Autosomal Recessive Cardiomyopathy Presenting as Acute Myocarditis

Abstract

Background: Myocarditis is inflammation of the heart muscle that can follow various viral infections. Why children only rarely develop life-threatening acute viral myocarditis (AVM), given that the causal viral infections are common, is unknown. Genetic lesions might underlie such susceptibilities. Mouse genetic studies demonstrated that interferon (IFN)-α/β immunity defects increased susceptibility to virus-induced myocarditis. Moreover, variations in human TLR3, a potent inducer of IFNs, were proposed to underlie AVM.

Objectives: This study sought to evaluate the hypothesis that human genetic factors may underlie AVM in previously healthy children.

Methods: We tested the role of TLR3-IFN immunity using human induced pluripotent stem cell-derived cardiomyocytes. We then performed whole-exome sequencing of 42 unrelated children with acute myocarditis (AM), some with proven viral causes.

Results: We found that TLR3- and STAT1-deficient cardiomyocytes were not more susceptible to Coxsackie virus B3 (CVB3) infection than control cells. Moreover, CVB3 did not induce IFN-α/β and IFN-α/β-stimulated genes in control cardiomyocytes. Finally, exogenous IFN-α did not substantially protect cardiomyocytes against CVB3. We did not observe a significant enrichment of rare variations in TLR3- or IFN-α/β-related genes. Surprisingly, we found that homozygous but not heterozygous rare variants in genes associated with inherited cardiomyopathies were significantly enriched in AM-AVM patients compared with healthy individuals (p = 2.22E-03) or patients with other diseases (p = 1.08E-04). Seven of 42 patients (16.7%) carried rare biallelic (homozygous or compound heterozygous) nonsynonymous or splice-site variations in 6 cardiomyopathy-associated genes (BAG3, DSP, PKP2, RYR2, SCN5A, or TNNI3).

Conclusions: Previously silent recessive defects of the myocardium may predispose to acute heart failure presenting as AM, notably after common viral infections in children.

Keywords: cardiomyocytes; children; genetics; immunity; sequencing; virus.

Copyright © 2017 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Figures

Central Illustration. Genetics of Acute Myocarditis

Acute myocarditis (AM), a rare, life-threatening disease that often results from common viral infections, occurs in ~1 in every 100,000 children annually. AM has been associated with the progression of myocardial dysfunction in certain inherited cardiomyopathies. Whole exome sequencing of 42 children with AM (red) were compared to healthy individuals (gray) from the 1000 Genomes database. While there was no significant (n.s.) difference between the groups with heterozygous rare genetic variants, homozygous damaging mutations were significantly enriched in the AM patients. *p

Figure 1. CVB3 Infection of Human iPSC-derived Cardiomyocytes

(A) Virus-induced cytotoxicity in representative lines of control, TLR3- and STAT1-deficient cardiomyocytes at 24 h post-infection with coxsackievirus B3 (CVB3) is compared at indicated multiplicities of infection (MOI). Percent cytotoxicity was determined by lactate dehydrogenase release assay and normalized to that of mock-infected cells for each induced pluripotent stem cells (iPSC) line, which is set as 1. CVB3 replication was determined by quantitative polymerase chain reaction in control and mutant cardiomyocyte lines at 24 h post-infection with CVB3 at various MOIs (B) and at different time-points post-infection at MOI 1 (C). Data (A–C) are mean ± SEM of several independent experiments performed in at least duplicates. Viral replication (D) and virus-induced cytotoxicity (E) were measured at 24 h post-infection with CVB3 at MOI 0.01 in control and mutant cardiomyocytes pre-treated with exogenous interferon (IFN)-α2b. Data are mean ± SEM of at least 2 independent experiments.

Figure 2. Familial Pedigrees

Shown are pedigrees of 7 families affected by myocarditis and variations in 6 cardiomyopathy-associated genes. Black = patients; white = healthy family members. When available, a gene’s mutation status is provided.

Figure 3. In silico Prediction of Damaging Impact of Disease-causing Mutations

Human Gene Mutation Database mutations associated with dominant (black) and recessive (blue) cardiomyopathies are shown for 6 genes: DSP, PKP2, and TNNI3 (A); and BAG3, RYR2, and SCN5A (B). Red = variations found in patients with acute or acute viral myocarditis. The line indicates the median (50th percentile) of Combined Annotation-Dependent Depletion (CADD) scores of all the mutations presented in each graph.