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Germline DLST Variants Promote Epigenetic Modifications in Pheochromocytoma-Paraganglioma

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Germline DLST Variants Promote Epigenetic Modifications in Pheochromocytoma-Paraganglioma Germline DLST Variants Promote Epigenetic Modifications in Pheochromocytoma-Paraganglioma Alexandre Buffet, Juan Zhang, Heggert Rebel, Eleonora Corssmit, Jeroen Jansen, Erik Hensen, Judith Bovée, Aurélien Morini, Anne-Paule Gimenez-Roqueplo, Frederik Hes, et al. To cite this version: Alexandre Buffet, Juan Zhang, Heggert Rebel, Eleonora Corssmit, Jeroen Jansen, et al.. Germline DLST Variants Promote Epigenetic Modifications in Pheochromocytoma-Paraganglioma. The Journal of Clinical Endocrinology & Metabolism, 2020, 10.1210/clinem/dgaa819. hal-03111589 HAL Id: hal-03111589 https://hal.archives-ouvertes.fr/hal-03111589 Submitted on 25 Jan 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. The Journal of Clinical Endocrinology & Metabolism, 2020, Vol. XX, No. XX, 1–13 doi:10.1210/clinem/dgaa819 Clinical Research Article Downloaded from https://academic.oup.com/jcem/advance-article/doi/10.1210/clinem/dgaa819/5979643 by Centre de Doc Medico Pharmaceutique user on 08 January 2021 Clinical Research Article Germline DLST Variants Promote Epigenetic Modifications in Pheochromocytoma-Paraganglioma Alexandre Buffet,*1,2 Juan Zhang,*3 Heggert Rebel,3 Eleonora P. M. Corssmit,4 Jeroen C. Jansen,5 Erik F. Hensen,5 Judith V. M. G. Bovée,6 Aurélien Morini,7 Anne-Paule Gimenez-Roqueplo,1,2 Frederik J. Hes,8 Peter Devilee,3,6 Judith Favier,**2 and Jean-Pierre Bayley**3 1Université de Paris, PARCC, INSERM, Equipe Labellisée par la Ligue contre le Cancer, F-75015 Paris, France; 2Genetic department, Adrenal Referral Center, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Européen Georges Pompidou, F-75015 Paris, France; 3Department of Human Genetics, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands; 4Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands; 5Department of Otorhinolaryngology, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands; 6Department of Pathology, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands; 7Assistance Publique-Hôpitaux de Paris (AP- HP), Hôpital Européen Georges Pompidou, Département d’anatomo-pathologie, F-75015 Paris, France; and 8Department of Clinical Genetics, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands ORCiD numbers: 0000-0002-4742-2708 (A. Buffet); 0000-0001-9836-9137 (E. P. M. Corssmit); 0000-0002-4393-7421 (E. F. Hensen); 0000-0003-1155-0481 (J. V. M. G. Bovée); 0000-0002-4816-670X (A.-P. Gimenez-Roqueplo); 0000-0002-8023-2009 (P. Devilee); 0000-0001-8190-5853 (J. Favier); 0000-0002-8288-0050 (J.-P. Bayley). *Joint first authors **Joint senior authors Abbreviations: 5hmC, 5-hydroxymethylcytosine; 5mC, 5-methyl cytosine;A450, absorbance at 450 nm;DLST, dihydrolipoamide S-succinyltransferase;DMEM, Dulbecco’s Modified Eagle Medium; ELISA, enzyme-linked immunosorbent assay; FFPE, formalin-fixed, paraffin-embedded;HIF, hypoxia-inducible factor;NADH, nicotinamide adenine dinucleotide;OGDH, oxoglutarate dehydrogenase enzyme complex;PCC, pheochromocytoma;PCR, polymerase chain reaction;PGL, paraganglioma;PPGL, pheochromocytoma/paraganglioma;TCA, tricarboxylic acid;TET, ten-eleven-translocation;WT, wild-type. Received: 25 March 2020; Editorial Decision: 31 October 2020; First Published Online: 12 November 2020; Corrected and Typeset: 15 December 2020. Abstract Context Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors in which altered central metabolism appears to be a major driver of tumorigenesis, and many PPGL genes encode proteins involved in the tricarboxylic acid (TCA) cycle. Objective/design While about 40% of PPGL cases carry a variant in a known gene, many cases remain unexplained. In patients with unexplained PPGL showing clear evidence of a familial burden or multiple tumors, we aimed to identify causative factors using genetic analysis ISSN Print 0021-972X ISSN Online 1945-7197 Printed in USA © The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: [email protected] https://academic.oup.com/jcem 1 The Journal of Clinical Endocrinology & Metabolism, 2020, Vol. XX, No. XX 2 of patient DNA and functional analyses of identified DNA variants in patient tumor material and engineered cell lines. Patients and Setting Downloaded from https://academic.oup.com/jcem/advance-article/doi/10.1210/clinem/dgaa819/5979643 by Centre de Doc Medico Pharmaceutique user on 08 January 2021 Patients with a likely familial cancer burden of pheochromocytomas and/or paragangliomas and under investigation in a clinical genetic and clinical research setting in university hospitals. Results While investigating unexplained PPGL cases, we identified a novel variant, c.1151C>T, p.(Pro384Leu), in exon 14 of the gene encoding dihydrolipoamide S-succinyltransferase (DLST), a component of the multi-enzyme complex 2-oxoglutarate dehydrogenase. Targeted sequence analysis of further unexplained cases identified a patient carrying a tumor with compound heterozygous variants in DLST, consisting of a germline variant, c.1121G>A, p.(Gly374Glu), together with a somatic missense variant identified in tumor DNA, c.1147A>G, p.(Thr383Ala), both located in exon 14. Using a range of in silico and functional assays we show that these variants are predicted to be pathogenic, profoundly impact enzyme activity, and result in DNA hypermethylation. Conclusions The identification and functional analysis of theseDLST variants further validates DLST as an additional PPGL gene involved in the TCA cycle. Key Words: Paraganglioma, pheochromocytoma, dihydrolipoamide S-succinyltransferase, DLST, 2-oxoglutarate de- hydrogenase, methylation Introduction shuttle (SLC25A11, GOT2), an endosomal signaling Paragangliomas (PGLs) are neuroendocrine tumors that factor (TMEM127), and a transcription factor (MAX). may arise in either the sympathetic or parasympathetic Nonetheless, most PPGLs can be attributed to variants in ganglia. Parasympathetic PGLs occur most commonly in genes involved in metabolism, currently including SDHA, the head and neck region (carotid body, jugular paraganglia, SDHB, SDHC, SDHD, SDHAF2, FH, MDH2, IDH1, and tympanic paraganglia), are generally benign and are IDH2, GOT2, and SLC25A11. rarely associated with the secretion of catecholamines. Although the mechanisms by which mutations in meta- PGLs arising from the sympathetic ganglia are mostly bolic enzymes promote tumor formation are not yet fully found in the abdomen and thorax, are frequently detected understood, inactivation of SDH, FH, and IDH1/2 leads due to endocrine-related hypertension associated with ex- to the accumulation of so-called “oncometabolites,” inter- cess secretion of catecholamines and are associated with mediary metabolites such as succinate, fumarate, and a higher risk of malignancy. Paragangliomas that arise in 2-hydroxyglutarate that are known to inhibit dioxygenases the chromaffin cells of the adrenal medulla, commonly re- (4-6), a large group of enzymes of diverse function. ferred to as pheochromocytomas (PCCs), are usually be- Perhaps the most important of these are the hypoxia- nign and are also frequently associated with hypertension inducible factor (HIF) prolyl hydroxylases, which when due to excessive catecholamine secretion (1). Collectively, disturbed cause HIF activation under normoxia (5), and these tumors are often referred to as pheochromocytomas other dioxygenases including histone demethylases and the and paragangliomas (PPGLs). TET (ten-eleven translocation) family of 5-methylcytosine PPGLs are associated with a heterogeneous collection of (5mC) hydroxylases (7-9) that result in altered patterns of both germline and somatic variants found in up to 19 genes histone methylation or hypermethylation known as a CpG- to date, although about half of all candidate PGL genes still island methylator-phenotype (CIMP) (10). have ill-defined pathogenicity (2, 3). PGL-associated genes In addition, PPGL tumors can be classified into 2 major belong to a wide range of functional categories that in- clusters in terms of gene expression patterns (11), to- clude kinase receptors (RET), regulators of signaling (NF1, gether with several minor clusters (12). Cluster 1 tumors, HRAS), hypoxia-related factors (VHL, EPAS1 [HIF2A]), which include tumors carrying variants in the succinate tricarboxylic acid cycle (TCA) enzymes involved in energy dehydrogenase (SDH) genes and VHL, were found to metabolism (SDHA, SDHB, SDHC, SDHD, SDHAF2, FH, differ markedly from cluster 2 (including RET and NF1) MDH2, IDH1, IDH2), members of the malate-aspartate pheochromocytomas, with cluster 1 tumors characterized 3 The Journal of Clinical Endocrinology & Metabolism, 2020, Vol. XX, No. XX by angiogenesis, hypoxia, extracellular matrix, and sup- Patient tissue samples pression of oxidoreductase enzymes activity, whereas Formalin-fixed paraffin-embedded (FFPE) pheochromocy- cluster 2 tumors show gene expression patterns related toma tissue samples (from
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