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
We evaluated the hypothesis that human genetic factors might underlie AVM in previously healthy children.
METHODS
We tested the role of TLR3-IFN immunity utilizing 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 etiologies.
RESULTS
We found that TLR3- and STAT1-deficient cardiomyocytes were not more susceptible to coxsackievirus 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 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.
Keywords: cardiomyocytes, children, genetics, immunity, sequencing, virus
Introduction
Acute myocarditis (AM) is an inflammatory disease of the myocardium. The estimated annual incidence of myocarditis is 1 per 100,000 children in the United States, corresponding to a prevalence of ~1/10,000 (1). The actual incidence of this heterogeneous disease is probably higher because of unrecognized cases.
Myocarditis most typically presents with sudden onset of congestive heart failure (CHF) or cardiogenic shock. Some patients fully recover spontaneously or following symptomatic treatment. Others suffer from cardiac sequelae such as dilated cardiomyopathy (DCM) (1), requiring long-term anticongestive therapy or heart transplantation. In some cases, sudden death occurs (1). Biopsy-proven myocarditis is reported in up to 46% of children with an identified cause of DCM (2). Although myocarditis has many causes, most cases apparently result from infection with viruses, including enteroviruses, adenoviruses, and parvoviruses (1). Such viruses are common, particularly during childhood; it is unclear, therefore, why only a very small proportion of children develop acute viral myocarditis (AVM). Of note, clinical severity is uncorrelated with viral etiology, suggesting a human genetic predisposition. Studies in inbred mice have implicated the major histocompatibility complex and other loci in genetic susceptibility to coxsackievirus-induced myocarditis (3,4). Overall, these observations suggest that human genetic variation, rendering the heart more sensitive to common viral infections, may contribute to the progression of myocarditis, at least in some children.
Severe childhood infectious diseases, including viral diseases striking healthy children, can result from single-gene, inborn errors of immunity (5,6). Some of these immunodeficiencies have tissue-specific phenotypes, which could reflect restricted viral tropism or tissue specificity of the inborn error. Herpes simplex encephalitis (HSE) and severe pulmonary influenza can be due to brain- and lung-intrinsic defects in interferon (IFN) immunity, respectively (6). Interestingly, heart, brain, and lungs are typically the only organs severely affected in patients with AVM, HSE, and severe influenza, respectively, despite the ability of the causal viruses to replicate in many tissues. Moreover, inborn errors of adaptive immunity do not predispose to any of these conditions (7).
Recently, an adult patient with enteroviral AVM carrying a rare, dominant-negative toll-like receptor 3 (TLR3) allele was reported (8). Similarly, mice lacking genes encoding key proteins in antiviral IFN immunity, such as IFN-β and TLR3, were more susceptible to coxsackievirus B3 (CVB3) infection and virus-induced myocardial injury (9,10). Whether variation in these human genes influences the outcome of infection by cardiotropic viruses remains unclear. To test the hypothesis that inborn errors of heart-intrinsic TLR3-IFN immunity may underlie AVM in previously healthy children, we explored the impact of mutations in TLR3-IFN-related genes on antiviral immunity of human cardiomyocytes derived from induced pluripotent stem cells (iPSC) and searched for TLR3-IFN-related gene mutations in AVM using whole exome sequencing (WES). As our results were inconsistent with the TLR3-IFN hypothesis, we then tested the hypothesis that hitherto silent cardiomyopathies may underlie AVM.
Methods
Control and mutant iPSCs carrying deleterious mutations in STAT1 (c.1928_1929insA/c.1928_1929insA) or TLR3 (p.P554S/p.E746*) were derived from primary dermal fibroblasts of healthy donors or patients with severe viral diseases other than AM, respectively (11,12). Using a modified version of the original protocol, iPSCs were differentiated into cardiomyocytes (13). Details of cardiomyocyte differentiation and CVB3 infection studies are described in the Online Appendix.
Patient Recruitment and Ethics
Our subject inclusion criteria were: age between 1 month and 16 years at the onset of cardiac symptoms; presentation with acute onset of chest pain, dyspnea, and/or CHF; cardiac magnetic resonance (CMR) imaging findings consistent with inflammation; recent history of febrile illness; absence of known family history of or metabolic disorder associated with cardiomyopathy; and histological evidence of inflammatory infiltrates within the myocardium on myocardial biopsy or cardiac explant when available, according to the Dallas classification (14). CMR criteria used to diagnose myocardial inflammation were modified from the Lake Louise criteria that apply in the adult population but are less sensitive in children, for whom early gadolinium enhancement (EGE) is difficult to quantify (15): 1) evidence of regional or global myocardial edema with T2 hyperintensity (T2 ratio: 2, where T2 ratio: Signal intensitymyocardium/Signal intensityskeletal muscle); 2) evidence of myocardial hyperemia and capillary leak with EGE on cine-steady-state free precession and/or T1-weighted images (acquired early after contrast injection) compared with the skeletal muscle; and 3) evidence of myocardial necrosis and fibrosis (visual assessment) with nonischemic regional distribution at late gadolinium enhancement imaging. AM was diagnosed when at least 2 of the CMR criteria were present. Overall, a total of 42 (23 female, 19 male) unrelated patients with AM, some with proven AVM, were recruited. Time from symptom onset to diagnosis varied, ranging from a few hours to 3 months. All patients were referred from the Necker Hospital for Sick Children in Paris, France, after a review of the clinical evidence for AM by a clinical expert (D.B.). Clinical history and biological specimens were obtained after consent from the patients and/or their participating family members under an approved protocol in accordance with the institutional, local, and national ethical guidelines.
WES and data analysis are described in the Online Appendix. We excluded polymorphisms with a minor allele frequency (MAF) of ≥1% in public databases: Exome Variant Server; 1000 Genomes; and Exome Aggregation Consortium (ExAC), including exclusion of commoner variants in each ethnic subpopulation in ExAC. Only damaging and missense mutations were retained. We then searched for: 1) heterozygous variations with MAF <0.01% in ExAC (excluding variants with MAF ≥0.1% in any ethnic subpopulation) under an autosomal dominant (AD) model; 2) homozygous or compound heterozygous variants with MAF <1% under an autosomal recessive (AR) model; and 3) monoallelic variations with MAF <0.01% in ExAC (excluding variants with MAF ≥0.1% in any ethnic subpopulation) or biallelic variations with MAF <1% under X-linked genetic models.
We performed an enrichment analysis of variations in 3 pathways: 1) TLR3 cascade; 2) IFN-α/β signaling; and 3) cardiomyopathy-associated genes (Online Tables 1 and 2) in our AM-AVM cohort compared to the 2 control cohorts of 1,164 healthy individuals from 1000 Genomes database and our in-house database of 2,324 exomes from healthy individuals and patients with severe infectious diseases of childhood other than myocarditis.
Statistical Analysis
The proportion of individuals with mutant alleles in each cohort were compared by means of logistic regression using the likelihood ratio test. To account for cohort ethnic heterogeneity, the 3 first principal components of the principal component analysis were systematically included in the logistic regression model (details in the Online Appendix). Bonferroni correction was applied to set the p value threshold for statistical significance: 8.33E-03 (= 0.05/6) as 3 gene lists were tested under 2 different genetic models. We also compared the proportion of individuals with variations in cardiomyopathy-associated genes in the AM cohort with each ethnic ExAC subpopulation using Fisher exact test, as principal components could not be obtained from available ExAC data. Finally, 1-tailed unpaired Student t test was used to compare the Combined Annotation-Dependent Depletion (CADD) scores of rare cardiomyopathy-associated alleles in AM-AVM patients to those of all variations known to cause inherited cardiomyopathy using p < 0.05 as the threshold for statistical significance.
Results
Human iPSC-derived cardiomyocytes are permissive to CVB3 infection (16). Here, we generated cardiomyocytes from iPSCs and selected the SIRPα+/CD90− population, achieving >95% cardiomyocyte purity. We infected control human iPSC-derived cardiomyocyte lines from 3 unrelated healthy individuals with CVB3 at increasing multiplicities of infection (MOI) to assess virus-induced cytotoxicity and viral replication at various time points upon infection. Cardiomyocyte viability was severely decreased upon CVB3 infection, with complete cell death occurring after 24 h post-infection at MOIs >1 (data not shown). CVB3 replication peaked at between 107 and 108 genome copies within 8 h, and remained unchanged up to 72 h post-infection at MOI 1 (data not shown). Moreover, we performed transcriptomic profiling of control cardiomyocytes upon infection with CVB3 or a mutant strain of vesicular stomatitis virus (mVSV), which is unable to attenuate host antiviral immunity (17), at MOI 5 for 8 h. We found that 88 and 155 genes were differentially expressed (>2-fold) in CVB3- and mVSV-infected cardiomyocytes, respectively (Online Figure 1A). Notably, CVB3 infection did not trigger IFNs or IFN-stimulated genes (ISG). Genes dysregulated upon CVB3 were disproportionately in pathways associated with unfolded protein response and cellular senescence, whereas mVSV-induced genes were significantly enriched within virus clearance pathways, indicating that antiviral IFN immunity is intact in human cardiomyocytes (Online Figures 1B and 1C).
We generated cardiomyocytes from iPSC lines carrying naturally occurring mutations in TLR3 or STAT1, genes relevant for IFN-mediated immunity. Of note, the TLR3-deficient line carries the same heterozygous dominant-negative allele (p.P554S) that was reported in a patient with enteroviral myocarditis (8). We infected TLR3- or STAT1-deficient cardiomyocyte lines with CVB3 at increasing MOIs (0.001, 0.01, 0.1, and 1). Although all lines were highly susceptible to CVB3, we did not observe dramatic differences in virus-induced cytotoxicity or viral replication at 24 h post-infection compared to the control (Figures 1A and 1B).
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.
We then assessed viral replication at different time points (2, 4, 8, 12, and 24 h) after infection at various MOIs (0.0001, 0.01, and 1). CVB3 genome copies were not different in TLR3- or STAT1-deficient cardiomyocytes compared to the control (Figure 1C and data not shown). We also measured CVB3 titers in supernatants, but did not observe any drastic changes in virus production over time among control and mutant cardiomyocytes (data not shown). Moreover, we tested whether exogenous IFN-α confers protection against CVB3 infection in cardiomyocytes. Upon pre-treatment with IFN-α2b (1,000 IU/ml) for 18 h, cardiomyocytes were infected with CVB3 at MOI 0.01 and analyzed at 24 h. CVB3 genome copies and virus-induced cytotoxicity were slightly reduced in both control and TLR3-deficient cardiomyocytes, but not altered in STAT1-deficient cardiomyocytes compared to untreated cells (Figures 1D and 1E). However, we did not detect any IFN-induced protection against CVB3 infection at MOI 1 in control or mutant cardiomyocytes (data not shown). Overall, these findings suggested no obvious impact of TLR3-IFN-mediated intrinsic immunity in cardiomyocytes during the course of CVB3 infection at early stages of AVM pathogenesis.
We performed WES of 42 unrelated children (21 European, 10 African, 9 North African, and 2 Middle Eastern). Principal component analysis performed on the WES data confirmed these patients’ ethnic origin (Online Appendix and Online Figure 2). We first searched for variations in the TLR3 cascade (91 genes) and IFN-α/β signaling (59 genes) pathways (Online Table 1), in the AM cohort under different autosomal genetic models. For comparison, and with respect to our patients’ ethnic origin, we applied the same analysis on the sequencing data of 1,164 healthy individuals from the 1000 Genomes who were of African (n = 661) or European (n = 503) origin. Using a Bonferroni corrected p value threshold of 8.33E-03, we did not find any statistically significant enrichment of heterozygous carriers with MAF <0.01% in TLR3 cascade (p = 0.846) or IFN-α/β signaling genes (p = 0.046) in the AM cohort (Table 1). Similarly, there was no statistically significant enrichment of homozygous variants with MAF <1% in the TLR3 cascade (p = 0.980) or IFN-α/β signaling genes (p = 0.807) in patients with AM-AVM (Table 1). Notably, we did not find any AM-AVM patients bearing compound heterozygous variations with MAF <1% in the TLR3 cascade or IFN-α/β signaling genes. Finally, there were no monoallelic (MAF <0.01%) or biallelic (MAF <1%) variations found in TLR3-IFN-α/β-related genes located on chromosome X in the AM cohort. Collectively, these data were inconsistent with the hypothesis that inborn errors of heart-intrinsic TLR3-IFN immunity may underlie AM-AVM.
Table 1.
No Enrichment of Variations in TLR3- or IFN-α/β-related Genes in Patients with AM
| Heterozygous Variations with MAF <0.01%* | |||
|---|---|---|---|
| Pathways | Acute Myocarditis (n = 42) | 1000 Genomes (n = 1,164) | p Value |
| TLR3 cascade | 13 (31.0) | 427 (36.7) | 8.46E-01 |
| IFN-α/β signaling | 15 (35.7) | 337 (29.0) | 4.56E-02 |
| Homozygous Variations with MAF <1%* | |||
| Pathways | Acute Myocarditis (n = 42) | 1000 Genomes (n = 1,164) | p Value |
| TLR3 cascade | 1 (2.4) | 3 (0.3) | 9.80E-01 |
| IFN-α/β signaling | 0 (0) | 6 (0.5) | 8.07E-01 |
Values show the number of individuals, as n (%), carrying heterozygous or homozygous variations in at least one gene.
AM = acute myocarditis; MAF = minor allele frequency.
The occurrence of AM has been associated with the progression of myocardial dysfunction in certain inherited cardiomyopathies, such as arrhythmogenic right ventricular cardiomyopathy (ARVC), left ventricular noncompaction, and Duchenne muscular dystrophy (DMD)-associated DCM (18–20). Therefore, we searched for alterations in 47 genes associated with ARVC, DCM, or left ventricular noncompaction (Online Table 2) in the AM and 1000 Genomes cohorts. We did not find a statistically significant enrichment of heterozygous variations with MAF <0.01% in cardiomyopathy-associated genes in patients with AM-AVM compared to 1000 Genomes cohort (p = 0.223). However, homozygous carriers (MAF <1%) were enriched significantly in the AM cohort compared to the 1000 Genomes, taking into account ethnic heterogeneity (p = 2.22E-03) (Central Illustration, Table 2). Next, we performed the same analyses on our in-house database of 2,324 exomes from healthy individuals and patients with diseases other than myocarditis. Like the analysis using the 1000 Genomes cohort, we found a statistically significant enrichment of homozygous (p = 1.08E-04), but not heterozygous (p = 0.947), individuals with cardiomyopathy-associated rare alleles in the AM cohort (Table 2). Enrichment of homozygous rare variants in cardiomyopathy-associated genes in patients with AM-AVM was further validated by Fisher exact test utilizing the ExAC database of a total 60,706 individuals, and comparing with each ethnic subpopulation (African: p = 1.22E-06; Finnish: p = 5.85E-08; non-Finnish European: p = 7.04E-08; South Asian: p = 1.45E-03; East Asian: p = 6.77E-07; and Latino: p = 3.65E-07) (Online Table 3). Strikingly, we identified 2 AM-AVM patients carrying compound heterozygous variations in cardiomyopathy-associated genes in addition to 5 homozygous carriers. Finally, we searched for X-linked alleles in cardiomyopathy-associated genes such as dystrophin (DMD) and tafazzin (TAZ) in males and females separately, but found no enrichment of monoallelic (MAF <0.01%) or biallelic (MAF <1%) carriers, respectively, in the AM cohort (data not shown). Overall, these findings demonstrated that biallelic, but not monoallelic, rare variations in cardiomyopathy-associated genes are dramatically enriched in patients with AM-AVM.
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 < 8.33E-03
Table 2.
Enrichment of Homozygous Rare Variations in Cardiomyopathy-associated Genes in Patients with AM
| Heterozygous variations with MAF <0.01% | |||
|---|---|---|---|
| Cardiomyopathy-associated genes | Acute Myocarditis (n = 42) | 1000 Genomes (n = 1,164) | In-house (n = 2,324) |
| Number of carriers | 18 (42.9) | 539 (46.3) | 996 (42.9) |
| Myocarditis vs. other cohorts p Value | 2.23E-01 | 9.47E-01 | |
| Homozygous variations with MAF <1% | |||
| Cardiomyopathy-associated genes | Acute Myocarditis (n = 42) | 1000 Genomes (n = 1,164) | In-house (n = 2,324) |
| Number of carriers | 5 (12) | 10 (0.9) | 29 (1.2) |
| Myocarditis vs. other cohorts p Value | 2.22E-03 | 1.08E-04 | |
Values are n (%).
Abbreviations as in Table 1.
We identified 7 (P1 through P7) of 42 patients (16.7%) carrying biallelic rare nonsynonymous or splice-site variants in 6 cardiomyopathy-associated genes (Table 3): P1 with compound heterozygous variations, p.P115S and p.R123Q, in BCL2-associated athanogene 3 (BAG3); P2 with homozygous p.R1458* alleles in desmoplakin (DSP); P3 with homozygous p.A840V substitutions in plakophilin-2 (PKP2); P4 with compound heterozygous variations, p.S688P and p.D829N, in PKP2; P5 with homozygous p.H464Q variants in ryanodine receptor 2, cardiac (RYR2); P6 with homozygous p.F1986L alleles in sodium channel, voltage gated, type V alpha subunit (SCN5A); and P7 with a homozygous, synonymous change, c.G150A, in troponin I type 3, cardiac (TNNI3), which was annotated as a splice-site variant. We validated these sequence alterations and their familial segregation by Sanger sequencing when parental samples were available (Figure 2 and Online Figure 3). The biallelic state of the compound heterozygous variants in BAG3 in P1 was also confirmed on the Integrative Genomics Viewer (Online Figure 3A). The PKP2 A840V substitution in P3 was absent from ExAC, and the remainder of these variants was listed only in heterozygous states with very low allele frequencies in that database (Table 3). Most alleles were not previously reported in any individual with inherited cardiomyopathy with the exception of the p.S688P (rs144601090) allele in P4, for which heterozygous variants had been found in some patients with ARVC (21). Although hereditary cardiomyopathies in humans have generally been associated with AD inheritance, AR traits have also been reported in patients with cardiomyopathies for DSP, PKP2, and TNNI3 (22,23). None of the 7 subjects whom we studied had any manifestation of cardiomyopathy before their AM presentations, which was unequivocal based on elevated troponin levels, CMR, and/or biopsy findings (Table 3). First-degree relatives (including heterozygous siblings) of these 7 individuals underwent electrocardiography and echocardiography when alive, and none had evidence of myocardial disease. The 2 patients (P2 and P7) homozygous for the most severe mutations (nonsense and splicing variants in DSP and TNNI3, respectively) died shortly after disease onset. The other 5 patients, who carry missense variants that might be less deleterious, either underwent heart transplantation (P3) or completely recovered with treatment (P1, P4, P5, and P6). No survivor has developed other cardiac problems to date (Table 3).
Table 3.
Characteristics of Patients with Biallelic Rare Variations in Cardiomyopathy-associated Genes
| General information | Genotype | Phenotype | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Patient | Sex | DOB | Origin | Gene | Mutation | Zygosity | MAF (ExAC)* |
CADD/ MSC† |
Age of Onset |
Troponin Levels |
ECG | Echocardiogram |
| P1 | F | 2009 | Morocco | BAG3 | c.C343T p.P115S |
Het | 8.25E-06 | 1.36/0.001 | 52 months | 2 N | Atrioventricular block | - LVEF 37% - LV dilation: normal dimensions |
| c.G368A p.R123Q |
Het | 2.48E-05 | 22.7/0.001 | |||||||||
| P2 | M | 2010 | Mayotte | DSP | c.C4372T p.R1458* |
Hom | 8.3E-06 | 38/0.001 | 32 months | 4 N | Normal | - LVEF 25% - LV dilation: severely dilated (Z-score: +6.1) - Global hypokinesia, no dyskinesia |
| P3 | F | 1997 | Maghreb/Central Europe | PKP2 | c.C2519T p.A840V |
Hom | 0 | 33/0.001 | 11 yrs | Not done | Normal | - LVEF 20% - LV dilation: severely dilated (Z-score: +4.2) - Global hypokinesia, no dyskinesia - Intra LV thrombosis |
| P4 | M | 1995 | Congo | PKP2 | c.T2062C p.S688P |
Het | 3.30E-05 | 29/0.001 | 16 yrs | 6 N | - Premature ventricular beats - Diffuse negative T waves |
- LVEF 55% - LV dilation: mildly dilated (Z-score: +2.3) - Hypokinesia apex and lateral wall |
| c.G2485A p.D829N |
Het | 4.53E-04 | 24.1/0.001 | |||||||||
| P5 | M | 2009 | France | RYR2 | c.C1392A p.H464Q |
Hom | 4.99E-05 | 1.51/13.58 | 1 month | 8 N | Supraventricular tachycardia | - LVEF 10% - LV dilation: mildly dilated (Z-score +2.3) - Global hypokinesia-hyperechogenicity of mitral papillary muscles |
| P6 | F | 2009 | La Reunion/France | SCN5A | c.T5956C p.F1986L |
Hom | 2.02E-03 | 1.35/0.003 | 9 months | 8 N | Normal | - LVEF 10% - LV dilation: mildly dilated (Z-score +5) - Moderate mitral regurgitation |
| P7 | F | 2009 | France | TNNI3 | c.G150A pK50K |
Hom | 4.21E-05 | 22.1/5.85 | 3 yrs | Not done | Negative T waves in V4, V5, V6 | - LVEF 13% - LV dilation: moderately dilated (Z-score +3.1) - Global hypokinesia |
| Phenotype | ||||||||
|---|---|---|---|---|---|---|---|---|
| Patient | CMR | Myocardial Biopsy |
Viral Etiology | Clinical Diagnosis |
Treatment at Diagnosis | Transplant | Family History |
Follow-up |
| P1 | Acute myocarditis (3 criteria - diffuse T2 signal hyperintensity) | Not done | None | Myocarditis | - Immunoglobulins + steroids - IV inotropes |
No | No | - LVEF 55% at 3 months, normal at 1 yr - Normal CMR |
| P2 | Acute myocarditis (3 criteria - diffuse T2 signal hyperintensity) | Not done | None | Myocarditis | - Immunoglobulins + steroids - IV inotropes |
No | No | - Death 3 months after diagnosis after intra LV thrombosis - No LV function recovery |
| P3 | Acute myocarditis (2 criteria - T2 signal hyperintensity + LGE) | Yes, myocarditis | CMV (Blood) | DCM/Myocarditis | - No specific treatment - IV inotropes |
Yes | No | - NA |
| P4 | Acute myocarditis (3 criteria - diffuse T2 signal hyperintensity) | Not done | None | Myocarditis | - Immunoglobulins + steroids - IV inotropes |
No | No | - Normal LVEF - Control CMR (10 months): LGE limited to 1 segment of lateral wall, normal LVEF, and normal RV |
| P5 | Acute myocarditis (2 criteria - diffuse EGE, diffuse LGE) | Not done | Enterovirus (Blood) | Myocarditis | - Immunoglobulins + steroids - IV inotropes |
No | No | - Normal LV function after 6 months - No control CMR |
| P6 | No CMR | Not done | None | Myocarditis | - No specific treatment - IV inotropes |
No | No | - Normal LVEF at last follow-up: 60 months |
| P7 | No CMR | Yes, myocarditis | Parvovirus B19 (LV, thymus, lymph nodes) | Fulminant myocarditis | - Immunoglobulins - IV Inotropes - Ventricular assist device |
No | No | - Death 6 months after diagnosis |
MAF in Exome Aggregation Consortium (ExAC) database with a total 60,706 individuals.
Any variant with a Combined Annotation-Dependent Depletion (CADD) score equal to or above the mutation significance cutoff (MSC) value for that specific gene is considered to be potentially damaging.
CMR = cardiac magnetic resonance; CMV = cytomegalovirus; DCM = dilated cardiomyopathy; DOB = date of birth; ECG = electrocardiogram; EGE = early gadolinium enhancement; Het = heterozygous; Hom = homozygous; IV = intravenous; LGE = late gadolinium enhancement; LV = left ventricular; LVEF = left ventricular ejection fraction; RV = right ventricular; other abbreviations as in Table 1.
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.
We investigated the functional impact of the c.G150A allele in TNNI3 which is a synonymous variation (p.K50K) located in exon 4, 1 bp from the exon 4-intron 4 junction (Online Figures 3G and 4A). In vitro exon-trapping experiments showed that the consensus splice donor site of intron 4 was utilized in most the wild-type splice-variants (79.1%), whereas this was rare for the mutant (0.6%) (Online Figure 4B). This suggested that the c.G150A substitution causes aberrant splicing of TNNI3 messenger ribonucleic acid consistent with its annotation. We then conducted an analysis of the impact of all AM-AVM-associated sequence variants (i.e., damaging versus benign), using an in silico prediction algorithm, CADD, together with the mutation significance cutoff (MSC) score, which provides a clinically and biologically relevant CADD cutoff value for genes (24,25). For each gene, any variant with a CADD score equal to or above the MSC score is considered potentially damaging. Of the 9 cardiomyopathy-associated variations found in the 7 AM-AVM patients studied, 8 had CADD scores above the MSCs of their respective genes; only the RYR2 p.H464Q allele was below its threshold (Table 3). The CADD scores of rare cardiomyopathy-associated alleles in AM-AVM patients were mostly among lower percentiles (<50th) of ARVC- and/or DCM-causing mutations obtained from the Human Gene Mutation Database (Figure 3) (26) and significantly lower than those of all cardiomyopathy-causing variations (p = 2.23E-03). Among the 6 genes, BAG3, RYR2, and SCN5A were associated with only AD traits, while DSP, PKP2, and TNNI3 were associated with both AD and AR traits (22,23).
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.
Interestingly, the mutations found in DSP, PKP2, and TNNI3 in the AM-AVM patients had CADD scores that were not significantly different from those of cardiomyopathy-causing variations (p = 0.44) (Figure 3A). The same result was observed when the comparison was restricted to biallelic variations of these genes known to cause AR cardiomyopathy (p = 0.48), and for which heterozygous carriers were known to be healthy. Conversely, for BAG3, RYR2, and SCN5A, the CADD scores of rare cardiomyopathy-associated alleles in the AM-AVM patients were markedly lower than those of all cardiomyopathy-causing variations (p = 4.06E-05) (Figure 3B). This suggested that AM-AVM-associated variants in these genes are probably milder than those causing AD cardiomyopathy, being asymptomatic in heterozygotes, and only manifesting clinically in biallelic carriers who developed AM or AVM.
Discussion
Separating AVM from DCM has long posed a significant clinical challenge (27,28). When AVM presents in childhood, as it often does, the meaning of a history of recent viral infections is dubious given their prevalence in that age group. Obtaining histologic proof of myocarditis is also difficult; there is significant risk associated with procuring the tissue in critically ill young patients (29) and, even when performed, the disease’s patchy nature reduces the diagnostic yield (30). Earlier attempts to document recent viral infections with acute and convalescing antibody titers or culturing of the agents from the myocardium or blood were minimally successful. More recent studies amplifying portions of viral genomes from myocardial biopsies have been somewhat more robust and have highlighted the shifting viral etiologies across time (28,31). Finally, AVM can present in post-acute phases when virus is no longer detectable and active inflammation has resolved, leaving scarring that is not readily differentiated from genetically mediated DCM. For these reasons, no virus has been unambiguously proven to cause AVM in humans, in the sense that herpes simplex virus can cause encephalitis. Accordingly, the search for viruses in children with AVM is not a standard diagnostic procedure and its result has unclear therapeutic implications. The data presented in our study provided further reason for believing that AVM and DCM are inextricably linked.
An extensive body of work supports the notion that viruses such as enteroviruses and adenovirus infect the myocardium, activating intrinsic (nonhematopoietic), innate (hematopoietic), and acquired immunity (27). Some patients who clear the virus from the myocardium completely recover without significant sequelae. For others, an inappropriate immune reaction ensues, leading to a chronic and irreversible cardiomyopathy. Given these drastically disparate outcomes and seeming dependence on immune responses, we considered whether variation in antiviral immunity genes might underlie the difference. The ongoing discovery of inborn errors of cell-intrinsic immunity underlying susceptibility to rare infections or rare complications of common infections, often in a tissue-specific fashion, provided a strong rationale for this possibility in AVM as did a recent report of a patient with enteroviral myocarditis who harbored a rare dominant-negative TLR3 allele (8). Despite that, the human genetic data we produced disclosed no significant burden of mutations altering innate and intrinsic immunity in patients with AM that was presumptively viral in etiology. Moreover, our in vitro models, in which normal human cardiomyocytes and those deficient in TLR3 or STAT1 were infected with CVB3, showed no induction of IFNs and ISGs in control cardiomyocytes and no enhanced susceptibility to infection in mutant cells compared with control cells. Therefore, the totality of our data pointed away from AM due to inborn errors of cardiomyocyte-intrinsic immunity in most affected individuals. Our previous findings with HSE and severe pulmonary influenza do not apparently apply to AVM (6).
In contrast, we discovered that rare alleles altering cardiac-specific genes previously associated with typically dominant genetic cardiomyopathies underlie AM but are mediated through an autosomal recessive mechanism. While unexpected, our findings have precedent. First, another severe disease of childhood, acute necrotizing encephalopathy, develops following common viral infections but has no obvious connection to antiviral immunity. Rather, familial acute necrotizing encephalopathy results from mutations in RANBP2, which encodes a component of the nuclear pore, Ran binding protein 2 (32). Second, AM has been shown to play a role in the progression of myocardial dysfunction in several inherited cardiomyopathies. A viral etiology has been directly implicated in acute deteriorations in hypertrophic cardiomyopathy (33) and DMD-associated DCM (34) through polymerase chain reaction identification of viral genomes in myocardial samples. For ARVC, myocarditis has been documented histologically in association with clinical deterioration (19), and viral genomes have been detected in ARVC myocardial samples at higher rates than in controls (35).
A critical question that arises from this work is whether AVM is merely a second hit revealing inherent myocardial abnormalities of a genetic nature or whether the genetic defects render the myocardium more susceptible to viral infections (36,37), perhaps through infectivity enhanced through a loss of membrane integrity. It has been shown that enteroviral protease 2A cleaves dystrophin, the protein altered in DMD, in a specific fashion, and that rendering dystrophin cleavage resistant through mutagenesis produces AVM resistance (38,39). These elegant mouse studies implied that disruption of a cardiac structural protein is critical for enteroviral-host myocardial interaction, whether by facilitating initial infection, viral replication, or viral exit from the cardiomyocytes. By extrapolation, boys with DMD are more susceptible to AVM since their dystrophin is already disrupted. Whether this mechanism is relevant for other viruses and other cardiac proteins remains to be determined.
Study Limitations
We were not able to document a viral etiology in all the AM cases for which mutations of cardiomyopathy genes were found due to a lack of available tissue. However, the patients in our AM cohort had clear evidence of myocardial inflammation (e.g., troponin leak, CMR findings) and our positive histological findings in those for whom we did have biopsies established the concept. We cannot exclude the possibility that some of the AM cases we studied were unrelated to a viral infection, perhaps attributable solely to the genetic defects. Another limitation in this study is the sample size for genetic analyses.
Conclusions
Our data suggest that AM is not primarily caused by inborn errors of TLR3-IFN immunity, but appears to be more commonly related to defects in cardiac structural proteins. Until now, AM has been classified as a nonfamilial, acquired disorder except when observed rarely in certain inherited cardiomyopathies, particularly ARVC; per the present study, this nosology might need to be revisited. Future work on AM is needed to determine if the myocardium of such patients is more susceptible to viral infections per se, in which case these defects would define novel mechanism of cell-intrinsic immunity operating in the heart, or whether common viral infections merely and indirectly destabilize inherently vulnerable hearts.
Supplementary Material
Supplemental Figure 1. Microarray analysis of CVB3- or mVSV-infected iPSC-derived cardiomyocytes. A. The representative heatmap shows differentially up- or down-regulated (>2-fold) genes in CVB3- or mVSV-infected CMs compared to the mock infected. B. Top 5 over-represented biological pathways (False discovery rate-, or FDR, adjusted P<0.05) among differentially regulated genes (>2-fold) in CVB3-infected CMs compared to the mock-infected cells. C. Top 10 over-represented biological pathways (FDR-adjusted P<0.05) among differentially regulated genes (>2-fold) in mVSV-infected CMs compared to the mock-infected CMs.
Supplemental Figure 2. Principal component analysis of the patients with acute myocarditis. Principal component (PC) analysis of 42 patients with acute myocarditis, 2324 individuals from our in-house database and 2504 healthy individuals of the 1000 Genomes database was performed with PLINK software v1.9. First two PCs (left), and first and third PCs (right) were plotted.
Supplemental Figure 3. Validation of alleles in patients with acute myocarditis and their familial segregation by Sanger sequencing. Sanger sequencing of variations in six cardiomyopathy-associated genes in patients are shown by chromatograms. A. Family 1. Biallelic state of compound heterozygous variations in BAG3 in P1 was also confirmed on the IGV by viewing as pairs (bottom panel). B. Family 2. C. Family 3. D. Family 4. E. Family 5. F. Family 6. G. Family 7. Carrier testing for some family members was not possible, as their samples were not available.
Supplemental Figure 4. TNNI3 c.G150A variation causes aberrant splicing of TNNI3 mRNA. A. Cloning of wild-type (WT) and mutant (MUT) TNNI3 genomic segments into pSPL3 vector for the in vitro exon-trapping experiment. B. Percentages of different splice variants (V1–V5) generated by WT or MUT TNNI3 clones are shown.
Supplemental Table 1. List of TLR3 cascade and IFN-α/β signaling genes.
Supplemental Table 2. List of genes associated with inherited cardiomyopathies: ARVC, DCM, and LVNC.
Supplemental Table 3. Enrichment of homozygous variations in cardiomyopathy-associated genes in patients with acute myocarditis compared to ExAC database.
PERSPECTIVES.
COMPETENCY IN MEDICAL KNOWLEDGE
Certain autosomal recessive defects in genes encoding various components of cardiac structure can predispose to acute myocarditis in young individuals.
TRANSLATIONAL OUTLOOK
Further studies are needed to elucidate the mechanisms by which mutations altering structural cardiac proteins are associated with acute myocarditis, often in response to common viral infections.
Acknowledgments
We gratefully thank the patients and their family members for their contribution to this project. We acknowledge the Flow Cytometry Center of Research Excellence at the Icahn School of Medicine at Mount Sinai and Genomics Resource Center at the Rockefeller University. We also thank Lei Shang, Bertrand Boisson, and Vimel Rattina for their assistance with bioinformatics.
ABBREVIATIONS AND ACRONYMS
- AM
acute myocarditis
- ARVC
arrhythmogenic right ventricular cardiomyopathy
- AVM
acute viral myocarditis
- CVB3
coxsackievirus B3
- DCM
dilated cardiomyopathy
- iPSC
induced pluripotent stem cells
- MAF
minor allele frequency
- WES
whole exome sequencing
Footnotes
Financial Disclosure: All authors have nothing to disclose. This study is supported in part by the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH) (#2R01NS072381), the French National Research Agency (ANR) (# IEIHSEER ANR-14-CE14-0008-01), and under the “Investments for the future” program (#ANR-10-IAHU-01), the Institut National de la Santé et de la Recherche Médicale (INSERM), Paris Descartes University, the St. Giles Foundation, the Rockefeller University, and the Howard Hughes Medical Institute (HHMI) (J.-L.C.), and the Association pour la Recherche en Cardiologie du Fœtus à l’Adulte (ARCFA) (D.B.). Dr. Belkaya is a recipient of Postdoctoral Fellowship (#16POST27260016) from the American Heart Association and an Early Career Award (#12775) from the Thrasher Research Fund. Dr. Kontorovich is a recipient of the Glorney-Raisbeck Fellowship in Cardiovascular Diseases from the Corlette Glorney Foundation and The New York Academy of Medicine. Funders have no role in the design and conduct of the study, in the collection, analysis, and interpretation of the data, and in the preparation, review, or approval of the manuscript.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplemental Figure 1. Microarray analysis of CVB3- or mVSV-infected iPSC-derived cardiomyocytes. A. The representative heatmap shows differentially up- or down-regulated (>2-fold) genes in CVB3- or mVSV-infected CMs compared to the mock infected. B. Top 5 over-represented biological pathways (False discovery rate-, or FDR, adjusted P<0.05) among differentially regulated genes (>2-fold) in CVB3-infected CMs compared to the mock-infected cells. C. Top 10 over-represented biological pathways (FDR-adjusted P<0.05) among differentially regulated genes (>2-fold) in mVSV-infected CMs compared to the mock-infected CMs.
Supplemental Figure 2. Principal component analysis of the patients with acute myocarditis. Principal component (PC) analysis of 42 patients with acute myocarditis, 2324 individuals from our in-house database and 2504 healthy individuals of the 1000 Genomes database was performed with PLINK software v1.9. First two PCs (left), and first and third PCs (right) were plotted.
Supplemental Figure 3. Validation of alleles in patients with acute myocarditis and their familial segregation by Sanger sequencing. Sanger sequencing of variations in six cardiomyopathy-associated genes in patients are shown by chromatograms. A. Family 1. Biallelic state of compound heterozygous variations in BAG3 in P1 was also confirmed on the IGV by viewing as pairs (bottom panel). B. Family 2. C. Family 3. D. Family 4. E. Family 5. F. Family 6. G. Family 7. Carrier testing for some family members was not possible, as their samples were not available.
Supplemental Figure 4. TNNI3 c.G150A variation causes aberrant splicing of TNNI3 mRNA. A. Cloning of wild-type (WT) and mutant (MUT) TNNI3 genomic segments into pSPL3 vector for the in vitro exon-trapping experiment. B. Percentages of different splice variants (V1–V5) generated by WT or MUT TNNI3 clones are shown.
Supplemental Table 1. List of TLR3 cascade and IFN-α/β signaling genes.
Supplemental Table 2. List of genes associated with inherited cardiomyopathies: ARVC, DCM, and LVNC.
Supplemental Table 3. Enrichment of homozygous variations in cardiomyopathy-associated genes in patients with acute myocarditis compared to ExAC database.