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No description available.

No description available.

Thesis (PhD ) -- Stellenbosch University, 2011. / Bibliography / ENGLISH ABSTRACT: The regulation of cardiac contractility is dependent on cooperative interaction between the thick and thin filaments, as well as their accessory proteins, within the cardiac sarcomere. Alteration in cardiac contractility due to a defective sarcomere typically results in cardiomyopathies, such as hypertrophic cardiomyopathy (HCM). One of the sarcomeric genes frequently mutated and which accounts for the second most common form of HCM encodes cardiac myosin binding protein C (cMyBPC), a thick filament accessory protein whose physiological function is poorly understood. However, studies have implicated cMyBPC in thick filament structure and function as well as in the regulation of contractility. The N-terminal region of cMyBPC houses the cMyBPC-motif, which contains three phosphorylation sites, between domains C1 and C2. The hierarchical phosphorylation of this motif, by first calcium/calmodulin kinase II (CamKII) and then by cyclic AMP-activated protein kinase (PKA), is cardinal in the role of cMyBPC in the regulation of cardiac contractility in response to ß-adrenergic stimulation. Moreover, phosphorylation of this motif is inversely correlated to cMyBPC proteolysis and has been shown to be cardioprotective. Thus, proteins that have an effect on cMyBPC function or turnover may also influence filament structure and hence affect contractility, which, in turn, affects the structure of the cardiac muscle. One such protein is the Copper metabolism MURR1-domain containing protein 4 (COMMD4), which was previously identified as a novel interactor of cMyBPC during a yeast two-hybrid (Y2H) library screen in our laboratory. COMMD4 binds specifically to the cMyBPC motif in a phosphorylation-dependent manner. The exact function of COMMD4 is unknown; however, it is a member of the COMM family of proteins that has been linked to copper metabolism as well as to the ubiquitin-proteasome pathway (UPS). Intriguingly, recent studies have shown that the UPS plays a role in cMyBPC-derived HCM, while dietary copper depletion is also known to cause cardiac hypertrophy. Based on these findings, COMMD4 was considered an interesting candidate regulator of sarcomeric function and contractility, and by extension, a candidate modifier of cardiac hypertrophy. Thus, the aim of the present study was two-fold. Firstly, COMMD4 was used as bait in a Y2H library screen to determine its distal ligands, with a view to further elucidate its function, particularly in the context of MyBPC functioning, and identified interactors were subjected to further in vitro and in vivo verification studies. Also, the phosphorylation-dependent nature of the interaction between COMMD4 and cMyBPC was further investigated using a domain/phosphorylation assay. Secondly, COMMD4 and its Y2H-identified putative interactors were assessed as possible modifiers of hypertrophy in a family-based association study, using three cohorts of South African HCM-families in which one of three founder mutations segregate. Six putative interactors, viz. cardiac actin (ACTC1), Down syndrome critical region 3 (DSCR3), enolase 1 (ENO1), F-box and leucine rich repeat protein 10 (FBXL10), legumain (LGMN) and sorting nexin3 (SNX3) were identified and confirmed as COMMD4 interactors using Y2H analyses, followed by in vitro and in vivo co-immunoprecipitation and 3D co-localisation assays. Moreover, as some COMMD protein family members and the newly-identified interactors of COMMD4 have previously been linked to the UPS, the functional effect of siRNA-mediated knockdown of COMMD4 on cMyBPC turnover was also investigated. Data revealed accumulation of cMyBPC in the endosomes upon COMMD4 knockdown, suggesting a functional role for COMMD4 in the turnover of cMyBPC. In addition, association analysis revealed strong evidence of association between various single nucleotide polymorphisms (SNPs) in SNX3 and a number of hypertrophy traits, thus suggesting a role for SNX3 as a candidate modifier of hypertrophy in HCM. No evidence of association was observed for any of the genes encoding the other COMMD4 interactors implicated in protein turnover. The present study demonstrates that COMMD4, a little understood member of the COMM family of proteins, binds to the cMyBPC motif of cMyBPC in a phosphorylation-dependent manner. Furthermore, based on the functions of its protein interactions, we hypothesise that COMMD4 plays a role in protein trafficking and turnover. More specifically, COMMD4 seems to help to facilitate formation of protein complexes with the Skp1-Cul1-Fbxl (SCF) E3 ubiquitin ligase and probably helps to stabilise the target substrate for subsequent ubiquitin-conjugation. As COMMD4 seems to affect the protein turnover of cMyBPC and possibly other sarcomeric proteins, such as actin, these results establish a novel association between the sarcomere, HCM and the UPS. In addition, identification of SNX3 as a hypertrophy modifier will allow for the improved understanding of HCM patho-aetiology. SNX3 thus adds to the growing body of sarcomeric modifier genes, which, eventually, may improve risk profiling in HCM. Furthermore, as genetic modifiers appear sufficient to completely prevent disease expression in some HCM carriers, the identification of SNX3 may point to the protein turnover pathway as a potential new target for intervention. / AFRIKAANSE OPSOMMING: Die regulering van kardiale kontraktiliteit is afhanklik van die koöperatiewe interaksie tussen die dik en dun filamente, asook hul geassosieerde proteïene, in die kardiale sarkomeer. Veranderinge in kardiale kontraktiliteit as gevolg van 'n defektiewe sarkomeer lei tot kardiomiopatieë soos hipertrofiese kardiomiopatie (HKM). Een van die sarkomeriese gene wat dikwels gemuteer is en wat verantwoordelik is vir die tweede algmeenste vorm van HKM,is dié van kardiale miosien-bindingsproteïen C (cMyBPC),'n proteïen geassosieer met die dik filament waarvan die fisiologiese funksie nog nie goed bekend is nie. Studies betrek cMyBPC in dik filament struktuur en funksie asook in die regulering van kontraktiliteit. Die N-terminale gebied van cMyBPC huisves die cMyBPC-motief, wat drie fosforilerings-setels tussen domeine C1 en C2 bevat. Die hiërargiese fosforilering van hierdie motief, eerstens deur kalsium/kalmodulien-gereguleerde kinase II (CamKII), gevolg deur siklies AMP-geaktiveerde proteïen kinase (PKA), is kardinaal in die rol van cMyBPC in die regulering van kardiale kontraktiliteit in reaksie op ß-adrenergiese stimulasie. Verder, fosforilering van hierdie motief is omgekeerd gekorreleer aan cMyBPC proteolise en is ook bewys om kardiobeskermend te wees. Dus, proteïene wat 'n uitwerking het op die funksie van cMyBPC mag ook filament struktuur en kontraktiliteit beïnvloed, wat op hul beurt die struktuur van die kardiale spier affekteer. Die koper metabolisme MURR1-domein bevattende protein 4 (COMMD4), was voorheen geïdentifiseer as 'n nuwe bindingsgenoot van cMyBPC tydens gis twee-hibried (G2H) analise in ons laboratorium. COMMD4 bind spesifiek aan die cMyBPC motief in 'n fosforilasie afhanklike wyse. Die presiese funksie van COMMD4 is onbekend; maar dit is 'n lid van die COMM domein familie van proteine wat geassosieerd is met koper metabolisme sowel as die “ubiquitin” proteosoom pad (UPP). Interesant genoeg, onlangse studies het bewys dat die UPP 'n rol speel in cMyBPC-afgeleide HKM, terwyl koper uitputting in die dieet ook bekend is om kardiale hipertrofie te veroorsaak. Gebaseer op hierdie bevindinge was COMMD4 oorweeg as 'n interessante kandidaat reguleerder van sarkomeries funksie en kontraktiliteit, asook 'n kandidaat modifiseerder van kardiale hipertrofie. Dus, die doel van die huidige studie was tweeledig. Eerstens, was COMMD4 as aas gebruik in 'n G2H biblioteek sifting om sy distale ligande te bepaal, met die oog om verdere lig te werp op sy funksie, veral in die konteks van MyBPC funksionering, en geïdentifiseerde bindingsgenote was onderwerp aan verdere 'in vitro’ en 'in vivo’ verifikasie studies. Daarbenewens was die fosforilering-afhanklike aard van die interaksie tussen COMMD4 en cMyBPC verder ondersoek met behulp van 'n domein/fosforilasie toets. Tweedens, COMMD4 en sy G2H-geïdentifiseerde vermeende bindingsgenote was geassesseer as moontlik modifiseerders van hipertrofie in 'n familie-gebaseerde assosiasie studie, met behulp van drie kohorte van Suid-Afrikaanse HKM-families waarin een van die drie stigter mutasies segregeer. Ses vermeende interaktors, nl. kardiale aktien (ACTC1), Down-sindroom kritiese streek 3 (DSCR3), enolase 1 (ENO1), F-boks en leusien ryke herhalings proteïen 10 (FBXL10), legumain (LGMN) en sorteer nexin3 (SNX3) is geïdentifiseer en bevestig as COMMD4 bindingsgenote deur G2H analises, gevolg deur in vitro en in vivo ko-immunopresipitasie en 3D ko-lokalisasie toetse. Die funksionele effek van siRNA-bemiddelde uitklop van COMMD4 op cMyBPC omset was ook ondersoek omdat 'n paar COMMD proteïen familielede, asook die nuut-geïdentifiseerde bindingsgenote van COMMD4, geassosieerd is met die UPP. Data toon ophoping van cMyBPC in die endosome by COMMD4 uitklop, wat dus aandui op 'n funksionele rol vir COMMD4 in die omset van cMyBPC. Daarbenewens, toon assosiasie analise sterk bewyse van assosiasie tussen die verskillende enkele nukleotied polimorfismes (SNPs) in SNX3 en 'n aantal hipertrofiese kenmerke,wat aandui op 'n rol vir SNX3 as 'n kandidaat modifiseerder van hipertrofie in HKM. Geen bewyse van assosiasie was waargeneem vir enige van die gene wat kodeer vir die ander COMMD4 bindingsgenote wat geïmpliseer word in die proteïen omset. Die huidige studie toon dat COMMD4, 'n min verstaande lid van die COMM familie van proteïene, aan die cMyBPC motief van cMyBPC in'n fosforilasie-afhanklike wyse bind. Verder, gebasseer op die funksies van die proteïen interaksies, hipotiseer ons dat COMMD4 'n rol speel in proteïen vervoer en omset. Meer spesifiek, COMMD4 blyk om die vorming van proteïene komplekse met die Skp1-Cul1-Fbxl (SCF) E3 "ubiquiti". ligase te fasiliteer en help waarskynlik om die teiken-substraat vir die daaropvolgende ubiquitin-konjugasie te stabiliseer. Omdat dit lyk asof COMMD4 die proteïen-omset van cMyBPC en moontlik ander sarkomeriese proteïene, soos aktien, ook beïnvloed, vestig die resultate dus 'n nuwe assosiasie tussen die sarkomeer, HKM en die UPP. Daarbenewens sal die identifisering van SNX3 as 'n hipertrofie modifiseerder voorsiening maak vir die verbeterde begrip van HKM pato-etiologie. SNX3 voeg dus by tot die groeiende ?getal van sarkomeriese modifiseerende gene, wat uiteindelik, die risiko-ontleding in HKM mag verbeter. Verder, omdat dit blyk dat genetiese modifiseerders voldoende is om die siekte-uitdrukking heeltemal te verhoed in sekere HKM draers, kan die identifikasie van SNX3 na die proteïen-omset roete dui as 'n potensiële nuwe teiken vir intervensie.

Cardiac myosin binding protein-C (cMyBP-C) is a structural and regulatory component of cardiac thick filaments. It is observed in electron micrographs as seven to nine transverse stripes in the central portion of each half of the A band. Its C-terminus binds tightly to the myosin rod and contributes to thick filament structure, while the N-terminus can bind both myosin S2 and actin, influencing their structure and function. Mutations in the MYBPC3 gene (encoding cMyBP-C) are commonly associated with hypertrophic cardiomyopathy (HCM). In cardiac cells there exists a population of myosin heads in the super-relaxed (SRX) state, which are bound to the thick filament core with a highly inhibited ATPase activity. This report examines the role cMyBP-C plays in regulating the population of the SRX state of cardiac myosin by using an assay that measures single ATP turnover of myosin. We report a significant decrease in the proportion of myosin heads in the SRX state in homozygous cMyBP-C knockout mice, however heterozygous cMyBP-C knockout mice do not significantly differ from the wild type. A smaller, non-significant decrease is observed when thoracic aortic constriction is used to induce cardiac hypertrophy in mutation negative mice. These results support the proposal that cMyBP-C stabilises the thick filament and that the loss of cMyBP-C results in an untethering of myosin heads. This results in an increased myosin ATP turnover, further consolidating the relationship between thick filament structure and the myosin ATPase. Crown Copyright (C) 2016 Published by Elsevier Ltd. All rights reserved.

Myosin subfragment-2 (S2) is a coiled coil linker between myosin subfragment-1 and light meromyosin (LMM). This dissertation examines whether the myosin S2 coiled coil could regulate the amount of myosin S1 heads available to bind actin thin filaments by modulating the stability of its coiled coil. A stable myosin S2 coiled coil would have less active myosin S1 heads compared to a more flexible myosin S2 coiled coil, thus causing increased force production through acto-myosin interaction. The stability of the myosin S2 coiled coil was modulated by the binding of a natural myosin S2 binding protein, myosin binding protein C (MyBPC), and synthetic myosin S2 binding proteins, stabilizer and destabilizer peptide, to myosin S2. Competitive enzyme linked immunosorbent assay (cELISA) experiments revealed the cross specificity and high binding affinity of the synthetic peptides to the myosin S2 of human cardiac and rabbit skeletal origins. Gravitational force spectroscopy (GFS) was performed to test the stability of myosin S2 coiled coil in the presence of these myosin S2 binding proteins. GFS experiments demonstrated the stabilization of the myosin S2 coiled coil by the binding of MyBPC and stabilizer peptide to myosin S2, while the binding of destabilizer peptide to the same resulted in a flexible myosin S2 coiled coil. The binding of MyBPC and stabilizer peptide respectively, resulted in 3.35 and 1.5 times increase in force required to uncoil the myosin S2, while the binding of destabilizer peptide resulted in 1.6 times decrease in force required to uncoil the myosin S2. The myofibrillar contractility assay was performed to test the effect of synthetic myosin S2 binding proteins on the sarcomere shortening in myofibrils. The stabilizer peptide resulted in decreased sarcomere shortening of myofibrils as a result of decreased acto-myosin interaction, on the other hand, the binding of destabilizer peptide caused an increase in sarcomere shortening. The in vitro motility assay was performed to test the effect of altered stability of myosin S2 by binding of these myosin S2 binding proteins on the motility of actin filaments sliding over myosin. The motility of actin filaments was hindered by treating myosin thick filaments with whole length skeletal MyBPC or by treating heavy meromyosin with stabilizer peptide, while the motility of actin filaments was enhanced when heavy meromyosin was treated with destabilizer peptide. This study demonstrates that the myosin S2 coiled coil stability influences the force produced by acto-myosin interaction in striated skeletal muscle. The myosin S2 coiled coil when stabilized by MyBPC and stabilizer peptide resulted in decreased force production by reduced acto-myosin interaction. While the binding of destabilizer resulted in a flexible myosin S2 coiled coil and increased force production by enhanced acto-myosin interaction. The potentially cooperative response of contractility to the instability of the S2 coiled coil promises that this biological mechanism may be the target of drugs to modulate muscle performance.

No description available.

Introdução: A cardiomiopatia hipertrófica (CH) é uma doença genética cardíaca primária, caracterizada por hipertrofia do ventrículo esquerdo, sem dilatação, geralmente assimétrica e predominantemente septal, com prevalência estimada em 1:500. Atualmente já foram descobertos 20 genes associados à doença, mas os genes mais frequentemente relacionados são o da Cadeia Pesada da ?-miosina (MYH7), Proteína C de Ligação da Miosina (MYBPC3) e Troponina T (TNNT2). O diagnóstico molecular dos pacientes é importante e custo-efetivo do ponto de vista de saúde pública, além de recomendado pela Sociedade Europeia de Cardiologia. Entretanto, o custo inicial do exame é muito alto, de forma que estudos tentam reduzir esse custo. Uma das propostas descritas utilizou o RNA extraído de leucócitos para sequenciamento com sucesso para o gene MYBPC3. Este trabalho tem como objetivo testar a metodologia de sequenciamento de RNA em larga escala e testar a aplicabilidade da mesma para os genes MYH7 e TNNT2, estimar as principais mutações envolvidas nos pacientes do estudo e estabelecer correlações entre os diferentes genótipos avaliados e os fenótipos dos grupos familiares portadores da doença. Métodos: Os sujeitos incluídos no estudo são portadores de cardiomiopatia hipertrófica nos quais foi feita uma coleta de sangue para extração de DNA e RNA. Foi utilizada nested PCR para amplificação do cDNA e PCR convencional para amplificação de DNA e posterior sequenciamento em sequenciador automático. As análises estatísticas dos dados clínicos foram realizadas utilizando-se ANOVA para comparação de médias e teste exato de Fisher para comparação de frequências. Resultados: O sequenciamento de RNA se mostrou problemático, com baixa taxa de amplificação, falsos positivos e possíveis falsos negativos, de forma que optou-se por utilizar o sequenciamento de DNA para identificação das alterações. Dos 268 pacientes estudados foi identificada uma mutação em 131 deles (48,8%). Das mutações encontradas, 78 (59,5%) estavam no gene MYH7, 50 (38,2%) no gene MYBPC3 e 3 (2,3%) no gene TNNT2. Foram identificadas 69 mutações diferentes, 24 delas ainda não descritas. Pacientes com mutação identificada possuíam em média idade de diagnóstico e idade atual menores, maior frequência cardíaca média e maior frequência de pacientes com taquicardia ventricular não sustentada quando comparados ao grupo sem mutação identificada. Pacientes com mutação no gene MYH7 possuíam maior tamanho de átrio esquerdo, maior frequência de fibrilação atrial e maior frequência de histórico familiar da doença do que pacientes com mutação em MYBPC3. Conclusões: A técnica sequenciamento de RNA extraído de leucócitos não é adequada para uma rotina de rastreamento em larga escala para CH devido à alta frequência de falsos positivos, possibilidade de falsos negativos e baixa taxa de amplificação, mas o sequenciamento de DNA, embora trabalhoso, se mostrou muito sensível para a análise. A identificação de uma mutação específica ainda não pode ser usada para prognóstico de gravidade da CH, mas as comparações de genótipo e fenótipo mostraram algumas relações interessantes que devem ser melhor avaliadas nos pacientes. Este é o primeiro trabalho a caracterizar a epidemiologia molecular da CH em pacientes brasileiros para os genes MYH7, MYBPC3 e TNNT2 / Introduction: Hypertrophic cardiomyopathy (HC) is a primary genetic cardiac disease characterized by left ventricular hypertrophy without dilation, usually asymmetric and predominantly septal, with an estimated prevalence of 1:500. There are 20 genes associated to the HC, but the ones more often related to the disease are ?-myosin heavy chain (MYH7), myosin binding protein C (MYBPC3) and troponin T (TNNT2). The molecular diagnosis of patients is important and cost-effective from the standpoint of public health, and recommended by the European Society of Cardiology. However, the initial cost of the exam is very high, so several studies are attempting to reduce it. One of the proposals described used the RNA extracted from leukocytes for sequencing successfully to the MYBPC3 gene. The present study aims to test the methodology for large-scale sequencing and test it\'s applicability to the MYH7 and TNNT2 genes, to estimate the main mutations involved in the studied patients and establish correlations between their genotypes and phenotypes. Methods: The subjects included in the study were patients previously diagnosed with hypertrophic cardiomyopathy in whom a blood sample was collected to DNA and RNA extraction. It was used nested PCR for cDNA amplification and conventional PCR for DNA amplification for posterior sequencing on an automatic sequencer. Statistical analyzes of clinical data were performed using ANOVA to compare means and Fisher\'s exact test for frequencies comparison. Results: The RNA sequencing was problematic with low rate of amplification, false positives and possible false negatives, so was used DNA sequencing to identify the mutations. Of the 268 patients studied a mutation was identified in 131 of them (48.8%). Among the mutations found, 78 (59.5%) were in the MYH7 gene, 50 (38.2%) in the MYBPC3 gene and three (2.3%) in the TNNT2 gene. We have identified 69 different mutations, 24 of them not yet described. Patients with an identified mutation had an average smaller age of diagnosis and current age, higher average cardiac frequency and higher frequency of patients with nonsustained ventricular tachycardia compared to those without an identified mutation. Patients with mutations in the MYH7 gene had larger left atrium size, higher frequency of atrial fibrillation and higher frequency of family history of the disease than patients with a mutation in the MYBPC3 gene. Conclusions: The technique of sequencing RNA extracted from leucocytes is not suitable for large scale routine screening for CH due to the high frequency of false positives, possibility of false negatives and low rate of amplification, but the DNA sequencing, though laborious, was very sensitive to the analysis. Identification of a specific mutation cannot yet be used for prognosis of the disease\'s severity, but comparisons of genotype and phenotype have shown some interesting relationships that should be further evaluated. The presente study is the first one to characterize the molecular epidemiology of hypertrophic cardiomyopathy in Brazilian patients for those three more important genes mentioned above

Introdução: A cardiomiopatia hipertrófica (CH) é uma doença genética cardíaca primária, caracterizada por hipertrofia do ventrículo esquerdo, sem dilatação, geralmente assimétrica e predominantemente septal, com prevalência estimada em 1:500. Atualmente já foram descobertos 20 genes associados à doença, mas os genes mais frequentemente relacionados são o da Cadeia Pesada da ?-miosina (MYH7), Proteína C de Ligação da Miosina (MYBPC3) e Troponina T (TNNT2). O diagnóstico molecular dos pacientes é importante e custo-efetivo do ponto de vista de saúde pública, além de recomendado pela Sociedade Europeia de Cardiologia. Entretanto, o custo inicial do exame é muito alto, de forma que estudos tentam reduzir esse custo. Uma das propostas descritas utilizou o RNA extraído de leucócitos para sequenciamento com sucesso para o gene MYBPC3. Este trabalho tem como objetivo testar a metodologia de sequenciamento de RNA em larga escala e testar a aplicabilidade da mesma para os genes MYH7 e TNNT2, estimar as principais mutações envolvidas nos pacientes do estudo e estabelecer correlações entre os diferentes genótipos avaliados e os fenótipos dos grupos familiares portadores da doença. Métodos: Os sujeitos incluídos no estudo são portadores de cardiomiopatia hipertrófica nos quais foi feita uma coleta de sangue para extração de DNA e RNA. Foi utilizada nested PCR para amplificação do cDNA e PCR convencional para amplificação de DNA e posterior sequenciamento em sequenciador automático. As análises estatísticas dos dados clínicos foram realizadas utilizando-se ANOVA para comparação de médias e teste exato de Fisher para comparação de frequências. Resultados: O sequenciamento de RNA se mostrou problemático, com baixa taxa de amplificação, falsos positivos e possíveis falsos negativos, de forma que optou-se por utilizar o sequenciamento de DNA para identificação das alterações. Dos 268 pacientes estudados foi identificada uma mutação em 131 deles (48,8%). Das mutações encontradas, 78 (59,5%) estavam no gene MYH7, 50 (38,2%) no gene MYBPC3 e 3 (2,3%) no gene TNNT2. Foram identificadas 69 mutações diferentes, 24 delas ainda não descritas. Pacientes com mutação identificada possuíam em média idade de diagnóstico e idade atual menores, maior frequência cardíaca média e maior frequência de pacientes com taquicardia ventricular não sustentada quando comparados ao grupo sem mutação identificada. Pacientes com mutação no gene MYH7 possuíam maior tamanho de átrio esquerdo, maior frequência de fibrilação atrial e maior frequência de histórico familiar da doença do que pacientes com mutação em MYBPC3. Conclusões: A técnica sequenciamento de RNA extraído de leucócitos não é adequada para uma rotina de rastreamento em larga escala para CH devido à alta frequência de falsos positivos, possibilidade de falsos negativos e baixa taxa de amplificação, mas o sequenciamento de DNA, embora trabalhoso, se mostrou muito sensível para a análise. A identificação de uma mutação específica ainda não pode ser usada para prognóstico de gravidade da CH, mas as comparações de genótipo e fenótipo mostraram algumas relações interessantes que devem ser melhor avaliadas nos pacientes. Este é o primeiro trabalho a caracterizar a epidemiologia molecular da CH em pacientes brasileiros para os genes MYH7, MYBPC3 e TNNT2 / Introduction: Hypertrophic cardiomyopathy (HC) is a primary genetic cardiac disease characterized by left ventricular hypertrophy without dilation, usually asymmetric and predominantly septal, with an estimated prevalence of 1:500. There are 20 genes associated to the HC, but the ones more often related to the disease are ?-myosin heavy chain (MYH7), myosin binding protein C (MYBPC3) and troponin T (TNNT2). The molecular diagnosis of patients is important and cost-effective from the standpoint of public health, and recommended by the European Society of Cardiology. However, the initial cost of the exam is very high, so several studies are attempting to reduce it. One of the proposals described used the RNA extracted from leukocytes for sequencing successfully to the MYBPC3 gene. The present study aims to test the methodology for large-scale sequencing and test it\'s applicability to the MYH7 and TNNT2 genes, to estimate the main mutations involved in the studied patients and establish correlations between their genotypes and phenotypes. Methods: The subjects included in the study were patients previously diagnosed with hypertrophic cardiomyopathy in whom a blood sample was collected to DNA and RNA extraction. It was used nested PCR for cDNA amplification and conventional PCR for DNA amplification for posterior sequencing on an automatic sequencer. Statistical analyzes of clinical data were performed using ANOVA to compare means and Fisher\'s exact test for frequencies comparison. Results: The RNA sequencing was problematic with low rate of amplification, false positives and possible false negatives, so was used DNA sequencing to identify the mutations. Of the 268 patients studied a mutation was identified in 131 of them (48.8%). Among the mutations found, 78 (59.5%) were in the MYH7 gene, 50 (38.2%) in the MYBPC3 gene and three (2.3%) in the TNNT2 gene. We have identified 69 different mutations, 24 of them not yet described. Patients with an identified mutation had an average smaller age of diagnosis and current age, higher average cardiac frequency and higher frequency of patients with nonsustained ventricular tachycardia compared to those without an identified mutation. Patients with mutations in the MYH7 gene had larger left atrium size, higher frequency of atrial fibrillation and higher frequency of family history of the disease than patients with a mutation in the MYBPC3 gene. Conclusions: The technique of sequencing RNA extracted from leucocytes is not suitable for large scale routine screening for CH due to the high frequency of false positives, possibility of false negatives and low rate of amplification, but the DNA sequencing, though laborious, was very sensitive to the analysis. Identification of a specific mutation cannot yet be used for prognosis of the disease\'s severity, but comparisons of genotype and phenotype have shown some interesting relationships that should be further evaluated. The presente study is the first one to characterize the molecular epidemiology of hypertrophic cardiomyopathy in Brazilian patients for those three more important genes mentioned above