RESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Cardiomiopatía Dilatada/genética , Variaciones en el Número de Copia de ADN/genética , Exones/genética , Estudio de Asociación del Genoma Completo , Mutación Puntual/genética , Adulto , Anciano , Animales , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Análisis Mutacional de ADN , Femenino , Técnicas de Silenciamiento del Gen , Heterocigoto , Humanos , Hibridación Genética , Masculino , Persona de Mediana Edad , Modelos Animales , Datos de Secuencia Molecular , Linaje , Fenotipo , Control de Calidad , Adulto Joven , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Riboflavin is a water-soluble vitamin that reduces the production of proinflammatory mediators and oxygen radicals. Because islet beta-cells are very sensitive to oxidative stress and to cytokines, we investigated the possible cytoprotective effects of riboflavin on insulinoma NIT-1 cells and on isolated rodent islets. NIT-1 cells and islets cultured in the presence or absence of 10 microM riboflavin were studied at baseline and after exposure to cytokines (TNF-alpha, IL-1beta, INF-gamma). Riboflavin treatment did not affect islet cell viability as assessed by flow cytometry for caspases activation. However, riboflavin prevented the cytokine-induced increase in IL-6 mRNA expression and p38 phosphorylation analyzed by real-time PCR and immunoassay, respectively. In summary, nontoxic doses of riboflavin prevent cytokines-induced p38 phosphorylation and IL-6 upregulation in islet cells. This observation, together with the safety profile of riboflavin in the clinical setting, makes it an appealing agent for islet cytoprotection in islet transplantation protocols.
Asunto(s)
Expresión Génica/efectos de los fármacos , Interleucina-6/biosíntesis , Islotes Pancreáticos/metabolismo , Riboflavina/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/terapia , Activación Enzimática/efectos de los fármacos , Interleucina-1beta/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Trasplante de Islotes Pancreáticos , Masculino , Ratones , Ratones Endogámicos NOD , Ratas , Riboflavina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Progressive graft dysfunction is commonly observed in recipients of islet allografts treated with high doses of rapamycin. This study aimed at evaluating the effect of rapamycin on pancreatic islet cell proliferation in vivo. METHODS: The murine pregnancy model was utilized, since a high rate of beta-cell proliferation occurs in a well-defined time frame. Rapamycin (0.2 mg/kg/day) was given to C57BL/6 mice for 5-7 days starting on day 7.5 of pregnancy. Cell proliferation was evaluated by detection of bromodeoxyuridine incorporation by immunohistochemistry. RESULTS: Pregnancy led to increased beta-cell proliferation and islet yield with skewing in islet size distribution as well as higher pancreatic insulin content, when compared to that of nonpregnant females. These effects of pregnancy on beta-cell proliferation and mass were significantly blunted by rapamycin treatment. Minimal effect of rapamycin was observed on islet function both in vivo and in vitro. Rapamycin treatment of islets in vitro resulted in reduced p70s6k phosphorylation, which was paralleled by increased ERK1/2 phosphorylation. CONCLUSIONS: Rapamycin treatment reduces the rate of beta-cell proliferation in vivo. This phenomenon may contribute to impair beta-cell renewal in transplanted patients and to the progressive dysfunction observed in islet graft recipients.
Asunto(s)
División Celular/efectos de los fármacos , Células Secretoras de Insulina/citología , Sirolimus/farmacología , Animales , Glucemia/metabolismo , Separación Celular/métodos , Inmunosupresores/farmacología , Insulina/análisis , Células Secretoras de Insulina/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Páncreas/citología , Páncreas/efectos de los fármacos , Proteínas Quinasas/metabolismoRESUMEN
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.
Asunto(s)
Cardiomiopatía Dilatada/genética , Lamina Tipo A/genética , Sustitución de Aminoácidos , Animales , Células COS , Cardiomiopatía Dilatada/patología , Núcleo Celular/ultraestructura , Chlorocebus aethiops , Estudios de Cohortes , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lamina Tipo A/metabolismo , Masculino , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Linaje , Fenotipo , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
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.
Asunto(s)
Cardiomiopatía Dilatada/genética , Mutación Missense/genética , Proteínas de Unión al ARN/genética , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , Proteínas de Unión al ARN/química , Adulto JovenRESUMEN
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.
Asunto(s)
Miosinas Cardíacas/genética , Cardiomiopatía Dilatada/genética , Proteínas Portadoras/genética , Cadenas Pesadas de Miosina/genética , Tropomiosina/genética , Troponina C/genética , Troponina I/genética , Sustitución de Aminoácidos , Estudios de Cohortes , Haplotipos , Humanos , Polimorfismo de Nucleótido Simple , Empalme del ARN , Análisis de Secuencia de ADNRESUMEN
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.