rare mutated RET gene. 14 CASE REPORT A 32-year-old woman presented with a right anterior neck mass. She had bumpy lips and multiple nodules on *Corresponding author: T. Usui, Clinical Research Institute, National Hospital Organization, Kyoto Medical Center, Mukaihata-cho Fukakusa, Fushimi-ku, Kyoto 612-8555, Japan. E-mail: tusui@kuhp.kyoto-u.ac.jp Contract grant sponsor: This work is part of the Ministry of Education, Culture, Sports, Science, and Technology KAKENHI 21500816 and a grant from the Smoking Research Foundation. HEAD & NECK—DOI 10.1002/HED MONTH 2012 1