Gain-of-function mutations in DNMT3A in patients with paraganglioma



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.


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.


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.


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.

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Fig. 1: Exome sequencing findings, trimethylated histone H3 interactions, and immunohistochemical staining.
Fig. 2: Arg318 location in a DNMT3A 3D structural model and methylation profile for DNMT3A-mutated tumors.


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This work was supported by the Instituto de Salud Carlos III (ISCIII), through the “Acción Estratégica en Salud” (AES) (projects PI15/00783 to A.C., PI14/00240 to M.R., and PI14/01884 to S.R.-P., cofounded by the European Regional Development Fund (ERDF)). M.C.-F. is a predoctoral fellow of the Severo Ochoa Program. We thank Antonio Galarreta for his help with the validation of the exome sequencing findings. We thank Maria Jesús Artiga and Manuel Morente for their help in obtaining tumor samples, collected from Spanish hospitals through the Spanish National Tumor Bank Network (CNIO).

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Correspondence to Alberto Cascón PhD.

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  • DNMT3A
  • paraganglioma
  • exome sequencing
  • hypermethylation
  • CRISPR/Cas9 gene editing

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