Thirteen apparently unrelated index cases with HCM (aged 40.7±14.6 years, 9 men [75%]) who were carriers of the same novel mutation p.Pro108Alafs*9 in the MYBPC3 gene (GenBank accession number 4607) were enrolled in the study. All patients underwent risk stratification and were managed following recommended guidelines.12 A pedigree was drawn for each patient, and first-degree relatives were screened using the same protocol. The phenotypes of p.Pro108Alafs*9 and c.2308+1 G>A were compared.

See the expanded “Methods” section in the supplementary material for a detail description of genetic study, RNA isolation, complementary DNA synthesis, MYBPC3 complementary DNA amplifications, puromycin analysis and statistical analysis.

The HCM phenotype was compared in 2 groups of patients carrying 2 different mutations; both of these mutations altered full-length MYBPC3 (Figure 2). The clinical characteristics of the 39 affected carriers of p.Pro108Alafs*9 were compared with those of the 61 affected carriers of c.2308+1G>A (Table 2).

Figure 2.

Summary of studies on mutations in MYBPC3 causing a potential truncated protein. HCM, hypertrophic cardiomyopathy; MLVH, maximal left ventricular wall thickness; NA, not available; SD, sudden death. aOnly affected carriers included. bUnpublished.

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The phenotype was similar in c.2308+1G>A and in p.Pro108Alafs*9. However, the proportion of patients with an electrocardiogram characteristic of HCM (55 [90.2%] vs 29 [74.4%]; P=.035), with 2 or more risk factors (27 [44.3%] vs 9 [23.1%]; P=.03) and a SD risk in 5 years of O’Mahony>6% (12 [19.7%] vs 2 [5.1%]; P=.041) were significantly higher in c.2308+1G>A carriers than in p.Pro108Alafs*9 carriers. The number of SD/ICD discharges during follow-up was similar in c.2308+1G>A and in p.Pro108Alafs*9 (7 [11.5%] vs 3 [7.8%]; P=.5).

The development of hypertrophy in the 2 carrier groups was similar over their life span, as reflected by the similarity between the 2 probability shapes of HCM diagnosis. Disease penetrance increased at a regular pace during life from 20 years of age onward, following a sigmoid shape (Figure 1A). When all living patients from the cohort and the SD cases (historical and prospective cases, see “Methods”) were included, SD/ICD discharge survival was significantly lower in the group with the c.2308+1G>A mutation than in the p.Pro108Alafs*9 group. The risk of SC/ICD discharge was higher in the c.2308+1G>A group than in the p.Pro108Alafs*9 group (hazard ratio [HR]=2.98; 95% confidence interval [95%CI], 1.13-7.81; P=.02) (Figure 1C).

On Kaplan-Meier analysis, SD/ICD survival was worst in men with c.2308+1G>A and best in women in the p.Pro108Alafs*9 group (Figure 1D). On multivariate analysis, male sex was a predictors of SD/ICD discharge (HR=3.84; 95%CI, 1.33-11.14; P=.013 and there was a trend toward higher risk with the c.2308+1G>A mutation (HR=2.45; 95%CI, 0.93-6.47, P=.07). There were no significant differences in survival free from heart failure (NHYA III-IV) or atrial fibrillation between the 2 groups or by sex (data not shown).

Twenty-three patients (8 with p.Pro108Alafs*9 and 15 with c.2308+1G>A) underwent cardiac magnetic resonance study (mean age 44±15 years, 17 men, 6 women). There was a trend toward a higher left ventricular mass in p.Pro108Alafs*9 compared with c.2308+1G>A (mean 269.1±138.4g vs 173.2±80.2g, P=.057, indexed P=.053) (Figure 3). Eighteen (78.3%) patients had late gadolinium enhancement (LGE) (7 [87.5%] p.Pro108Alafs*9 and 11 [73.3%] c.2308+1G>A], P=.9). Patterns of LGE were similar in the 2 groups. There was no significant difference in the fibrosis pattern between the 2 groups. A right-left ventricular junction pattern predominated in carriers of p.Pro108Alafs*9 (50.0%) while a septal diffuse pattern predominated in c.2308+1G>A carriers (40.0%). There were no differences in LGE mass and percentage of LGE mass between the groups (39.9±46.8g vs 24.6±31.3g, P=.3; 12.4±11.5% vs 13.9±11.2%, P=.8 for p.Pro108Alafs*9 and c.2308+1G>A respectively) (Figure 4).

Figure 3.

Comparison of left ventricular mass in carriers of 2 mutations by sex. The box defines the interquartile range with the median indicated by the crossbar. LV, left ventricular.

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Figure 4.

Correlation between LGE with left ventricular mass in carriers of 2 mutations. LGE, late gadolinium enhancement.

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The presence of the p.Pro108Alafs*9 was an independent predictor of LV mass (absolute and indexed) (HR=2.31; 95%CI, 4.8-96.1; P=.032 for indexed LV mass). The type of mutation was not a predictor for absolute LGE or percentage of LGE.

Two (8.7%) patients with a cardiac magnetic resonance study had an arrhythmic event (1 resuscitated cardiac arrest and 1 ICD discharge). Both patients were c.2308+1G>A carriers and had a diffuse septal fibrosis pattern of LGE (10% and 33% of LGE from total LV mass).

Six (26.0%) patients had nonsustained ventricular tachycardia on Holter monitoring. Three of them (50.0%) had LGE. The risk score for SD was not associated with the presence of LGE (SD score, 4.2±2.3 vs 3.2±1.6 for absence and presence of LGE respectively; P=.3). Neither nonsustained ventricular tachycardia nor SD risk score was associated with LV mass or LGE variables.

Histopathological study from an explanted heart from a female p.Pro108Alafs*9 carrier showed severe concentric hypertrophy and diffuse interstitial fibrosis (Figure A, Figure B, and Figure C of the supplementary material). This patient was diagnosed with HCM at age 44 years. She showed no evidence of obstruction. Echocardiograms prior to transplant revealed a small LV cavity and severe diastolic dysfunction, which was thought to be the cause of the limiting symptoms. Despite medical therapy, she received a transplant when she was 68 years old.

The histological study from a transplanted male c.2308+1G>A carrier demonstrated large transmural and heterogeneous scars (Figure D, Figure E, and Figure F of the supplementary material). The patient was diagnosed with HCM when he was 26 years old, with symptoms associated with obstruction. After 23 years of follow-up, he developed systolic dysfunction and received a heart transplant at 49 years of age. Examination revealed a dilated heart with extensive fibrosis, disarray, and hypertrophy.

Apart from a couple of illustrative cases, the attractive hypothesis that carriers of a malignant mutation would develop a particular pattern of fibrosis could not be demonstrated from cardiac magnetic resonance results.

To investigate which transcripts were produced by c.2308+1G>A and to determine whether the truncated transcripts were degraded by nonsense-mediated RNA decay, lymphocytes of carriers of 2 mutations were cultured with the translation inhibitor puromycin.

c.2308+1G>A is a G>A transition in the 3 prime splice donor site of intron 23 of MYBPC3 that inactivates this splicing site. This mutation produced alternative splice donor sites, resulting in 2 transcripts of longer sizes (4515 and 4761 pb) than wildtype (4217 pb) when lymphocytes were cultured with puromycin. Nevertheless, only the wildtype allele was shown when lymphocytes were cultured without puromycin, indicating that aberrant transcripts generated by this variant were degraded independently of their lengths. The same occurred when we analyzed the messenger RNA of a p.Pro108Alafs*9 carrier; the wildtype allele was expressed in blood only.

The p.Pro108Alafs*9 mutation is an insertion of the GCTGGCCCCTGCC nucleotides in position 29 of exon 3. This insertion also produces a frameshift: the aberrant complementary DNA results in 107 normal MYBPC3 residues and then 8 novel amino acids, followed by a premature stop codon in the proline-alanine (Ala-Pro)-rich region. This should produce a large truncated protein of 116 amino acids (-91%) lacking the MYBPC3 motif containing the phosphorylation sites and the titin and myosin binding sites rather than the wildtype protein consisting of 1275 amino acids.

Figure 2 shows distinct previously described mutations in MYBPC3, each of which produce peptides of different lengths.10,11,14–17 Penetrance varies from 62% to 82% with no association with the position of the mutation in MYBPC3. On the contrary, a trend toward an increased prevalence of SD proportional with the length of the transcript can be seen, being 13% for the shortest (p.Pro108Alafs*9) and maximum (67%) for the second longest (IVS20-2A>G). The longest Q1061* does not fit with the rest and the prevalence of SD is low.

The pathogenicity of p.Pro108Alafs*9 was studied using the modified criteria used previously by Van Spaendonck-Zwarts et al.18 A list of mutation-specific features based on in silico analysis with the mutation interpretation software MutationTaster and the frecuency in a control population predicted this variant as disease causing with a probability of 1.19 Familial study was required to study the cosegregation and finally to classify the variant as pathogenic.

In the present study, we describe a novel mutation identified in 54 carriers from 13 families. Overall, 72% of our carriers of p.Pro108Alafs*9 had HCM. Our study confirms the association between the p.Pro108Alafs*9 mutation in the MYBPC3 gene and the development of HCM with a family cosegregation of 100%.

Patients affected by p.Pro108Alafs*9 are characterized by asymmetric septal hypertrophy with the presence of obstruction in approximately 25%, a high penetrance, and middle age at disease onset (43±17 years old). Heart failure symptoms are predominant while SD is a rare complication.

In keeping with the literature, our carrier patients exhibited an age-related penetrance,20 with similar shapes for both mutations. Interestingly, and according to 2 other reported series10,21 there was a clear male predominance in affected carriers of p.Pro108Alafs*9, who were also younger than the women at diagnosis. However, differences in penetrance and a delay in the onset of disease between the sexes have not been demonstrated in large populations of patients with HCM.15,22–24

Similar to other series, most affected carriers were in NYHA class I-II, less than 20% reported syncope, and less than 30% had chest pain.25 On average, the women were in a worse NYHA functional class and more frequently had chest pain. The percentage of patients with atrial fibrillation in our series (41%) was higher than that reported by other authors.24,26

This report describes the comparison of the phenotypical characteristics of the 2 most prevalent mutations found in our region, p.Pro108Alafs*9 and c.2308+1G>A. This latest series includes one of the largest with the same mutation in MYBPC3 reported to date.10 p.Pro108Alafs*9 was also a founder mutation in our cohort but, in contrast with c.2308+1G>A, it produced a lower rate of arrhythmic events in the affected patients. Survival free from SD/ICD discharge (historical and prospective) was clearly lower in c.2308+1G>A than in p.Pro108Alafs*9. Other founder mutations have been described in MYBPC3 located in different regions predicting a C-terminal truncated protein due to a premature termination codon, although each expresses a different phenotype.11,12,14–17,27,28 Dominant mutations are generally responsible for negative selection pressure and tend to disappear after several generations. Some of these mutations, such as c.2308+1G>A, escape this selection pressure and are transmitted over generations because disease expression is delayed beyond reproductive age.16

The results of genotype-phenotype correlation studies in HCM have been confounded by the small size of the families, the low frequency of each casual mutation (< 5%), and the small number of families with identical mutations.29 Generally, MYBPC3 mutations are associated with a later average age at symptom onset, a lower incidence of SD, and a relatively benign clinical course, although no differences in clinical phenotype have been attributable to the specific type of MYBPC3 mutation.30 Therefore, the high prevalence of these founder mutations provides an opportunity to define their clinical profiles.

The nonsense-mediated decay pathway is a messenger RNA surveillance system that typically degrades transcripts containing premature termination codons to prevent the translation of unnecessary or aberrant transcripts. A relatively milder phenotype may be caused by nonsense mutations that activate the nonsense-mediated decay, thereby reducing dominant-negative expression and resulting in haploinsufficiency.20,31,32 It is known that truncated cMYBPCs are preferentially degraded by the ubiquitin-proteasome system, which may impair the proteolytic capacity of the ubiquitin-proteasome system.33,34

Length of messenger RNA transcript might play a role in the different risk profile of distinct mutations. Important differences have been observed at the cellular level in infected cardiac cells with mutant MYBPC3 regarding the length of the transcript protein: a) overexpression of human truncated cMYBP-Cs in transgenic mice resulted in markedly lower protein levels, being directly correlated to the size of the protein; b) larger proteins are more likely to be incorporated into the sarcomere, and c) smaller proteins are more prone to block the ubiquitin-proteasome system degradation system leading to the formation of truncated protein aggregates and an increase in the cytosolic concentration of other proteins involved in muscle growth and atrophy and apoptosis.33,35 The ubiquitin-proteasome system also plays a role in degradation of β2 adrenergic receptors and ion channels.

Interestingly, the pathogenicity of mutations in titin has recently been associated with the length of the transcript protein.36

We can discern from our results that the length of the transcript does not affect the severity of the hypertrophy or disease penetrance, but there were differences in risk profile and in SD prognosis, which was worse in the longer transcript group. When we analyzed in detail other founder mutations that truncate the MYBPC3 gene, we observed that the number of SD was proportional to the length of transcript, except in the case of p.Gln1061* (P=.081) (Table 2, in “total SD cases/total HCM affected”).

Late gadolinium enhancement has been associated with nonsustained ventricular tachycardia and SD risk profile in HCM.26,37–39 The right-to-LV junction pattern is the most frequent and is believed to have a more benign prognosis than diffuse or transmural patterns.25 Extensive LGE (> 20% or>30% from left ventricular mass) is indicative of a worse arrhythmic substrate.39,40

Although patients with c.2308+1G>A in our study had a significantly worse SD score (SD risk profile obtained from O’Mahony model) and survival free of SD compared with those with p.Pro108Alafs*9, the supposed arrhythmic substrate was not seen in the subgroup of patients who underwent cardiac magnetic resonance study. The degree and pattern of LGE were similar in patients with p.Pro108Alafs*9 and c.2308+1G>A. Moreover, and contrary to what could be expected, LV mass indexed by sex and age was higher in carriers of p.Pro108Alafs*9 than in c.2308+1G>A carriers (P=.032).