RESUMO
Dilated cardiomyopathy commonly causes heart failure and is the most frequent precipitating cause of heart transplantation. Familial dilated cardiomyopathy has been shown to be caused by rare variant mutations in more than 30 genes but only ~35% of its genetic cause has been identified, principally by using linkage-based or candidate gene discovery approaches. In a multigenerational family with autosomal dominant transmission, we employed whole-exome sequencing in a proband and three of his affected family members, and genome-wide copy number variation in the proband and his affected father and unaffected mother. Exome sequencing identified 428 single point variants resulting in missense, nonsense, or splice site changes. Genome-wide copy number analysis identified 51 insertion deletions and 440 copy number variants > 1 kb. Of these, a 8733 bp deletion, encompassing exon 4 of the heat shock protein cochaperone BCL2-associated athanogene 3 (BAG3), was found in seven affected family members and was absent in 355 controls. To establish the relevance of variants in this protein class in genetic DCM, we sequenced the coding exons in BAG3 in 311 other unrelated DCM probands and identified one frameshift, two nonsense, and four missense rare variants absent in 355 control DNAs, four of which were familial and segregated with disease. Knockdown of bag3 in a zebrafish model recapitulated DCM and heart failure. We conclude that new comprehensive genomic approaches have identified rare variants in BAG3 as causative of DCM.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Cardiomiopatia Dilatada/genética , Variações do Número de Cópias de DNA/genética , Éxons/genética , Estudo de Associação Genômica Ampla , Mutação Puntual/genética , Adulto , Idoso , Animais , Proteínas Reguladoras de Apoptose , Sequência de Bases , Análise Mutacional de DNA , Feminino , Técnicas de Silenciamento de Genes , Heterozigoto , Humanos , Hibridização Genética , Masculino , Pessoa de Meia-Idade , Modelos Animais , Dados de Sequência Molecular , Linhagem , Fenótipo , Controle de Qualidade , Adulto Jovem , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: LMNA cardiomyopathy presents with electrocardiogram (ECG) abnormalities, conduction system disease (CSD), and/or arrhythmias before the onset of dilated cardiomyopathy (DCM). Knowing the time interval between the onset of CSD and its progression to DCM would help to guide clinical care. METHODS AND RESULTS: We evaluated family members from 16 pedigrees previously identified to carry LMNA mutations for the ages of onset of ECG abnormalities, CSD, or arrhythmia and of left ventricular enlargement (LVE) and/or systolic dysfunction. Of 103 subjects, 64 carried their family LMNA mutation, and 51 (79%) had ECG abnormalities with a mean age of onset of 41.2 years (range 18-76). Ventricular dysfunction was observed in 26 with a mean age of onset of 47.6 years (range 28-82); at diagnosis 9 had systolic dysfunction but no LVE, 5 had LVE but no systolic dysfunction, and 11 had DCM. Of 16 subjects identified with ECG abnormalities who later developed ventricular dysfunction, the median ages of onset by log-rank analyses were 41 and 48 years, respectively. CONCLUSIONS: ECG abnormalities preceded DCM with a median difference of 7 years. Clinical surveillance should occur at least annually in those at risk for LMNA cardiomyopathy with any ECG findings.
Assuntos
Cardiomiopatia Dilatada/genética , Eletrocardiografia/tendências , Sistema de Condução Cardíaco/fisiologia , Lamina Tipo A/genética , Disfunção Ventricular/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/epidemiologia , Feminino , Seguimentos , Sistema de Condução Cardíaco/patologia , Humanos , Lipodistrofia/diagnóstico , Lipodistrofia/epidemiologia , Lipodistrofia/genética , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Disfunção Ventricular/diagnóstico , Disfunção Ventricular/epidemiologia , Adulto JovemRESUMO
The goal of the Familial Dilated Cardiomyopathy (FDC) Research Project, initiated in 1993, has been to identify and characterize FDC genetic cause. All participating individuals have been consented for the return of genetic results, an important but challenging undertaking. Since the inception of the Project we have enrolled 606 probands, and 269 of these had 1670 family members also enrolled. Each subject was evaluated for idiopathic dilated cardiomyopathy (IDC) and pedigrees were categorized as familial or sporadic. The coding regions of 14 genes were resequenced in 311 to 324 probands in five studies. Ninety-two probands were found to carry nonsynonymous rare variants absent in controls, and with Clinical Laboratory Improvement Amendment of 1988 (CLIA) compliant protocols, relevant genetic results were returned to these probands and their consented relatives by study genetic counselors and physicians in 353 letters. In 10 of the 51 families that received results >1 year ago, at least 23 individuals underwent CLIA confirmation testing for their family's rare variant. Return of genetic results has been successfully undertaken in the FDC Research Project. This report describes the methods utilized in the process of returning research results. We use this information as a springboard for providing guidance to other genetic research groups and proposing future directions in this arena.
Assuntos
Cardiomiopatia Dilatada/genética , Predisposição Genética para Doença , Aconselhamento Genético , Testes Genéticos , Humanos , Estados UnidosRESUMO
TNNC1, which encodes cardiac troponin C (cTnC), remains elusive as a dilated cardiomyopathy (DCM) gene. Here, we report the clinical, genetic, and functional characterization of four TNNC1 rare variants (Y5H, M103I, D145E, and I148V), all previously reported by us in association with DCM (Hershberger, R. E., Norton, N., Morales, A., Li, D., Siegfried, J. D., and Gonzalez-Quintana, J. (2010) Circ. Cardiovasc. Genet. 3, 155-161); in the previous study, two variants (Y5H and D145E) were identified in subjects who also carried MYH7 and MYBPC3 rare variants, respectively. Functional studies using the recombinant human mutant cTnC proteins reconstituted into porcine papillary skinned fibers showed decreased Ca(2+) sensitivity of force development (Y5H and M103I). Furthermore, the cTnC mutants diminished (Y5H and I148V) or abolished (M103I) the effects of PKA phosphorylation on Ca(2+) sensitivity. Only M103I decreased the troponin activation properties of the actomyosin ATPase when Ca(2+) was present. CD spectroscopic studies of apo (absence of divalent cations)-, Mg(2+)-, and Ca(2+)/Mg(2+)-bound states indicated that all of the cTnC mutants (except I148V in the Ca(2+)/Mg(2+) condition) decreased the α-helical content. These results suggest that each mutation alters the function/ability of the myofilament to bind Ca(2+) as a result of modifications in cTnC structure. One variant (D145E) that was previously reported in association with hypertrophic cardiomyopathy and that produced results in vivo in this study consistent with prior hypertrophic cardiomyopathy functional studies was found associated with the MYBPC3 P910T rare variant, likely contributing to the observed DCM phenotype. We conclude that these rare variants alter the regulation of contraction in some way, and the combined clinical, molecular, genetic, and functional data reinforce the importance of TNNC1 rare variants in the pathogenesis of DCM.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Mutação de Sentido Incorreto , Miofibrilas/metabolismo , Troponina C/metabolismo , Animais , Cardiomiopatia Dilatada/genética , Feminino , Humanos , Masculino , Miofibrilas/genética , Estrutura Secundária de Proteína , Troponina C/química , Troponina C/genéticaRESUMO
PURPOSE OF REVIEW: This review examines the application of next-generation sequencing (NGS) technologies in the identification of the causation of nonsyndromic genetic cardiomyopathies. RECENT FINDINGS: NGS sequencing of the entire genetic coding sequence (the exome) has successfully identified five novel genes and causative variants for cardiomyopathies without previously known cause within the last 12 months. Continual rapidly decreasing costs of NGS will shortly allow cost-effective sequencing of the entire genomes of affected individuals and their relatives to include noncoding and regulatory variant discovery and epigenetic profiling. Despite this rapid technological progress with sequencing, analysis of these large data sets remains challenging, particularly for assigning causality to novel rare variants identified in DNA samples from patients with cardiomyopathy. SUMMARY: NGS technologies are rapidly moving to identify novel rare variants in patients with cardiomyopathy, but assigning pathogenicity to these novel variants remains challenging.
Assuntos
Cardiomiopatias/genética , Exoma/genética , Análise de Sequência de DNA , Cardiomiopatias/epidemiologia , Biologia Computacional , Epigenômica , Deleção de Genes , Variação Genética/genética , Humanos , Biologia Molecular , Medição de RiscoRESUMO
BACKGROUND: The term peripartum cardiomyopathy (PPCM) describes dilated cardiomyopathy (DCM) without known cause that occurs during the last month of pregnancy to 5 months postpartum. A related term, pregnancy-associated cardiomyopathy (PACM), refers to DCM onset earlier in pregnancy. Multiple studies have focused on inflammatory, immunologic, and environmental causes. An alternative hypothesis is that PPCM and PACM result, in part, from a genetic cause. In this study, we sought to test the hypothesis that rare DCM-associated mutations underlie a proportion of PACM or PPCM cases. METHODS AND RESULTS: A systematic search of our DCM database designed for family-based genetic studies was undertaken for cases associated with pregnancy and the postpartum period; in the identified cases, clinical and molecular genetic data, including exonic and near intron/exon boundaries of DCM genes, were analyzed. Of 4110 women from 520 pedigrees in the Familial Dilated Cardiomyopathy Research Project database, we identified 45 cases of PPCM/PACM. Evidence of familial clustering with DCM was present in 23 unrelated cases. Of the 45 cases, 19 had been resequenced for known DCM genes, and 6 carried mutations. Five had PPCM, of which 3 were familial with mutations found in MYH7, SCN5A, and PSEN2, and 2 were sporadic with mutations in MYH6 and TNNT2. One case had PACM and carried a mutation in MYBPC3. CONCLUSIONS: These findings suggest that a proportion of PPCM/PACM cases results from a genetic cause.
Assuntos
Cardiomiopatia Dilatada/genética , Mutação , Complicações na Gravidez/genética , Transtornos Puerperais/genética , Adulto , Cardiomiopatia Dilatada/fisiopatologia , Sistema Cardiovascular/fisiopatologia , Feminino , Humanos , Linhagem , Gravidez , Adulto JovemRESUMO
Dilated cardiomyopathy (DCM) in infants and children can be partially explained by genetic cause but the catalogue of known genes is limited. We reviewed our database of 41 cases diagnosed with DCM before 18 years of age who underwent detailed clinical and genetic evaluation, and summarize here the evidence for mutations causing DCM in these cases from 15 genes (PSEN1, PSEN2, CSRP3, LBD3, MYH7, SCN5A, TCAP, TNNT2, LMNA, MYBPC3, MYH6, TNNC1, TNNI3, TPM1, and RBM20). Thirty-five of the 41 pediatric cases had relatives with adult-onset DCM. More males (66%) were found among children diagnosed after 1 year of age with DCM. Nineteen mutations in 9 genes were identified among 15 out of 41 patients; 3 patients (diagnosed at ages 2 weeks, 9 and 13 years) had multiple mutations. Of the 19 mutations identified in 12 families, mutations in TPM1 (32%) and TNNT2 (21%) were the most commonly found. Of the 6 patients diagnosed before 1 year of age, 3 had mutations in TPM1 (including a set of identical twins), 1 in TNNT2, 1 in MYH7, and 1 with multiple mutations (MYH7 and TNNC1). Most DCM was accompanied by advanced heart failure and need for cardiac transplantation. We conclude that in some cases pediatric DCM has a genetic basis, which is complicated by allelic and locus heterogeneity as seen in adult-onset DCM. We suggest that future prospective comprehensive family-based genetic studies of pediatric DCM are indicated to further define mutation frequencies in known genes and to discover novel genetic cause.
RESUMO
BACKGROUND: Lamin A/C mutations are a well-established cause of dilated cardiomyopathy (DCM), although their frequency has not been examined in a large cohort of patients. We sought to examine the frequency of mutations in LMNA, the gene encoding lamin A/C, in patients with idiopathic (IDC) or familial dilated cardiomyopathy (FDC). METHODS: Clinical cardiovascular data, family histories, and blood samples were collected from 324 unrelated IDC probands, of whom 187 had FDC. DNA samples were sequenced for nucleotide alterations in LMNA. Likely protein-altering mutations were followed up by evaluating additional family members, when possible. RESULTS: We identified 18 protein-altering LMNA variants in 19 probands or 5.9% of all cases (7.5% of FDC; 3.6% of IDC). Of the 18 alterations, 11 were missense (one present in 2 kindreds), 3 were nonsense, 3 were insertion/deletions, and 1 was a splice site alteration. Conduction system disease and DCM were common in carriers of LMNA variants. Unexpectedly, in 6 of the 19 kindreds with a protein-altering LMNA variant (32%), at least one affected family member was negative for the LMNA variant. CONCLUSIONS: Lamin A/C variants were observed with a frequency of 5.9% in probands with DCM. The novel observation of FDC pedigrees in which not all affected individuals carry the putative disease-causing LMNA mutation suggests that some protein-altering LMNA variants are not causative or that some proportion of FDC may be because of multiple causative factors. These findings warrant increased caution in FDC research and molecular diagnostics.
Assuntos
Cardiomiopatia Dilatada/genética , Predisposição Genética para Doença/epidemiologia , Heterozigoto , Mutação de Sentido Incorreto , Adulto , Cardiomiopatia Dilatada/epidemiologia , Cardiomiopatia Dilatada/patologia , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Regulação da Expressão Gênica , Humanos , Lamina Tipo A/genética , Masculino , Pessoa de Meia-Idade , Lâmina Nuclear/genética , Linhagem , Reação em Cadeia da Polimerase , Prognóstico , Índice de Gravidade de Doença , Análise de SobrevidaRESUMO
Positional cloning is a comprehensive genetic strategy used to identify a disease-causing gene without any prior knowledge of the pathogenesis or protein defects involved in the disease process. The basic process involves collection of accurately diagnosed patients and their family members, genotyping DNAs with polymorphic DNA markers mapped to specific regions on chromosomes, genetic linkage analysis to determine markers that are in close proximity to the chromosome location of the disease gene and to define the critical region by haplotype analysis, identification and selection of candidate genes residing in the critical region, and, eventually, identification of the disease-causing DNA sequence variants by various methods. Many molecular techniques are utilized in positional cloning. Bioinformatics and computation analysis are significant and indispensable components of such a study, and are detailed elsewhere in this volume. This chapter presents a few basic laboratory protocols for conducting positional cloning: genomic DNA preparation, genotyping polymorphic markers, DNA sequencing, and related procedures that serve on fluorescent-labeled and capillary electrophoresis-based semi-automated genetic analysis systems.
Assuntos
Doenças Cardiovasculares/genética , Mapeamento Cromossômico/métodos , Clonagem Molecular/métodos , Biologia Computacional/métodos , Bases de Dados Genéticas , Eletroforese Capilar , Ligação Genética/genética , Marcadores Genéticos/genética , Genoma Humano/genética , Genótipo , Humanos , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND- Familial dilated cardiomyopathy (DCM) is a genetically heterogeneous disease with >30 known genes. TTN truncating variants were recently implicated in a candidate gene study to cause 25% of familial and 18% of sporadic DCM cases. METHODS AND RESULTS- We used an unbiased genome-wide approach using both linkage analysis and variant filtering across the exome sequences of 48 individuals affected with DCM from 17 families to identify genetic cause. Linkage analysis ranked the TTN region as falling under the second highest genome-wide multipoint linkage peak, multipoint logarithm of odds, 1.59. We identified 6 TTN truncating variants carried by individuals affected with DCM in 7 of 17 DCM families (logarithm of odds, 2.99); 2 of these 7 families also had novel missense variants that segregated with disease. Two additional novel truncating TTN variants did not segregate with DCM. Nucleotide diversity at the TTN locus, including missense variants, was comparable with 5 other known DCM genes. The average number of missense variants in the exome sequences from the DCM cases or the ≈5400 cases from the Exome Sequencing Project was ≈23 per individual. The average number of TTN truncating variants in the Exome Sequencing Project was 0.014 per individual. We also identified a region (chr9q21.11-q22.31) with no known DCM genes with a maximum heterogeneity logarithm of odds score of 1.74. CONCLUSIONS- These data suggest that TTN truncating variants contribute to DCM cause. However, the lack of segregation of all identified TTN truncating variants illustrates the challenge of determining variant pathogenicity even with full exome sequencing.
Assuntos
Cardiomiopatia Dilatada/genética , Exoma/genética , Genoma Humano , Proteínas Musculares/genética , Proteínas Quinases/genética , Adolescente , Adulto , Idoso , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Cromossomos Humanos Par 9 , Conectina , Feminino , Heterogeneidade Genética , Ligação Genética , Loci Gênicos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Razão de Chances , Linhagem , Análise de Sequência de DNA , Adulto JovemRESUMO
BACKGROUND: Human exome sequencing is a recently developed tool to aid in the discovery of novel coding variants. Now broadly applied, exome sequencing data sets provide a novel opportunity to evaluate the allele frequencies of previously published pathogenic rare variants. METHODS AND RESULTS: We examined the exome data set from the National Heart, Lung and Blood Institute Exome Sequencing Project and compared this data set with a catalog of 197 previously published rare variants reported as causative of dilated cardiomyopathy (DCM) from familial and sporadic cases. Of these 197, 33 (16.8%) were also present in the Exome Sequencing Project database, raising the question of whether they were uncommon polymorphisms. Supporting functional data has been published for 14 of the 33 (42%), suggesting they are unlikely to be false-positives. The frequencies of these functional variants in the Exome Sequencing Project data set ranged from 0.02 to 1.33% (median 0.04%), which when applied as a cutoff to filter variants in a DCM pedigree identified an additional DCM candidate gene. A greater proportion of sporadic DCM cases had variants that were present in the Exome Sequencing Project data set versus novel variants (ie, not in the Exome Sequencing Project; 44% versus 21%; P=0.002), suggesting some of the variants identified as disease causing in sporadic DCM are either false-positives or low penetrance alleles in human populations. CONCLUSIONS: Rare nonsynonymous variants identified in DCM subjects also present at very low frequencies in public databases are likely relevant for DCM. Allele frequencies >0.04% are of less certain pathogenicity, especially if identified in sporadic cases, although this cutoff should be viewed as preliminary.
Assuntos
Cardiomiopatia Dilatada/genética , Exoma , Variação Genética , Éxons , Feminino , Humanos , Masculino , LinhagemRESUMO
The contribution of copy number variation (CNV) to dilated cardiomyopathy (DCM) is unknown. However, estimates have suggested that CNVs could constitute 15% of mutations underlying Mendelian disease. This is of particular relevance to DCM, where only approximately 35% of genetic cause has been identified. We have previously reported 19 point mutations in LMNA, the gene encoding Lamin A/C, in a cohort of 324 unrelated DCM probands (5.9%), making it the most common genetic cause of DCM. Recently a large deletion was reported in LMNA in 1 of 25 DCM probands. To further assess the contribution of CNVs in LMNA cardiomyopathy, we used Multiplex Ligation Probe Amplification (MLPA) to screen for large deletions and duplications in 58 DCM probands negative for point mutations in LMNA. Despite excellent quality control and robust MLPA results, our study failed to identify any deletions or duplications. We conclude that at least for LMNA, point mutations are the major source of DCM causation.
Assuntos
Cardiomiopatia Dilatada/genética , Variações do Número de Cópias de DNA/genética , Lamina Tipo A/genética , Adolescente , Adulto , Idoso , Humanos , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Mutations in the LMNA gene, encoding lamins A/C, represent a significant cause of dilated cardiomyopathy. We recently identified 18 protein-altering LMNA variants in a cohort of 324 unrelated patients with dilated cardiomyopathy. However, at least one family member with dilated cardiomyopathy in each of 6 pedigrees lacked the LMNA mutation (nonsegregation), whereas small sizes of 5 additional families precluded definitive determinations of segregation, raising questions regarding contributions by those variants to disease. METHODS AND RESULTS: We have consequently expressed, in COS7 cells, GFP-prelamin A (GFPLaA) fusion constructs incorporating the 6 variants in pedigrees with nonsegregation (R101P, A318T, R388H, R399C, S437Hfsx1, and R654X), the 4 variants in pedigrees with unknown segregation (R89L, R166P [in 2 families], I210S, R471H), and 3 additional missense variants (R190Q, E203K, and L215P) that segregated with disease. Confocal immunofluorescence microscopy was used to characterize GFP-lamin A localization and nuclear morphology. Abnormal phenotypes were observed for 10 of 13 (77%) variants (R89L, R101P, R166P, R190Q, E203K, I210S, L215P, R388H, S437Hfsx1, and R654X), including 4 of 6 showing nonsegregation and 3 of 4 with uncertain segregation. All 7 variants affecting coil 1B and the lamin A-only mutation, R654X, exhibited membrane-bound GFP-lamin A aggregates and nuclear shape abnormalities. Unexpectedly, R388H largely restricted GFP-lamin A to the cytoplasm. Equally unexpected were unique streaked aggregates with S437Hfsx1 and giant aggregates with both S437Hfsx1 and R654X. CONCLUSIONS: This work expands the recognized spectrum of lamin A localization abnormalities in dilated cardiomyopathy. It also provides evidence supporting pathogenicity of 10 of 13 tested LMNA variants, including some with uncertain or nonsegregation.
Assuntos
Cardiomiopatia Dilatada/genética , Lamina Tipo A/genética , Substituição de Aminoácidos , Animais , Células COS , Cardiomiopatia Dilatada/patologia , Núcleo Celular/ultraestrutura , Chlorocebus aethiops , Estudos de Coortes , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lamina Tipo A/metabolismo , Masculino , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Linhagem , Fenótipo , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The genetic basis of most of dilated cardiomyopathy (DCM) cases remains unknown. A recent study indicated that mutations in a highly localized five amino acid hotspot in exon 9 of RBM20, a gene encoding a ribonucleic acid-binding protein, caused aggressive DCM. We undertook this study to confi rm and extend the nature of RBM20 mutations in another DCM cohort. Clinical cardiovascular data, family histories, and blood samples were collected from patients with idiopathic DCM. DNA from 312 DCM probands was sequenced for nucleotide alterations in exons 6 through 9 of RBM20, and additional family members as possible. We found six unique RBM20 rare variants in six unrelated probands (1.9%). Four mutations, two of which were novel (R634W and R636C) and two previously identified (R634Q and R636H), were identified in a five amino acid hotspot in exon 6. Two other novel variants (V535I in exon 6 and R716Q in exon 9) were outside of this hotspot. Age of onset and severity of heart failure were variable, as were arrhythmias and conduction system defects, but many subjects suffered severe heart failure resulting in early death or cardiac transplantation. This article concludes that DCM in patients with RBM20 mutations is associated with advanced disease.
Assuntos
Cardiomiopatia Dilatada/genética , Mutação de Sentido Incorreto/genética , Proteínas de Ligação a RNA/genética , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Sequência de Bases , Criança , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Proteínas de Ligação a RNA/química , Adulto JovemRESUMO
BACKGROUND: Rare variants in >30 genes have been shown to cause idiopathic or familial dilated cardiomyopathy (DCM), but the frequency of genetic causation remains poorly understood. We have previously resequenced 9 genes in a cohort of idiopathic or familial DCM probands for rare variants, and now we report resequencing results for 5 more genes with established relationships to DCM. METHODS AND RESULTS: Blood samples were collected, and DNA specimens were prepared from 312 patients, 181 with familial DCM and 131 with idiopathic DCM. Genomic DNA underwent bidirectional sequencing, and DNA of additional family members underwent analysis when a rare variant was identified. We identified rare variants in 34 probands (10.9% overall), including 29 unique protein-altering rare variants and 2 splicing variants that were absent in 246 control subjects (492 chromosomes). These variants were 12 MYBPC3 (myosin-binding protein C) in 13 (4.2%) probands, 8 MYH6 (alpha-myosin heavy chain) in 10 (3.2%), 6 TPM1 (tropomyosin) in 6 (1.9%), 4 TNNC1 (cardiac troponin C) in 4 (1.3%), and 1 TNNI3 (cardiac troponin I) in 2 (0.6%). Variants were classified as likely or possibly disease causing in 13 and 20 probands, respectively (n=33; 10.6% overall). One MYH6 variant was classified as unlikely to be disease causing. CONCLUSIONS: Rare variants in these 5 genes likely or possibly caused 10.6% of DCM in this cohort. When combined with our prior resequencing reports, approximately 27% of DCM probands had possible or likely disease-causing variants identified.
Assuntos
Miosinas Cardíacas/genética , Cardiomiopatia Dilatada/genética , Proteínas de Transporte/genética , Cadeias Pesadas de Miosina/genética , Tropomiosina/genética , Troponina C/genética , Troponina I/genética , Substituição de Aminoácidos , Estudos de Coortes , Haplótipos , Humanos , Polimorfismo de Nucleotídeo Único , Splicing de RNA , Análise de Sequência de DNARESUMO
Obtaining functional data with newly identified rare variants increases certainty that the variant identified is relevant for dilated cardiomyopathy (DCM) causation. Two novel SCN5A rare variants, R222Q and I1835T, segregated with DCM in two families with affected individuals homozygous or heterozygous for the common SCN5A polymorphism H558R. cDNAs with each rare variant were constructed in the common Q1077del or Q1077 splice variant backgrounds with and without the H558R polymorphism and expressed in HEK293 cells. Sodium current (I(Na) ) was studied for each using whole-cell voltage clamp. In the Q1077del background I(Na) densities of R222Q and I1835T were not different from wild type, but the combined variants of R222Q/H558R, I1835T/H558R caused approximately 35% and approximately 30% reduction, respectively, and each showed slower recovery from inactivation. In the Q1077del background R222Q and R222Q/H558R also exhibited a significant negative shift in both activation and inactivation while I1835T/H558R showed a significant negative shift in inactivation that tended to decrease window current. In contrast, expression in the Q1077 background showed no changes in peak I(Na) densities, decay, or recovery from inactivation for R222Q/H558R and I1835T/H558R. We conclude that the biophysical findings, dependent upon common SCN5A variants, provide further evidence that these novel SCN5A rare variants are relevant for DCM.
Assuntos
Processamento Alternativo/genética , Ativação do Canal Iônico/genética , Proteínas Musculares/genética , Polimorfismo de Nucleotídeo Único/genética , Canais de Sódio/metabolismo , Adolescente , Adulto , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/fisiopatologia , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.5 , Linhagem , Fenótipo , Canais de Sódio/genética , Adulto JovemRESUMO
Mutations in TNNT2, encoding cardiac troponin T, commonly shows early onset, aggressive dilated cardiomyopathy (DCM). This observation may influence the decision of whether to undertake clinical genetic testing for TNNT2 in later onset DCM. Further, the trigger for late onset DCM remains enigmatic. A 70-year-old woman, previously healthy with a left ventricular ejection fraction of 50%-55% at age 69, presented with DCM of unknown cause and a 4-month history progressive heart failure requiring cardiac transplantation. Clinical genetic testing revealed a novel TNNT2 R139H mutation but no relevant variants in 18 other DCM genes. Her explanted heart showed partial fatty replacement in the right ventricle. Sequencing for five arrhythmogenic right ventricular dysplasia genes was negative. Functional studies in porcine cardiac skinned fibers reconstituted with the mutant R139H troponin T protein showed decreased Ca(2+) sensitivity at pH 7, characteristic of DCM. Because fatty infiltration may acidify the myocellular environment, maximal force development examined at pH 6.5 was diminished, suggesting a possible environmental trigger. We conclude that the TNNT2 R139H mutation was likely to be disease causing. Further, later age of onset may not be relevant to exclude genetic testing for TNNT2 mutations.
Assuntos
Cardiomiopatia Dilatada/genética , Mutação/genética , Miocárdio/patologia , Troponina T/genética , Idade de Início , Idoso , Substituição de Aminoácidos/genética , Animais , Cardiomiopatia Dilatada/patologia , Éxons/genética , Feminino , Heterozigoto , Humanos , Íntrons/genética , Masculino , Linhagem , Sus scrofaRESUMO
BACKGROUND: A key issue for cardiovascular genetic medicine is ascertaining if a putative mutation indeed causes dilated cardiomyopathy (DCM). This is critically important as genetic DCM, usually presenting with advanced, life-threatening disease, may be preventable with early intervention in relatives known to carry the mutation. METHODS AND RESULTS: We recently undertook bidirectional resequencing of TNNT2, the cardiac troponin T gene, in 313 probands with DCM. We identified 6 TNNT2 protein-altering variants in 9 probands, all who had early onset, aggressive disease. Additional family members of mutation carriers were then studied when available. Four of the 9 probands had DCM without a family history, and 5 probands had familial DCM. Only 1 mutation (Lys210del) could be attributed as definitively causative from previous reports. Four of the 5 missense mutations were novel (Arg134Gly, Arg151Cys, Arg159Gln, and Arg205Trp), and one was previously reported with hypertrophic cardiomyopathy (Glu244Asp). Based on the clinical, pedigree, and molecular genetic data, these 5 mutations were considered possibly or likely disease causing. To further clarify their potential pathophysiologic impact, we undertook functional studies of these mutations in cardiac myocytes reconstituted with mutant troponin T proteins. We observed decreased Ca(2+) sensitivity of force development, a hallmark of DCM, in support of the conclusion that these mutations are disease causing. CONCLUSIONS: We conclude that the combination of clinical, pedigree, molecular genetic, and functional data strengthen the interpretation of TNNT2 mutations in DCM.
Assuntos
Cardiomiopatia Dilatada/genética , Troponina T/genética , Adolescente , Adulto , Idoso , Substituição de Aminoácidos , Cálcio/metabolismo , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Hipertrófica/genética , Criança , Pré-Escolar , Predisposição Genética para Doença , Humanos , Lactente , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , LinhagemRESUMO
Idiopathic dilated cardiomyopathy (IDC) is characterized by left ventricular (LV) enlargement with systolic dysfunction, other causes excluded. When inherited, it represents familial dilated cardiomyopathy (FDC). We hypothesized that IDC or FDC would show with cardiac magnetic resonance (CMR) increased myocardial accumulation of gadolinium contrast at steady state and decreased baseline myocardial blood flow (MBF) due to structural alterations of the extracellular matrix compared with normal myocardium. CMR was performed in nine persons affected with IDC/FDC. Healthy controls came from the general population (n = 6) or were unaffected family members of FDC patients (n = 3) without signs or symptoms of IDC/FDC or any structural cardiac abnormalities. The myocardial partition coefficient for gadolinium contrast (lambda(Gd)) was determined by T1 measurements. LV shape and function and MBF were assessed by standard CMR methods. lambda(Gd) was elevated in IDC/FDC patients vs. healthy controls (lambda(Gd) = 0.56 +/- 0.15 vs. 0.41 +/- 0.06; P = 0.002), and correlated with LV enlargement (r = 0.61 for lambda(Gd) vs. end-diastolic volume indexed by height; P < 0.01) and with ejection fraction (r = -0.80; P < 0.001). The extracellular volume fraction was higher in IDC patients than in healthy controls (0.31 +/- 0.05 vs. 0.24 +/- 0.03; P = 0.002). Resting MBF was lower in IDC patients (0.64 +/- 0.13 vs. 0.91 +/- 0.22; P = 0.01) than unaffected controls and correlated with both the partition coefficient (r = -0.57; P = 0.012) and the extracellular volume fraction (r = -0.56; P = 0.019). The expansion of the extracellular space correlated with reduced MBF and ventricular dilation. Expansion of the extracellular matrix may be a key contributor to contractile dysfunction in IDC patients.
Assuntos
Cardiomiopatia Dilatada/patologia , Circulação Coronária/fisiologia , Coração/fisiologia , Miocárdio/patologia , Adulto , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Simulação por Computador , Meios de Contraste/metabolismo , Espaço Extracelular/metabolismo , Feminino , Gadolínio DTPA/farmacocinética , Humanos , Interpretação de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Modelos Estatísticos , Mutação/genética , Miocárdio/metabolismoRESUMO
BACKGROUND: More than 20 genes have been reported to cause idiopathic and familial dilated cardiomyopathy (IDC/FDC), but the frequency of genetic causation remains poorly understood. METHODS AND RESULTS: Blood samples were collected and DNA prepared from 313 patients, 183 with FDC and 130 with IDC. Genomic DNA underwent bidirectional sequencing of six genes, and mutation carriers were followed up by evaluation of additional family members. We identified in 36 probands, 31 unique protein-altering variants (11.5% overall) that were not identified in 253 control subjects (506 chromosomes). These included 13 probands (4.2%) with 12 beta-myosin heavy chain (MYH7) mutations, nine probands (2.9%) with six different cardiac troponin T (TNNT2) mutations, eight probands (2.6%) carrying seven different cardiac sodium channel (SCN5A) mutations, three probands (1.0%) with three titin-cap or telethonin (TCAP) mutations, three probands (1.0%) with two LIM domain binding 3 (LDB3) mutations, and one proband (0.3%) with a muscle LIM protein (CSRP3) mutation. Four nucleotide changes did not segregate with phentoype and/or did not alter a conserved amino acid and were therefore considered unlikely to be disease-causing. Mutations in 11 probands were assessed as likely disease-causing, and in 21 probands were considered possibly disease-causing. These 32 probands included 14 of the 130 with IDC (10.8%) and 18 of 183 with FDC (9.8%) CONCLUSIONS: Mutations of these six genes each account for a small fraction of the genetic cause of FDC/IDC. The frequency of possible or likely disease-causing mutations in these genes is similar for IDC and FDC.