Rapid molecular genetic diagnosis of hypertrophic cardiomyopathy by semiconductor sequencing

For the HCM resequencing panel targeted genes selection, recent ten years’ literatures, including prior genetic detection technique articles, reviews and case-reports of HCM, were carefully accessed. To recruit a maximum coverage of the mutation spectrum of this polygenetic disorder, we designed a currently most comprehensive HCM-specific resequencing panel including 30 causal genes that most frequently affected in patients with HCM (Table 1). Then, primers of overlapping amplicons covering the CDS-region and flanking sequences of each targeted gene were automated designed by Ion AmpliSeq™ Ready-to-Use custom designer platform following guide of the website (https://​www.​ampliseq.​com/​protected/​dashboard.​action) (Primers for Semiconductor sequencing are presented in Additional file1: Table S1). With the ability to perform ultrahigh-multiplex PCR reaction in one tube parallelly, the primers were mixed and provided (Life Technologies, Carlsbad, California, USA) in two primer-pools. Eventually, 97.96% of the targeted region (64.06 kb) was overlapped by 690 about 200 bp-length amplicons.

Ion Torrent adapter-ligated libraries were builded using Ion Ampliseq™ Library Kit 2.0 (Life Technologies) following the manufacturer’s protocol within about 5 hours. Briefly, 20 ng gDNA for every sample was quantitated by Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) for multiplex PCR amplification with each of the two primer-pools, respectively. The resulting amplicons of the two primer-pools were mixed together, and then ligated to barcodes and Ion Torrent adapters (Life Technologies). Subsequently libraries were purified with AMPure XP beads (Beckman Coulter, Brea, CA, USA) using 5-cycles of PCR amplification and further purification, followed by quantification by Qubit 2.0 fluorometer. In order to increase efficiency and reduce costs, sixteen uniquely barcoded libraries were combined together with equal molar ratios for one 318 chip. Subsequent emulsion PCR and enrichment of the sequencing beads of the pooled libraries was performed using the OneTouch system (Life Technologies) according to the manufacturer’s protocol within about 5 hours. Finally, 500 Flows (125 cycles) sequencing was done on the 318- chip using Ion PGM 200 Sequencing Kit (Life Technologies) on the Ion Torrent Personal Genome Machine (PGM) (Life Technologies) (Figure 1).

Raw data from 4.5 hours’ PGM runs were initially processed using the Ion Torrent platform-specific software Torrent Suite v3.6.2 to generate sequence reads, trim adapter sequences, align to the hg19 human reference genome, analyze coverage and call variants (Variants Caller parameter settings see Additional file1: Table S2). Then, all variants were annotated with an online-software Variant Effect Predictor (http://​asia.​ensembl.​org/​info/​docs/​variation/​vep/​index.​html). To predict possible impact of detected non-synonymous variants in exons, all missense substitutions were scored and in-silico-function-predicted by SIFT (http://​sift.​jcvi.​org/​www/​SIFT_​BLink_​submit.​html) and PolyPhen-2 (http://​genetics.​bwh.​harvard.​edu/​pph2/​). The putative novel pathogenic variants were further confirmed that were reported neither in the 1000-Genome project database nor in the NCBI dbSNP database. To calculate the importance of novel mutations, conservation test was performed in patients and thirteen other species around the mutated position by COBALT algorithm (http://​www.​ncbi.​nlm.​nih.​gov/​tools/​cobalt/​cobalt.​cgi).

To determine a functional variant, the variant should be a Sanger sequencing validated coding variant. If the variant is a previously reported variant, it should be causal mutations in NCBI ClinVar database or HGMD database. If the variant is a novel variant, it should in accordance with none of the following database: the 1000-Genome project database, the NCBI dbSNP database, UCSC common SNP database and the 5000 Exmoes database in Exome Sequencing Project (ESP). If the variant is a missense substitution, it should be evolutionarily conserved and predicted functional damaged by either SIFT or Polyphen-2.

One-hundred-and-twenty unrelated patients with HCM were studied. The mean age of male (85/120) was 45 ± 16 years (1, 78) and the maximal wall thickness was 18.34 ± 5.1 mm (14, 46). For female (35/120) the mean age was 51 ± 17 years (15, 86) and the maximal wall thickness was 19.73 ± 4.8 mm (13, 41). The mean left ventricular ejection fractions of male and female are 58 ± 14 and 62 ± 13, respectively. In the 120 HCM patients, 24.2% (29/120) are hypertrophic obstructive cardiomyopathy and 19.2% (23/120) have positive family history. (The detailed characteristics of HCM Patients were shown in Additional file1: Table S3).

The sequencing of selected regions of 30 HCM-associated genes on the Ion torrent PGM achieved an average output of 595628 mapped reads and 95.51% on target per sample in the 120 HCM specimens. In summary, 99.55% of all target amplicons was covered at least once, 96.98% amplicons was covered at least 20 times, 91.95% amplicons was covered at least 100 times. The mean uniformity of base coverage is 93.24% in this panel. The average read depth in the 64.06 K target region across the 120 samples was ~490 folds (Figure 2). Moreover, chip-loading-rate was improved shortly and polyclonal-rate was reduced significantly after few trails in the beginning of experiments, which result in an increase in mean coverage.

The Ion Torrent platform-specific software Torrent Suite v3.6.2 and online software Variant Effect Predictor were employed to align the reads sequences to the human reference genome build hg19, call variants and bioinformatical annotate. Criteria for variant identification were a read coverage of higher than 30-fold. All together, in the 120 patients, 458 known or novel variants were detected by Semiconductor sequencing and on average 80 variants per sample. After Sanger sequencing validation, except 25 variants, 433 variants were determined truly exist. Most of the 25 false-positive miscalls are insertions or deletions and detected in more than one sample. Of these 433 variants, 345 (80%) are predicted to be noncoding or synonymous, whereas 88 (20%) are non-synonymous, including missense mutation and small insertion/deletion, resulting in the change of amino acids (Table 2). Notably, we identified at least one functional variant in 104/120 (87%) HCM patients and found more than one functional variants in 12/120 (10%) HCM patients.Furthermore, the two known positive pathogenic mutations (rs121913641 and rs121913637) in the two probands were successfully identified.

To further assess the sensitivity and specificity of this HCM panel, direct Sanger sequencing of seven randomly selected genes was performed in eight selected subjects. Finally, 38 variants were detected by semiconductor sequencing, including 35 known variants and 3 novel variants (Table 3). Compared with Sanger sequencing results, 2 variants were failed to be validated. Therefore, the sensitivity of this HCM panel was evaluated as 100% and the false-positive-rate was evaluated as 5%.