Germline mutations in FH confer predisposition to malignant pheochromocytomas and paragangliomas

Human Molecular Genetics, 2014, Vol. 23, No. 9 doi:10.1093/hmg/ddt639 Advance Access published on December 13, 2013 2440–2446 Germline mutations in FH confer predisposition to malignant pheochromocytomas and paragangliomas Luis Jaime Castro-Vega1,2,{, Alexandre Buffet1,2,3,{, Aguirre A. De Cubas4,{, Alberto Cascón4,5, Mélanie Menara1,2, Emmanuel Khalifa1,2,3, Laurence Amar1,2,6, Sharona Azriel7, Isabelle Bourdeau8, Olivier Chabre9, Maria Currás-Freixes4, Valérie Franco-Vidal10, Marine Guillaud-Bataille11, Christophe Simian3, Aurélie Morin1,2, Rocı́o Letón4,5, Álvaro Gómez-Graña4, Patrick J. Pollard12, Pierre Rustin13, Mercedes Robledo4,5,{, Judith Favier1,2,{ and Anne-Paule Gimenez-Roqueplo1,2,3,{, ∗ INSERM, UMR970, Paris-Cardiovascular Research Center, F-75015, Paris, France, 2Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, F-75006 Paris, France, 3Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Service de Génétique, F-75015 Paris, France, 4Hereditary Endocrine Cancer Group, Spanish National Cancer Centre (CNIO), Madrid, Spain, 5Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain, 6Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Unité d’Hypertension Artérielle, F-75015 Paris, France, 7Infanta Sofı́a Hospital, Endocrinology Service, San Sebastian de los Reyes, Madrid, Spain, 8Division of Endocrinology, Department of Medicine, Research Centre, Centre Hospitalier de l’Université de Montréal (CRCHUM), Montréal, Québec, Canada, 9Department of Endocrinology, University Hospital Albert Michallon, F-38043 Grenoble, France, 10Department of Otolaryngology and Skull Base Surgery, Pellegrin University Hospital, University Bordeaux Segalen, F-33000 Bordeaux, France, 11Service de Génétique, Institut de Cancérologie Gustave Roussy, 94805 Villejuif, France, 12Cancer Biology and Metabolism Group, Edinburgh Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK and 13 INSERM U676, Hôpital Robert Debré, F-75019 Paris, France Received October 28, 2013; Revised and Accepted December 10, 2013 Malignant pheochromocytoma (PCC) and paraganglioma (PGL) are mostly caused by germline mutations of SDHB, encoding a subunit of succinate dehydrogenase. Using whole-exome sequencing, we recently identified a mutation in the FH gene encoding fumarate hydratase, in a PCC with an ‘SDH-like’ molecular phenotype. Here, we investigated the role of FH in PCC/PGL predisposition, by screening for germline FH mutations in a large international cohort of patients. We screened 598 patients with PCC/PGL without mutations in known PCC/PGL susceptibility genes. We searched for FH germline mutations and large deletions, by direct sequencing and multiplex ligation-dependent probe amplification methods. Global alterations in DNA methylation and protein succination were assessed by immunohistochemical staining for 5-hydroxymethylcytosine (5-hmC) and S-(2succinyl) cysteine (2SC), respectively. We identified five pathogenic germline FH mutations (four missense and one splice mutation) in five patients. Somatic inactivation of the second allele, resulting in a loss of fumarate hydratase activity, was demonstrated in tumors with FH mutations. Low tumor levels of 5-hmC, resembling those in SDHB-deficient tumors, and positive 2SC staining were detected in tumors with FH mutations. Clinically, metastatic phenotype (P 5 0.007) and multiple tumors (P 5 0.02) were significantly more frequent in patients with FH ∗ To whom correspondence should be addressed at: Service de Génétique, Hôpital Européen Georges Pompidou, 20-40 rue Leblanc, 75015 Paris, France. Tel: +33 156093881; Fax: +33 156093884; Email: † These authors contributed equally to this work. ‡ These authors contributed equally to this work. # The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: 1 Human Molecular Genetics, 2014, Vol. 23, No. 9 2441 mutations than those without such mutations. This study reveals a new role for FH in susceptibility to malignant and/or multiple PCC/PGL. Remarkably, FH-deficient PCC/PGLs display the same pattern of epigenetic deregulation as SDHB-mutated malignant PCC/PGL. Therefore, we propose that mutation screening for FH should be included in PCC/PGL genetic testing, at least for tumors with malignant behavior. INTRODUCTION RESULTS A large international series of 598 patients, collected in France (n ¼ 359) and Spain (n ¼ 239), diagnosed with PCC/PGL without mutations in any of the 11 known PCC/PGL susceptibility genes were analyzed for germline mutations of FH (Table 1). This population consisted of 151 patients (25%) diagnosed with PCC/PGL before the age of 35 years, 85 patients (14%) with multiple PCC/PGL and 59 patients (10%) with metastatic PCC/PGL. Forty-one different heterozygous variants covering the entire length of the FH gene were identified (Fig. 1; Supplementary Material, Table S1). We detected 18 single nucleotide polymorphisms (SNPs): 12 with frequencies similar to those reported in the general population, and 6 for which no frequency value was available. We also identified 23 other variants: 14 (61%) were intronic, 6 (26%) were missense and 3 (13%) were synonymous. The presence of copy number variations was excluded by multiplex ligation-dependent probe amplification (MLPA). Five of the identified variants were classified as pathogenic, either because they had already been described as causal variants, or on the basis of in silico prediction (Supplementary Material, Table S2). None of these variants was found in public SNP databases. We previously described the patient carrying the missense mutation (c.349G.C; p.Ala117Pro) in exon 3 (10), which had already been identified as pathogenic by two independent groups in patients with a diagnosis of multiple cutaneous and uterine leiomyomatosis (MCUL) (15). A second variant affecting an essential splicing site (c.268-2A.G; p.?) had previously been found in a patient with FH deficiency and reported to be pathogenic in the TCA Cycle Gene Mutation Database (16). The remaining three FH missense variants identified in this study are previously unknown mutations, located within exon 8 (c.1142C.T; p.Thr381Ile), exon 7 (c.986A.G; p.Asn329Ser) and exon 5 (c.580G.A; p.Ala194Thr). The clinical and genetic features of individuals carrying FH mutations are presented in Table 2. Three of these patients had metastatic PCC/PGL and three developed multiple tumors. The first patient, described in a previous study (10), presented metastatic PCC at the age of 63 years and died at 73 years old. Subsequent analysis of her clinical record revealed that she had a history of hysterectomy at the age of 35 years for hemorrhagic fibromas highly suggestive of uterine leiomyoma. The second patient was diagnosed with left adrenal PCC at the a (...truncated)