Coverage and diagnostic yield of Whole Exome Sequencing for the Evaluation of Cases with Dilated and Hypertrophic Cardiomyopathy

www.nature.com/scientificreports OPEN Received: 21 November 2017 Accepted: 4 July 2018 Published: xx xx xxxx Coverage and diagnostic yield of Whole Exome Sequencing for the Evaluation of Cases with Dilated and Hypertrophic Cardiomyopathy Timothy Shin Heng Mak 1, Yee-Ki Lee2, Clara S. Tang Pak-Chung Sham 1,5,6 & Hung-Fat Tse2,7,8,9 3,4 , JoJo S. H. Hai2, Xinru Ran2, Targeted next generation sequencing of gene panels has become a popular tool for the genetic diagnosis of hypertrophic (HCM) and dilated cardiomyopathy (DCM). However, it is uncertain whether the use of Whole Exome Sequencing (WES) represents a more effective approach for diagnosis of cases with HCM and DCM. In this study, we performed indirect comparisons of the coverage and diagnostic yield of WES on genes and variants related to HCM and DCM versus 4 different commercial gene panels using 40 HCM and DCM patients, assuming perfect coverage in those panels. We identified 6 pathogenic or likely pathogenic among 14 HCM patients (diagnostic yield 43%). 3 pathogenic or likely pathogenic were found among the 26 DCM patients (diagnostic yield 12%). The coverage was similar to that of four existing commercial gene panels due to the clustering of mutation within MYH7, MYBPC3, TPM1, TNT2, and TTN. Moreover, the coverage of WES appeared inadequate for TNNI3 and PLN. We conclude that most of the pathogenic variants for HCM and DCM can be found within a small number of genes which were covered by all the commercial gene panels, and the application of WES did not increase diagnostic yield. Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are important causes of heart failure1 and sudden cardiac deaths2. It has been estimated that HCM and DCM affect at least 1/500 and 1/2500 persons, respectively3,4. Clinically, DCM is characterized by the dilatation and dysfunction of the left ventricle (LV), while HCM is characterized by the hypertrophy of LV. Both HCM and DCM can be caused by genetic or non-genetic factors, and there are significant overlaps in their clinical phenotypes and etiologies5. In particular, mutations in several genes have been reported to cause both DCM and HCM. In the past, genetic testing for cardiomyopathy was usually done by targeted Sanger sequencing of a small number of genes. However, the increased availability of high-throughput genotyping and next generation sequencing (NGS) means that a much larger number of genes can be interrogated at the same time and thus potentially increase the yield in genetic diagnosis3,6,7. NGS can be applied in three ways: targeted sequencing for a number of genes, whole-exome sequencing (WES), and whole-genome sequencing (WGS)8. The advantage of targeted sequencing is that the region of sequencing can be highly specific and tailored to the specific application. The region can also be covered at great depth and many samples can be analysed at the same time. For diseases in which only a small number of genes are involved, the cost of targeted sequencing is considerably less than WES and WGS. Indeed, different commercial panels of targeted sequencing have been developed for particular diseases. In contrast, WES attempts to capture and sequence all protein-coding regions in the genome, the capture regions being predesigned through commercial capture kits. 1 Centre for Genomic Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China. Department of Medicine, the University of Hong Kong, Hong Kong, China. 3Department of Surgery, the University of Hong Kong, Hong Kong, China. 4The University of Hong Kong – Karolinska Institutet Collaboration in Regenerative Medicine, Hong Kong, China. 5Department of Psychiatry, the University of Hong Kong, Hong Kong, China. 6State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Hong Kong, China. 7Department of Medicine, Shenzhen Hong Kong University Hospital, Shenzhen, China. 8Hong Kong-Guangdong Joint Laboratory on Stem Cell and Regenerative Medicine, the University of Hong Kong, Hong Kong, China. 9Shenzhen Institutes of Research and Innovation, the University of Hong Kong, Hong Kong SAR, China. Correspondence and requests for materials should be addressed to P.-C.S. (email: ) or H.-F.T. (email: ) 2 SCIenTIFIC REPOrTS | (2018) 8:10846 | DOI:10.1038/s41598-018-29263-3 1 www.nature.com/scientificreports/ The advantage of this approach is that it covers the entire exome and can be used for discovery purposes. However, due to the difficulties in the design of probes, typically the coverage of the exome is not complete. WGS, though more costly than WES, has better performance than WES in terms of the coverage of the genome, even for the exonic regions and also includes the intron regions. However, WES may be sufficient for the genetic diagnosis of cardiomyopathy if the majority of the associated mutations are found in protein coding regions. More recently, Cirino et al.9 compared the use of WGS and target sequencing in genetic tests of HCM. They found that WGS was able to identify most of the pathogenic or likely pathogenic variants identified using a targeted HCM panel, and also a number not included in the gene panel. However, one variant from the gene panel was missed due to low coverage. For diseases other than cardiomyopathies, a number of studies recently found that targeted sequencing provided a better coverage than WES for genetic diagnosis10,11. Here, we add to this literature by comparing between WES and targeted sequencing used by 4 commercial panels for genetic diagnosis of HCM and DCM. Methods Study Population. We recruited consecutive Chinese patients with well-characterized phenotypes of DCM or HCM who were followed up in our Cardiac Arrhythmia and Device outpatient clinic at Queen Mary Hospital, Hong Kong between January 2013 and December 2015. The clinical diagnosis of DCM and HCM were made based on current guidelines12,13 and verified by two independent cardiologists (JJH and HFT). All patients meeting the diagnositic criteria for DCM and HCM and agreed to genetic testing were recruited. Blood samples were collected for DNA extraction upon recruitment from all patients. This study was approved by the Institutional Review Board of the University of Hong Kong and the Hospital Authority Hong Kong West Cluster, and written informed consent was obtained from each patient. Whole Exome Sequencing. All of the libraries were prepared based on the protocols of KAPA Hyper Prep Kit (KR0961-V1.14). Exome capture was prepared based on the protocols of Roche SeqCap EZ Library SR User’s Guide version 4.1. Before hybridization, 6 libraries were normalized and combined with different indices into a single pool prior to enrichment. The pooled DNA libraries were mixed with capture probes of targeted regions using the SeqCap EZ Exome + UTR capture kit. The hybridization was performed at 47 °C for 64–72 hours to ensure targeted regions bind to the capture probes (...truncated)