Lamin A/C–mediated neuromuscular junction defects in Emery-Dreifuss muscular dystrophy

The LMNA gene encodes lamins A and C, two intermediate filament-type proteins that are important determinants of interphase nuclear architecture. Mutations in LMNA lead to a wide spectrum of human diseases including autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD), which affects skeletal and cardiac muscle. The cellular mechanisms by which mutations in LMNA cause disease have been elusive. Here, we demonstrate that defects in neuromuscular junctions (NMJs) are part of the disease mechanism in AD-EDMD. Two AD-EDMD mouse models show innervation defects including misexpression of electrical activity–dependent genes and altered epigenetic chromatin modifications. Synaptic nuclei are not properly recruited to the NMJ because of mislocalization of nuclear envelope components. AD-EDMD patients with LMNA mutations show the same cellular defects as the AD-EDMD mouse models. These results suggest that lamin A/C–mediated NMJ defects contribute to the AD-EDMD disease phenotype and provide insights into the cellular and molecular mechanisms for the muscle-specific phenotype of AD-EDMD

Genetic modifiers of ambulation in the cooperative international Neuromuscular Research Group Duchenne natural history study

OBJECTIVE: We studied the effects of LTBP4 and SPP1 polymorphisms on age at loss of ambulation (LoA) in a multiethnic Duchenne muscular dystrophy (DMD) cohort. METHODS: We genotyped SPP1 rs28357094 and LTBP4 haplotype in 283 of 340 participants in the Cooperative International Neuromuscular Research Group Duchenne Natural History Study (CINRG-DNHS). Median ages at LoA were compared by Kaplan-Meier analysis and log-rank test. We controlled polymorphism analyses for concurrent effects of glucocorticoid corticosteroid (GC) treatment (time-varying Cox regression) and for population stratification (multidimensional scaling of genome-wide markers). RESULTS: Hispanic and South Asian participants (n=18, 41) lost ambulation 2.7 and 2 years earlier than Caucasian subjects (p=0.003, <0.001). The TG/GG genotype at SPP1 rs28357094 was associated to 1.2-year-earlier median LoA (p=0.048). This difference was greater (1.9 years, p=0.038) in GC-treated participants, whereas no difference was observed in untreated subjects. Cox regression confirmed a significant effect of SPP1 genotype in GC-treated participants (hazard ratio = 1.61, p=0.016). LTBP4 genotype showed a direction of association with age at LoA as previously reported, but it was not statistically significant. After controlling for population stratification, we confirmed a strong effect of LTBP4 genotype in Caucasians (2.4 years, p =0.024). Median age at LoA with the protective LTBP4 genotype in this cohort was 15.0 years, 16.0 for those who were treated with GC. INTERPRETATION: SPP1 rs28357094 acts as a pharmacodynamic biomarker of GC response, and LTBP4 haplotype modifies age at LoA in the CINRG-DNHS cohort. Adjustment for GC treatment and population stratification appears crucial in assessing genetic modifiers in DMDFil: Bello, Luca. Children's National Medical Center; Estados Unidos. Università di Padova; ItaliaFil: Kesari, Akanchha. Children's National Medical Center; Estados UnidosFil: Gordish Dressman, Heather. Children's National Medical Center; Estados UnidosFil: Cnaan, Avital. Children's National Medical Center; Estados Unidos. The George Washington University; Estados UnidosFil: Morgenroth, Lauren P.. Children's National Medical Center; Estados UnidosFil: Punetha, Jaya. Children's National Medical Center; Estados Unidos. The George Washington University; Estados UnidosFil: Duong, Tina. Children's National Medical Center; Estados UnidosFil: Henricson, Erik K.. University of California at Davis; Estados UnidosFil: Pegoraro, Elena. Università di Padova; ItaliaFil: McDonald, Craig M.. University of California at Davis; Estados UnidosFil: Hoffman, Eric P.. Children's National Medical Center; Estados Unidos. The George Washington University; Estados UnidosFil: Dubrovsky, Alberto. Cooperative International Neuromuscular Research Group Investigators; ArgentinaFil: Andreone, Luz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Cooperative International Neuromuscular Research Group Investigators; Argentina. Fundación Favaloro; ArgentinaFil: Cooperative International Neuromuscular Research Group Investigators. No especifica

SMN1 dosage analysis in spinal muscular atrophy from India

BACKGROUND: Spinal muscular atrophy (SMA) represents the second most common fatal autosomal recessive disorder after cystic fibrosis. Due to the high carrier frequency, the burden of this genetic disorder is very heavy in developing countries like India. As there is no cure or effective treatment, genetic counseling becomes very important in disease management. SMN1 dosage analysis results can be utilized for identifying carriers before offering prenatal diagnosis in the context of genetic counseling. METHODS: In the present study we analyzed the carrier status of parents and sibs of proven SMA patients. In addition, SMN1 copy number was determined in suspected SMA patients and parents of children with a clinical diagnosis of SMA. RESULTS: wenty nine DNA samples were analyzed by quantitative PCR to determine the number of SMN1 gene copies present, and 17 of these were found to have one SMN1 gene copy. The parents of confirmed SMA patients were found to be obligate carriers of the disease. Dosage analysis was useful in ruling out clinical suspicion of SMA in four patients. In a family with history of a deceased floppy infant and two abortions, both parents were found to be carriers of SMA and prenatal diagnosis could be offered in future pregnancies. CONCLUSION: SMN1 copy number analysis is an important parameter for identification of couples at risk for having a child affected with SMA and reduces unwarranted prenatal diagnosis for SMA. The dosage analysis is also useful for the counseling of clinically suspected SMA with a negative diagnostic SMA test

Osteopontin is linked with AKT, FoxO1, and myostatin in skeletal muscle cells

Introduction: Osteopontin (OPN) polymorphisms are associated with muscle size and modify disease progression in Duchenne muscular dystrophy (DMD). We hypothesized that OPN may share a molecular network with myostatin (MSTN). Methods: Studies were conducted in the golden retriever (GRMD) and mdx mouse models of DMD. Follow-up in-vitro studies were employed in myogenic cells and the mdx mouse treated with recombinant mouse (rm) or human (Hu) OPN protein. Results: OPN was increased and MSTN was decreased and levels correlated inversely in GRMD hypertrophied muscle. RM-OPN treatment led to induced AKT1 and FoxO1 phosphorylation, microRNA-486 modulation, and decreased MSTN. An AKT1 inhibitor blocked these effects, whereas an RGD-mutant OPN protein and an RGDS blocking peptide showed similar effects to the AKT inhibitor. RMOPN induced myotube hypertrophy and minimal Feret diameter in mdx muscle. Discussion: OPN may interact with AKT1/MSTN/FoxO1 to modify normal and dystrophic muscle

Asynchronous remodeling is a driver of failed regeneration in Duchenne muscular dystrophy

We sought to determine the mechanisms underlying failure of muscle regeneration that is observed in dystrophic muscle through hypothesis generation using muscle profiling data (human dystrophy and murine regeneration). We found that transforming growth factor β-centered networks strongly associated with pathological fibrosis and failed regeneration were also induced during normal regeneration but at distinct time points. We hypothesized that asynchronously regenerating microenvironments are an underlying driver of fibrosis and failed regeneration. We validated this hypothesis using an experimental model of focal asynchronous bouts of muscle regeneration in wild-type (WT) mice. A chronic inflammatory state and reduced mitochondrial oxidative capacity are observed in bouts separated by 4 d, whereas a chronic profibrotic state was seen in bouts separated by 10 d. Treatment of asynchronously remodeling WT muscle with either prednisone or VBP15 mitigated the molecular phenotype. Our asynchronous regeneration model for pathological fibrosis and muscle wasting in the muscular dystrophies is likely generalizable to tissue failure in chronic inflammatory states in other regenerative tissues

Mutations in GDP-mannose pyrophosphorylase b cause congenital and limb-girdle muscular dystrophies associated with hypoglycosylation of α-dystroglycan

Congenital muscular dystrophies with hypoglycosylation of α-dystroglycan (α-DG) are a heterogeneous group of disorders often associated with brain and eye defects in addition to muscular dystrophy. Causative variants in 14 genes thought to be involved in the glycosylation of α-DG have been identified thus far. Allelic mutations in these genes might also cause milder limb-girdle muscular dystrophy phenotypes. Using a combination of exome and Sanger sequencing in eight unrelated individuals, we present evidence that mutations in guanosine diphosphate mannose (GDP-mannose) pyrophosphorylase B (GMPPB) can result in muscular dystrophy variants with hypoglycosylated α-DG. GMPPB catalyzes the formation of GDP-mannose from GTP and mannose-1-phosphate. GDP-mannose is required for O-mannosylation of proteins, including α-DG, and it is the substrate of cytosolic mannosyltransferases. We found reduced α-DG glycosylation in the muscle biopsies of affected individuals and in available fibroblasts. Overexpression of wild-type GMPPB in fibroblasts from an affected individual partially restored glycosylation of α-DG. Whereas wild-type GMPPB localized to the cytoplasm, five of the identified missense mutations caused formation of aggregates in the cytoplasm or near membrane protrusions. Additionally, knockdown of the GMPPB ortholog in zebrafish caused structural muscle defects with decreased motility, eye abnormalities, and reduced glycosylation of α-DG. Together, these data indicate that GMPPB mutations are responsible for congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-DG. © 2013 The American Society of Human Genetics.Funding for UK10K was provided by the Wellcome Trust under award WT091310

Mutation analysis in spinal muscular atrophy using allele-specific polymerase chain reaction

439-441Polymerase chain reaction (PCR), followed by restriction digestion is universally used for molecular diagnosis of spinal muscular atrophy (SMA). In the present study, we have used a modified strategy based on amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) to develop a rapid and reliable method for mutation detection and prenatal diagnosis in SMA patients. The telomeric (SMN1) and centromeric (SMN2) copies of exon 7 of the survival motor neuron (SMN) gene were amplified by ARMS-PCR, using primers specific to SMN1 and SMN2 nucleotide sequence with the exonic mismatch G (for SMN1) and A (for SMN2) at the 3’ end. The PCR products were analyzed on agarose gels. All the patients who had homozygous deletion of exon 7 of SMN1 gene by conventional PCR-restriction fragment length polymorphism (PCR-RFLP) method showed the same deletion status by ARMS-PCR. This procedure showed a 100% concordance between PCR-RFLP and ARMS-PCR methods for the detection of SMN1/SMN2 status in patients with SMA. An artifact due to incomplete digestion is not a problem while using ARMS-PCR. The modified protocol is specific, rapid and highly reliable for use in prenatal diagnosis as well

Analysis of VNTR loci, ApoB 3' HVR and D1S80 in North Indians

358-362Polymorphic markers like VNTRs at Apolipoprotein B 3' and locus D1S80 hypervariable region have been used extensively for population studies through out the world. In the present study, the polymorphism data in North Indian population at these VNTR loci was reported. The allele distributions and their genotype frequencies at the VNTR loci, Apo B and D1S80 were reported in 86 (172 chromosomes) and 75 (150 chromosomes) unrelated normal individuals, respectively. Genomic DNA was extracted from blood samples and amplified by polymerase chain reaction. The respective alleles and their sizes were determined, 19 and 24 different alleles making up 51 and 50 genotypes of Apo B and D1S80 respectively were identified in the North Indian population. As per 2 analysis, the allele and genotype frequencies for both VNTRs were in Hardy-Weinberg equilibrium. The most frequent allele of Apo B (allele 6) corresponded to 40 repeats, and D1S80 (allele 12) to 28 repeats. The frequencies were 0.087 and 0.17 and observed heterozygosities were 55 and 57% for Apo B and D1S80, respectively. This information may have implications in disease diagnostics, forensics, paternity analysis, and for ruling out maternal contamination in fetal samples during prenatal diagnosis of genetic disorders