Identification of a novel hypertrophic cardiomyopathy-associated mutation using targeted next-generation sequencing

Abstract

Hypertrophic cardiomyopathy (HCM), one of the most common forms of myocardial diseases, is the major cause of sudden cardiac death in young adults and competitive athletes. Analyses of gene mutations associated with HCM are valuable for its molecular diagnosis, genetic counseling, and management of familial HCM. To dissect the relationship between the clinical presentation and gene mutations of HCM, the genetic characterizations of 19 HCM-related genes in 18 patients (8 cases from 6 pedigrees with familial HCM and 10 cases without familial HCM) were detected using next-generation sequencing (NGS). As a result, 12 disease-related mutations were identified in the 18 subjects, including 6 single mutations and 3 double mutations [MYBPC3 (p.Gln998Glu) plus TNNI3 (p.Arg145Gly), PRKAG2 (p.Gly100Ser) plus MYBPC3 (p.Lys1209Serfs*28) and TNNI3 (p.Glu124Gln) plus GLA (p.Trp47*)]. The 3 heterozygous double mutations were discovered for the first time in the malignant familial HCM patients. Of the 6 single mutations, a novel mutation was found in tafazzin (TAZ, p.Ile208Val), and a mutation in β-myosin heavy chain gene (MYH7, p.Arg54Gln), which was reported as rare in the general population, was firstly found in one HCM patient. Identification of novel and rare mutations in HCM patients have added new data to the spectrum of gene mutations associated with this disease. These findings provide an essential basis for the molecular diagnosis and better management of family members at risk of familial HCM.

Figure 1

Sequencing coverage of mutation sites in the coding region of 19 hypertrophic cardiomyopathy (HCM)-related genes. The sequence coverages for each gene ranged from 30 to 498× in 16 HCM patients, and the mean coverage was 165.4× for overall selected target genes.

Figure 2

The genetic analyses of target gene mutations in patients from 6 pedigrees with familial hypertrophic cardiomyopathy (HCM) (families A, B, C, D, E and F). Squares, male family members; circles, female family members; open symbols, normal individuals; solid symbols, affected individuals; black arrow, proband; plus (+) signs, the presence of disease mutation; minus (−) signs, the absence of disease mutation. Both (+) and (−) indicate members for whom the PCR and Sanger sequencing validation were carried out.

Figure 3

Two-dimensional echocardiography of hypertrophic cardiomyopathy (HCM) patients. (A and B) The echocardiogram of the four chambers of proband family F (patient II: 1) and patient A14, respectively. White arrows indicate areas of hypertrophy.

Figure 4

Identification of potential disease mutations in hypertrophic cardiomyopathy (HCM). Nucleotide mutation sites are shown with arrows. (A) A double heterozygous mutation at the nucleotide position c. (730T>C, 732C>T) of the β-myosin heavy chain gene (MYH7) gene in HCM patient 8. (B) A mutation at the nucleotide position c.161G>A of the MYH7 gene in HCM patient A14. (C) A heterozygous mutation at the nucleotide position c.1087A>T of the α-myosin heavy chain (MYH6) gene in patient 9. (D) A heterozygous mutation at the nucleotide position c.3575G>A of the sodium channel, voltage-gated type V α-subunit (SCN5A) gene in patient 25 and 26.

Figure 5

As determined using Clustal W, (A and B) the synonymous mutations -p.Ile208Val and p.Arg54Gln involved an amino acid in tafazzin (TAZ) and β-myosin heavy chain (MYH7) genes that were highly conserved across many species, respectively.

Figure 6

The structure modeling of predicted β-myosin heavy chain gene (MYH7) with wild-type and mutant. (A and B) Wild-type MYH7 (codon 1 to 841) and mutant p.Arg54Gln, respectively. The wild-type and mutated site are emphasized by a red circle and are locally zoomed.