Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is clinically characterized by muscle weakness. However, the mechanisms underlying this weakness are poorly understood. Here, we studied the contractile phenotype of skeletal muscle from NM patients with nebulin mutations (NEM2). SDS-PAGE and Western blotting studies revealed markedly reduced nebulin protein levels in muscle from NM patients, whereas levels of other thin filament-based proteins were not significantly altered. Muscle mechanics studies indicated significantly reduced calcium sensitivity of force generation in NM muscle fibers compared to control fibers. In addition, we found slower rate constant of force redevelopment, as well as increased tension cost, in NM compared to control fibers, indicating that in NM muscle the rate of cross-bridge attachment is reduced, whereas the rate of cross-bridge detachment is increased. The resulting reduced fraction of force generating cross-bridges is expected to greatly impair the force generating capacity of muscle from NM patients. Thus, the present study provides important novel insights into the pathogenesis of muscle weakness in nebulin-based NM.

Cardiac sodium channel Na(v)1.5 plays a critical role in heart excitability and conduction. The molecular mechanism that underlies the expression of Na(v)1.5 at the cell membrane is poorly understood. Previous studies demonstrated that cytoskeleton proteins can be involved in the regulation of cell surface expression and localization of several ion channels. We performed a yeast two-hybrid screen to identify Na(v)1.5-associated proteins that may be involved in channel function and expression. We identified alpha-actinin-2 as an interacting partner of the cytoplasmic loop connecting domains III and IV of Na(v)1.5 (Na(v)1.5/LIII-IV). Co-immunoprecipitation and His(6) pull-down assays confirmed the physical association between Na(v)1.5 and alpha-actinin-2 and showed that the spectrin-like repeat domain is essential for binding of alpha-actinin-2 to Na(v)1.5. Patch-clamp studies revealed that the interaction with alpha-actinin-2 increases sodium channel density without changing their gating properties. Consistent with these findings, coexpression of alpha-actinin-2 and Na(v)1.5 in tsA201 cells led to an increase in the level of expression of Na(v)1.5 at the cell membrane as determined by cell surface biotinylation. Lastly, immunostaining experiments showed that alpha-actinin-2 was colocalized with Na(v)1.5 along the Z-lines and in the plasma membrane. Our data suggest that alpha-actinin-2, which is known to regulate the functional expression of the potassium channels, may play a role in anchoring Na(v)1.5 to the membrane by connecting the channel to the actin cytoskeleton network.

Diamond-Blackfan Anemia (DBA) is characterized by a defect of erythroid progenitors and, clinically, by anemia and malformations. DBA exhibits an autosomal dominant pattern of inheritance with incomplete penetrance. Currently nine genes, all encoding ribosomal proteins (RP), have been found mutated in approximately 50% of patients. Experimental evidence supports the hypothesis that DBA is primarily the result of defective ribosome synthesis. By means of a large collaboration among six centers, we report here a mutation update that includes nine genes and 220 distinct mutations, 56 of which are new. The DBA Mutation Database now includes data from 355 patients. Of those where inheritance has been examined, 125 patients carry a de novo mutation and 72 an inherited mutation. Mutagenesis may be ascribed to slippage in 65.5% of indels, whereas CpG dinucleotides are involved in 23% of transitions. Using bioinformatic tools we show that gene conversion mechanism is not common in RP genes mutagenesis, notwithstanding the abundance of RP pseudogenes. Genotype-phenotype analysis reveals that malformations are more frequently associated with mutations in RPL5 and RPL11 than in the other genes. All currently reported DBA mutations together with their functional and clinical data are included in the DBA Mutation Database.

In 2004, Anderson et al. reported a homozygous 2502 bp deletion including exon 55 of the nebulin gene in five Ashkenazi Jewish probands with nemaline myopathy. We determined the occurrence of this deletion in a world-wide series of 355 nemaline myopathy probands with no previously known mutation in other genes and found the mutation in 14 probands, two of whom represented families previously ascertained by Anderson et al. Two of the families were not of known Ashkenazi Jewish descent but they had the haplotype known to segregate with this mutation. In all but two of eight homozygous patients, the clinical picture was more severe than in typical nemaline myopathy.

Inclusion body myositis (IBM) is an inflammatory disease of skeletal muscle of unknown cause. To further understand the nature of the tissue injury in this disease, we developed methods for large-scale detection and quantitation of proteins in muscle biopsy samples and analyzed proteomic data produced by these methods together with histochemical, immunohistochemical, and microarray data. Twenty muscle biopsy samples from patients with inflammatory myopathies (n = 17) or elderly subjects without neuromuscular disease (n = 3) were profiled by proteomic studies using liquid chromatographic separation of peptides followed by mass spectrometry. Thirteen of the diseased samples additionally underwent microarray studies. Seventy muscle specimens from patients with a range of neuromuscular disorders were examined by ATPase histochemical methods. Smaller numbers of samples underwent immunohistochemical and immunoblot studies. Mass spectrometric studies identified and quantified approximately 300 total distinct proteins in each muscle sample. In IBM and to a lesser extent in polymyositis, proteomic studies confirmed by histochemical, immunohistochemical, and immunoblot studies showed loss of many fast-twitch specific structural proteins and glycolytic enzymes despite relative preservation of transcript levels. Increased abundance of a nuclear membrane protein, immunoglobulins, and two calpain-3 substrates were present. The atrophy present in IBM muscle is accompanied by preferential loss of fast-twitch structural proteins and glycolytic enzymes, particularly glycogen debranching enzyme, with relative preservation of the abundance of their respective transcripts. Although muscle atrophy has long been recognized in IBM, these studies are the first to report specific proteins which are reduced in quantity in IBM muscle.

The ACTA1 gene encodes skeletal muscle alpha-actin, which is the predominant actin isoform in the sarcomeric thin filaments of adult skeletal muscle, and essential, along with myosin, for muscle contraction. ACTA1 disease-causing mutations were first described in 1999, when a total of 15 mutations were known. In this article we describe 177 different disease-causing ACTA1 mutations, including 85 that have not been described before. ACTA1 mutations result in five overlapping congenital myopathies: nemaline myopathy; intranuclear rod myopathy; actin filament aggregate myopathy; congenital fiber type disproportion; and myopathy with core-like areas. Mixtures of these histopathological phenotypes may be seen in a single biopsy from one patient. Irrespective of the histopathology, the disease is frequently clinically severe, with many patients dying within the first year of life. Most mutations are dominant and most patients have de novo mutations not present in the peripheral blood DNA of either parent. Only 10% of mutations are recessive and they are genetic or functional null mutations. To aid molecular diagnosis and establishing genotype-phenotype correlations, we have developed a locus-specific database for ACTA1 variations (http://waimr.uwa.edu.au).

Nemaline myopathy (NM) is the most common non-dystrophic congenital myopathy. Clinically the most important feature of NM is muscle weakness; however, the mechanisms underlying this weakness are poorly understood. Here, we studied the muscular phenotype of NM patients with a well-defined nebulin mutation (NM-NEB), using a multidisciplinary approach to study thin filament length regulation and muscle contractile performance. SDS-PAGE and western blotting revealed greatly reduced nebulin levels in skeletal muscle of NM-NEB patients, with the most prominent reduction at nebulin's N-terminal end. Muscle mechanical studies indicated approximately 60% reduced force generating capacity of NM-NEB muscle and a leftward-shift of the force-sarcomere length relation in NM-NEB muscle fibers. This indicates that the mechanism for the force reduction is likely to include shorter and non-uniform thin filament lengths in NM-NEB muscle compared with control muscle. Immunofluorescence confocal microscopy and electron microscopy studies indicated that average thin filament length is reduced from approximately 1.3 microm in control muscle to approximately 0.75 microm in NM-NEB muscle. Thus, the present study is the first to show a distinct genotype-functional phenotype correlation in patients with NM due to a nebulin mutation, and provides evidence for the notion that dysregulated thin filament length contributes to muscle weakness in NM patients with nebulin mutations. Furthermore, a striking similarity between the contractile and structural phenotypes of nebulin-deficient mouse muscle and human NM-NEB muscle was observed, indicating that the nebulin knockout model is well suited for elucidating the functional basis of muscle weakness in NM and for the development of treatment strategies.

BACKGROUND: One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive.These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels.The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. RESULTS: An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper. CONCLUSION: This automated process allows laboratories to discover DNA variations in a short time and at low cost.

An important subset of the sudden infant death syndrome (SIDS) is associated with multiple serotonergic (5-HT) abnormalities in regions of the medulla oblongata. The mouse ortholog of the fifth Ewing variant gene (FEV) is critical for 5-HT neuronal development. A putatively rare intronic variant [IVS2-191_190insA, here referred to as c.128-(191_192)dupA] has been reported as a SIDS-associated mutation in an African-American population. We tested this association in an independent dataset: 137 autopsied cases (78 SIDS, 59 controls) and an additional 296 control DNA samples from Coriell Cell Repositories. In addition to the c.128-(191_192)dupA variant, we observed an associated single-base deletion [c.128-(301-306)delG] in a subset of the samples. Neither of the two FEV variants showed significant association with SIDS in either the African-American subgroup or the overall cohort. Although we found a significant association of c.128-(191_192)dupA with SIDS when San Diego Hispanic SIDS cases were compared with San Diego Hispanic controls plus Mexican controls (p = 0.04), this became nonsignificant after multiple testing correction. Among Coriell controls, 33 of 99 (33%) African-American and 0 of 197 (0%) of the remaining controls carry the polymorphism (c.128-(191_192)dupA). The polymorphism seems to be a common, likely nonpathogenic, variant in the African-American population.

Skeletal muscle contraction is triggered by the excitation-contraction (E-C) coupling machinery residing at the triad, a membrane structure formed by the juxtaposition of T-tubules and sarcoplasmic reticulum (SR) cisternae. The formation and maintenance of this structure is key for muscle function but is not well characterized. We have investigated the mechanisms leading to X-linked myotubular myopathy (XLMTM), a severe congenital disorder due to loss of function mutations in the MTM1 gene, encoding myotubularin, a phosphoinositide phosphatase thought to have a role in plasma membrane homeostasis and endocytosis. Using a mouse model of the disease, we report that Mtm1-deficient muscle fibers have a decreased number of triads and abnormal longitudinally oriented T-tubules. In addition, SR Ca(2+) release elicited by voltage-clamp depolarizations is strongly depressed in myotubularin-deficient muscle fibers, with myoplasmic Ca(2+) removal and SR Ca(2+) content essentially unaffected. At the molecular level, Mtm1-deficient myofibers exhibit a 3-fold reduction in type 1 ryanodine receptor (RyR1) protein level. These data reveal a critical role of myotubularin in the proper organization and function of the E-C coupling machinery and strongly suggest that defective RyR1-mediated SR Ca(2+) release is responsible for the failure of muscle function in myotubular myopathy.