The Cardiac Myosin Binding Protein C Arg502Trp Mutation: A Common Cause of Hypertrophic Cardiomyopathy

Abstract

Rationale

MYBPC3 variant Arg502Trp has been identified in multiple HCM cases, but compelling evidence to support or refute the pathogenicity of this variant is lacking.

Objective

To determine the prevalence, origin and clinical significance of the MYBPC3 Arg502Trp variant.

Methods and Results

The prevalence of MYBPC3 Arg502Trp was ascertained in 1414 sequential Caucasian HCM patients. Segregation of MYBPC3 Arg502Trp with clinical status was assessed in family members. Disease haplotypes were examined in 17 families using two loci flanking MYBPC3. MYBPC3 Arg502Trp was identified in 34 of 1414 unrelated HCM patients. Family studies identified an additional 43 variant carriers, many with manifest disease, yielding a calculated odds ratio of 11,000:1 for segregation of MYBPC3 Arg502Trp with HCM. Analyses in 17 families showed at least 4 independent haplotypes flanked MYBPC3 Arg502Trp. Eight individuals (4 probands and 4 family members) also had another sarcomere protein gene mutation. Major adverse clinical events occurred in approximately 30% of MYBPC3 Arg502Trp carriers by age 50; these were significantly more likely (p≤0.0001) when another sarcomere mutation was present.

Conclusion

MYBPC3 Arg502Trp is the most common and recurrent pathogenic mutation in a diverse Caucasian HCM cohort, occurring in 2.4% of patients. MYBPC3 Arg502Trp conveys a 340-fold increased risk for HCM by 45 years of age, when more than 50% of carriers have overt disease. HCM prognosis worsens when MYBPC3 Arg502Trp occurs in the setting of another sarcomere protein gene mutation.

INTRODUCTION

HCM is an autosomal dominant disorder characterized by unexplained LVH in a non-dilated ventricle. HCM displays marked variability in penetrance and expressivity. Some patients exhibit massive hypertrophy with heart failure, arrhythmias, and sudden cardiac death while others have mild hypertrophy, late onset disease, and minimal symptoms^1–3. To date, over 800 different mutations in 10 sarcomere protein genes responsible for HCM have been identified^{4, 5}. The prevalence of sarcomere protein gene mutations is estimated to be 1 in 300, making HCM a very common monogenic cardiovascular disorder⁶. Disease-causing genotype influences clinical course⁷, as is illustrated by the particularly severe manifestations of HCM in individuals with compound mutations^{1, 8}. Despite recognized correlations between clinical phenotypes and HCM mutations (e.g., delayed expression of MYBPC3 mutations and early-onset severe disease of specific MYH7 mutations)^{7, 9}, defining clinical consequences of most mutations has been thwarted by the large number of unique pathogenic mutations and the intrinsic genetic and life-style diversity even among related patients who share an identical HCM mutation.

A variant (1504C>T) in exon 17 of MYBPC3 encoding Arg502Trp has been repeatedly recognized^{1, 8, 10–12}, but its pathogenicity in HCM remains untested. Here we demonstrate that MYBPC3 Arg502Trp is a pathogenic missense mutation and the most common cause of HCM in a diverse Caucasian population. We report the clinical consequences of this mutation alone and in combination with other HCM mutations.

METHODS

Standard methods for DNA isolation and analyses were employed and are described in the expanded Methods section available in the Online Data Supplement at http://circres.ahajournals.org.

RESULTS

Thirty-four of 1414 (2.4%) consecutive unrelated HCM probands who underwent genetic testing (Supplement Methods), harbored the MYBPC3 Arg502Trp variant. MYBPC3 residue 502 has been highly conserved during evolution (Online Figure I). Insertion of tryptophan at position 502 substitutes a nonpolar hydrophobic residue for the polar uncharged residue arginine.

MYBPC3 Arg502Trp co-segregated with HCM in families harboring only this sarcomere protein gene variant (Online Figure II). MYBPC3 Arg502Trp was absent in 545 healthy control subjects (n=95, this study; n=150¹; n=100 Caucasian Americans and n=100 African Americans⁸; n=100¹¹). The combined calculated LOD score for HCM in MYBPC3 Arg502Trp families = 4.04 (θ = 0, assuming penetrance = 85% in subjects ≥ 20 years of age, and allele frequency = 0.001); the likelihood that MYBPC3 Arg502Trp and HCM co-segregated by random chance was less than 1/10,000. We conclude that MYBPC3 Arg502Trp is a pathogenic HCM mutation.

Haplotype analyses were performed in 17 of the 34 MYBPC3 Arg502Trp families (Online Methods and Figure II) to determine if a founding mutation in a common ancestor accounted for its high prevalence, or if MYBPC3 Arg502Trp arose repeatedly in unrelated HCM families. Haplotypes were assessed using short tandem repeat (STR) sequences that flank MYBPC3, 102 kilobases (kb) downstream and 188 kb upstream. Four unique haplotypes were identified (Table 1) with the most frequent haplotype, designated 4,5, seen in 13 families.

Table 1.

Haplotypes flanking the MYBPC3 Arg502Trp mutation.

STR 1	STR 2	Number of families
4	5	13
4/5	3	1
1	5	1
3	4/5	2

Loci STR 1 chr11: 47497698 and STR 2 chr11:47207103 flank the MYBPC3 gene. The allele at each locus was determined by segregation analysis. In three families the allele at one locus could not be unambiguously defined (noted as 4/5).

Forty-three of 56 at-risk relatives in 34 families carried the MYBPC3 Arg502Trp mutation. Clinical information was available for 58 of 77 subjects (probands and family members) with the MYBPC3 Arg502Trp mutation (Online Table I). 45 of 77 mutation carriers (78%) had clinical evidence of HCM, including hypertrophy, sudden cardiac death (SCD), or implanted cardioverter defibrillators (ICD).

The age of HCM diagnosis for Arg502Trp carriers relative to MYBPC3 truncation mutations and to MYH7 Arg719Trp, a mutation that causes severe HCM, carriers was compared (Figure 1a). There was no significant difference in ages at diagnosis for 48 subjects who carried only the MYBPC3 Arg502Trp mutation versus carriers of MYBPC3 truncation mutations (p=0.5). However, the age at diagnosis of HCM in carriers of MYH7 Arg719Trp was significantly earlier than for carriers of MYBPC3 Arg502Trp (p<0.0001). While overt disease was recognized in nearly all carriers of MYH7 Arg719Trp by age 30, approximately 50% of MYBPC3 Arg502Trp carriers lacked clinical evidence of HCM until after age 45 (Figure 1a).

Figure 1.

A) Kaplan-Meier curves comparing the age of HCM onset (based upon imaging studies, sudden cardiac death, or ICD implant) of individuals with MYBPC3 Arg502Trp mutation (-□-), MYH7 Arg719Trp mutation (-▽-), and individuals with truncation MYBPC3 mutations (-▲-). The curves for MYBPC3 Arg502Trp and truncation mutations were not significantly different (p=0.2568). MYH7 Arg719Trp mutation carriers were significantly more likely to develop HCM at at a younger age than MYBPC3 Arg502Trp mutation carriers (p<0.0001). B) Kaplan-Meier curves comparing the age of first adverse cardiac event (sudden cardiac death, ICD implant, transplant or myectomy) for MYBPC3 Arg502Trp mutation carriers (-□-), MYH7 Arg719Trp mutation (-▽-) carriers and MYBPC3 Arg502Trp plus another sarcomere gene mutation (-×-) carriers. Individuals with MYH7 Arg719Trp mutation or with MYBPC3 Arg502Trp plus another sarcomere mutation were significantly more likely to have early adverse events than individuals with the MYBPC3 Arg502Trp mutation alone (p<0.0001).

Eight HCM subjects harbored both MYBPC3 Arg502Trp and another sarcomere protein gene mutation (Online Table III and Online Figure IIb). Based upon patient age at the time of a significant adverse event (e.g., age at sudden cardiac death, ICD implantation, myectomy, or cardiac transplantation), the clinical course of subjects with compound MYBPC3 Arg502Trp mutations was similar to subjects with the severe MYH7 Arg719Trp mutation, and was significantly worse (p≤0.0001) than the clinical course of subjects with an isolated MYBPC3 Arg502Trp mutation (Figure 1b). Approximately 30% of HCM patients with an isolated MYBPC3 Arg502Trp mutation had a significant cardiac event by age 50, with 10% of these occurring before age 20. In comparison 75% of carriers of MYBPC3 Arg502Trp and another sarcomere protein gene mutations experienced an adverse cardiac event by age 20.

DISCUSSION

We demonstrate that MYBPC3 Arg502Trp is a prevalent pathogenic mutation occurring in 2.4% of 1414 unrelated Caucasian patients. Haplotype data indicates that this incidence is accounted for by recurrent mutation of this residue rather than a founding MYBPC3 Arg502Trp mutation. Clinical manifestations in mutation carriers indicate that in isolation, MYBPC3 Arg502Trp causes relatively mild HCM, with expression that can be delayed until middle age. However, when MYBPC3 Arg502Trp is combined with another sarcomere protein gene mutation, HCM is severe with adverse clinical outcomes occurring at young ages. Recognition of this prognostic information is particularly important given the prevalence of MYBPC3 Arg502Trp.

A few founding HCM mutations have been defined, and all occur in the MYBPC3 gene. The population prevalence of founding MYBPC3 mutations that have been identified in Europe (2373_2374insG), India (25-bp deletion in intron 32), and Finland (Gln1061X)^{11, 13, 14} are attributed to neutral evolutionary selection. While analyses of the MYBPC3 Arg502Trp mutation revealed one prevalent haplotype (4,5; Table 1) in 75% of mutation carriers, the 4,5 haplotype was also common throughout this study cohort (data not shown). As such, we could not determine if one founding mutation accounted for disease in multiple families or if multiple independent mutations arose on this common haplotype. Regardless, because MYBPC3 Arg502Trp was also identified on 3 other haplotypes, our data are consistent with independent mutations that occurred at least four times. Further evidence that MYBPC3 codon 502 is a mutational hotspot comes from two other HCM mutations that alter arginine502 to either glutamine (Arg502Gln)⁷ or to leucine (Arg502Leu) (unpublished). The cause for multiple mutational events at residue 502 is likely a CG dinucleotide, which has been associated with other mutational hotspots¹⁵.

Some founding mutations provide a selective advantage, such as the hemoglobin mutation Glu6Val, which causes sickle cell disease but also conveys resistance to malarial infection¹⁶, therein accounting for its prevalence in the general population. Unlike these mutations, MYBPC3 Arg502Trp has only been associated with adverse cardiac events (Figure 1b) that can reduce life expectancy. Moreover, MYBPC3 Arg502Trp has a low prevalence in the general population (estimated at 1/20,000 individuals, based upon a 2.4% frequency in this HCM cohort, and the prevalence of HCM). Taken together with haplotype data, we suggest that the deleterious consequences of MYBPC3 Arg502Trp result in negative selection, but new mutation increases its’ prevalence among HCM patients. A prediction of this model is that MYBPC3 Arg502Trp will also be a relatively common cause of HCM in non-Caucasian patients.

The clinical course of HCM observed in more than 80 patients with MYBPC3 Arg502Trp reported here and elsewhere^{1, 8, 10, 11} appears comparable to disease caused by MYBPC3 truncation mutations and carries a better prognosis than that associated with several MYH7 mutations (e.g., MYH7 Arg719Trp; Figure 2a). We note however, that even this milder spectrum of HCM has important impact on health, as 30% of MYBPC3 Arg502Trp carriers had an adverse cardiac event by age 50 (Figure 1b).

The high prevalence (2.4%) of MYBPC3 Arg502Trp mutation contributed to finding compound mutations in four families. In comparison to patients with only MYBPC3 Arg502Trp, patients with a second sarcomere protein gene mutation had more severe disease, and approximately 75% had an ICD, SCD, or a myectomy before 20 years of age. Although the clinical consequences associated with these particular sarcomere mutations (MYBPC3 Ser858Asn, MYBPC3 Glu542Gln, MYBPC3 Gly148Arg or MYH7 Asp587Asn) are unknown, they are more likely to cause typical rather than severe HCM. Rather, we suggest that a high dosage of mutant protein, due to two mutations, accounts for disease severity, as has been previously observed with other compound mutations¹. Consistent with this model, we note that segregation analyses in families F001849 and HCM-345 indicated that the 2 MYBPC3 mutations occurred in trans, which predicts that no normal MYBPC3 protein would be found in these patients’ hearts.

In conclusion, MYBPC3 Arg502Trp is a common pathogenic mutation that increases the risk for HCM by 340-fold. Identification of MYBPC3 Arg502Trp as an isolated HCM mutation or in combination with another sarcomere gene mutation provides prognostic information that should guide patient management and counseling.

Novelty and Significance.

What is known?

• More than 800 different sarcomere protein gene mutations, of which >200 are in the MYBPC3 gene, cause hypertrophic cardiomyopathy (HCM).

• Some sarcomere protein gene mutations cause worse disease than others.

What new information does this article contribute?

• The MYBPC3 Arg502Trp mutation is the most common HCM-causing mutation among individuals of European descent from several North American clinics, and has a penetrance of about 50% (increased relative risk of 340) by age 45.

• The high frequency of this mutation reflects a high mutation frequency rather than a founder effect.

Mutations in genes encoding components of the heart’s contractile apparatus cause the inherited human disease HCM, most commonly recognized as the cause of 50% of deaths that occur on the athletic field. While some HCM-causing mutations cause severe disease, other mutations in the same genes cause milder disease. The MYBPC3 Arg502Trp mutation, the most common HCM-causing mutation found in a North American population, causes milder disease than many other HCM mutations. However 30% of mutation carriers have a serious event by age 40. The high frequency of mutation carriers reflects new mutations rather than a founder mutation.

Non-standard Abbreviations and Acronyms

HCM

Hypertrophic Cardiomyopathy

MYBPC3

Myosin binding protein C isoform 3

Arg502Trp

Arginine to tryptophan mutation at residue 502 of MYBPC3

LVH

Left ventricular hypertrophy