Molecular analysis of inherited cardiomyopathy using next generation semiconductor sequencing technologies

Patients diagnosed with IC phenotypes as HCM, DCM, RCM, ARVC/D, LVNC, and overlapping or undefined phenotypes were included in this study. HCM, DCM, RCM, ARVC/D and LVNC were defined based on guidelines described by a the American Heart Association [1]. Overlapping or undefined cardiomyopathies were defined as cardiac manifestations exhibited at least two phenotypes (such as a significantly thickened interventricular septum together with left ventricular dilation) that cannot be ascribed to a single classical phenotype.

Exclusion criteria included secondary cardiomyopathies or cardiomyopathies with pathogenic mechanisms that have already been described, including inflammatory (myocarditis), stress-provoked (Tako-tsubo), peripartum, ischemic, hypertensive, valvular, hyperthyroid/hypothyroid, alcoholic, and diabetic cardiomyopathies.

A total of 110 unrelated patients diagnosed with IC, including HCM (n = 34), DCM (n = 22), RCM (n = 13), ARVC/D (n = 7), LVNC (n = 9), and overlapping or undefined cardiomyopathies (n = 25), were identified and enrolled at the Cardiology Clinic at the Peking Union Medical College from January 2012 to December 2015. All patients were determined to not have secondary cardiomyopathy. Of these patients, 18 had a family history of cardiomyopathy or sudden death, while 92 were sporadic cases. Clinical evaluation consisted of a medical history, family history, physical examination, 12-lead echocardiogram (ECG), transthoracic and/or transesophageal ECG, and/or cardiac magnetic resonance imaging. Peripheral blood samples were collected from patients and their family members (if available). Genomic DNA was extracted from peripheral blood leukocytes using a QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The study was approved by the Peking Union Medical College Hospital Institutional Review Board, and all individuals signed a written informed consent.

According to Online Mendelian Inheritance in Man (OMIM, http://​omim.​org) and PubMed literature retrieval, 64 candidate genes have been reported to be causes of inherited cardiomyopathy, and were selected for panel design (Additional file 1: Table S1). Primers of overlapping amplicons covering the coding sequence (CDS) region, untranslated regions (UTR), and flanking sequences (padding +25 base pairs) of each targeted gene were automatically generated by Ion AmpliSeq designer software. This produced 2231 amplicons, which were divided into 2 primer pools (Life Technologies; Thermo Fisher Scientific). Although Titin (TTN) is a major causative gene for DCM [3, 4], it was not included in the 64-gene panel. As such, we used a separate 6-gene panel (including DMD, TTN, OBSCN, FBN1, TGFBR2 and TGFBR1) for 23 patients with DCM, including one patient with overlapping DCM phenotype. Amplicon libraries were prepared using the Ion AmpliSeq Library Kit v2.0 and custom designed primer pools, according to the manufacturer’s instructions. DNA fragments from different samples were ligated with barcoded sequencing adaptors using the Ion Xpress Barcode Adapter 1-16 Kit. The library was quantified with a Qubit 2.0 fluorometer (Invitrogen; Thermo Fisher Scientific).

Fifteen barcoded samples were pooled in equimolar amounts. Amplified libraries were subjected to emulsion polymerase chain reaction performed on the Ion OneTouch system using the Ion PGM Hi-Q OT2 Kit. Next, ion sphere particles (ISPs) were recovered and enriched using the Ion OneTouch ES system. Enriched template-positive ISPs were sequenced on an Ion 318 V2 chip using the Ion PGM HI-Q SEQ Kit by the Ion Torrent PGM. Data from PGM runs were processed using Ion Torrent Suite 4.4 software to generate sequence reads. After sequence alignment and variant calling, synonymous variants, intronic variants far away from the exon/intron boundaries, and variants with a minor allelic frequency (MAF) ≥ 1% in the 1000 Genomes Project, the dbSNP database, and the Exome Aggregation Consortium (ExAC) database were removed from further analysis. Variants were subsequently selected according to the prevalence of each type of cardiomyopathy (for example, MAF < 0.4%, < 0.3%, < 0.2%, < 0.05% for DCM, LVNC, HCM, and ARVC, respectively) in the general population [5, 6]. NGS reads were visualized using an integrated genomic viewer (IGV).

Sequence variants were confirmed by bidirectional Sanger sequencing. Annotation of the variants was performed using ANNOVAR (http://​wannovar.​wglab.​org/​) and Seattleseq (http://​snp.​gs.​washington.​edu/​SeattleSeqAnnota​tion137/​). Polyphen2 (http://​genetics.​bwh.​harvard.​edu/​pph2/​), SIFT (http://​sift.​jcvi.​org/​) and MutationTaster (http://​www.​mutationtaster.​org/​) were used to predict the function of amino acid substitution. Berkeley Drosophila Genome Project (BDGP) (http://​www.​fruitfly.​org/​seq_​tools/​splice.​html) and Human Splicing Finder (HSF) (http://​www.​umd.​be/​HSF/​) were used predict the effect of intronic variants on splicing efficiency.

We placed verified variants into the following categories according to guidelines from the American College of Medical Genetics and Genomics (ACMG) and Association of Molecular Pathology (AMP) [7]: pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB) and benign (B).

In total, 1352 fragments with a target size of about 409.7 kb were simultaneously generate about 2231 amplicons designed based on the exons and 25 bp exon–intron boundaries of 64 cardiomyopathy-associated genes. Amplicon sizes ranged from 63 to 189 bp, with a mean amplicon length of 145 bp. The custom designed panel covered 93.73% of the bases in the target regions. For each sample, coverage of the targeted region was approximately 96.5%, with an average depth of 100. Target base coverage at 20× was above 94%, with a mean read length of 130 bp. After variant calling by Ion Torrent Suite 4.4, more than 200 variants were detected in each sample (data available upon request). After variants were filtered by allele frequency and mutation type, only zero to two variants required verification by Sanger sequencing in each sample (Additional file 2: Figure S1). All variants’ annotation information verified in this study listed in Additional file 3: Table S2.

We analyzed 110 patients with different primary cardiomyopathies, and detected mutations in 64 cardiomyopathy-associated genes using the NGS method. In total, 78 distinct variants from 30 genes were identified in 73 patients after filtering NGS data and verification using Sanger sequencing. Five patients had two variants in different genes (Table 2). Clinical characteristics of these patients were showed in Additional file 4: Table S3. A pathogenic or likely pathogenic variant was identified in 26 out of 110 independent cases (23.6%) (Table 1). In 45 additional patients (40.9%), VUS were found and the pathogenicity of which need further study in future (Additional file 5: Table S4). Two variants in two patients previously reported as damaging-mutation, were reclassified as B and LB. Higher detection rates of 50% (9/18) were observed in patients with a family history of cardiac disease. Among 26 pathogenic or likely pathogenic variant, 15 were recorded in HGMD or the ClinVar database and 11 were novel. These variants consisted of 15 missense, 6 nonsense, 2 frameshift, 2 splicing, and 1 stop-loss mutations.

The prevalence of disease alleles among the cardiomyopathy genes was not equally distributed. Twenty-six pathogenic or likely pathogenic mutations involved in 10 genes, and MYH7(8/26) and MYBPC3 (6/26) mutations accounted for more than 50% of the variants found in cardiomyopathy cases (Fig. 1). Most of pathogenic or likely pathogenic variants were heterozygous except one homozygous pathogenic variant in the SLC25A4 gene. All of these mutations found in our study are private, identified in a single proband. These data further confirm the genetic heterogeneity of cardiomyopathy.

For the consideration of sequencing cost, we put the largest causative gene (TTN) of DCM in a small panel and performed sequencing only in DCM patients. Of the total number of patients recruited in our study, there were 23 patients with DCM, including overlapping phenotypes. Due to the extremely large size of TTN, missense variants have been commonly observed in genetic screening, but no obviously frequency differences between DCM patients and healthy controls have been noted [4]. Thus, we focused on the truncating variants of TTN found in DCM patients. TTN truncations (two frameshifts and one nonsense variant, Table 1) were found in three patients, which accounted for 13% of the DCM patients in this study.

Five patients had two rare variants (Table 2). In patient 50 (MYBPC3: c.527C>T p.Ala176Val; MYPN: c.411G>C p.Arg137Ser), both variants were classified into VUS according to ACMG guidelines. In patient 56 (BAG3: c.772C>T p.Arg258Trp; VCL: c.133G>T, p.Ala45Ser), the BAG3 variant is regarded as a damaging mutation in HGMD, but was reclassified as “likely benign” in this study based on the following criteria: BS1 (allele frequency is greater than expected for disorder) and BP6 (reputable source recently reported variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation). Patient 62 (PRKAG2: c.1589A>G, p.His530Arg; LAMA4: c.241C>T p.Pro81Ser) was 26 years old and had suffered from heart failure for 2 years. Electrocardiogram showed sinus bradycardia with absolute left bundle branch block and Wolff–Parkinson–White syndrome. Echocardiography revealed hypertrophic non-obstructive cardiomyopathy, uniform thickness in the left ventricular (LV) wall, and severe LV dysfunction (EF 19%). Given that three family members, including the patient’s father, suffered sudden death, blood samples from these individuals were not available. Clinical manifestations in patient 62 were consistent with the features of PRKAG2-related cardiomyopathy. The missense mutation p.His530Arg in PRKAG2 was reported as a pathogenic variant in HGMD and the ClinVar database. Although this variant likely contributed to the phenotype in patient 62, whether the LAMA4 variant produced a “double dose” gene mutation effect will require further study. Genetic screening of first-degree relatives of the proband is important in clinical diagnosis and decision-making. In patient 87 (RBM20: c.3545G>A, p.Arg1182His; SCN5A: c.2962C>T, p.Arg988Trp), both variants were reported as damaging in HGMD. The 23-year-old patient presented with generalized cardiac enlargement with heart failure, but no signs of electrical disorders. Thus, both variants were reclassified as VUS in this patient; In patient 92 (VCL: c.2630C>T, p.Pro877Leu; PSEN2: c.998A>G, p.Glu333Gly), the VCL variant was likely benign because it was observed in a healthy adult individual for recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorders, with full penetrance expected at an early age. The pathogenicity of the PSEN2 variant is uncertain significance.