Estudio preliminar de correlación fenotipo-genotipo en miocardiopatías de pacientes derivados a un centro de alta complejidad del conurbano bonaerense / A Preliminary Study of the Phenotype-Genotype Correlation in Cardiomyopathies in Patients Referred to a Tertiary Healthcare Center in the Suburbs of Buenos Aires

RESUMEN Introducción : Las miocardiopatías se definen como un trastorno del miocardio en el que el músculo cardíaco es estructural y funcionalmente anormal, en ausencia de enfermedad arterial coronaria, hipertensión arterial (HTA), enfermedad valvular y enfermedad cardíaca congénita. Estas enfermedades son relativamente frecuentes, y suponen una importante causa de morbimortalidad a nivel global. Aunque el estudio genético se recomienda para el cribado familiar, la falta de datos robustos sobre asociaciones genotipo-fenotipo específicas ha reducido su impacto en el manejo clínico. Objetivos : El objetivo de este estudio es analizar la frecuencia de mutaciones en una población de pacientes con miocardiopatía derivados a un centro de alta complejidad y el análisis de la correlación genotipo-fenotipo en las mutaciones identificadas. Material y métodos: Se estudiaron en forma prospectiva 102 pacientes con sospecha de miocardiopatía hipertrófica (MCH) familiar, de los cuales 70 constituían casos índices, de una cohorte ambispectiva de pacientes con miocardiopatías controladas en un hos pital público de alta complejidad de tercer nivel de atención de la provincia de Buenos Aires, desde enero 2012 al 30 agosto 2022. Resultados : De 102 pacientes 83 fueron considerados afectados. De eelos, 31 eran MCH y 52 fenocopias, sin diferencia en el pronóstico. Se realizó estudio genético en 77 pacientes, de los cuales 57 presentaron mutaciones reconocibles, en el 80% de los casos coincidentes con un Score de Mayo ≥3. Se detectaron 28 variantes de significado incierto. Conclusiones : Se comprobó que realizar estudio molecular guiado por el Score de Mayo permitió obtener un alto grado de probabilidad de detectar mutaciones. Se evidenció la importancia del estudio molecular debido a la existencia de solapamiento fenotípico y genotípico de las miocardiopatías. El conocimiento de la variante genética causal actualmente no afecta el manejo clínico de la mayoría de los pacientes con MCH, pero es de ayuda ante un pequeño grupo de genes que tienen opciones de tratamiento.

ABSTRACT Background : Cardiomyopathies are defined as a disorder of the myocardium in which the heart muscle is structurally and functionally abnormal, in the absence of coronary artery disease, hypertension (HT), valvular heart disease and congenital heart disease. These diseases are relatively common and a major cause of morbidity and mortality worldwide. Although genetic testing is recommended for family screening, lack of solid data on specific genotype-phenotype associations has reduced its impact on clinical management. Objectives : This study aims to analyze the frequency of mutations in a population of patients with cardiomyopathy referred to a tertiary healthcare center and to analyze the genotype-phenotype correlation of the identified mutations. Methods : We prospectively included 102 patients with suspected familial hypertrophic cardiomyopathy (HCM), 70 of which were index cases, from an ambispective cohort of patients with cardiomyopathies treated in a tertiary healthcare public hos pital in the province of Buenos Aires, from January 2012 to August 30, 2022. Results : Of 102 patients, 83 were considered affected. Of these, 31 were HCM and 52 were phenocopies, with no difference in prognosis. A genetic study was carried out in 77 patients, of whom 57 presented recognizable mutations, in 80% of the cases coinciding with a Mayo Score ≥3. Twenty-eight variants of uncertain significance were detected. Conclusions : It was confirmed that molecular testing guided by the Mayo Score provided high probability of detecting mutations. Molecular testing proved to be important due to the phenotypic and genotypic overlap in cardiomyopathies. Understanding the causative genetic variant, nowadays, does not affect the clinical management of most HCM patients, but is helpful in a small group of genes with treatment options.

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 / Prevalência e Expressão Fenotípica das Mutações nos Genes MYH7, MYBPC3 e TNNT2 em Famílias com Cardiomiopatia Hipertrófica no Sul do Brasil: Um Estudo Transversal

Abstract Background: Mutations in sarcomeric genes are found in 60-70% of individuals with familial forms of hypertrophic cardiomyopathy (HCM). However, this estimate refers to northern hemisphere populations. The molecular-genetic profile of HCM has been subject of few investigations in Brazil, particularly in the south of the country. Objective: To investigate mutations in the sarcomeric genes MYH7, MYBPC3 and TNNT2 in a cohort of HCM patients living in the extreme south of Brazil, and to evaluate genotype-phenotype associations. Methods: Direct DNA sequencing of all encoding regions of three sarcomeric genes was conducted in 43 consecutive individuals of ten unrelated families. Results: Mutations for CMH have been found in 25 (58%) patients of seven (70%) of the ten study families. Fourteen (56%) individuals were phenotype-positive. All mutations were missense, four (66%) in MYH7 and two (33%) in MYBPC3. We have not found mutations in the TNNT2 gene. Mutations in MYH7 were identified in 20 (47%) patients of six (60%) families. Two of them had not been previously described. Mutations in MYBPC3 were found in seven (16%) members of two (20%) families. Two (5%) patients showed double heterozygosis for both genes. The mutations affected different domains of encoded proteins and led to variable phenotypic expression. A family history of HCM was identified in all genotype-positive individuals. Conclusions: In this first genetic-molecular analysis carried out in the south of Brazil, we found mutations in the sarcomeric genes MYH7 and MYBPC3 in 58% of individuals. MYH7-related disease was identified in the majority of cases with mutation.

Resumo Fundamento: Mutações em genes do sarcômero são encontradas em 60-70% dos indivíduos com formas familiares de cardiomiopatia hipertrófica. (CMH). Entretanto, essa estimativa refere-se a populações de países do hemisfério norte. O perfil genético-molecular da CMH foi tema de poucos estudos no Brasil, particularmente na região sul do país. Objetivo: Realizar a pesquisa de mutações dos genes sarcoméricos MYH7, MYBPC3 e TNNT2 numa coorte de CMH estabelecida no extremo sul do Brasil, assim como avaliar as associações genótipo-fenótipo. Métodos: Sequenciamento direto do DNA de todas as regiões codificantes dos três genes sarcoméricos foi realizada em 43 indivíduos consecutivos de dez famílias não-relacionadas. Resultados: Mutações para CMH foram encontradas em 25 (58%) indivíduos de sete (70%) das dez famílias estudadas, sendo 14 (56%) deles fenótipo-positivos. Todas as mutações eram missense, quatro (66%) no gene MYH7 e duas (33%) no gene MYBPC3. Não foram encontradas mutações no gene TNNT2. Mutações em MYH7 foram identificadas em 20 (47%) indivíduos de seis (60%) famílias. Duas delas não haviam sido previamente relatadas. Mutações de MYBPC3 foram detectadas em sete (16%) membros de duas (20%) famílias. Dois (5%) indivíduos apresentaram dupla heterozigose com mutações em ambos os genes. As mutações acometeram distintos domínios das proteínas codificadas e produziram expressão fenotípica variável. História familiar de CMH foi identificada em todos os indivíduos genótipo-positivos. Conclusões: Nessa primeira análise genético-molecular da CMH realizada no sul do Brasil, foram encontradas mutações nos genes sarcoméricos MYH7 e MYBPC3 em 58% dos indivíduos. Doença relacionada ao gene MYH7 foi identificada na maioria dos casos com mutação.

Efeitos do ultrassom terapêutico associados ao alongamento estático sobre parâmetros histomorfométricos longitudinais de sóleos imobilizados de ratos / Effects of therapeutic ultrasound associated with static stretching on longitudinal histomorphometric parameters of immobilized soleus of rats

O músculo é um tecido dotado de plasticidade que se adapta a diferentes estímulos. A imobilização causa danos ao sistema muscular incluindo atrofia, perda da extensibilidade e resistência muscular. O alongamento muscular e o ultrassom terapêutico são modalidades utilizadas para acelerar o processo de reparo muscular, provendo aumento da síntese proteica e melhora da extensibilidade. OBJETIVO: Comparar o uso do ultrassom terapêutico, associado ao alongamento, na remobilização de músculo sóleo, de ratos, submetido ao encurtamento muscular, sobre os aspectos histomorfométricos longitudinais. MATERIAIS E MÉTODOS: Vinte e oito ratos Wistar foram imobilizados por 15 dias e, após liberados do aparato de imobilização, distribuídos em quatro grupos: grupo (GA) apenas remobilizado por alongamento durante 10 dias; e os demais foram submetidos a 10 dias de intervenção terapêutica do ultrassom de 1MHz a 1,0W/cm² (GAUS 1,0); 0,5W/cm² (GAUS 0,5); e 0,2W/cm² (GAUS 0,2), e posterior alongamento dos músculos sóleos. Ao final do tratamento, os animais foram eutanasiados e tiveram seus músculos removidos para posterior análise histológica dos parâmetros longitudinais (contagem de sarcômeros). RESULTADOS: Na análise intragrupo, quanto ao comprimento muscular, apenas o grupo GAUS 0,5 não teve diferença significativa. Quanto à contagem de sarcômeros, os grupos GA e GAUS 0,2 tiveram diferença significativa. Quanto ao tamanho dos sarcômeros, nenhum grupo teve diferença significativa. Na análise intergrupos, nenhum grupo apresentou diferença significativa. CONCLUSÃO: O alongamento foi insuficiente para reverter os efeitos da imobilização. Quando associado ao ultrassom terapêutico, a dose 0,5W/cm² recuperou o comprimento muscular, e as doses 1,0 e 0,5W/cm² contribuíram para o aumento da quantidade dos sarcômeros em série.

The muscle tissue is endowed with plasticity that adapts to different stimuli. Immobilization causes damage to the musculature including atrophy, loss of muscle strength and extensibility. The stretching and ultrasound treatment modalities are used to speed up muscle repair process as they can increase protein synthesis and improve extensibility. OBJECTIVE: To compare the use of therapeutic thermal and non thermal ultrasound, associated with stretching, in the remobilization of the soleus muscle of rats subjected to position of muscle shortening on aspects histomorphometric longitudinal muscle.} METHODS: 28 rats were immobilized for 15 days, later released from the apparatus and divided into four groups: group AG only remobilized by stretching for 10 days and the others were subjected to 10 days of therapeutic intervention 1MHz of ultrasound at 1.0W/cm² (GAUS 1.0), 0.5W/cm² (GAUS 0.5), and 0.2 W/cm² (GAUS 0.2), and further stretching to the soleus. At the end of treatment, the animals were sacrificed and their soleus muscles were removed for later histological analysis of longitudinal parameters (count of sarcomeres). RESULTS: At intragroup analysis on the muscle length, only the group GAUS 0.5 did not present significant difference. The count of sarcomeres in the groups GA and GAUS 0.2 was statistically different. The size of the sarcomeres in both groups had no statistically significant difference. In inter-group analysis both groups had no statistically significant difference for any of the variables. CONCLUSION: The stretching was insufficient to reverse the effects of immobilization. When associated with therapeutic ultrasound, the dose 0.5 W/cm² recovered muscle length significantly, and the doses 1.0 and 0.5 W/cm² contributed to the significant increase of the number of sarcomeres in immobilized muscles.

The intimate nature of oculomotor muscles contracture / A natureza íntima da contratura do músculo oculomotor

The author makes comments about the shortening and loss of elasticity of the oculomotor muscle that remains slack for some time (contracture), by means of a reasoning based on the Hooke´s law and on the papers carried out to demonstrate that a muscle that remains relaxed for some time suffers a shortening due to loss of sarcomeres on the longitudinal direction and the increase of the cross-sectional area due to the increase of collagen tissue in the perimysium and the endomysium.

O autor procura demonstrar a razão da perda de elasticidade e do encurtamento do músculo oculomotor que permanece relaxado durante certo tempo (contratura), mediante raciocínio baseado na lei de Hooke e nos trabalhos que demonstram que o músculo oculomotor que permanece frouxo por algum tempo sofre encurtamento devido à perda de sarcômeros no sentido longitudinal e ao aumento da área da secção transversa, devida ao aumento do tecido colágeno do perimísio e do endomísio.

Influência do alongamento passivo em três repetições de 30 segundos a cada 48 horas em músculo sóleo imobilizado de ratos / Influence of passive stretching in three 30-second sets at every 48 hours in immobilized soleum muscle of rats

O músculo esquelético tem a propriedade de adaptar-se a diferentes estímulos, alterando o número de sarcômeros em série, de acordo com o comprimento a que está submetido. Este trabalho tem como objetivo comparar as adaptações de peso e comprimento do músculo sóleo de ratos, além de estimativa do total de sarcômeros em série, quando submetidos a alongamento intermitente durante imobilização em plantiflexão. Foram utilizados 24 ratos (Wistar) machos de 14 ± 2 semanas de idade, divididos em 4 grupos: C (n = 6) - controle; A (n = 6) - músculo sóleo esquerdo (MSE) alongado; I - (n = 6) MSE imobilizado; IA (n = 6) MSE imobilizado e alongado. O protocolo utilizado foi 3 séries de alongamento com duração de 30s a cada 48h; e período total de imobilização de 21 dias. Foram comparadas as variações observadas entre os músculos sóleus direito (MSD) e o MSE com relação a peso muscular, comprimento muscular e estimativa do número de sarcômeros em série (ENSS), através da contagem em 300mm. Nas variáveis analisadas ao comparar o MSD (controle) e o MSE (submetido a alongamento e/ou imobilização) foram observados: peso muscular: C = -1,59 por cento, (p = 0,67); A = +5,41 por cento (p = 0,05); I = -41,48 por cento (p = 0,01); IA = -32,46 por cento (p = 0,01); comprimento muscular: C = -2,63 por cento (p = 0,43); A = +7,54 por cento (p = 0,07); I = -12,74 por cento (p = 0,01); IA = -10,42 por cento (p = 0,11); ENSS: C = -3,81 por cento (p = 0,09); A = + 2,73 por cento (p = 0,56); I = -12,20 por cento (p = 0,01); IA = -12,21 por cento (p = 0,21). Conclui-se que o alongamento durante a imobilização preservou o comprimento muscular e a quantidade de sarcômeros em série, mas não evitou a perda do peso muscular.

The skeletal muscle has the property to adapt to different stimuli, alternating the number of sarcomeres in series, according to the length to which it is submitted to. This investigation has the aim to compare weight and length adaptations of the soleus muscle in rats, besides the total estimate of the sarcomeres in series, when submitted to intermittent stretching during immobilization in plantarflexion. Twenty-four 14 ± 2 weeks old Wistar male rats were used. They were divided in 4 groups, namely: C (n = 6) - control; A (n = 6) - stretched left soleus muscle (LSM); I (n = 6) - immobilized left soleus muscle; IA (n = 6) - immobilized and stretched LSM. The used protocol was of 3 stretching sets with 30 s duration at every 48 h, and total immobilization period of 21 days. The variations observed were compared between right soleus muscle (RSM) and LSM concerning muscular weight, muscular length and estimate of number of sarcomeres in series (ENSS), through counting in 300 mm. In the analyzed variables, when RSM (control) and LSM were compared (submitted to stretching and/or immobilization) the following were observed: muscular weight: C = -1.59 percent (p = 0.67); A = +5.41 percent (p = 0.05); I = -41.48 percent (p = 0.01); IA = -32.46 percent (p = 0.01); muscular length: C = -2.63 percent (p = 0.43); A = +7.54 percent (p = 0.07); I = -12.74 percent (p = 0.01); IA = -10.42 percent (p = 0.11); ENSS: C = -3.81 percent (p = 0.09); A = +2.73 percent (p = 0.56); I = -12.20 percent (p = 0.01); IA = -12.21 percent (p = 0.21). It is concluded that stretching during immobilization has preserved muscular length and the amount of sarcomeres in series; however, it did not prevent muscular weight loss.

Adaptability of Extraocular Muscle of Rabit in Experimental Exotropia

Fusion loss or anomaly of eye muscle and its nerve is a cause of strabismus. This induces adaptation of vergence and muscle length, the mechanisms of which are normally responsible for long-term maintenance of ocular alignment and orthophorization. To determine variability of muscle length adaptation according to age, we divided white rabbits into three groups: Group I(less than 8 weeks of age and 15000gm of body weight), Group II(10~14 weeks and 1500~2000gm), GroupIII(more than 16 weeks and 2500gm). Under local anesthesia with intravenous pentobarbital sodium, the right eyeball was fixed in exotropic position by suturing through the periosteum of the lateral canthal region with 5-o polypropylene. The muscles of left eye were not operated and served as a control. We examined electron microscopically the changes in sarcomere of the experimental right medial rectus(RMR) and right lateral rectus(RLR) after sacrificing four rabbits of each group at 1, 2 and 4 weeks, respectively. Regardless of age, when the muscle(RLR) was immobilized in the lengthened position, sarcomeres were added on and its length decreased, and when the muscle(RMR) was immobilized in the shortened position, sarcomeres were lost and its length increased. In conclusion, the study revealed that experimental extraocular muscle of rabbit is adapted in the direction to produce maximal power of its muscle and maintain ocular alignment constantly.