Mutaciones patogénicas en el gen CAPN3 en pacientes con fenotipo inespecífico de LGMD2

Tesis para optar al grado de magíster en genéticaLas distrofias de cintura (LGMD) son un grupo de distrofias musculares caracterizadas por presentar un deterioro progresivo de los músculos de las cinturas escapular, pélvica y proximales de las extremidades. La LGMD2A es causada por mutaciones en el gen CAPN3, que codifica la proteína calpaína-3, y su diagnóstico se basa principalmente en el análisis de la proteína mediante Western Blot (WB) en tejido muscular y análisis genético. Sin embargo, alrededor del 20-30% de los pacientes con LGMD2A muestra cuantificación normal de la proteína, pero funcionalmente inactiva, debido a mutaciones que afectan su capacidad autocatalítica. Objetivo: Detectar mutaciones en el gen CAPN3 en pacientes con fenotipo de LGMD2 indeterminado y comprobar su efecto sobre actividad autocatalítica. Metodología: Se estudió genéticamente un grupo de 13 pacientes chilenos con características clínicas de LGMD2 que, luego de la evaluación clínica, radiológica y/o histopatológica, no pudieron ser subclasificados en una forma específica de LGMD. El análisis genético se realizó mediante secuenciación masiva de 15 genes causales de LGMD y el análisis proteico se realizó mediante ensayo funcional observado por WB Resultado: En 2 pacientes se encontraron 3 mutaciones posiblemente patogénicas en el gen CAPN3, cuyos pacientes son portadores de los genotipos heterocigotos compuestos c.107delG/c.2362_2363delAGinsTCATCT y c.2105C>T/c.2362_2363delAGinsTCATCT. En el análisis proteico del paciente portador de la mutación missense c.2105C>T, se observaron las bandas de degradación autocatalítica a los 60 y 30 kDa. Discusión: La mutación c.107delG del gen CAPN3 no ha sido reportada previamente, a diferencia de la mutación c.2362_2363delAGinsTCATCT, que ha sido reportada y encontrada en otros pacientes chilenos. La mutación missense c.2105C>T ha sido reportada como posiblemente patogénica; sin embargo la actividad autocatalítica de CAPN3 se encuentra conservada. Conclusión: Los resultados permiten concluir que las mutaciones encontradas no ejercerían su efecto patogénico a través de la alteración de la función autocatalítica de CAPN3.The limb-girdle muscular dystrophies (LGMD) are a group of muscular dystrophies characterized by progressive weakness and wasting of pelvic and shoulder girdles, and proximal limb muscles. LGMD2A is caused by mutations in the CAPN3 gene, that encodes the protein calpain-3, and its diagnosis is mainly based on Western Blot (WB) analysis of the protein in muscular tissue and genetic analysis. Nevertheless, around 20-30% of patients with LGMD2A diagnosis show a normal protein quantification, but functionally deficient due mutations that affect its autocatalytic properties. Aims: To detect CAPN3 gene mutations in patients with undetermined LGMD2 phenotype and to assess the autocatalytic activity. Methods: A group of 13 Chileans patients with clinical features of LGMD2 that, after the clinical, radiological and/or histopathological evaluation could not be subclassified in a specific form of LGMD was genetically studied. The genetic analysis was done by massive sequencing of 15 genes causative of LGMD, and the protein analysis was done by WB. Results: In two patients 3 possibly pathogenic mutations were found in the CAPN3 gene, patients showing compound heterozygous genotypes c.107delG/c.2362_2363delAGinsTCATCT and c.2105C>T/c.2362_2363delAGinsTCATCT were found. Protein analysis by WB of the patient carrying the missense mutation c.2105C>T, the autocatalytic degradation bands were observed at 60 and 30 kDa. Discussion: The c.107delG mutations of the CAPN3 gene has not been previously reported, unlike the c.2362_2363delAGinsTCATCT mutation, that has been previously reported, in addition to having been found in other Chilean patients. The missense c.2105C>T mutation has been reported as possibly pathogenic; however the autocatalytic activity of CAPN3 is conserved. Conclusion: These results allow us to conclude that the pathogenic mutations described would not exert their pathogenic effect through the alteration of the autocatalytic function of CAPN3

Toward an objective measure of functional disability in dysferlinopathy

Artículo de publicación ISIIntroduction: Understanding the natural history of dysferlinopathy is essential to design and quantify novel therapeutic protocols. Our aim in this study was to assess, clinically and functionally, a cohort of patients with dysferlinopathy, using validated scales. Methods: Thirty-one patients with genetically confirmed dysferlinopathy were assessed using the motor function measure (MFM), Modified Rankin Scale (MRS), Muscle Research Council (MRC) scale, serum creatine kinase (CK) assessment, baseline spirometry data, and echocardiographic and electrophysiologic studies. Results: MFM and MRC scores showed a significant negative correlation with disease duration and inverse correlation with MRS, but not with onset age, clinical phenotype, or CK levels. Percent forced vital capacity (% FVC) correlated negatively with disease duration and onset age. Eight known pathogenic mutations were identified recurrently, 4 of which accounted for 79% of the total. Conclusions: The results suggest that MFM is a reliable outcome measure that may be useful for longitudinal follow-up in dysferlinopathy. Recurrent mutations suggest a founder effect in the Chilean population

Genetic Profile of Patients with Limb-Girdle Muscle Weakness in the Chilean Population

Hereditary myopathies are a group of genetically determined muscle disorders comprising more than 300 entities. In Chile, there are no specific registries of the distinct forms of these myopathies. We now report the genetic findings of a series of Chilean patients presenting with limb-girdle muscle weakness of unknown etiology. Eighty-two patients were explored using high-throughput sequencing approaches with neuromuscular gene panels, establishing a definite genetic diagnosis in 49 patients (59.8%) and a highly probable genetic diagnosis in eight additional cases (9.8%). The most frequent causative genes identified were DYSF and CAPN3, accounting for 22% and 8.5% of the cases, respectively, followed by DMD (4.9%) and RYR1 (4.9%). The remaining 17 causative genes were present in one or two cases only. Twelve novel variants were identified. Five patients (6.1%) carried a variant of uncertain significance in genes partially matching the clinical phenotype. Twenty patients (24.4%) did not carry a pathogenic or likely pathogenic variant in the phenotypically related genes, including five patients (6.1%) presenting an autoimmune neuromuscular disorder. The relative frequency of the different forms of myopathy in Chile is like that of other series reported from different regions of the world with perhaps a relatively higher incidence of dysferlinopathy