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Hypertrophic remodelling in cardiac regulatory myosin light chain (MYL2) founder mutation carriers

Godelieve R.F. Claes, Florence H.J. van Tienen, Patrick Lindsey, Ingrid P.C. Krapels, Apollonia T.J.M. Helderman-van den Enden, Marije B. Hoos, Yvette E.G. Barrois, Johanna W.H. Janssen, Aimée D.C. Paulussen, Jan-Willem E.M. Sels, Simone H.H. Kuijpers, J. Peter van Tintelen, Maarten P. van den Berg, Wilfred F. Heesen, Pablo Garcia-Pavia, Andreas Perrot, Imke Christiaans, Simone Salemink, Carlo L.M. Marcelis, Hubert J.M. Smeets, Han G. Brunner, Paul G.A. Volders, Arthur van den Wijngaard
DOI: http://dx.doi.org/10.1093/eurheartj/ehv522 ehv522 First published online: 24 October 2015

Abstract

Aims Phenotypic heterogeneity and incomplete penetrance are common in patients with hypertrophic cardiomyopathy (HCM). We aim to improve the understanding in genotype–phenotype correlations in HCM, particularly the contribution of an MYL2 founder mutation and risk factors to left ventricular hypertrophic remodelling.

Methods and results We analysed 14 HCM families of whom 38 family members share the MYL2 c.64G > A [p.(Glu22Lys)] mutation and a common founder haplotype. In this unique cohort, we investigated factors influencing phenotypic outcome in addition to the primary mutation. The mutation alone showed benign disease manifestation with low penetrance. The co-presence of additional risk factors for hypertrophy such as hypertension, obesity, or other sarcomeric gene mutation increased disease penetrance substantially and caused HCM in 89% of MYL2 mutation carriers (P = 0.0005). The most prominent risk factor was hypertension, observed in 71% of mutation carriers with HCM and an additional risk factor.

Conclusion The MYL2 mutation c.64G > A on its own is incapable of triggering clinical HCM in most carriers. However, the presence of an additional risk factor for hypertrophy, particularly hypertension, adds to the development of HCM. Early diagnosis of risk factors is important for early treatment of MYL2 mutation carriers and close monitoring should be guaranteed in this case. Our findings also suggest that the presence of hypertension or another risk factor for hypertrophy should not be an exclusion criterion for genetic studies.

  • Hypertrophy
  • Hypertrophic cardiomyopathy
  • Hypertension
  • Risk factors
  • Genetics
  • Mutation

Clinical summary

This study investigates the influence of additional genetic and environmental risk factors on the phenotypic outcome of patients with the same genetic mutation. In a large cohort of MYL2 founder mutation carriers, the mutation on its own was not sufficient to develop HCM in most patients. The presence of an additional risk factor for hypertrophy, particularly hypertension, adds to the development of HCM. These findings have important consequences for clinical practice.

Introduction

Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium characterized by abnormal and often asymmetric myocardial thickening without obvious cause. It is typically inherited in an autosomal dominant fashion with variable penetrance. Prevalence is estimated at 0.13–0.17%.1,2 Clinical manifestations can range from asymptomatic to severe disease at early age.3,4

To date, more than 50 genes have been associated with HCM5 and a heterozygous mutation is identified in 35–60% of HCM patients.4,610 Most of the genes involved encode proteins of the sarcomere, the contractile unit of cardiomyocytes. Myosin light chains (MLCs) stabilize and regulate contraction of cardiomyocytes.

In this study, we investigate the myosin regulatory light chains (RLCs) expressed mainly in the human ventricle and encoded by the MYL2 (MLC2v) gene. The importance of RLC in heart function and structure has been illustrated by the identification of MYL2 mutations in patients with HCM.11 The pathogenicity of several of these MYL2 mutations has been studied thoroughly.1216 Specifically, the MYL2 p.(Glu22Lys) mutation (p.(E22K) in the remainder of this manuscript) introduced structural changes in the protein that inhibited phosphorylation and greatly reduced Ca2+ binding to the EF hand motif disturbing normal MYL2 protein function13 and altered interactions between actin and myosin.16 Family members carrying the MYL2 p.(E22K) mutation showed diverse clinical presentations, ranging from asymptomatic and very mild to severe. The mutation was first reported in two brothers with HCM and one unrelated individual who presented with severe HCM.11 Later, the mutation was identified in a German family with only mild to moderate septal hypertrophy and low penetrance, as only four of seven mutation carriers were classified as affected.17 Finally, the mutation was reported homozygously in two Spanish patients with end-stage heart failure due to HCM who underwent heart transplantation at age 41 (diagnosis at 31) and 53 (diagnosis at 30).18 Heterozygous mutation carriers in this family were asymptomatic. This variability in clinical presentation is not uncommon in HCM.19

Here, we report the identification of the MYL2 p.(E22K) mutation in 14 apparently unrelated HCM families. We examined whether this mutation represents a founder mutation and we evaluated the clinical impact in 38 mutation carriers compared with 25 non-carriers. In this analysis, we particularly focused on additional risk factors that could explain the difference in severity of hypertrophy and variability in clinical presentation.

Materials and methods

Materials and methods are in the Supplementary material online.

Results

Diagnostic mutation analysis

From 1996 to 2012, a total of 860 index patients were referred to the Maastricht University Medical Centre (MUMC) for diagnostic genetic testing because of confirmed or suspected HCM. Informed consent was obtained for all patients. Genetic analysis of HCM patients always involves Sanger sequencing analysis of the two major genes MYH7 and MYBPC3. Analysis of six other HCM-implicated genes (TNNT2, TNNI3, TPM1, MYL2, MYL3, and PLN) is performed whenever no pathogenic mutation is found in the two major genes, the pathogenicity of the identified variant is unclear, the variant does not segregate with the disease in the family, or when the patient has such a severe and/or early-onset phenotype that a second mutation is suspected. Therefore, in 267 of these patients, the entire MYL2 coding region was analysed, revealing the c.64G > A substitution which predicts a p.(E22K) missense mutation in 11 HCM index patients. Compared with the reported 1% prevalence of MYL2 mutations in the population,20 we found a relatively high frequency of this MYL2 mutation in our cohort (4%; 11/267). We were able to include three additional families with the same mutation from two other European centres bringing the total to 14 families.

Haplotype analysis and age estimation

The relatively high frequency of the MYL2 mutation together with the observation that most patients live in the same area in the Netherlands (see Supplementary material online, Figure S1) led us to assume this mutation is a founder. Therefore, we analysed seven short tandem repeat (STR) markers and two single nucleotide polymorphisms (SNPs) to determine the haplotype surrounding the MYL2 mutation (see Supplementary material online, Figure S2). This revealed a common haplotype of 2.00 Mb in all 14 index patients (see Supplementary material online, Figure S3A), which provided evidence for a common ancestry for these families.

The p.(E22K) mutation in MYL2 has been previously reported in other HCM families from Spain and Germany. We obtained DNA from mutation carriers of the two families reported by Garcia-Pavia et al.18 and Kabaeva et al.17 Haplotype analysis revealed that both families shared part of the ‘Dutch’ haplotype (see Supplementary material online, Figure S3B) and therefore share the same common ancestor.

By calculating the age of origin of the mutation (see Supplementary material online), we found that it originated between 17 and 38 generations ago. Assuming 25 years per generation, the p.(E22K) mutation has arisen between 420 and 940 years ago. Using genealogy, we could follow nine families up to nine generations ago and were able to connect three and two families (see Supplementary material online, Figure S4).

Family studies and clinical findings

We studied 107 individuals from the 14 families. A cohort of 63 family members was used for statistical modelling. Informed consent was obtained for all participants. For the remaining 44 family members, either clinical diagnosis (based on echocardiographic or cardiac magnetic resonance imaging data) was not available or genetic information was missing. They were, however, included in pedigrees because they are (obligate) mutation carrier and/or have had a major adverse cardiac event.

After initial Sanger sequencing of the index, we sequenced all eight HCM-implicated genes using Sanger or next-generation sequencing for 52 family members, for seven all genes except for PLN were screened, and three allowed limited screening of familial variants only (n = 63).

In 38 of 63 family members for whom both clinical and genetical data were available, the MYL2 c.64G > A mutation was present. An additional (likely) pathogenic mutation was identified in family 10 019 [MYH7 c.5779A > T, p.(Ile1927Phe), n = 2], 8748 [MYBPC3 c.1696T > C, p.(Cys566Arg), n = 13], 17 437 [MYBPC3 c.94G > A, p.(Glu32Lys), n = 3], and 15 662 [MYH7 c.2890G > C, p.(Val964Leu), n = 1]. A summary of clinical characteristics is provided in Table 1, and detailed genetic and clinical information in pedigrees (see Supplementary material online, Figure S5) and tables (see Supplementary material online, Tables S1–S4). Clinical descriptions and data of the families are summarized in the Supplementary material online.

View this table:
Table 1

Summary of clinical characteristics

MYL2 wild-typeMYL2 p.(E22K)P-value
Sex (male/female, %)41/5958/420.17
NYHA functional class (I/II/III, %)95/5/070/11/19
Angina pectoris (%)10140.71
Major adverse cardiac event (%)7380.004
 Sudden death and/or OHCA (%)0220.008
 Myocardial infarction (%)0110.07
 Syncope (%)10150.53
 Documented ventricular tachycardia (%)0160.07
Echocardiography
 Left atrial diameter (mm)39 ± 442 ± 50.21
 Interventricular septum (mm)10 ± 2a15 ± 22 × 10−5
 Left ventricular ejection fraction (%)65 ± 560 ± 60.16
Electrocardiogram
 Inferolateral Q waves (%)26290.84
 Increased QRS voltage (Sokolow–Lyon positive, %)11140.70
 ST-segment deviation (%)14180.70
 T-wave inversion (%)6360.02
 Atrial fibrillation (%)0350.002
Other sarcomeric mutation (%)40240.17
Additional parameters
 Hypertension (%)31430.31
 Obesity (%)8180.29
 Hyperlipidaemia (%)12140.86
 Diabetes mellitus (%)050.26
  • Data for family members for whom clinical diagnosis and MYL2 genotype were available are included (n = 72) but complete data were not available for each characteristic. Significant P-values are in bold.

  • NYHA, New York Heart Association; OHCA, out-of-hospital cardiac arrest; HCM, hypertrophic cardiomyopathy; IVS, interventricular septum.

  • aThe average of 10 mm WTh for MYL2 wild-type is compatible with mild hypertrophy (10–12 mm), which is explained by the presence of five HCM patients in this group.

In 27 of the 38 MYL2 mutation carriers, at least one of the traditional Framingham Heart Study (FHS) risk factors previously associated with increased cardiac dimensions (hypertension, obesity, hyperlipidaemia, or diabetes mellitus)21 or another sarcomeric gene mutation9 was documented (Figure 1A).

Figure 1

(A) Venn diagram showing the presence of additional risk factors for hypertrophy in MYL2 mutation carriers and wild-type family members. Other mutation: additional variant in MYH7 or MYBPC3. Other risk factors: obesity, hyperlipidaemia, and diabetes. (B and C) Normalized stacked column chart showing the percentage of disease penetrance of hypertrophic cardiomyopathy (HCM; shaded). Number of family members in each group is indicated in red for HCM (interventricular septum (IVS) or maximum wall thickness (MWTh) ≥13 mm) and black for an IVS or MWTh <13 mm. (B) If only hypertension is considered as a risk factor for hypertrophy, the differences between groups are significantly different. *P = 0.02; **P = 0.0002. (C) When all risk factors are considered, the differences between groups are even more significantly different. *P = 0.0005; **P = 0.0001. Student's t-tests were used to compute the P-values.

When we consider only hypertension as a risk factor for hypertrophy, the association in MYL2 p.(E22K) mutation carriers with HCM is highly significant (P = 0.02; combined OR: 18.80; Figure 1B). Considering the other risk factors separately, the association was not significant, underscoring the importance of hypertension. If we add other sarcomeric mutation and obesity as risk factors in addition to hypertension, the model predicting HCM improves. Hyperlipidaemia and diabetes do not improve the model further. This lack of improvement is most probably due to the fact that hyperlipidaemia and diabetes were present only in small numbers of patients. Because these were established as risk factors for hypertrophy in the FHS,21 we still included them.

Considering all risk factors together, 89% (24/27) of mutation carriers with a risk factor had HCM, compared with only 36% (4/11) without such a risk factor (Figure 1C). The presence of this additional risk factor for hypertrophy in MYL2 mutation carriers was significantly associated with HCM (P = 0.0005; combined OR: 39.47). When we exclude indexes, the association remains statistically significant for all risk factors combined (P = 0.002; combined OR: 48.52) and for hypertension only as a risk factor for hypertrophy, it is borderline non-significant (P = 0.06; combined OR: 11.37). Statistical calculations are provided in Supplementary material online, Table S5.

MYL2 p.(E22K) mutation carriers had a significantly larger interventricular septum (IVS) or maximum wall thickness (MWTh) compared with MYL2 wild-type family members (Table 1). The relatively large WTh in the MYL2 wild-type group is explained by five MYL2 wild-type family members who had echocardiographic features of HCM with an IVS WTh ≥13 mm. Noteworthy, four of these five had a mutation in another sarcomeric gene and the fifth had hypertension.

For the majority of MYL2 mutation carriers with HCM (n = 28), the IVS was most thickened (23/28). In the remaining five, concentric LVH was observed in four and predominant apical hypertrophy in one.

The WTh increases due to the presence of the MYL2 mutation [P = 2 × 10−6; +3.37 mm; 95% confidence interval (CI): 1.55–5.18] or an additional risk factor for hypertrophy (P = 3 × 10−8; +5.12 mm; 95% CI: 3.24–6.99). They both contribute independently to the severity of the HCM; therefore, the presence of both the MYL2 mutation and an additional risk factor for hypertrophy has an additive effect on the size of the WTh (Figure 2). The different risk factors individually are not significantly associated with the size of the WTh. We have also looked whether the number of additional risk factors for hypertrophy was contributing to the diagnosis or WTh. This was significant, but not as good as taking into account whether the patient had additional risk factors or not (see Supplementary material online, Tables S5 and S6). In these models, the family effect was significant and therefore taken into account.

Figure 2

Boxplot showing interventricular septum (IVS) or maximum wall thickness (MWTh) measurements in MYL2 p.(E22K) mutation carriers and wild-type family members with or without an additional risk factor for hypertrophy. Black lines in the boxes indicate the median (second quartile); box limits show P75 (third quartile, top) and P25 (first quartile, bottom). Outliers (1.5× interquartile range) are indicated as circles. Maximum and minimum of measurements that are not outliers are indicated by the top and bottom whisker ends, respectively. Number of patients in each category is indicated below each plot. The dashed line divides between MYL2 p.(E22K) mutation carriers and MYL2 wild-type family members. Significant differences are indicated with asterisks. *P = 2 × 10−6; **P = 3 × 10−8. Student's t-tests were used to compute the P-values.

With regard to diastolic function, 13 of 14 index patients had a left ventricular diastolic dysfunction type II with an average E/E′ ratio of 11.3 ± 2.9. The E/A ratio was not significantly different between MYL2 mutation carriers and wild-type family members. Other characteristics are summarized in Table 1.

Although there was no significant difference in current age or age of diagnosis between MYL2 mutation carriers or wild-type family members with or without HCM (Table 2), or males and females (Table 3), a Kaplan–Meier curve shows that male MYL2 mutation carriers seemed to be diagnosed at a younger age than females (P = 0.07; Figure 3), and appeared to be affected more often with HCM compared with female mutation carriers. However, these differences were not statistically significant.

View this table:
Table 2

Comparison of current age or age of diagnosis between different groups of MYL2 p.(E22K) mutation carriers and MYL2 wild-type individuals

Age
IVS or MWTh ≥13 mm (HCM)IVS or MWTh <13 mm
MYL2 p.(E22K) mutation carriers52.8 ± 15.554.0 ± 12.9
MYL2 wild-type individuals52.0 ± 12.646.8 ± 11.8
  • For patients with HCM, age of diagnosis was considered, for individuals with IVS or MWTh <13 mm current age was considered.

  • IVS, interventricular septum; MWTh, maximum wall thickness; HCM, hypertrophic cardiomyopathy.

View this table:
Table 3

Comparison of sex between different groups of MYL2 p.(E22K) mutation carriers and MYL2 wild-type individuals

Sex
IVS or MWTh ≥13 mm (HCM)IVS or MWTh <13 mm
MYL2 p.(E22K) mutation carriers (male/female)19/93/7
MYL2 wild-type individuals (male/female)3/18/13
  • IVS, interventricular septum; MWTh, maximum wall thickness; HCM, hypertrophic cardiomyopathy.

Figure 3

Kaplan–Meier survival curve showing disease penetrance of hypertrophic cardiomyopathy (HCM) in male (♂) and female (♀) MYL2 p.(E22K) mutation carriers.

In addition to the FHS risk factors for hypertrophy, we investigated if new risk factors contributed to hypertrophy in MYL2 mutation carriers and analysed if smoking, thyroid function (hyper-/hypothyroidism), obstructive sleep apnoea syndrome, aortic valve dysfunction, and atrial fibrillation (AF) were associated with HCM (see Supplementary material online, Table S7). None of the investigated presumed risk factors were significant, except for AF that showed a significant relation with HCM with a highly significant P-value, but at present we do not believe that AF is causal to HCM.22

Discussion

The genetic basis of HCM has been extensively described, however, the genotype–phenotype correlation is poorly understood, and incomplete penetrance and phenotypic heterogeneity are often reported. To our knowledge, this is the first study to clearly demonstrate the effect of additional risk factors on the phenotypic outcome in HCM patients carrying the same genetic mutation.

Previous studies reported on smaller genetically homogenous cohorts carrying an HCM founder mutation in TNNI3, MYBPC3, MYH7, and TPM1.2327 Additional risk factors were not investigated for these founder mutation carriers, except for other sarcomeric mutations identified in some patients of one study.27 The association of physiological risk factors and sarcomeric mutations with cardiac morphology and function was assessed in a large genetically heterogeneous cohort, namely the FHS.21 More genetic complexity and epigenetic or environmental modifiers to explain the observed phenotype, or lack thereof, in families carrying a pathogenic mutation has been suggested.

Our patient cohort is relatively large and genetically homogenous and therefore very suitable to investigate the genetic and non-genetic factors involved in hypertrophic remodelling influencing the phenotype in addition to the primary mutation.

The MYL2 p.(E22K) mutation is a founder mutation

We identified the MYL2 p.(E22K) mutation in 14 HCM families not known to be related and originating from the south of the Netherlands. The common haplotype in all Dutch patients and in two previously reported HCM families from Spain and Germany demonstrates that this is a founder mutation. The presence of the mutated founder haplotype in distant populations, the Netherlands and Spain, can be explained by European history. During the 80 years Dutch Revolt from 1568 to 1648, many Spanish soldiers were deployed in the Netherlands. Specifically, the south of the Netherlands was attacked, overtaken and destroyed several times by the Spaniards between 1583 and 1629. This timeframe matches with the calculated date for the origin of the mutation, 420–940 years ago. Furthermore, the Spanish army fought in different places in Germany during the Thirty Years’ War (1618–1648) which was a series of wars principally fought in Central Europe between Catholics and Protestants. The identification of the founder mutation in German individuals also fits well with these historical events and the calculated time frame.

In addition to the genetic proof, we could demonstrate genealogically that five families have common ancestors around the year 1650.

Risk factors for hypertrophy influence the phenotypic outcome in MYL2 p.(E22K) mutation carriers

We investigated the role of risk factors for hypertrophy (hypertension, obesity, hyperlipidaemia, diabetes, and other mutation in another sarcomeric gene) in the development of HCM in MYL2 p.(E22K) mutation carriers. We hypothesized that an additional risk factor for hypertrophy adds to the development of the HCM phenotype. We demonstrated that hypertension had an important contribution to the expression of HCM in MYL2 mutation carriers. However, additional risk factors for hypertrophy further improved the prediction of our hypothesis. We therefore strongly recommend that all risk factors are taken into account in the clinic.

Hypertension is the most common risk factor in MYL2 p.(E22K) mutation carriers with hypertrophic cardiomyopathy

In 71% (17/24) of MYL2 mutation carriers with HCM and an additional risk factor for hypertrophy, hypertension was documented. The presence of hypertension in MYL2 p.(E22K) mutation carriers is significantly associated with HCM (P = 0.02; combined OR: 18.80).

Hypertensive patients are often excluded from genetic studies of HCM because of confounding effects on hypertrophy. For example, in a recent paper, HCM patients with hypertension were only included in the genetic study if they had a ratio of septal to posterior WTh ≥1.5.7 Had we applied this criterion to our MYL2 mutation carriers with hypertension (n = 18), only four had a ratio ≥1.5, which implies that most of our patients would not have been offered genetic screening due to the presence of hypertension. Our study clearly shows that such individuals can have a genetic mutation and that their phenotypic expression is influenced by both the mutation and hypertension. Another argument underscoring this is the observation that 94% (17/18) of p.(E22K) mutation carriers with hypertension in our families developed HCM, whereas only 17% (1/6) MYL2 wild-type family members with hypertension developed HCM (P = 4 × 10−5; OR: 14.70; 95% CI: 4.40–60.64). This patient also had a likely pathogenic mutation in MYBPC3.

Limitations

Our study was retrospective and complete data were not available for all parameters in all patients. Only age of diagnosis was available and not age of onset, which may have influenced the data. In addition, duration of the hypertension, obesity, hyperlipidaemia, or diabetes and medication will have an influence on the development of the phenotype but was unknown.

Conclusion

To our knowledge, systematic analysis of risk factors for hypertrophy in patients carrying the same genetic mutation has not been reported before. In our cohort of MYL2 p.(E22K) founder mutation carriers, the presence of an additional risk factor for hypertrophy increased the disease penetrance of HCM (P = 0.0005; combined OR: 39.47), with hypertension being the most prominent risk factor.

The information presented in this paper has direct implications for clinical practice. It suggests that the presence of an additional risk factor for hypertrophy is a very important indication for the cardiologist regarding the follow-up of the MYL2 mutation carriers. Early diagnosis of risk factors, such as hypertension, should lead to early treatment. Our findings also suggest that individuals who have hypertension or another risk factor should not always be excluded from genetic studies of hypertrophy. It remains to be examined if the striking gene–environment interaction demonstrated for MYL2 p.(E22K) also applies to mutations in other HCM-implicated genes.

Authors’ contributions

P.L.: performed statistical analysis. A.v.d.W., H.G.B., H.J.M.S.: handled funding and supervision. G.R.F.C., F.H.J.v.T., I.P.C.K., A.T.J.M.H.-v.d.E., M.B.H., Y.E.G.B., J.W.H.J., A.D.C.P., J.E.M.S., S.H.H.K., J.P.v.T., M.P.v.d.B., W.F.H., P.G.-P., A.P., I.C., S.S., C.L.M.M., and P.G.A.V.: acquired the data. A.v.d.W. and G.R.F.C.: conceived and designed the research. G.R.F.C.: drafted the manuscript. F.H.J.v.T., A.D.C.P., P.G.A.V., A.v.d.W., H.G.B., and P.L.: made critical revision of the manuscript for key intellectual content.

Funding

We acknowledge the support from the Netherlands CardioVascular Research Initiative (PREDICT) for P.G.A.V.: the Dutch Heart Foundation, Dutch Federation of University Medical Centres, the Netherlands Organisation for Health Research and Development, and the Royal Netherlands Academy of Sciences.

Conflict of interest: none declared.

Acknowledgements

We are grateful to the families for their participation in this study, and to Dr Z. Kabaeva and Prof. K.J. Österziel for helping us obtain DNA samples from the German families. We also wish to thank Dr N. Epstein for the efforts in contacting the family from his research. We would like to thank our genetic counsellors V.A.M. Hovers and Dr J.M.J. de Vos-Houben for counselling family members from the MUMC. Special thanks to R.J.E. Jongbloed and our genealogist T. Hendriks for assisting J.W.H. Janssen in connecting our families.

References

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