The Ser96Ala variant in histidine-rich calcium-binding protein is associated with life-threatening ventricular arrhythmias in idiopathic dilated cardiomyopathy

Abstract

Aims: To investigate whether genetic variants of the histidine-rich calcium (HRC)-binding protein are associated with idiopathic dilated cardiomyopathy (DCM) and its progression.

Methods and results: We screened 123 idiopathic DCM patients and 96 healthy individuals by single-strand conformation polymorphism analysis and direct sequencing for genetic variants in HRC. Six polymorphisms were detected: Leu35Leu (A/G), Ser43Asn (G/A), Ser96Ala (T/G), Glu202_Glu203insGlu (-/GAG), Asp261del (GAT/-), and an in-frame insertion of 51 amino acids at His321. The analysis of their frequencies did not reveal any significant correlation with DCM development. However, the Ser96Ala polymorphism exhibited a statistically significant correlation with the occurrence of life-threatening ventricular arrhythmias. During a follow-up of 4.02 +/- 2.4 years, the risk for ventricular arrhythmias was higher (HR, 9.620; 95% CI, 2.183-42.394; P = 0.003) in the Ala/Ala patients, compared with Ser/Ser homozygous patients. On multivariable Cox regression analysis, the Ser96Ala polymorphism was the only significant genetic arrythmogenesis predictor in DCM patients (HR, 4.191; 95% CI, 0.838-20.967; P = 0.018).

Conclusion: The Ser96Ala genetic variant of HRC is associated with life-threatening ventricular arrhythmias in idiopathic DCM and may serve as an independent predictor of susceptibility to arrhythmogenesis in the setting of DCM.

Figure 1

Intracardiac electrograms showing recording of monomorphic (A) or polymorphic (B) ventricular tachycardia that terminated after implantable cardioverter-defibrillator firing, from two patients with Ala/Ala at codon 96. Their resting ECGs are also presented (C and D, respectively).

Figure 2

Representative example of single strand conformational polymorphism analysis of HRC exon 1 with primer set 7, where the insertion of 51 amino acid residues was found. (A) Single strand conformational polymorphism analysis on 0.5× MDE polyacrylamide gels of three wild types (lanes 1–3), and one heterozygote for the insertion (lane 4). (B) Same set of DNA products (lanes 1–4) analysed in a 2% agarose gel, stained with ethidium bromide; M, 100 bp ladder; second to fourth lanes, wild-type samples; fifth lane, heterozygote for the insertion. (C) DNA sequences: top line, amino acid residues; bottom line, nucleotide sequence. (D) Alignment of the 10 histidine-rich and acidic tandem repeats of the wild-type histidine-rich calcium. The dashed lines indicate gaps for the best fit of the alignment. (E) Alignment of the histidine-rich and acidic tandem repeats of the mutant histidine-rich calcium with the 51 amino acids insertion (in bold, highlighted). The dashed lines indicate gaps for the best fit of the alignment. The in-frame insertion at position 321 adds two extra repeats to the protein.

Figure 3

Group analysis of the relative risk of dilated cardiomyopathy development associated with each of the histidine-rich calcium genotypes detected in dilated cardiomyopathy patients and healthy controls. The number of dilated cardiomyopathy cases and controls with a specific genotype are presented. Each genotype was compared with the rest, as indicated. None of the differences between polymorphism frequencies was statistically significant.

Figure 4

The histidine-rich calcium polymorphism at position 96 is associated with the history of sustained ventricular tachycardia or ventricular fibrillation of dilated cardiomyopathy patients prior to study entry. Percentages of dilated cardiomyopathy patients with Ser/Ser, Ser/Ala, and Ala/Ala at codon 96 in need of implantable cardioverter-defibrillator implantation during study entry (*, χ² = 11.710, df = 2, P = 0.003).

Figure 5

Relative risk of dilated cardiomyopathy patients for implantable cardioverter-defibrillator implantation (history of sustained ventricular tachycardia or ventricular fibrillation prior to study entry) with respect to histidine-rich calcium polymorphisms. The numbers of dilated cardiomyopathy patients with and without an implantable cardioverter-defibrillator, respectively, with a specific genotype are presented. Each genotype was compared with the rest as indicated. Asterisks indicate statistically significant difference. Homozygosity for alanine of the histidine-rich calcium Ser96Ala polymorphism was associated with an increased risk for malignant ventricular arrhythmias in dilated cardiomyopathy patients prior to study entry, while homozygosity for serine was protective.

Figure 6

Kaplan–Meier plots for the probability of survival from: (A) life-threatening ventricular arrhythmic events including sudden cardiac death and episodes of unstable VT (>180 b.p.m.) or ventricular fibrillation, which were recorded by an implantable cardioverter-defibrillator device and (B) cardiac death from any cause, including pump failure, transplantation, sudden cardiac death, and episodes of unstable VT (>180 b.p.m.) or ventricular fibrillation, which were recorded by an implantable cardioverter-defibrillator device. Each event is depicted as a step down. Each censored case [due to (A) death from other causes except sudden cardiac death, heart transplantation, and study termination and (B) death from non-cardiac aetiology and study termination] is marked with a cross. The table at the bottom of the plots indicates the number of dilated cardiomyopathy patients in risk for each year of the follow-up study. The Ala/Ala homozygotes for the Ser96Ala polymorphism were statistically more susceptible to ventricular arrhythmic events, compared with Ser/Ala heterozygotes and Ser/Ser homozygotes.