Cardiovascular Genetics: A News Round-Up Exome Sequencing in Congenital Heart Disease Points to Importance of DNA Methylation Rajat M. Gupta, MD 1. Bruneau BG. The developmental genetics of congenital heart disease. Nature. 2008;451:943–948. 2. Oyen N, Poulsen G, Boyd HA, Wohlfahrt J, Jensen PK, Melbye M. Recurrence of congenital heart defects in families. Circulation. 2009;120:295–301. 3. Pierpont ME, Basson CT, Benson DW Jr, Gelb BD, Giglia TM, Goldmuntz E, et al. Genetic basis for congenital heart defects: current knowledge: a scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics. Circulation. 2007;115:3015–3038. 4. Zaidi S, Choi M, Wakimoto H, Ma L, Jiang J, Overton JD, et al. De novo mutations in histone-modifying genes in congenital heart disease. Nature. 2013;498:220–223.

Study Hypothesis Although congenital heart disease (CHD) comprises many different and seemingly divergent diseases, there does seem to be a shared genetic component for the general category.1 Studies have demonstrated an inherited risk in a few families2; however, the vast majority of cases occur sporadically, pointing to de novo mutations.3 The literature is full of case reports in which sequencing has uncovered a novel de novo mutation thought to be causal, but no large-scale efforts have been undertaken to date. Despite the heterogeneity of the CHD category, it was hypothesized that exome sequencing in a well ascertained case–control study can identify a novel set of potentially causal genes and elucidate shared pathways important to cardiac development.

How Was the Hypothesis Tested? Zaidi et al4 compared the incidence of de novo mutations in 362 severe CHD cases and 264 controls (from an existing autism cohort) by exome-sequencing patient-offspring trios. Targeted bases in each sample were sequenced a mean of 107 times, and family relationships were reconfirmed with sequence data. The authors demonstrated that the sequencing yields and quality were similar between the 2 groups. Although a large number of de novo mutations were identified in both cases and controls (0.88 and 0.85 per subject, respectively), the authors partitioned out the 4169 genes with highest heart expression (based on RNA-sequencing data from mouse embryos at day E14.5) for their analyses.

Principal Findings In genes with high heart expression during embryonic development, there were a higher number of mutations (both

synonymous and nonsynonymous) in CHD trios compared with controls. Although the difference met the threshold for significance (P=0.05), the difference was accentuated when only those mutations that were predicted to be severe and damaging were considered (P=0.0005). The authors define severe and damaging as all de novo mutations without missense mutations, leaving only premature termination, splice site, and frameshift mutations. This admittedly straightforward approach was confirmed with a computational approach (PolyPhen-2) to predict protein-altering mutations. To validate that the correct set of high heart expression genes was ascertained, the analysis was repeated in genes with high expression in E9.5 mouse embryos with similar results. Using the relatively long list of interesting and potentially disease-causing mutations in CHD subjects, the authors conducted pathway analysis that uncovered a significant association with the H3K4 methylation pathway. They also found deleterious mutations in genes previously linked to Mendelian syndromes; however, they found that the cohort subjects with these mutations had unique phenotypes and did not share the typical features of their Mendelian counterparts (with diseases such as congenital deafness syndromes or neurofibromatosis).

Implications This study is noteworthy because it validates exome sequencing as an approach to uncover potentially causal mutations and pathways for CHD. The authors acknowledge that many interesting disease-causing genes may have been inappropriately discarded by their use of RNA-sequencing data from mouse embryos, particularly those in which mouse–human orthologs do not exist. Nonetheless, their final list includes genes and pathways not previously known to be associated with CHD, such as H3K4 methylation. Given the diverse cardiac phenotypes seen in patients with similar mutations, the important work of determining how genotype contributes to phenotype remains, but the comprehensive analysis performed by the authors should serve as a useful starting point.

Disclosures Dr Gupta is a member of the Early Career Committee of the American Heart Association Functional Genomics and Translational Biology Council. Key Words: congenital heart disease ◼ genetics ◼ humans

From the Division of Cardiovascular Medicine, Brigham and Women’s Hospital, Boston, MA. Correspondence to Rajat Gupta, MD, Division of Cardiovascular Medicine, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115. E-mail [email protected] (Circ Cardiovasc Genet. 2013;6:522.) © 2013 American Heart Association, Inc. Circ Cardiovasc Genet is available at http://circgenetics.ahajournals.org

DOI: 10.1161/CIRCGENETICS.113.000325

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Exome Sequencing in Congenital Heart Disease Points to Importance of DNA Methylation Rajat M. Gupta Circ Cardiovasc Genet. 2013;6:522 doi: 10.1161/CIRCGENETICS.113.000325 Circulation: Cardiovascular Genetics is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2013 American Heart Association, Inc. All rights reserved. Print ISSN: 1942-325X. Online ISSN: 1942-3268

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Exome sequencing in congenital heart disease points to importance of DNA methylation.

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