Abstract

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disease caused by mutations in genes encoding sarcomere proteins. It is the major cause of sudden cardiac death in young high-level athletes. Studies have demonstrated a poorer prognosis when associated with specific mutations. The association between HCM genotype and phenotype has been the subject of several studies since the discovery of the genetic nature of the disease.

This study shows the effect of a MYBPC3 compound variant on the phenotypic HCM expression.

A family in which a young man had a clinical diagnosis of HCM underwent clinical and genetic investigations. The coding regions of the MYH7, MYBPC3 and TNNT2 genes were sequenced and analyzed.

The proband present a malignant manifestation of the disease, and is the only one to express HCM in his family. The genetic analysis through direct sequencing of the three main genes related to this disease identified a compound heterozygous variant (p.E542Q and p.D610H) in MYBPC3. A family analysis indicated that the p.E542Q and p.D610H alleles have paternal and maternal origin, respectively. No family member carrier of one of the variant alleles manifested clinical signs of HCM.

We suggest that the MYBPC3-biallelic heterozygous expression of p.E542Q and p.D610H may cause the severe disease phenotype seen in the proband.

Keywords
Hypertrophic cardiomyopathy; sarcomere genes; compound variant; MYBPC3 gene

Resumo

A cardiomiopatia hipertrófica (CMH) é uma doença autossômica dominante causada por mutações em genes que codificam as proteínas dos sarcômeros. É a principal causa de morte súbita cardíaca em atletas jovens de alto nível. Estudos têm demonstrado um pior prognóstico associado a mutações específicas. A associação entre genótipo e fenótipo em CMH tem sido objeto de diversos estudos desde a descoberta da origem genética dessa doença.

Este trabalho apresenta o efeito de uma mutação composta em MYBPC3 na expressão fenotípica da CMH.

Uma família na qual um jovem tem o diagnóstico clínico de CMH foi submetida à investigação clínica e genética. As regiões codificadoras dos genes MYH7, MYBPC3 e TNNT2 foram sequenciadas e analisadas.

O probando apresenta uma manifestação maligna da doença e é o único em sua família a desenvolver CMH. A análise genética pelo sequenciamento direto dos três principais genes relacionados à essa doença identificou uma variante em heterozigose composta (p.E542Q e p.D610H) em MYBPC3. A análise da família mostrou que os alelos p.E542Q e p.D610H tem origem paterna e materna, respectivamente. Nenhum familiar portador de um dos alelos variantes manifestou sinais clínicos de CMH.

Sugerimos que a expressão heterozigótica bialélica de p.E542Q e p.D610H pode ser responsável pelo fenótipo severo da doença encontrada no probando.

Palavras-chave
Cardiomiopatia Hipertrófica; Genes Sarcoméricos; Mutação Composta; MYBPC3

Introduction

Hypertrophic cardiomyopathy (HCM) is a genetic myocardial disorder characterized by ventricular hypertrophy (VH), which is frequently asymmetrical in the interventricular septum and can lead to a dynamic obstruction of the left ventricle (LV) outflow tract.¹1 Arola A, Jokinen E, Ruuskanen O, Saraste M, Pesonen E, Kuusela AL, et al. Epidemiology of idiopathic cardiomyopathies in children and adolescents. A nationwide study in Finland. Am J Epidemiol. 1997;146(5):385-93. It is the main cause of sudden cardiac death (SCD) in young people, with a 2-4% annual mortality rate in adults and 6% in adolescents and children.²2 Maron BJ, Olivotto I, Spirito P, Casey SA, Bellone P, Gohman TE, et al. Epidemiology of hypertrophic cardiomyopathy-related death: revisited in a large non-referral-based patient population. Circulation. 2000;102(8):858-64. A benign outcome of HCM may also occur, such as late onset, mild hypertrophy, and a history of non-malignant events.³3 Niimura H, Patton KK, McKenna WJ, Soults J, Maron BJ, Seidman JG, et al. Sarcomere protein gene mutations in hypertrophic cardiomyopathy of the elderly. Circulation. 2002;105(4):446-51. Modifier genes, environmental influences, genetic variant diversity and the effect of multiple variants could explain the great clinical heterogeneity between individuals of the same family or from different families.⁴4 Richard P, Charron P, Carrier L, Ledeuil C, Cheav T, Pichereau C, et al; EUROGENE Heart Failure Project. Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation. 2003;107(17):2227-32. Erratum in: Circulation. 2004;109(25):3258.

HCM is a relatively common (0.2%) Mendelian disorder, caused mainly by mutations in sarcomere protein genes, most commonly those encoding β-myosin heavy chain (MYH7), myosin-binding protein C (MYBPC3) and troponin T (TNNT2).⁵5 Maron BJ, Ommen SR, Semsarian C, Spirito P, Olivotto I, Maron MS. Hypertrophic cardiomyopathy: present and future, with translation into contemporary cardiovascular medicine. J Am Coll Cardiol. 2014;64(1):83-99. Erratum in: J Am Coll Cardiol. 2014;64(11):1188 Recent studies suggest that this prevalence is even higher, around 1:200, in the general population,⁶6 Semsarian C, Ingles J, Maron MS, Maron BJ. New perspectives on the prevalence of hypertrophic cardiomyopathy. J Am Coll Cardiol. 2015;65(12):1249-54. and around 5% of those who have HCM carry more than one disease-causing gene variant.⁷7 Kelly M, Semsarian C. Multiple mutations in genetic cardiovascular disease: a marker of disease severity? Circ Cardiovasc Genet. 2009;2(2):182-90.

8 Van Driest SL, Vasile VC, Ommen SR, Will ML, Tajik AJ, Gersh BJ, et al. Myosin binding protein C mutations and compound heterozygosity in hypertrophic cardiomyopathy. J Am Coll Cardiol. 2004;44(9):1903-10.^-99 Ingles J, Doolan A, Chiu C, Seidman J, Seidman C, Semsarian C. Compound and double mutations in patients with hypertrophic cardiomyopathy: implications for genetic testing and counselling. J Med Genet. 2005;42(10):e59. The hypothesis of gene dosage effects in patients with multiple variants is supported by some authors who have reported a more severe clinical feature, with greater risk of SCD, major LV hypertrophy, and earlier onset of HCM.⁷7 Kelly M, Semsarian C. Multiple mutations in genetic cardiovascular disease: a marker of disease severity? Circ Cardiovasc Genet. 2009;2(2):182-90.^,1010 Wang J, Wang Y, Zou Y, Sun K, Wang Z, Ding H, et al. Malignant effects of multiple rare variants in sarcomere genes on the prognosis of patients with hypertrophic cardiomyopathy. Eur J Heart Fail. 2014;16(9):950-7.

In this context, we present a case herein in which a compound heterozygous variant led to a HCM manifestation with disease phenotype magnification.

Methods

Subjects

The proband with clinical HCM diagnosis was referred to genetic analysis at the National Cardiology Institute (Instituto Nacional de Cardiologia - INC) in Rio de Janeiro. A genealogical tree, including the highest possible number of generations, was built based on his family history. Family members were submitted to clinical assessments and genetic investigations. The local ethics committee approved this study. Written informed consent was obtained for every analyzed family member.

Clinical assessment

The proband underwent clinical and cardiovascular examination, including a 12-lead electrocardiogram (ECG), transthoracic echocardiography (TTE) and 24-hour Holter monitoring. Diagnosis of HCM was based on TTE: major echo diagnostic criteria were defined by a maximal LV end-diastolic wall thickness ≥ 15 mm. The same clinical examination was performed for the phenotypic analyses of all family members, and cardiac magnetic resonance imaging (CMR) was requested as a complementary exam.

A risk score proposed by the European Cardiac Society (ESC) was used to predict the risk for SCD in five years for patients with HCM.¹¹11 O'Mahony C, Jichi F, Pavlou M, Monserrat L, Anastasakis A, Rapezzi C, et al; Hypertrophic Cardiomyopathy Outcomes Investigators. A novel clinical risk prediction model for sudden cardiac death in hypertrophic cardiomyopathy (CMH risk-SCD). Eur Heart J. 2014;35(30):2010-20.

Genetic analysis

Sanger sequencing

The genetic analysis of the proband was performed through direct sequencing of the three sarcomere genes: MYH7, MYBPC3 and TNNT2. Genomic DNA obtained from leukocytes according to Miller et al.¹²12 Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16(3):1215. was submitted to a polymerase chain reaction (PCR) of all coding exons, using previously described primers and others designed by us (Tables 1, 2 and 3), and the same amplification program. PCR products were cleaned-up with EXOSAP-IT (Affymetrix, Santa Clara, CA), subjected to the sequencing reaction using the BigDye^® Terminator v3.1 reagent (Thermo Fisher Scientific, Waltham, MA) and subsequently analyzed on a ABI 3500xL genetic analyzer (Thermo Fisher Scientific, Waltham, MA). Sequence analyses were performed using the Geneious^® v.6.1.6 software package (Biomatters, Auckland, NZ). The family was submitted to a mutation-specific screening according to the HRS/EHRA expert consensus statement.¹³13 Ackerman MJ, Priori SG, Willems S, Berul C, Brugada R, Calkins H, et al; Heart Rhythm Society (HRS); European Heart Rhythm Association (EHRA). HRS/EHRA expert consensus statement on the state of genetic testing for the channelopathies and cardiomyopathies: this document was developed as a partnership between the Heart Rhythm Society (HRS) and the European Heart Rhythm Association (EHRA). Europace. 2011;13(8):1077-109. Erratum in: Erratum in: Europace. 2012;14(2):277.

Variant pathogenicity prediction

Effects of missense mutations were predicted by using the PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT/PROVEAN (http://SIFT.jcvi.org/) and PredictProtein (http://predictprotein.org/home) tools. A5YM48 and Q14896 were used as MYBPC3 reference sequences (UniProtKB).

Results

A seventeen-year-old (y) male proband presenting with a clinical manifestation of HCM and syncope history was submitted to a cardioverter-defibrillator implantation for syncope primary prevention. The diagnosis was based on TTE and showed a reverse curve asymmetric septal hypertrophy, with 39-mm thickness with preserved LV systolic function and normal LV ejection fraction (Figure 1). Additionally, diastolic type II dysfunction, maximum gradient LV/Aorta of 25 mmHg, systolic anterior motion of the mitral valve, obstruction of the LV outflow tract, and enlarged left atrium (46 mm) were also present. The ECG showed LV and LA overload and 24-hour Holter monitoring failed to document the presence of ventricular tachycardia. The risk of SCD was considered high, at 7.69%. The genetic analysis identified a compound heterozygous missense variant, c.1624G>C (p.E542Q) and c.1828G>C (p.D610H) in MYBPC3 (Figure 2). The variant p.E542Q (rs121909374) has been associated with HCM in ClinVar and in the Human Gene Mutation Database (HGMD). The in silico analysis performed by PolyPhen-2 predicts this variant as possibly harmful, while SIFT/PROVEAN and PredictProtein classify this mutation as tolerable. On the other hand, p.D610H (rs371564200) is classified as a variant of uncertain significance (VUS), although pathogenicity prediction tools rank p.D610H as probably deleterious/harmful. Both variants affect conserved residues in the polypeptide chain (Figure 2).

The proband is the only member that manifests the HCM phenotype in his family. His father was adopted, so only maternal ascendants are known. The constructed heredogram revealed 30 relatives, over five generations, in which only one unexplained death of a 30-year-old female with no HCM diagnosis was detected (Figure 2).¹⁴14 Mattos BP, Scolari FL, Torres MA, Simon L, Freitas VC, Giugliani R, et al. Prevalence and phenotypic expression of mutations in the MYH7, MYBPC3 and TNNT2 genes in families with hypertrophic cardiomyopathy in the south of Brazil: a cross-sectional study. Arq Bras Cardiol. 2016;107(3):257-65.

Genotyping of maternal family members - grandmother (59y), aunt (29y), uncle (35y) and mother (39y) - detected the p.D610H variant. All family members were asymptomatic, with normal TTE and ECG, with no evidence of VH. On the other hand, the allele p.E542Q was detected in the father (40y) and a paternal sibling (8y), both with normal clinical assessment results (Table 4). CMR was performed in the mother, aunt, and father, and resulted in normal findings, specifically normal LV wall thickness and no signs of fibrosis (Figure 1).

Discussion

The present study reports on a young individual with severe HCM who carries a compound trans-heterozygous variant in the MYBPC3 gene, with one allele - p.D610H - inherited from the mother and the other - p.E542Q - inherited from the father.

Individuals with a single variant did not show any HCM phenotype. The p.E542Q variant, found in the paternal relatives, is associated to HCM, with good prognosis and moderate wall hypertrophy, although only a few studies mentioning this mutation are available¹⁰10 Wang J, Wang Y, Zou Y, Sun K, Wang Z, Ding H, et al. Malignant effects of multiple rare variants in sarcomere genes on the prognosis of patients with hypertrophic cardiomyopathy. Eur J Heart Fail. 2014;16(9):950-7.^,1515 Marsiglia JD, Credidio FL, de Oliveira TG, Reis RF, Antunes Mde O, de Araujo AQ, et al. Screening of MYH7, MYBPC3, and TNNT2 genes in Brazilian patients with hypertrophic cardiomyopathy. Am Heart J. 2013;166(4):775-82.

16 Fokstuen S, Munoz A, Melacini P, Iliceto S, Perrot A, Ozcelik C, et al. Rapid detection of genetic variants in hypertrophic cardiomyopathy by custom DNA resequencing array in clinical practice. J Med Genet. 2011;48(8):572-6.^-1717 Rodríguez-García MI, Monserrat L, Ortiz M, Fernández X, Cazón L, Núñez L, et al. Screening mutations in myosin binding protein C3 gene in a cohort of patients with Hypertrophic Cardiomyopathy. BMC Med Genet. 2010;11:67.. Pathogenicity prediction of p.E542Q is in agreement with literature data¹⁸18 Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7(4):248-9.

19 Ng PC, Henikoff S. Predicting deleterious amino acid substitutions. Genome Res. 2001;11(5):863-74.

20 Ng PC, Henikoff S. Accounting for human polymorphisms predicted to affect protein function. Genome Res. 2002;12(3):436-46.^-2121 Choi Y, Sims GE, Murphy S, Miller JR, Chan AP. Predicting the functional effect of amino acid substitutions and indels. PLoS One. 2012;7(10):e46688..

Moreover, the p.D610H variant, identified in the maternal relatives, also did not manifest any HCM phenotype, even in the oldest investigated familiar member (59y). The association between p.D610H and HCM remains uncertain, despite the fact that pathogenicity predicting tools classified this as probably pathogenic. Only a single study in the literature has identified this mutation, although it did not correlate it with the disease²²22 Olivotto I, Girolami F, Sciagrà R, Ackerman MJ, Sotgia B, Bos JM, et al. Microvascular function is selectively impaired in patients with hypertrophic cardiomyopathy and sarcomere myofilament gene mutations. J Am Coll Cardiol. 2011;58(8):839-48..

In general, a single HCM-heterozygous mutation is sufficient to affect myocardial function and lead to hypertrophy; however, early studies have associated variants in the MYBPC3 gene with incomplete penetrance, mild VH, low SCD risk and benign clinical evolution²³23 Niimura H, Bachinski LL, Sangwatanaroj S, Watkins H, Chudley AE, McKenna W, et al. Mutations in the gene for cardiac myosin-binding protein C and late-onset familial hypertrophic cardiomyopathy. N Engl J Med. 1998;338(18):1248-57.

24 Cardim N, Perrot A, Santos S, Morgado P, Pádua M, Ferreira S, et al. Hypertrophic cardiomyopathy in a Portuguese population: mutations in the myosin-binding protein C gene. Rev Port Cardiol. 2005;24(12):1463-76.^-2525 Charron P, Dubourg O, Desnos M, Isnard R, Hagege A, Millaire A, et al. Diagnostic value of electrocardiography and echocardiography for familial hypertrophic cardiomyopathy in a genotyped adult population. Circulation. 1997;96(1):214-9..

In conclusion, it is suggested that, individually, the p.E542Q and p.D610H variants generate mild changes in protein structure/function, insufficient to cause a strong phenotype. However, the expression of these variants in trans may be responsible for early disease onset, a more severe clinical phenotype and increased risk of malignant events in the proband. In other words, double or compound variants by themselves are not decisive for a poorer HCM prognosis, but the allelic composition of these variants may be determinant in this regard.

Study limitations

The present study investigated the three major HCM-genes that account for approximately 60-70% of HCM cases⁵5 Maron BJ, Ommen SR, Semsarian C, Spirito P, Olivotto I, Maron MS. Hypertrophic cardiomyopathy: present and future, with translation into contemporary cardiovascular medicine. J Am Coll Cardiol. 2014;64(1):83-99. Erratum in: J Am Coll Cardiol. 2014;64(11):1188^,1414 Mattos BP, Scolari FL, Torres MA, Simon L, Freitas VC, Giugliani R, et al. Prevalence and phenotypic expression of mutations in the MYH7, MYBPC3 and TNNT2 genes in families with hypertrophic cardiomyopathy in the south of Brazil: a cross-sectional study. Arq Bras Cardiol. 2016;107(3):257-65.. However, several other genes have already been associated to this disease⁵5 Maron BJ, Ommen SR, Semsarian C, Spirito P, Olivotto I, Maron MS. Hypertrophic cardiomyopathy: present and future, with translation into contemporary cardiovascular medicine. J Am Coll Cardiol. 2014;64(1):83-99. Erratum in: J Am Coll Cardiol. 2014;64(11):1188^,1414 Mattos BP, Scolari FL, Torres MA, Simon L, Freitas VC, Giugliani R, et al. Prevalence and phenotypic expression of mutations in the MYH7, MYBPC3 and TNNT2 genes in families with hypertrophic cardiomyopathy in the south of Brazil: a cross-sectional study. Arq Bras Cardiol. 2016;107(3):257-65., which are yet to be investigated.

References

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