Myosin-binding Protein C Compound Heterozygous Variant Effect on the Phenotypic Expression of Hypertrophic Cardiomyopathy

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.


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. 1 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. 2 A benign outcome of HCM may also occur, such as late onset, mild hypertrophy, and a history of non-malignant events. 3 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. 4 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). 5 Recent studies suggest that this prevalence is even higher, around 1:200, in the general population, 6 and around 5% of those who have HCM carry more than one disease-causing gene variant. [7][8][9] 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. 7,10 In this context, we present a case herein in which a compound heterozygous variant led to a HCM manifestation with disease phenotype magnification.

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.

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. 12 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. 13

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). 14 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 available 10,[15][16][17] . Pathogenicity prediction of p.E542Q is in agreement with literature data [18][19][20][21] .
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 disease 22 .
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 evolution 23-25 . 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 cases 5,14 . However, several other genes have already been associated to this disease 5,14 , which are yet to be investigated.

Potential Conflict of Interest
No potential conflict of interest relevant to this article was reported.

Sources of Funding
This study was funded by Instituto Nacional de Cardiologia and Fundação Pró Coração (Fundacor).

Study Association
This article is part of the thesis of Master submitted by Julianny Freitas Rafael, from Instituto Nacional de Cardiologia.