Arq Bras Cardiol abc Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia - SBC njwLCw4dvpvwS7T4TYNLtVs S0066-782X2017000400354 10.5935/abc.20170045 Brief Communication Myosin-binding Protein C Compound Heterozygous Variant Effect on the Phenotypic Expression of Hypertrophic Cardiomyopathy Rafael Julianny Freitas 1 Cruz Fernando Eugênio dos Santos Filho 1 Carvalho Antônio Carlos Campos de 1 Gottlieb Ilan 1 2 Cazelli José Guilherme 2 Siciliano Ana Paula 1 Dias Glauber Monteiro 1 Instituto Nacional de Cardiologia Rio de Janeiro RJ Brazil Instituto Nacional de Cardiologia; Rio de Janeiro, RJ - Brazil Casa de Saúde São José Rio de Janeiro RJ Brazil Casa de Saúde São José, Rio de Janeiro, RJ - Brazil Mailing Address: Glauber Monteiro Dias, Rua das Laranjeiras, 374. 5º andar. Postal Code 22240-006, Laranjeiras, RJ - Brazil. E-mail: glauber.dias@gmail.com

Author contributions

Conception and design of the research: Cruz Filho FES, Dias GM; Acquisition of data: Rafael JF, Gottlieb I, Cazelli JG, Siciliano AP, Dias GM; Analysis and interpretation of the data: Rafael JF, Cruz Filho FES, Gottlieb I, Dias GM; Obtaining funding: Dias GM; Writing of the manuscript: Rafael JF, Dias GM; Critical revision of the manuscript for intellectual content: Cruz Filho FES, Carvalho ACC, Dias GM.

Potential Conflict of Interest

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

 04 2017 108 4 354 360 21 07 2016 29 11 2016 20 12 2016 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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 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.7-9 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.

 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.11

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.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

Primers for MYH7 sequencing
Exon Forward Primer 5'-3' Reverse Primer 5'-3' Amplicon† A.T.‡
3 TCTTGACTCTTGAGCATGGTGCTA TCTGTCCACCCAGGTGTACAGGTG 381 bp 62ºC
4 AGGAAGGAGGGAAAGCCCAGGCTG TCTGCATGCACTCAATCTGAGTAA 380 bp 62ºC
5 ATCTTTCTCTAACTCCCAAAATCA ACTCACGTGATCAGGATGGACTGG 398 bp 60ºC
6 TGTCACCGTCAACCCTTACAAGTG GAGGCTGAGTCTATGCCTCGGGG 394 bp 62ºC
7 CTTGCTGGTCTCCAGTAGTATTGT CTGCGGTACAGGACCTTGGAGGGC 198 bp 62ºC
8 GCCCTCCAAGGTCCTGTACCGCAG GTCCAAGTCCCAAGGCCAAGGTCA 200 bp 62ºC
9 GACAACTCCTCCCGCTTCGTG AACAGAGGGAGGGAGGGGAGAG 281 bp 62ºC
10 CCTTTTGCTTGCTACATTTATCAT GCCACAAGCAGAGGGGACCAG 252 bp 60ºC
11 CTGCTTCCTCAGGCCATGTGCTGT ACCAATGGCCAGAGTCTTAGCTCT 284 bp 62ºC
12 CACAGGGATTAAGGAGACAAGTTT TTACAGCTGCCCCAAGAATC 273 bp 58ºC
13 AGTCATCTCTTTACCAACTTTGCTA ATTATCATCTGAAGATGGACCCACC 186 bp 62ºC
14 CAAGTTCACTCTTCCCAACAACCCT ATGTGGGAGCGAGTGAGTGATTGTT 258 bp 62ºC
15 ACTCACACCCACTTTCTGACTGCTC GAATTCAGGTGGTAAGGCCAAAGAG 247 bp 62ºC
16 ATAACTGTACTCAGAGCTGAGCCTA TCCATCCCACTGAGTCTGTAAACCT 578 bp 62ºC
17 GCAAATGCCAGCAAGGATGTAAAG AGAGAAGGGAGATGGGAAGTAA 359 bp 58ºC
18 CATCTCTGTGACTTCTCGAATTCT CACTGTGGTGGTAGGTAGGGAGAT 300 bp 60ºC
19 ACAAAGCCAGGATCAGAACCCAGA GTCCAGAGTCACCCATGCTCTGCA 323 bp 62ºC
20 TGGGTATGAGGGTGCACCAGAGCT GCATCAGAGGAGTCAATGGAAAAG 330 bp 62ºC
21 TAGGCTGTTACCCTTCCTAAGGTA GCCTCTGACCCTGTGACTGCAGTG 374 bp 62ºC
22 GGACCTCAGGTAGGAAGGAGGCAG TGTGCAGGGAGGTGCAGGGTTGTG 390 bp 62ºC
23 TCCTATTTGAGTGATGTGCCTCTC ATGGTCTGAGAGTCCTGATGAGAC 390 bp 62ºC
24 AGATGGCACCAAGCTGGTGACCTT TCTGGGCACAGATAGACATGGCAT 290 bp 62ºC
25 GGCAATCTCACAGTCCCCTAATAA TTTTTGCCAGGGAGGACCATCTAA 508 bp 60ºC
26 ACTCTTTACCTGTATCATTACCAT GCCTCCATGGACACATAATCAGTT 306 bp 60ºC
27a* AGCCGAGAGCCTTTTAGAGCCG GTCCCGCCGCATCTTCTGGA 274 bp 64ºC
27b* TCCAGAAGATGCGGCGGGAC AGGGGAGGTGGGAGGAGGAAGT 266 bp 64ºC
28 TCCCACTTCCCTTCCTCTGCCT CAGCACTCCTCTCTATCCCCACCT 438 bp 56ºC
29 GGTGGGGATAGAGAGGAGTGCTGA TGTGGCAGGGTTTGGGCTGT 315 bp 64ºC
30 GAGAAGGGCAAGGGTGGGGT CCTGAGAGGAGAAGGAGGTGGG 422 bp 58ºC
31 TTGTCCCCATCCACACCCTCCA GCTCCGACTGCGACTCCTCATACT 469 bp 56ºC
32 GCTGAAGAGTGAGCCTTGTCCC TCCGCTGGAACCCAACTGCT 396 bp 56ºC
33 AGTATGAGGAGTCGCAGTCGGA GGGGATGAGAACAGGGAGCCAA 500 bp 60ºC
34 CTGCCCTGTGCCCTGACTGT CCAGCCTCGGTTCCCTTCACT 500 bp 64ºC
35 GTGAAGGGAACCGAGGCTGGC GTTGGGCAGAGCAGGAAAAGCA 364 bp 62ºC
36 TCCGTGCCAACGACGACCTGAA GTCCTCACACACTTGCTGCCCA 497 bp 60ºC
37 TGGGCAGCAAGTGTGTGAGGA GGTTGTCACTGTGGCTATGGTGC 391 bp 62ºC
38 / 39 ACCTTCTATGACTGTGCCATCTTCAC GTTTGAGGGTGCTCTGTCTGG 464 bp 62ºC
40 ATGCCCTGTCCCTGCCCAATAC TTTCCACCTCCCCTATGCCAGACC 268 bp 60ºC

Necessary more than one primer pair to cover the exon;

Size of the amplified fragment;

Annealing temperature

 Primers for MYBPC3 sequencing
Exon Forward Primer 5'-3' Reverse Primer 5'-3' Amplicon* A.T.†
2 GACCTCAGCTCTCTGGAATTCATC GCTCAGAGGCCACGTCCTCGTCAA 311 bp 62ºC
3 GTGCACGCTCCAACCAG CAGCAAAGGCAAGAAAGTGTG 429 bp 65ºC
4 CTGGGACGGGGAGGAGAATGTG GCTTTTGAGACCTGCCCTGGAC 385 bp 62ºC
5 GGGCACCTGCGGTCCCAGCTAACT ACGCGGGCTGAGAAGGTGATG 378 bp 62ºC
6 CTACCCCTGGAGCCCCCGATGACC TGCCTCCCAGATTCCCCACACC 449 bp 62ºC
7 CTGGAGCTCCTGGTCTTATGTGAT GGAGCCGTGACACCAAGATGATAA 528 bp 62ºC
8 GCTTCTCAAACGGCCCCCTCTG AGCTCCGCCCCGCAAATCATCC 213 bp 62ºC
9 GGGCTGGGGATGATTTG GGAGGGAGAAAGGGACACTA 226 bp 63ºC
10 AATCTGGCTAGTGTCCCTTTCTCC AGCCCTTTAACTCCTTCCACACTG 322 bp 62ºC
11 TCGGCCCAACTGACTTA CCCATGGGCCTTTACTT 389 bp 58ºC
12 CGGCTCCCCACGGACAG CCCAGGCCAGGCAGGACT 405 bp 67ºC
13 TCCCCAGCCCCTCTTCA GCCGGACTCCGCTCTTT 515 bp 62ºC
14 GGCGGCACAGAGGGGATTG ACCGGCAGGAGCAAAAGGATG 402 bp 62ºC
15 ATCCGGCTGACCGTGGAACT CAGTGCGCCCCGTGATAATC 375 bp 65ºC
16 AACACTTCAACGGCCCCTTCTG GCCCCCTCCTCCGATACTTCACAC 451 bp 62ºC
17 CGGACGACGCAGCCTACCAGT GTCAGCTCCACCCCGTCCTTCA 366 bp 62ºC
18 GGAGGAGGGGGCGCAAGTCAAAT GTCAAAGGCCCAAGGTCACAGAGG 400 bp 62ºC
19 ACAGGCACACGTGTTTTCAC CAGTCTCCACCTGTCCCATC 345 bp 61ºC
20 AGAATACCAACAAGCCAGGACAAG GCGGGAAAGTGAGCAGAACC 402 bp 62ºC
21 TGCCTTTGCCCCCGTGCTACTTG GCCCCAGGACCCCCACTTTTGAT 187 bp 62ºC
22 TCCTCCTGGCTCTCCCGTTTCTCT GCGCCCCTCTGCTGCTTCTTC 379 bp 62ºC
23 GCTCCTCTGCTCCCTACTTCC ATGGCCATCAGCACACTTCAC 310 bp 62ºC
24 TCGGTGCCACAGAGATGATTTTGA GGCTGCCCCTCTGTGTTCTCCA 367 bp 62ºC
25 CCTGTGGCGGTTAGTTGG CACCGGTAGCTCTTCTTCTTCTTG 350 bp 62ºC
26 CCGAGGGAAGGTGGTGTGG TCTGTAAAATGCGGCTGAGTATCC 404 bp 62ºC
27 GGAAGTGCCCCCTATGT TCGCACTGCTCAAAGAAG 457 bp 62ºC
28 TCAGAGGAGTGGGCAGTGGGAGTG CTGGGGTGTCAATGGCGGGTCTT 292 bp 62ºC
29 GCCTGGAGTTGCTGTGTTAG GGCTGCCCCTCTTTGGTC 467 bp 62ºC
30 GCGGCCGGCCCTTGGAGT TGGAAAATGTGAGCTGTGGGTTGG 356 bp 62ºC
31 GCATTCAGGCACTTACCAGGTGACG CACGGTGAGGACAGTGAAGGGTAGC 527 bp 60ºC
32 GGCCGCAGCTACCCTTCAC GGCCCCTCTCCCTGTTCC 392 bp 65ºC
33 GGCCTCTCGGTACCAAGTCCTGTC CAACGTCGGGGCCTGTGAGC 232 bp 65ºC
34 GCAGGGCCATGGTACTCACTCTTG CCGCCCGCTCTTCCCATCTC 404 bp 62ºC
35 CACAGTGACATGGCCTCCTCTTCT GCCCCTACAGCCTCCCATTTACT 159 bp 62ºC

Size of the amplified fragment;

Annealing temperature.

 Primers for TNNT2 sequencing
Exon Forward Primer 5'-3' Reverse Primer 5'-3' Amplicon* A.T.†
2 ACAGCTCATGAGGGGTGGAACTA GTGCTCTGCCTGGGATCTACAACC 376 bp 65ºC
3 / 4 ATGAGAACGGCAGGCCAGGCTAGTG GTTTGCCTCAAGACCCGAGCAACC 506 bp 65ºC
5 GTGGCGGGAGGTAGCCGACAGT TGGGCAATCAATGGTTGAATCTTA 403 bp 65º C
6 TTGACCCAGCGCTTCTCTTGTGTC ACTGGGTGCCACCAATGCAACTTC 449 bp 65º C
7 CCAGTGCCGGGAGGGACTCAC CAGCCCGTGTCCACTGCACCATAC 262 bp 65º C
8 GGATCAGGGGCCCTGCCTGTCCTGACA TCCTCCTCCTCTTTCTTCCTGTTCT 538 bp 62º C
9 GCCAGGCCCTGCCAGAGGTCTT CCCTGGGGGAGGCCTGAAACAG 494 bp 70º C
10 ACGTCCGTGGAGCTGGTTGAAAGT CCCGGCCAATATTGTCTCTTGACT 373 bp 62º C
11 TGGGAGCTACCCTCTCAGAA CACAGCAGCTGGGAATCTCT 369 bp 60º C
12 GTAAACCCGGCTGACTACAG AGCCAGCCCAATCTCTTCAC 258 bp 62º C
13 CAGGGGGTTTGGGGAGGGTTAG GTGGGGCACCTGCTCAGTTCTCT 402 bp 60º C
14 GGAGGGCCCTTTCTTACTGGAC CCGGACCCAGTGAACCAGGAGGAG 207 bp 68º C
15 GCCCCTCCTGACCCTTAACTATCC CGGAGGAGCCAGAGAAGGAAACCT 353 bp 62º C
16 GGGGGTGAAATGTGGGGCGGAGAA GTGTGGGGGCAGGCAGGAGTGGTG 383 bp 62º C

Size of the amplified fragment;

Annealing temperature.

 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).

TTE of the proband and CMR of the family. A) TTE image of the four heart chambers and aorta revealing the reverse curve septal hypertrophy. B) Parasternal short-axis view showing the septal hypertrophy. C) Parasternal long-axis view displaying the LV and septal hypertrophy and the enlarged left atrium. The white arrow shows the systolic anterior motion of the mitral valve. D) TTE image showing the obstruction and the turbulence in the outflow tract of the left ventricle (white arrow). Mild mitral regurgitation in the left atrium is visible. CMR of the proband's father (E), aunt (F) and mother (G), showing no hypertrophy or fibrosis signs. CMR in the inversion-recovery sequence (delayed enhancement) in 4CH axes (E1, F1, G1), LVSV (E2, F2, G2) and 2CH (E3, F3, G3). RA: right atrium; RV: right ventricle; LA: left atrium; LV: left ventricle; Ao: aorta.

A) Pedigree showing five generations of the maternal family. The proband is the only HCM-affected member. The family variant allele carriers are indicated by E542Q+ and D610H+. B) Electropherograms of the compound missense variant regions of the MYBPC3 gene of the proband. C) Multiple species alignment of the myosin-binding protein C amino acid sequence for residues 538 to 546 and 606 to 614. The conserved residues, glutamic acid and aspartic acid, are indicated by a rectangle.

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).

Clinical assessment data of the individuals
Epidemiology ECG TTE
ID Age (Y) Sex HCM Variant LAO LVO ABN T wave LVH + LVH type Form Max LVWT (mm) LVOG mmHg LVSD LVDD SAM LA size (mm)
III.8 59 F No D610H No No No No - - 10 No No No No 28
IV.2 40 M No E542Q No No No No - - 9 No No No No 35
IV.3 39 F No D610H No No No No - - 9 No No No No 37
IV.6 29 F No D610H No No No No - - 8 No No No No 32
IV.7 35 M No D610H No No No No - - 8 No No No No 36
V.1 8 M No E542Q No No No No - - 7 No No No No 37
V.2 17 M Yes D610H E542Q Yes Yes Yes Yes Septal Reverse Curve 39 25 No Type I No 46

The identification numbering (ID) of individuals follows the standard adopted in the pedigree charts (Figure 2); ECG: electrocardiography; TTE: Transthoracic echocardiography; (Y): years; HCM: hypertrophic cardiomyopathy; LAO: left atrial overload; LVO: left ventricular overload; ABN T wave: abnormal T wave; LVH + : left ventricular hypertrophy showed by echo; LVH type: type of the left ventricular hypertrophy; Max LVWT: maximal thickness of the left ventricular wall; LVOG: left ventricular outflow gradient; LVSD: left ventricular systolic dysfunction; LVDD: left ventricular diastolic dysfunction; SAM: systolic anterior motion; LA size: left atrial size.

 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 available10,15-17. Pathogenicity prediction of p.E542Q is in agreement with literature data18-21.

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 disease22.

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 evolution23-25.

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 cases5,14. However, several other genes have already been associated to this disease5,14, which are yet to be investigated.

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.

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Ackerman MJ Priori SG Willems S Berul C Brugada R Calkins H Heart Rhythm Society European Heart Rhythm Association 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 1109 Erratum in: Erratum in: Europace. 2012;14(2):277 14 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. Mattos BP Scolari FL Torres MA Simon L Freitas VC Giugliani R 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 3 257 265 15 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. Marsiglia JD Credidio FL de Oliveira TG Reis RF Antunes Mde O de Araujo AQ Screening of MYH7, MYBPC3, and TNNT2 genes in Brazilian patients with hypertrophic cardiomyopathy Am Heart J 2013 166 4 775 782 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. Fokstuen S Munoz A Melacini P Iliceto S Perrot A Ozcelik C Rapid detection of genetic variants in hypertrophic cardiomyopathy by custom DNA resequencing array in clinical practice J Med Genet 2011 48 8 572 576 17 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. Rodríguez-García MI Monserrat L Ortiz M Fernández X Cazón L Núñez L Screening mutations in myosin binding protein C3 gene in a cohort of patients with Hypertrophic Cardiomyopathy BMC Med Genet 2010 11 67 67 18 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. Adzhubei IA Schmidt S Peshkin L Ramensky VE Gerasimova A Bork P A method and server for predicting damaging missense mutations Nat Methods 2010 7 4 248 249 19 Ng PC, Henikoff S. Predicting deleterious amino acid substitutions. Genome Res. 2001;11(5):863-74. Ng PC Henikoff S Predicting deleterious amino acid substitutions Genome Res 2001 11 5 863 874 20 Ng PC, Henikoff S. Accounting for human polymorphisms predicted to affect protein function. Genome Res. 2002;12(3):436-46. Ng PC Henikoff S Accounting for human polymorphisms predicted to affect protein function Genome Res 2002 12 3 436 446 21 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. 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 22 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. Olivotto I Girolami F Sciagrà R Ackerman MJ Sotgia B Bos JM Microvascular function is selectively impaired in patients with hypertrophic cardiomyopathy and sarcomere myofilament gene mutations J Am Coll Cardiol 2011 58 8 839 848 23 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. Niimura H Bachinski LL Sangwatanaroj S Watkins H Chudley AE McKenna W Mutations in the gene for cardiac myosin-binding protein C and late-onset familial hypertrophic cardiomyopathy N Engl J Med 1998 338 18 1248 1257 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. Cardim N Perrot A Santos S Morgado P Pádua M Ferreira S Hypertrophic cardiomyopathy in a Portuguese population: mutations in the myosin-binding protein C gene Rev Port Cardiol 2005 24 12 1463 1476 25 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. Charron P Dubourg O Desnos M Isnard R Hagege A Millaire A Diagnostic value of electrocardiography and echocardiography for familial hypertrophic cardiomyopathy in a genotyped adult population Circulation 1997 96 1 214 219 Comunicação Breve Mutação em Hetrozigose Composta no Gene da Proteína C Ligante de Miosina e sua Expressão Fenotípica na Cardiomiopatia Hipertrófica Rafael Julianny Freitas 1 Cruz Fernando Eugênio dos Santos Filho 1 Carvalho Antônio Carlos Campos de 1 Gottlieb Ilan 1 2 Cazelli José Guilherme 2 Siciliano Ana Paula 1 Dias Glauber Monteiro 1 Instituto Nacional de Cardiologia; Rio de Janeiro, RJ - Brasil Casa de Saúde São José, Rio de Janeiro, RJ - Brasil Correspondência: Glauber Monteiro Dias, Rua das Laranjeiras, 374. 5º andar. CEP 22240-006, Laranjeiras, RJ – Brasil. E-mail: glauber.dias@gmail.com

Contribuição dos autores

Concepção e desenho da pesquisa: Cruz Filho FES, Dias GM; Obtenção de dados: Rafael JF, Gottlieb I, Cazelli JG, Siciliano AP, Dias GM; Análise e interpretação dos dados: Rafael JF, Cruz Filho FES, Gottlieb I, Dias GM; Obtenção de financiamento: Dias GM; Redação do manuscrito: Rafael JF, Dias GM; Revisão crítica do manuscrito quanto ao conteúdo intelectual importante: Cruz Filho FES, Carvalho ACC, Dias GM.

Potencial conflito de interesses

Declaro não haver conflito de interesses pertinentes.

 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 Introdução

A cardiomiopatia hipertrófica (CMH) é um transtorno miocárdico genético caracterizado por hipertrofia ventricular (HV), frequentemente assimétrica no septo interventricular e que pode levar a uma obstrução dinâmica da via de saída do ventrículo esquerdo (VE).1 É a principal causa de morte súbita cardíaca (MSC) em jovens, com uma mortalidade anual de 2-4% em adultos e 6% em adolescentes e crianças.2 Um resultado benigno da CMH também pode ocorrer, como início tardio, hipertrofia leve e histórico sem eventos malignos.3 Genes modificadores, influências ambientais, diversidade de variantes genéticas e o efeito de múltiplas variantes poderiam explicar a grande heterogeneidade clínica entre indivíduos de uma mesma família ou de famílias diferentes.4

A CMH é um distúrbio mendeliano relativamente comum (0,2%), causado principalmente por mutações nos genes das proteínas do sarcômero, mais comumente aqueles que codificam a cadeia pesada da β-miosina (MYH7), a proteína C ligante de miosina (MYBPC3) e a troponina T (TNNT2).5 Estudos recentes sugerem que essa prevalência é ainda maior, em torno de 1: 200 na população em geral,6 e cerca de 5% daqueles que têm CMH possuem mais de uma variante genética causadora da doença.7-9 A hipótese de efeitos da dosagem gênica em pacientes com múltiplas variantes é sustentada por alguns autores que relataram quadro clínico mais grave, com maior risco de MSC, maior hipertrofia do VE e início mais precoce da CMH.7,10

Neste contexto, apresentamos aqui um caso no qual uma variante em heterozigose composta conduz à uma manifestação da CMH com exacerbação do fenótipo da doença.

 Métodos Amostra

O probando com diagnóstico clínico de CMH foi encaminhado para análise genética no Instituto Nacional de Cardiologia - INC, no Rio de Janeiro. Uma árvore genealógica, incluindo o maior número de gerações possíveis, foi construída com base em sua história familiar. Os membros da família foram submetidos a avaliações clínicas e investigações genéticas. O comitê de ética local aprovou este estudo. Obteve-se consentimento informado por escrito para cada membro avaliado da família.

 Avaliação clínica

O probando foi submetido a exame clínico e cardiovascular, incluindo eletrocardiograma de 12 derivações (ECG), ecocardiograma transtorácico (ETT) e monitoramento de 24h por Holter. O diagnóstico de CMH foi baseado no ETT: os principais critérios diagnósticos do ecocardiograma foram definidos por uma espessura máxima diastólica final do VE ≥ 15 mm. O mesmo exame clínico foi realizado para as análises fenotípicas de todos os membros da família, e a ressonância magnética cardíaca (RMC) foi solicitada como exame complementar.

Foi utilizado um escore de risco proposto pela European Cardiac Society (ESC) para prever o risco de MSC em cinco anos para os pacientes com CMH.11

Análise genética Sequenciamento de Sanger

A análise genética do probando foi realizada através do sequenciamento direto de três genes do sarcômero: MYH7, MYBPC3 e TNNT2. O DNA genômico obtido de leucócitos, de acordo com Miller et al.,12 foi submetido à reação em cadeia da polimerase (PCR) de todos os exons codificadores, utilizando primers previamente descritos e outros projetados por nós (Tabelas 1, 2 e 3), e o mesmo programa de amplificação. Os produtos da PCR foram purificados com EXOSAP-IT (Affymetrix, Santa Clara, CA), submetidos à reação de sequenciamento utilizando o reagente BigDye® Terminator v3.1 (Thermo Fisher Scientific, Waltham, MA) e subsequentemente analisados em um analisador genético ABI 3500xL (Thermo Fisher Scientific, Waltham, MA). As análises de sequências foram realizadas utilizando-se o pacote de software Geneious® v.6.1.6 (Biomatters, Auckland, NZ). A família foi submetida a um teste de mutação específica de acordo com a declaração de consenso de especialistas HRS/EHRA.13

Primers para o sequenciamento do MYH7
Forward Primer 5'-3' Reverse Primer 5'-3' Amplicon† A.T.‡
3 TCTTGACTCTTGAGCATGGTGCTA TCTGTCCACCCAGGTGTACAGGTG 381 bp 62ºC
4 AGGAAGGAGGGAAAGCCCAGGCTG TCTGCATGCACTCAATCTGAGTAA 380 bp 62ºC
5 ATCTTTCTCTAACTCCCAAAATCA ACTCACGTGATCAGGATGGACTGG 398 bp 60ºC
6 TGTCACCGTCAACCCTTACAAGTG GAGGCTGAGTCTATGCCTCGGGG 394 bp 62ºC
7 CTTGCTGGTCTCCAGTAGTATTGT CTGCGGTACAGGACCTTGGAGGGC 198 bp 62ºC
8 GCCCTCCAAGGTCCTGTACCGCAG GTCCAAGTCCCAAGGCCAAGGTCA 200 bp 62ºC
9 GACAACTCCTCCCGCTTCGTG AACAGAGGGAGGGAGGGGAGAG 281 bp 62ºC
10 CCTTTTGCTTGCTACATTTATCAT GCCACAAGCAGAGGGGACCAG 252 bp 60ºC
11 CTGCTTCCTCAGGCCATGTGCTGT ACCAATGGCCAGAGTCTTAGCTCT 284 bp 62ºC
12 CACAGGGATTAAGGAGACAAGTTT TTACAGCTGCCCCAAGAATC 273 bp 58ºC
13 AGTCATCTCTTTACCAACTTTGCTA ATTATCATCTGAAGATGGACCCACC 186 bp 62ºC
14 CAAGTTCACTCTTCCCAACAACCCT ATGTGGGAGCGAGTGAGTGATTGTT 258 bp 62ºC
15 ACTCACACCCACTTTCTGACTGCTC GAATTCAGGTGGTAAGGCCAAAGAG 247 bp 62ºC
16 ATAACTGTACTCAGAGCTGAGCCTA TCCATCCCACTGAGTCTGTAAACCT 578 bp 62ºC
17 GCAAATGCCAGCAAGGATGTAAAG AGAGAAGGGAGATGGGAAGTAA 359 bp 58ºC
18 CATCTCTGTGACTTCTCGAATTCT CACTGTGGTGGTAGGTAGGGAGAT 300 bp 60ºC
19 ACAAAGCCAGGATCAGAACCCAGA GTCCAGAGTCACCCATGCTCTGCA 323 bp 62ºC
20 TGGGTATGAGGGTGCACCAGAGCT GCATCAGAGGAGTCAATGGAAAAG 330 bp 62ºC
21 TAGGCTGTTACCCTTCCTAAGGTA GCCTCTGACCCTGTGACTGCAGTG 374 bp 62ºC
22 GGACCTCAGGTAGGAAGGAGGCAG TGTGCAGGGAGGTGCAGGGTTGTG 390 bp 62ºC
23 TCCTATTTGAGTGATGTGCCTCTC ATGGTCTGAGAGTCCTGATGAGAC 390 bp 62ºC
24 AGATGGCACCAAGCTGGTGACCTT TCTGGGCACAGATAGACATGGCAT 290 bp 62ºC
25 GGCAATCTCACAGTCCCCTAATAA TTTTTGCCAGGGAGGACCATCTAA 508 bp 60ºC
26 ACTCTTTACCTGTATCATTACCAT GCCTCCATGGACACATAATCAGTT 306 bp 60ºC
27a* AGCCGAGAGCCTTTTAGAGCCG GTCCCGCCGCATCTTCTGGA 274 bp 64ºC
27b* TCCAGAAGATGCGGCGGGAC AGGGGAGGTGGGAGGAGGAAGT 266 bp 64ºC
28 TCCCACTTCCCTTCCTCTGCCT CAGCACTCCTCTCTATCCCCACCT 438 bp 56ºC
29 GGTGGGGATAGAGAGGAGTGCTGA TGTGGCAGGGTTTGGGCTGT 315 bp 64ºC
30 GAGAAGGGCAAGGGTGGGGT CCTGAGAGGAGAAGGAGGTGGG 422 bp 58ºC
31 TTGTCCCCATCCACACCCTCCA GCTCCGACTGCGACTCCTCATACT 469 bp 56ºC
32 GCTGAAGAGTGAGCCTTGTCCC TCCGCTGGAACCCAACTGCT 396 bp 56ºC
33 AGTATGAGGAGTCGCAGTCGGA GGGGATGAGAACAGGGAGCCAA 500 bp 60ºC
34 CTGCCCTGTGCCCTGACTGT CCAGCCTCGGTTCCCTTCACT 500 bp 64ºC
35 GTGAAGGGAACCGAGGCTGGC GTTGGGCAGAGCAGGAAAAGCA 364 bp 62ºC
36 TCCGTGCCAACGACGACCTGAA GTCCTCACACACTTGCTGCCCA 497 bp 60ºC
37 TGGGCAGCAAGTGTGTGAGGA GGTTGTCACTGTGGCTATGGTGC 391 bp 62ºC
38 / 39 ACCTTCTATGACTGTGCCATCTTCAC GTTTGAGGGTGCTCTGTCTGG 464 bp 62ºC
40 ATGCCCTGTCCCTGCCCAATAC TTTCCACCTCCCCTATGCCAGACC 268 bp 60ºC

Necessário mais de um par de primers para cobrir o exon;

Tamanho do fragmento amplificado;

Temperatura de anelamento.

 Primers para o sequenciamento de MYBPC3
Exon Forward Primer 5'-3' Reverse Primer 5'-3' Amplicon* A.T.†
2 GACCTCAGCTCTCTGGAATTCATC GCTCAGAGGCCACGTCCTCGTCAA 311 bp 62ºC
3 GTGCACGCTCCAACCAG CAGCAAAGGCAAGAAAGTGTG 429 bp 65ºC
4 CTGGGACGGGGAGGAGAATGTG GCTTTTGAGACCTGCCCTGGAC 385 bp 62ºC
5 GGGCACCTGCGGTCCCAGCTAACT ACGCGGGCTGAGAAGGTGATG 378 bp 62ºC
6 CTACCCCTGGAGCCCCCGATGACC TGCCTCCCAGATTCCCCACACC 449 bp 62ºC
7 CTGGAGCTCCTGGTCTTATGTGAT GGAGCCGTGACACCAAGATGATAA 528 bp 62ºC
8 GCTTCTCAAACGGCCCCCTCTG AGCTCCGCCCCGCAAATCATCC 213 bp 62ºC
9 GGGCTGGGGATGATTTG GGAGGGAGAAAGGGACACTA 226 bp 63ºC
10 AATCTGGCTAGTGTCCCTTTCTCC AGCCCTTTAACTCCTTCCACACTG 322 bp 62ºC
11 TCGGCCCAACTGACTTA CCCATGGGCCTTTACTT 389 bp 58ºC
12 CGGCTCCCCACGGACAG CCCAGGCCAGGCAGGACT 405 bp 67ºC
13 TCCCCAGCCCCTCTTCA GCCGGACTCCGCTCTTT 515 bp 62ºC
14 GGCGGCACAGAGGGGATTG ACCGGCAGGAGCAAAAGGATG 402 bp 62ºC
15 ATCCGGCTGACCGTGGAACT CAGTGCGCCCCGTGATAATC 375 bp 65ºC
16 AACACTTCAACGGCCCCTTCTG GCCCCCTCCTCCGATACTTCACAC 451 bp 62ºC
17 CGGACGACGCAGCCTACCAGT GTCAGCTCCACCCCGTCCTTCA 366 bp 62ºC
18 GGAGGAGGGGGCGCAAGTCAAAT GTCAAAGGCCCAAGGTCACAGAGG 400 bp 62ºC
19 ACAGGCACACGTGTTTTCAC CAGTCTCCACCTGTCCCATC 345 bp 61ºC
20 AGAATACCAACAAGCCAGGACAAG GCGGGAAAGTGAGCAGAACC 402 bp 62ºC
21 TGCCTTTGCCCCCGTGCTACTTG GCCCCAGGACCCCCACTTTTGAT 187 bp 62ºC
22 TCCTCCTGGCTCTCCCGTTTCTCT GCGCCCCTCTGCTGCTTCTTC 379 bp 62ºC
23 GCTCCTCTGCTCCCTACTTCC ATGGCCATCAGCACACTTCAC 310 bp 62ºC
24 TCGGTGCCACAGAGATGATTTTGA GGCTGCCCCTCTGTGTTCTCCA 367 bp 62ºC
25 CCTGTGGCGGTTAGTTGG CACCGGTAGCTCTTCTTCTTCTTG 350 bp 62ºC
26 CCGAGGGAAGGTGGTGTGG TCTGTAAAATGCGGCTGAGTATCC 404 bp 62ºC
27 GGAAGTGCCCCCTATGT TCGCACTGCTCAAAGAAG 457 bp 62ºC
28 TCAGAGGAGTGGGCAGTGGGAGTG CTGGGGTGTCAATGGCGGGTCTT 292 bp 62ºC
29 GCCTGGAGTTGCTGTGTTAG GGCTGCCCCTCTTTGGTC 467 bp 62ºC
30 GCGGCCGGCCCTTGGAGT TGGAAAATGTGAGCTGTGGGTTGG 356 bp 62ºC
31 GCATTCAGGCACTTACCAGGTGACG CACGGTGAGGACAGTGAAGGGTAGC 527 bp 60ºC
32 GGCCGCAGCTACCCTTCAC GGCCCCTCTCCCTGTTCC 392 bp 65ºC
33 GGCCTCTCGGTACCAAGTCCTGTC CAACGTCGGGGCCTGTGAGC 232 bp 65ºC
34 GCAGGGCCATGGTACTCACTCTTG CCGCCCGCTCTTCCCATCTC 404 bp 62ºC
35 CACAGTGACATGGCCTCCTCTTCT GCCCCTACAGCCTCCCATTTACT 159 bp 62ºC

Tamanho do fragmento amplificado;

Temperatura de anelamento.

 Primers para o sequenciamento de TNNT2
Exon Forward Primer 5'-3' Reverse Primer 5'-3' Amplicon* A.T.†
2 ACAGCTCATGAGGGGTGGAACTA GTGCTCTGCCTGGGATCTACAACC 376 bp 65ºC
3 / 4 ATGAGAACGGCAGGCCAGGCTAGTG GTTTGCCTCAAGACCCGAGCAACC 506 bp 65ºC
5 GTGGCGGGAGGTAGCCGACAGT TGGGCAATCAATGGTTGAATCTTA 403 bp 65º C
6 TTGACCCAGCGCTTCTCTTGTGTC ACTGGGTGCCACCAATGCAACTTC 449 bp 65º C
7 CCAGTGCCGGGAGGGACTCAC CAGCCCGTGTCCACTGCACCATAC 262 bp 65º C
8 GGATCAGGGGCCCTGCCTGTCCTGACA TCCTCCTCCTCTTTCTTCCTGTTCT 538 bp 62º C
9 GCCAGGCCCTGCCAGAGGTCTT CCCTGGGGGAGGCCTGAAACAG 494 bp 70º C
10 ACGTCCGTGGAGCTGGTTGAAAGT CCCGGCCAATATTGTCTCTTGACT 373 bp 62º C
11 TGGGAGCTACCCTCTCAGAA CACAGCAGCTGGGAATCTCT 369 bp 60º C
12 GTAAACCCGGCTGACTACAG AGCCAGCCCAATCTCTTCAC 258 bp 62º C
13 CAGGGGGTTTGGGGAGGGTTAG GTGGGGCACCTGCTCAGTTCTCT 402 bp 60º C
14 GGAGGGCCCTTTCTTACTGGAC CCGGACCCAGTGAACCAGGAGGAG 207 bp 68º C
15 GCCCCTCCTGACCCTTAACTATCC CGGAGGAGCCAGAGAAGGAAACCT 353 bp 62º C
16 GGGGGTGAAATGTGGGGCGGAGAA GTGTGGGGGCAGGCAGGAGTGGTG 383 bp 62º C

Tamanho do fragmento amplificado;

Temperatura de anelamento.

 Previsão de patogenicidade da variante

Os efeitos de mutações missense foram previstos utilizando-se as ferramentas PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), SIFT/PROVEAN (http://SIFT.jcvi.org/) e PredictProtein (http:/predictprotein.org/home). A5YM48 e Q14896 foram utilizados como sequências de referência para MYBPC3 (UniProtKB).

 Resultados

Um paciente de dezessete anos de idade apresentando manifestação clínica de CMH e histórico de síncope foi submetido ao implante de cardioversor-desfibrilador para prevenção primária de síncope. O diagnóstico foi baseado no ETT e mostrou hipertrofia septal assimétrica de curva reversa, de 39 mm de espessura, com função sistólica do VE preservada e fração de ejeção normal do VE (Figura 1). Além disso, também apresentou disfunção diastólica tipo II, gradiente máximo VE/Aorta de 25 mmHg, movimento sistólico anterior da valva mitral, obstrução do trato de saída do VE e aumento do átrio esquerdo (46 mm). O ECG mostrou uma sobrecarga no VE e AE e o Holter de 24h não documentou a presença de taquicardia ventricular. O risco de MSC foi considerado elevado, 7,69%. A análise genética identificou uma variante missense em heterozigose composta, c.1624G>C (p.E542Q) e c.1828G>C (p.D610H) em MYBPC3 (Figura 2). A variante p.E542Q (rs121909374) já foi associada com CMH no ClinVar e no banco de dados Human Gene Mutation Database (HGMD). A análise in silico realizada com PolyPhen-2 prevê essa variante como possivelmente prejudicial, enquanto o SIFT/PROVEAN e PredictProtein classificam essa mutação como tolerável. Por outro lado, a variante p.D610H (rs371564200) é classificada como uma variante de significado incerto (VSI), embora as ferramentas de previsão de patogenicidade classifiquem a p.D610H como provavelmente prejudicial/nociva. Ambas as variantes afetam os resíduos conservados na cadeia polipeptídica (Figura 2).

ETT do probando e RMC da família. A) Imagem do ETT das quatro câmaras cardíacas e aorta revelando a hipertrofia septal de curva reversa. B) Vista do eixo curto paraesternal mostrando a hipertrofia septal. C) Vista do eixo longitudinal paraesternal mostrando o VE e a hipertrofia septal e o átrio esquerdo aumentado. A seta branca mostra o movimento sistólico anterior da válvula mitral. D) Imagem de ETT mostrando a obstrução e a turbulência no trato de saída do ventrículo esquerdo (seta branca). Regurgitação mitral leve no átrio esquerdo é visível. RMC do pai (E), tia (F) e mãe (G) do probando, sem sinais de hipertrofia ou fibrose. RMC na sequência de inversão-recuperação (atraso do realce) no eixo de 4 câmaras (E1, F1, G1), LVSV (E2, F2, G2) e 2 câmaras (E3, F3, G3). AD: átrio direito; VD: ventrículo direito; AE: átrio esquerdo; VE: ventrículo esquerdo; Ao: aorta.

A) Pedigree mostrando cinco gerações da família materna. O probando é o único membro afetado pela CMH. Os portadores de alelos variantes da família são indicados por E542Q+ e D610H+. B) Eletroferogramas das regiões das variantes missense do gene MYBPC3 do probando. C) Alinhamento de múltiplas espécies da sequência de aminoácidos da proteína C ligante de miosina dos resíduos 538 a 546 e 606 a 614. Os resíduos conservados, ácido glutâmico e ácido aspártico, são indicados por um retângulo.

O probando é o único membro que manifesta um fenótipo de CMH em sua família. Seu pai foi adotado, de modo que só os ascendentes maternos são conhecidos. O heredograma construído revelou 30 parentes, ao longo de cinco gerações, nos quais ocorreu apenas uma morte inexplicável de uma mulher de 30 anos sem diagnóstico de CMH (Figura 2).14

A genotipagem dos familiares maternos - avó (59 anos), tia (29 anos), tio (35anos) e mãe (39 anos) - detectou a variante p.D610H. Todos os membros da família eram assintomáticos, com ETT e ECG normais, sem evidência de HV. Por outro lado, o alelo p.E542Q foi detectado no pai (40 anos) e em um irmão por parte de pai (8 anos), ambos com resultados normais na avaliação clínica (Tabela 4). A RMC foi realizada na mãe, tia e pai, e resultou em achados normais, especificamente a espessura normal de parede do VE e sem sinais de fibrose (Figura 1).

Dados de avaliação clínica dos indivíduos
Epidemiologia ECG TTE
ID Idade (A) Sexo CMH Variante SAE SVE Onda T AN HVE + HVE tipo Forma Max EPVE (mm) GSVE mmHg DSVE DDVE MAS Tamanho AE (mm)
III.8 59 F Não D610H Não Não Não Não Não - - 10 Não Não Não Não 28
IV.2 40 M Não E542Q Não Não Não Não Não - - 9 Não Não Não Não 35
IV.3 39 F Não D610H Não Não Não Não Não - - 9 Não Não Não Não 37
IV.6 29 F Não D610H Não Não Não Não Não - - 8 Não Não Não Não 32
IV.7 35 M Não D610H Não Não Não Não Não - - 8 Não Não Não Não 36
V.1 8 M Não E542Q Não Não Não Não Não - - 7 Não Não Não Não 37
V.2 17 M Sim D610H E542Q Sim Sim Sim Sim Septal Curva reversa 39 25 Não Tipo I Não 46

A numeração de identificação (ID) dos indivíduos segue o padrão adotado nos quadros de Pedigree (Figura 2); ECG: eletrocardiograma; ETT: ecocardiograma transtorácico; (A) - anos; CMH: cardiomiopatia hipertrófica; SAE: sobrecarga atrial esquerda; SVE: sobrecarga ventricular esquerda; onda T NA: onda T anormal; HVE+ : hipertrofia ventricular esquerda mostrada por eco; Tipo HVE: tipo de hipertrofia ventricular esquerda; Max EPVE: máxima espessura da parede ventricular esquerda; GSVE: gradiente de saída do ventrículo esquerdo; DSVE: diâmetro sistólico do ventrículo esquerdo; DDVE: diâmetro diastólico do ventrículo esquerdo; MAS: movimento anterior sistólico; Tamanho AE: tamanho do átrio esquerdo.

 Discussão

O presente estudo relata o caso de um jovem com CMH grave, portador de uma variante trans em heterozigose composta no gene MYBPC3, com um alelo - p.D610H - herdado da mãe e outro - p.E542Q - herdado do pai.

Indivíduos com apenas uma única variante não apresentaram qualquer fenótipo de CMH. A variante p.E542Q, encontrada nos familiares paternos, está associada à CMH, com prognóstico bom e hipertrofia moderada na parede ventricular, embora poucos estudos citando essa mutação estejam disponíveis.10,15-17 A previsão de patogenicidade do p.E542Q está de acordo com os dados da literatura.18-21

Além disso, a variante p.D610H, identificada nos familiares maternos, também não manifestou qualquer fenótipo de CMH, mesmo no membro mais velho da família investigado (59 anos). A associação entre p.D610H e CMH permanece incerta, apesar do fato de que as ferramentas de previsão de patogenicidade a tenham classificado como provavelmente patogênica. Apenas um único estudo na literatura identificou essa mutação, embora não a tivesse correlacionado com a doença.22

Em geral, uma única mutação heterozigótica para CMH é suficiente para afetar a função miocárdica e levar à hipertrofia, porém os estudos iniciais associaram variantes no gene MYBPC3 com penetrância incompleta, HV leve, baixo risco de MSC e evolução clínica benigna.23-25

Em conclusão, sugere-se que, individualmente, as variantes p.E542Q e p.D610H resultam em leves alterações na estrutura/função da proteína, insuficientes para causar um fenótipo forte. Contudo, a expressão destas variantes em trans pode ser responsável pelo início precoce da doença, um fenótipo clínico mais grave e um risco aumentado de eventos malignos no probando. Em outras palavras, as variantes duplas e compostas por si só não são decisivas para um prognóstico pior da CMH, mas a composição alélica destas variantes pode ser determinante para este desfecho.

Limitações do estudo

O presente estudo investigou os três principais genes da CMH que representam aproximadamente 60-70% dos casos de CMH.5,14 No entanto, vários outros genes já foram associados a esta doença,5,14 os quais ainda não foram investigados.

Fontes de financiamento

O presente estudo foi financiado pelo Instituto Nacional de Cardiologia and Fundação Pró Coração (Fundacor).

Vinculação acadêmica

Este artigo é parte de dissertação de Mestrado de Julianny Freitas Rafael pelo Instituto Nacional de Cardiologia.