MYBPC3's alternate ending: consequences and therapeutic implications of a highly prevalent 25 bp deletion mutation

Abstract

Hypertrophic cardiomyopathy (HCM) is the most common form of inherited cardiac disease and the leading cause of sudden cardiac death in young people. HCM is caused by mutations in genes encoding contractile proteins. Cardiac myosin binding protein-C (cMyBP-C) is a thick filament contractile protein that regulates sarcomere organization and cardiac contractility. About 200 different mutations in the cMyBP-C gene (MYBPC3) have thus far been reported as causing HCM. Among them, a 25 base pair deletion in the branch point of intron 32 of MYBPC3 is widespread, particularly in South Asia, where it affects ≈4% of South Asian descendants worldwide. This polymorphic mutation results in skipping of exon 33 and a reading frame shift, which, in turn, replaces the last 65 amino acids of the C-terminal C10 domain of cMyBP-C (cMyBP-C^C10mut) with a novel sequence of 58 residues. Carriers of the 25 base pair deletion mutation are at increased risk of developing cardiomyopathy and heart failure. Because of the high prevalence of this mutation in certain populations, genetic screening of at-risk groups might be beneficial. Scientifically, the functional consequences of C-terminal mutations and the precise mechanisms leading to HCM should be defined using induced pluripotent stem cells and engineered heart tissue in vitro, or mouse models in vivo. Most importantly, therapeutic strategies that include pharmacology, gene repair and gene therapy should be developed to prevent the adverse clinical effects of cMyBP-C^C10mut. This review article aims to examine the effects of cMyBP-C^C10mut on cardiac function, emphasizing the need for the development of genetic testing and expanded therapeutic strategies.

Introduction

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. HCM has a prevalence of 1:500 in the general population [24] and is defined by the presence of left ventricular hypertrophy (LVH) that cannot be explained by cardiac or systemic diseases [9]. The clinical phenotype of HCM is heterogeneous and ranges from diastolic dysfunction and atrial fibrillation to heart failure and sudden cardiac death [22,13]. Interestingly, sudden death among athletes is predominantly associated with HCM [23,20]. Characteristic morphological and histological changes observed in HCM include LVH, which is defined as LV wall thickness greater than, or equal to, 15 mm (although 13–14 mm is considered borderline) [9]. Typically, LVH is not uniform, with most HCM patients only showing hypertrophy of the interventricular septum [18]. Apart from hypertrophy, cardiomyocyte disarray and interstitial fibrosis are observed, which may potentially contribute to the impairment of diastolic function and arrhythmias [25].

HCM is caused by mutations in genes encoding for sarcomeric proteins. These mutations are autosomal dominant, and most patients are heterozygous carriers of the disease-causing mutation. In particular, mutations in MYBPC3 (encoding cardiac myosin binding protein-C) and MYH7 (encoding β-myosin heavy chain) each contribute 30–40% of HCM-associated mutations. Discovering that mutations in MYBPC3 lead to HCM [2,47] has hastened the pace of research aimed at understanding the role of cMyBP-C. Studies recently reported a polymorphic HCM-causing mutation [46,5], a 25 base pair (bp) deletion, in MYBPC3 that is highly prevalent in South Asian countries [41] and is estimated to affect 55 million people. However, the pathogenic mechanism of the mutation is completely unknown. Therefore, this review article will examine this mutation, describe the current literature, and emphasize the need for systematic studies to determine the cause of HCM, develop new diagnostic methods and advance therapeutic care.

Mutations in MYBPC3 cause HCM

Mutations in MYBPC3 are the most common cause of HCM and account for around 40% of all reported mutations [34]. Thus far, at least 197 HCM-associated mutations in MYBPC3 have been identified [15]. Most MYBPC3 mutations are predicted to result in protein truncation of the C-terminus, either by interfering with normal mRNA splicing, leading to exon skipping, or by causing a reading frame shift resulting in a nonsense coding sequence followed by a premature stop in translation [2,47]. Mutations in other sarcomeric genes typically result in the expression and incorporation of dominant negative mutant proteins, or poison polypeptides, into the sarcomere where they affect function. In human patients harboring a truncation mutation, mutant MYBPC3 is transcribed to mRNA; however, mutant cMyBP-C proteins are not detectable, and, overall, cMyBP-C in the sarcomere is significantly reduced [35,26,44]. These findings strongly support the idea that most MYBPC3 mutations cause HCM through haploinsufficiency. Certain mouse models of MYBPC3 truncation mutations provide additional evidence that haploinsufficiency is sufficient to cause HCM, as these animals express no, or very little, mutant protein, causing reduced overall levels of cMyBP-C. Moreover, these mouse models exhibit hypertrophy along with mild septal thickening [3,45]. In contrast, other animal models demonstrate either robust expression of truncated protein along with sarcomere disorganization [49], very low expression of truncated protein with no reduction of wild-type (WT) protein [50], or reduced expression of total cMyBP-C with incorporation of mutant protein into the sarcomere [17,29]. Furthermore, in mice hemizygous for cMyBP-C (+/−) or heterozygous for a functionally null allele (+/t), sarcomere organization and contractile function are normal [28,27,14,32]. Thus, further investigation is necessary to clearly distinguish the HCM causing mechanisms of individual mutations in order to tailor therapeutic approaches.

Role of C-terminal Region of cMyBP-C in thick filament organization

cMyBP-C is a thick filament-associated protein in the cardiac sarcomere that plays both regulatory and structural roles in cardiomyocyte contraction [39,40,37,38,11,12,36,21]. The cardiac isoform of MyBP-C consists of 12 domains, including one phosphorylation (M) domain, eight immunoglobulin (IgC2)-like domains, and three fibronectin type-III (FN3) domains. In mouse models where cMyBP-C is knocked out or not expressed, animals are viable, but they exhibit abnormal misaligned sarcomeric structure, contractile dysfunction, and cardiomyopathy [14,28]. While these data suggest that cMyBP-C is not essential for cardiac development, it is necessary for normal sarcomere structure and cardiac function. The precise positioning of cMyBP-C in the sarcomere is unclear; however, two models, including the trimeric collar model and the axial model, have been proposed to explain the specific arrangement of cMyBP-C and the interaction of its C-terminus with myosin and titin. The collar model proposes that three molecules of cMyBP-C form collar-like rings every 43 nm around the thick filament which are stabilized by domain interactions in C5–C10 [31,6,7]. The axial model proposes that the C-terminal domains of cMyBP-C run parallel along the thick filament with the N-terminus extended towards the thin filament [42]. Supporting data exist for both models, but more high-resolution structural data are needed to confirm which model is correct.

While the N-terminal region of cMyBP-C functions as a critical regulator of contractile function, the C-terminal region is thought to play a structural anchoring role. Domains C7–C10 of the C-terminal region of cMyBP-C bind to the thick filament and are required for incorporation into the sarcomere [10]. The C10 domain of cMyBP-C interacts with the light meromyosin (LMM) portion of myosin rods, forming the backbone of the thick filament [7]. Domains C8–C10 of cMyBP-C have been shown to bind to immunoglobulin domains of titin that are repeated approximately every 42 nm within the C zone of the thick filament, and it is likely that the position of cMyBP-C in the thick filament is dictated by the localization of these titin domains [19]. Any mutation producing a truncation or modification of the myosin and titin binding sites within the C-terminus of cMyBP-C would therefore be expected to result in a reduced or aberrant incorporation of the mutant protein into the sarcomere.

A highly prevalent C-terminal mutation in cMyBP-C

Previously, we described a polymorphic deletion of 25 base pairs in intron 32 of the cMyBP-C gene (MYBPC3^Δ25bp) that is associated with hypertrophic and dilated cardiomyopathies [46]. Interestingly, the distribution of this mutation is almost exclusively restricted to South Asian countries [5,41]. It is estimated that 55 million people in South Asian populations are affected by this deletion and are at increased risk of developing contractile dysfunction and heart failure. To date, this mutation has been found to occur primarily in India, Pakistan, Sri Lanka, Malaysia, and Indonesia [5,33,41]. While most young and middle-aged individuals exhibit a mild phenotype or are asymptomatic, individuals over the age of 40 years present with more moderate to severe symptoms. Out of 28 unrelated carrier families, 90% of the oldest members were found to be symptomatic. The fact that disease symptoms are typically dormant until the third decade of life, generally a time beyond child-bearing years, helps to explain the high prevalence of this mutation, which originated at least 10,000 years ago [48]. Carrying the mutation leads to increased susceptibility to worse outcome following cardiac disease. For example, patients with coronary artery disease also carrying the deletion had significantly worse systolic function [43].

Transcriptional studies demonstrated that MYBPC3^Δ25bp causes the skipping of exon 33 and a reading frame shift, resulting in the replacement of 65 wild-type amino acid residues with a novel sequence of 58 residues in the C10 domain (cMyBP-C^C10mut) [46] (Figure 1A–C). Expression of cMyBP-C^C10mut protein in neonatal cardiomyocytes resulted in the aberrant incorporation of mutant protein in the sarcomere and the disruption of normal sarcomeric structure by a still unknown mechanism [5]. The most likely consequence of the cMyBP-C^C10mut mutation is an alteration of the interaction with LMM (Figure 1D). Miyamoto et al. showed that 5 charged amino acids spread throughout the C10 domain are needed for cMyBP-C/LMM interaction (bold in Figure 1C), all of which are conserved between MyBP-C isoforms [30]. In cMyBP-C^C10mut, only 1 out of 5 of these key residues is conserved, suggesting a decreased affinity for LMM binding. Given that this MYBPC3^Δ25bp mutation affects nearly 1% of the world’s population, it is clinically urgent to fully elucidate the function of this cMyBP-C mutant protein. Further in vitro and in vivo investigation into the mechanisms by which the MYBPC3^Δ25bp mutation leads to disorganization of sarcomere structure and cardiomyopathy will greatly contribute to an understanding of the pathology of other cMyBP-C mutations and sarcomeric gene mutations in general.

Figure 1. The MYBPC3^Δ25bp mutation and cMyBP-C^C10mut protein.

(A) 3’ end of normal MYBPC3 that codes for the C-terminal C9 and C10 domains of cMyBP-C. Coding exons in bright orange. Normal stop codon exists at the beginning of exon 34. (B) Location of MYBPC3^Δ25bp mutation is shown in intron 32, at the branch point, which causes skipping of exon 33 and subsequent reading frame shift. This leads to translation of the entire exon 34 and part of the 3’ UTR. This mutation leads to the expression of cMyBP-C^C10mut, which has a partly altered C10 domain (indicated in red). (C) Amino acid sequences of wild-type (WT)-C10 and cMyBP-C^C10mut domains. First 29 amino acids are encoded by exon 32 and are therefore identical between WT-C10 and cMyBP-C^C10mut (black). The mutation leads to the replacement of the last 65 amino acids with a novel sequence of 58 amino acids. In the WT-C10 residues, the 5 charged amino acids that were indispensable for LMM binding [30] are indicated bold and underlined. Only 1 out of 5 of these was preserved in the cMyBP-C^C10mut residues. (D) Schematic illustration of cMyBP-C/myosin heavy chain (MHC) interaction. The MHC S2 domain interacts with the M-domain (red) of cMyBP-C on the N-terminal end of the protein. The C10 domain interacts with the LMM domain of MHC, and the WT-C10 domain and putative cMyBP-C^C10mut interactions with LMM are enlarged on the insert. We propose that the cMyBP-C^C10mut has weaker interaction with the LMM domain. On cMyBP-C, IgC2 domains are displayed in blue, FN3 domains in off-white, M-domain in red and proline-alanine-rich linker in green.

Necessity for genetic testing

The role of genotyping in inherited cardiomyopathies has been a hot button issue. Arguments can be made that knowing the mutation has no implications for treatment in these patients and therefore does not justify the costs; because 1) HCM can be caused by mutations in >11 genes and 2) the frequency of individual causal mutations is generally low [34]. In current clinical practice, genetic testing is used for screening of family members, rather than screening of the general population [4]. Although most mutations are infrequent, a number of mutations occur with much more frequency than others in some populations. Examples of these are the MYBPC3^Δ25bp mutation and the 2373InsG founder mutation in MYBPC3 that accounts for nearly 25% of all HCM patients in the Netherlands [1]. The high prevalence of the MYBPC3^Δ25bp mutation in South Asia [5,41] may make general population screening for this particular mutation both beneficial and feasible. With a relatively simple PCR-based screening method, this mutation can be easily detected (Figure 2). Identification of MYBPC3^Δ25bp carriers at a presymptomatic stage would enable unprecedented monitoring opportunities. Although therapeutic interventions to prevent the onset of symptoms are still not available in patients, recent studies in animal models show that prevention of hypertrophy and cardiac dysfunction [8,16] might be achievable in the not too distant future.

Figure 2. Genetic testing to determine the presence of the 25 bp deletion mutation.

(A) Schematic representation of part of MYBPC3 gene representing wild-type (WT), heterozygous and homozygous mutation carries. The arrows indicate both forward and reverse primers used in the PCR reaction (Forward primer: 5'-GTT TCC AGC CTT GGG CAT AGT C-3' and Reverse primer: 5'-GAG GAC AAC GGA GCA AAG CCC-3') to obtain a 403 bp and 378 bp from WT and mutant alleles, respectively. (B) Schematic representation of expected PCR products run on an acrylamide gel (7%) with silver staining as previously described [46]. Non-mutation carriers have a 403 bp PCR product, while homozygous MYBPC3^Δ25bp carriers have only the shorter 378 bp PCR product. Heterozygous MYBPC3^Δ25bp carriers have both products. In contrast, when PCR products were resolved in 1% agarose gel, the difference of 25 bp is not resolved (C). However, by digesting them with BglI enzyme (D), MYBPC3^Δ25bp carriers can be detected. Within the 403 bp sequence, two BglI sites are present, resulting in three fragments (234, 101 and 68 bp). However, MYBPC3^Δ25bp loses one of the two BglI sites, resulting in two fragments (310 and 68 bp). Blank reaction resulted in the absence of PCR products (Blank). Uncut PCR products without digestion with BglI are shown (Uncut). Marker lanes IV and X indicate Roche IV and Roche X markers, respectively. DNA sequences of wild-type (E) and mutant (F) alleles are shown. PCR products from heterozygous carriers were cloned into pCR-Blunt II TOPO vector using Zero Blunt TOPO PCR cloning kit (Invitrogen) and then sequenced using T7 forward and M13 reverse primers.

Conclusion

cMyBP-C is a key regulator of cardiac contractility. Although mutations in the gene encoding cMyBP-C are a leading cause of HCM, little is known about the molecular mechanisms underlying the disease process. The availability of cardiac tissue from myectomy operations has resulted in a relatively large body of evidence on contractile function in human HCM patients. Nonetheless, additional experimentation, including animal models, induced pluripotent stem cells and engineered heart tissue, is required to clearly establish the detrimental effects of mutated proteins, all of which comprise the focus of current studies in the authors’ laboratory. Gaining more insight into these fundamental questions will lead to the development of novel therapeutic strategies for the treatment of HCM and save millions of patients from heart failure.

Abbreviations

HCM

Hypertrophic cardiomyopathy

LV

left ventricle

LVH

left ventricular hypertrophy

MYH7

myosin heavy chain gene

MYBPC3

cardiac myosin binding protein-C gene

cMyBP-C

cardiac myosin binding protein-C

MYBPC3^Δ25bp

25 basepair deletion mutation in MYBPC3

cMyBP-C^C10mut

protein product of MYBPC3^Δ25bp gene