Laura Remacha1, Maria Currás-Freixes1, Raúl Torres-Ruiz2, Francesca Schiavi3, Rafael Torres-Pérez1, Bruna Calsina1, Rocío Letón1, Iñaki Comino-Méndez1, Juan M Roldán-Romero1, Cristina Montero-Conde1, María Santos1, Lucía Inglada Pérez1, Guillermo Pita4, María R Alonso4, Emiliano Honrado5, Susana Pedrinaci6, Benedicto Crespo-Facorro7, Antonio Percesepe8, Maurizio Falcioni9, Sandra Rodríguez-Perales2, Esther Korpershoek10, Santiago Ramón-Maiques11, Giuseppe Opocher3, Cristina Rodríguez-Antona1,12, Mercedes Robledo1,12, Alberto Cascón13,14. 1. Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain. 2. Molecular Cytogenetics Unit, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain. 3. Veneto Institute of Oncology, Istituto di Ricovero e Cura a Carattere Scientifico, Padua, Italy. 4. Human Genotyping Unit-CeGen, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain. 5. Anatomical Pathology Service, Hospital de León, León, Spain. 6. Department of Genetics, Hospital Universitario Virgen de las Nieves, Granada, Spain. 7. University of Cantabria and HU Marqués de Valdecilla-IDIVAL, CIBER Mental Health Santander, Santander, Spain. 8. Medical Genetics, Department of Medicine and Surgery, University of Parma, Parma, Italy. 9. Otolaryngology and Otoneurosurgery Department, Azienda Ospedaliero-Universitaria di Parma, Parma, Italy. 10. Department of Pathology, Erasmus Medical Center Cancer Institute, University Medical Center Rotterdam, Rotterdam, The Netherlands. 11. Structural Bases of Genome Integrity Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain. 12. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain. 13. Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain. acascon@cnio.es. 14. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain. acascon@cnio.es.
Abstract
PURPOSE: The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors. METHODS: Whole-exome sequencing was applied to the germlines of a parent-proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed. RESULTS: We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma. CONCLUSION: Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.
PURPOSE: The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors. METHODS: Whole-exome sequencing was applied to the germlines of a parent-proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed. RESULTS: We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma. CONCLUSION: Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.
Authors: Daniel N Weinberg; Phillip Rosenbaum; Xiao Chen; Douglas Barrows; Cynthia Horth; Matthew R Marunde; Irina K Popova; Zachary B Gillespie; Michael-Christopher Keogh; Chao Lu; Jacek Majewski; C David Allis Journal: Nat Genet Date: 2021-05-13 Impact factor: 38.330