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Rapid molecular genetic diagnosis of hypertrophic cardiomyopathy by semiconductor sequencing.

ABSTRACT: BACKGROUND: Rapidly determining the complex genetic basis of Hypertrophic cardiomyopathy (HCM) is vital to better understanding and optimally managing this common polygenetic cardiovascular disease. METHODS: A rapid custom Ion-amplicon-resequencing assay, covering 30 commonly affected genes of HCM, was developed and validated in 120 unrelated patients with HCM to facilitate genetic diagnosis of this disease. With this HCM-specific panel and only 20 ng of input genomic DNA, physicians can, for the first time, go from blood samples to variants within a single day. RESULTS: On average, this approach gained 595628 mapped reads per sample, 95.51% reads on target (64.06 kb), 490-fold base coverage depth and 93.24% uniformity of base coverage in CDS regions of the 30 HCM genes. After validation, we detected underlying pathogenic variants in 87% (104 of 120) samples. Tested seven randomly selected HCM genes in eight samples by Sanger sequencing, the sensitivity and false-positive-rate of this HCM panel was 100% and 5%, respectively. CONCLUSIONS: This Ion amplicon HCM resequencing assay provides a currently most rapid, comprehensive, cost-effective and reliable measure for genetic diagnosis of HCM in routinely obtained samples.

SUBMITTER: Li Z

PROVIDER: S-EPMC4072843 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Rapid molecular genetic diagnosis of hypertrophic cardiomyopathy by semiconductor sequencing.

Li Zongzhe Z Huang Jin J Zhao Jinzhao J Chen Chen C Wang Hong H Ding Hu H Wang Dao Wu DW Wang Dao Wen DW

Journal of translational medicine 20140617

<h4>Background</h4>Rapidly determining the complex genetic basis of Hypertrophic cardiomyopathy (HCM) is vital to better understanding and optimally managing this common polygenetic cardiovascular disease.<h4>Methods</h4>A rapid custom Ion-amplicon-resequencing assay, covering 30 commonly affected genes of HCM, was developed and validated in 120 unrelated patients with HCM to facilitate genetic diagnosis of this disease. With this HCM-specific panel and only 20 ng of input genomic DNA, physician ...[more]

PMID: 24938736

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Project description:BackgroundClinical interpretation of the large number of rare variants identified by high throughput sequencing (HTS) technologies is challenging. The aim of this study was to explore the clinical implications of a HTS strategy for patients with hypertrophic cardiomyopathy (HCM) using a targeted HTS methodology and workflow developed for patients with a range of inherited cardiovascular diseases. By comparing the sequencing results with published findings and with sequence data from a large-scale exome sequencing screen of UK individuals, we sought to quantify the strength of the evidence supporting causality for detected candidate variants.Methods and results223 unrelated patients with HCM (46±15 years at diagnosis, 74% males) were studied. In order to analyse coding, intronic and regulatory regions of 41 cardiovascular genes, we used solution-based sequence capture followed by massive parallel resequencing on Illumina GAIIx. Average read-depth in the 2.1 Mb target region was 120. Rare (frequency<0.5%) non-synonymous, loss-of-function and splice-site variants were defined as candidates. Excluding titin, we identified 152 distinct candidate variants in sarcomeric or associated genes (89 novel) in 143 patients (64%). Four sarcomeric genes (MYH7, MYBPC3, TNNI3, TNNT2) showed an excess of rare single non-synonymous single-nucleotide polymorphisms (nsSNPs) in cases compared to controls. The estimated probability that a nsSNP in these genes is pathogenic varied between 57% and near certainty depending on the location. We detected an additional 94 candidate variants (73 novel) in desmosomal, and ion-channel genes in 96 patients (43%).ConclusionsThis study provides the first large-scale quantitative analysis of the prevalence of sarcomere protein gene variants in patients with HCM using HTS technology. Inclusion of other genes implicated in inherited cardiac disease identifies a large number of non-synonymous rare variants of unknown clinical significance.

Project description:Dilated cardiomyopathy (DCM) is a complex myocardial disease of multifactorial etiologies, including enlarged cardiac chambers and contractile dysfunction. It has been suggested that the inheritance of DCM‑associated mutations predominates its onset. Therefore, the present study investigated the pathogenesis of DCM via pedigree analysis and genetic diagnosis by massive whole‑exome screening, and targeted exon capture. To study the familial gene‑phenotype association, the exon and splice sites of 325 hereditary disease‑associated genes in the proband with familial dilated cardiomyopathy (FDC), including 61 cardiac disease‑associated genes, such as the lamins A/C (LMNA), were analyzed by ultra‑high multiplex polymerase chain reaction and the Ion AmpliSeq™ Inherited Disease Panel. The present study also conducted Sanger DNA Sequencing for family members with global minor allele frequencies <1% to verify potential pathogenic mutation sites. A total of three rare missense mutations were detected, including heterozygous c.244G>A in LMNA, c.546C>G in potassium voltage‑gated channel subfamily KQT (KCNQ4) and c.1276G>A in EYA transcriptional coactivator and phosphatase 1 (EYA1), indicating a glutamic acid to lysine substitution at amino acid 82 (p.E82K) in LMNA, a p.F182L in KCNQ4 (a mutation associated with pathogenic deafness) and p.G426S in EYA1 (associated with Branchiootorenal syndrome 1 and Branchiootic syndrome 1 pathogenesis). In the present study, a carrier with slight hearing impairment was detected in the family analyzed; however, no patients with deafness or branchiootorenal syndrome were observed. LMNA p.E82K revealed SIFT and PolyPhen‑2 scores of 0 and 1, respectively. In the second generation, 3 patients with DCM underwent permanent pacemaker implantation due to sick sinus syndrome, atrioventricular block and unstable cardiac electrophysiology. The present study suggested that LMNA p.E82K may contribute to the pathogenesis of FDC and concomitant atrioventricular block. At present, only three families with DCM resulting from similar mutations have been reported. The present study demonstrated the strong pathogenic effects of LMNA p.E82K on DCM.

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Rapid molecular genetic diagnosis of hypertrophic cardiomyopathy by semiconductor sequencing.

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Rapid molecular genetic diagnosis of hypertrophic cardiomyopathy by semiconductor sequencing.

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