Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.
Molecular Cytogenetics Unit, Human Cancer Genetics Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.
Genet Med. 2018 Dec;20(12):1644-1651. doi: 10.1038/s41436-018-0003-y. Epub 2018 May 8.
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.
由于携带种系突变的患者比例很高，因此嗜铬细胞瘤/副神经节瘤是所有肿瘤中遗传性最高的肿瘤。然而，仍有一些病例无法用已知基因的突变来解释。我们旨在确定强烈怀疑患有遗传性肿瘤的患者的疾病遗传原因。
对一个先证者-父母三人的种系进行全外显子组测序。还进行了全基因组甲基化组分析、RNA-seq、CRISPR/Cas9 基因编辑和靶向测序。
我们在 DNMT3A 中发现了一个新的从头种系突变，该突变影响了一个高度保守的残基，该残基靠近结合三甲基化组蛋白 H3 的芳香笼。DNMT3A 突变的肿瘤表现出同源盒基因的显著高甲基化，表明该突变具有激活作用。CRISPR/Cas9 介导的 HeLa 细胞中的基因敲入导致了甲基化的全局变化，为 DNMT3A 改变功能提供了证据。靶向测序揭示了另外六个副神经节瘤中的亚克隆体细胞突变。最后，在一名有嗜铬细胞瘤家族史的患者中发现了第二个种系 DNMT3A 突变，也导致了肿瘤 DNA 的全局高甲基化。
我们的研究结果表明，DNMT3A 可能是副神经节瘤的易感基因，如果在未来的研究中得到证实，将代表第一个影响参与癌症易感性的 DNA 甲基转移酶基因的功能获得性突变的例子。
