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Sarcolipin (SLN) is a small regulatory protein that inhibits the sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) pump. When bound to SERCA, SLN reduces the apparent Ca 2+ affinity of SERCA and uncouples SERCA Ca 2+ transport from its ATP consumption. As such, SLN plays a direct role in altering skeletal muscle relaxation and energy expenditure. Interestingly, the expression of SLN is dynamic during times of muscle adaptation, in that large increases in SLN content are found in response to development, atrophy, overload, and disease. Several groups have suggested that increases in SLN, especially in dystrophic muscle, are deleterious as it may reduce muscle function and exacerbate already abhorrent intracellular Ca 2+ levels. However, there is also significant evidence to show that increased SLN content is a beneficial adaptive mechanism that protects the SERCA pump and activates Ca 2+ signaling and adaptive remodeling during times of cell stress. In this review, we first discuss the role for SLN in healthy muscle during both development and overload, where SLN has been shown to activate Ca 2+ signaling to promote mitochondrial biogenesis, fiber-type shifts, and muscle hypertrophy. Then, with respect to muscle disease, we summarize the discrepancies in the literature as to whether SLN upregulation is adaptive or maladaptive in nature. This review is the first to offer the concept of SLN hormesis in muscle disease, wherein both too much and too little SLN are detrimental to muscle health. Finally, the underlying mechanisms which activate SLN upregulation are discussed, specifically acknowledging a potential positive feedback loop between SLN and Ca 2+ signaling molecules.

Protein kinases are essential for signal transduction and control of most cellular processes, including metabolism, membrane transport, motility, and cell cycle. Despite the critical role of kinases in cells and their strong association with diseases, good coverage of their interactions is available for only a fraction of the 535 human kinases. Here, we present a comprehensive mass-spectrometry-based analysis of a human kinase interaction network covering more than 300 kinases. The interaction dataset is a high-quality resource with more than 5,000 previously unreported interactions. We extensively characterized the obtained network and were able to identify previously described, as well as predict new, kinase functional associations, including those of the less well-studied kinases PIM3 and protein O-mannose kinase (POMK). Importantly, the presented interaction map is a valuable resource for assisting biomedical studies. We uncover dozens of kinase-disease associations spanning from genetic disorders to complex diseases, including cancer., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Ankyrin repeat and kinase domain containing 1 (ANKK1) gene has been widely related to neuropsychiatry disorders. The localization of ANKK1 in neural progenitors and its correlation with the cell cycle has suggested its participation in development. However, ANKK1 functions still need to be identified. Here, we have further characterized the ANKK1 localization in vivo and in vitro, by using immunolabeling, quantitative real-time PCR and Western blot in the myogenic lineage. Histologic investigations in mice and humans revealed that ANKK1 is expressed in precursors of embryonic and adult muscles. In mice embryos, ANKK1 was found in migrating myotubes where it shows a polarized cytoplasmic distribution, while proliferative myoblasts and satellite cells show different isoforms in their nuclei and cytoplasm. In vitro studies of ANKK1 protein isoforms along the myogenic progression showed the decline of nuclear ANKK1-kinase until its total exclusion in myotubes. In adult mice, ANKK1 was expressed exclusively in the Fast-Twitch muscles fibers subtype. The induction of glycolytic metabolism in C2C12 cells with high glucose concentration or treatment with berberine caused a significant increase in the ANKK1 mRNA. Similarly, C2C12 cells under hypoxic conditions caused the increase of nuclear ANKK1. These results altogether show a relationship between ANKK1 gene regulation and the metabolism of muscles during development and in adulthood. Finally, we found ANKK1 expression in regenerative fibers of muscles from dystrophic patients. Future studies in ANKK1 biology and the pathological response of muscles will reveal whether this protein is a novel muscle disease biomarker.

Protein O-mannosyltransferase 1 (POMT1) is a glycosyltransferase involved in α-dystroglycan glycosylation. POMT1 mutations cause a wide spectrum of clinical conditions from Walker-Warburg syndrome (WWS), which involves muscle, eye and brain abnormalities, to mild forms of limb-girdle muscular dystrophy with mental retardation. We aimed to elucidate the impact of different POMT1 mutations on the clinical phenotype. We report five Chinese patients with POMT1 mutations: one had a typical clinical manifestation of WWS, and the other four were diagnosed with congenital muscular dystrophy with mental retardation of varying severity. We analyzed the influence of the POMT1 mutations on POMT activity by assaying the patients' muscles and cultured skin fibroblasts. We demonstrated different levels of decreased POMT activity that correlated highly with decreased α-dystroglycan glycosylation. Our results suggest that POMT activity is inversely proportional to clinical severity, and demonstrate that skin fibroblasts can be used for differential diagnosis of patients with α-dystroglycanopathies. We have provided clinical, histological, enzymatic and genetic evidence of POMT1 involvement in five unrelated Chinese patients.

Glycosylation is an essential post-translational modification that underlies many biological processes and diseases. α-dystroglycan (α-DG) is a receptor for matrix and synaptic proteins that causes muscular dystrophy and lissencephaly upon its abnormal glycosylation (α-dystroglycanopathies). Here we identify the glycan unit ribitol 5-phosphate (Rbo5P), a phosphoric ester of pentose alcohol, in α-DG. Rbo5P forms a tandem repeat and functions as a scaffold for the formation of the ligand-binding moiety. We show that enzyme activities of three major α-dystroglycanopathy-causing proteins are involved in the synthesis of tandem Rbo5P. Isoprenoid synthase domain-containing (ISPD) is cytidine diphosphate ribitol (CDP-Rbo) synthase. Fukutin and fukutin-related protein are sequentially acting Rbo5P transferases that use CDP-Rbo. Consequently, Rbo5P glycosylation is defective in α-dystroglycanopathy models. Supplementation of CDP-Rbo to ISPD-deficient cells restored α-DG glycosylation. These findings establish the molecular basis of mammalian Rbo5P glycosylation and provide insight into pathogenesis and therapeutic strategies in α-DG-associated diseases., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Differentiation among types of muscular dystrophy (MD) has remained challenging. In this retrospective study, we sought to develop a methodology for differentiation of MD types using analysis of serum enzyme profiles., Methods: The serum levels of enzymes from 232 patients, including 120 with DMD, 36 with BMD, 36 with FSHD, 46 with LGMD, and 11 with EDMD, were evaluated., Results: The characteristic profiles of serum enzymes facilitated differentiation of these five types of MD. DMD was characterized by simultaneous elevation of ALT, AST, LDH, and ALP; BMD and LGMD were characterized by elevation of ALT, AST, and LDH; and FSHD and EDMD were characterized by a lack of abnormal serum enzyme levels. We further developed discriminant functions to distinguish BMD and LGMD. For LGMD, LGMD2B patients had significantly higher ALP levels than non-LGMD2B patients (98 ± 59 U/L versus 45 ± 9 U/L, resp., p < 0.05)., Conclusions: Our approach enabled the determination of MD subtypes using serum enzyme profiles prior to genetic testing, which will increase the chance a mutation will be found in the first gene analyzed.

Mutations in the LARGE gene have been identified in congenital muscular dystrophy (CMD) patients with brain abnormalities. Both LARGE and its paralog, LARGE2 (also referred to as GYLTL1B) are bifunctional glycosyltransferases with xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, and are capable of forming polymers consisting of [-3Xyl-α1,3GlcAβ1-] repeats. LARGE-dependent modification of α-dystroglycan (α-DG) with these polysaccharides is essential for the ability of α-DG to act as a receptor for ligands in the extracellular matrix. Here we report on the endogenous enzymatic activities of LARGE and LARGE2 in mice and humans, using a newly developed assay for GlcA-T activity. We show that normal mouse and human cultured cells have endogenous LARGE GlcA-T, and that this activity is absent in cells from the Large(myd) (Large-deficient) mouse model of muscular dystrophy, as well as in cells from CMD patients with mutations in the LARGE gene. We also demonstrate that GlcA-T activity is significant in the brain, heart, and skeletal muscle of wild-type and Large2(-/-) mice, but negligible in the corresponding tissues of the Large(myd) mice. Notably, GlcA-T activity is substantial, though reduced, in the kidneys of both the Large(myd) and Large2(-/-) mice, consistent with the observation of α-DG/laminin binding in these contexts. This study is the first to test LARGE activity in samples as small as cryosections and, moreover, provides the first direct evidence that not only LARGE, but also LARGE2, is vital to effective functional modification of α-DG in vivo., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Congenital muscular dystrophies (CMD) with hypoglycosylation of α-dystroglycan are clinically and genetically heterogeneous disorders that are often associated with brain malformations and eye defects. Presently, 16 proteins are known whose dysfunction impedes glycosylation of α-dystroglycan and leads to secondary dystroglycanopathy., Objective: To identify the cause of CMD with secondary merosin deficiency, hypomyelination and intellectual disability in two siblings from a consanguineous family., Methods: Autozygosity mapping followed by whole exome sequencing and immunochemistry were used to discover and verify a new genetic defect in two siblings with CMD., Results: We identified a homozygous missense mutation (c.325C>T, p.Q109*) in protein O-mannosyl kinase (POMK) that encodes a glycosylation-specific kinase (SGK196) required for function of the dystroglycan complex. The protein was absent from skeletal muscle and skin fibroblasts of the patients. In patient muscle, β-dystroglycan was normally expressed at the sarcolemma, while α-dystroglycan failed to do so. Further, we detected co-localisation of POMK with desmin at the costameres in healthy muscle, and a substantial loss of desmin from the patient muscle., Conclusions: Homozygous truncating mutations in POMK lead to CMD with secondary merosin deficiency, hypomyelination and intellectual disability. Loss of desmin suggests that failure of proper α-dystroglycan glycosylation impedes the binding to extracellular matrix proteins and also affects the cytoskeleton.

Mutations in several known or putative glycosyltransferases cause glycosylation defects in α-dystroglycan (α-DG), an integral component of the dystrophin glycoprotein complex. The hypoglycosylation reduces the ability of α-DG to bind laminin and other extracellular matrix ligands and is responsible for the pathogenesis of an inherited subset of muscular dystrophies known as the dystroglycanopathies. By exome and Sanger sequencing we identified two individuals affected by a dystroglycanopathy with mutations in β-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2). B3GALNT2 transfers N-acetyl galactosamine (GalNAc) in a β-1,3 linkage to N-acetyl glucosamine (GlcNAc). A subsequent study of a separate cohort of individuals identified recessive mutations in four additional cases that were all affected by dystroglycanopathy with structural brain involvement. We show that functional dystroglycan glycosylation was reduced in the fibroblasts and muscle (when available) of these individuals via flow cytometry, immunoblotting, and immunocytochemistry. B3GALNT2 localized to the endoplasmic reticulum, and this localization was perturbed by some of the missense mutations identified. Moreover, knockdown of b3galnt2 in zebrafish recapitulated the human congenital muscular dystrophy phenotype with reduced motility, brain abnormalities, and disordered muscle fibers with evidence of damage to both the myosepta and the sarcolemma. Functional dystroglycan glycosylation was also reduced in the b3galnt2 knockdown zebrafish embryos. Together these results demonstrate a role for B3GALNT2 in the glycosylation of α-DG and show that B3GALNT2 mutations can cause dystroglycanopathy with muscle and brain involvement., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

The severe phenotypic effects of altered glycosylation in the congenital muscular dystrophies, including Walker-Warburg syndrome, muscle-eye-brain disease, Fukuyama congenital muscular dystrophy, and congenital muscular dystrophy 1D, are caused by mutations resulting in altered glycans linked to proteins through O-linked mannose. A glycosyltransferase that branches O-Man, N-acetylglucosaminyltransferase Vb (GnT-Vb), is highly expressed in neural tissues. To understand the expression and function of GnT-Vb, we studied its expression during neuromorphogenesis and generated GnT-Vb null mice. A paralog of GnT-Vb, N-acetylglucosaminyltransferase (GnT-V), is expressed in many tissues and brain, synthesizing N-linked, β1,6-branched glycans, but its ability to synthesize O-mannosyl-branched glycans is unknown; conversely, although GnT-Vb can synthesize N-linked glycans in vitro, its contribution to their synthesis in vivo is unknown. Our results showed that deleting both GnT-V and GnT-Vb results in the total loss of both N-linked and O-Man-linked β1,6-branched glycans. GnT-V null brains lacked N-linked, β1,6-glycans but had normal levels of O-Man β1,6-branched structures, showing that GnT-Vb could not compensate for the loss of GnT-V. By contrast, GnT-Vb null brains contained normal levels of N-linked β1,6-glycans but low levels of some O-Man β1,6-branched glycans. Therefore, GnT-V could partially compensate for GnT-Vb activity in vivo. We found no apparent change in α-dystroglycan binding of glycan-specific antibody IIH6C4 or binding to laminin in GnT-Vb null mice. These results demonstrate that GnT-V is involved in synthesizing branched O-mannosyl glycans in brain, but the function of these branched O-mannosyl structures is unresolved using mice that lack these glycosyltransferases.

Activation of transcription factor NF-kappaB, the major regulator of the inflammatory response, depends on the inhibitor of NF-kappaB kinase (IKK) complex, which is composed of 2 catalytic subunits, IKK1 and IKK2 (also known as IKKalpha and IKKbeta), and a regulatory subunit, IKKgamma (also known as NEMO). In this issue of the JCI, Mourkioti et al. show that muscle-specific disruption in mice of the gene encoding IKK2 prevents NF-kappaB activation in response to denervation or toxin-induced injury (see the related article beginning on page 2945). Importantly, this genetic manipulation prevents muscle wasting, thereby providing strong evidence in support of a major pathogenic role for inflammation in a variety of muscular dystrophies characterized by progressive muscle fiber degeneration.

Background: Persistent creatine kinase elevation is occasionally encountered in subjects without any clinical manifestation of a neuromuscular disorder or any condition known to be associated with increased serum CK levels. It is still unresolved whether extensive investigations and specifically a muscle biopsy should be performed in clinically normal individuals with elevated CK levels., Objective: To study the muscle pathology of patients with asymptomatic or minimally symptomatic hyperCKemia., Methods: The clinical and laboratory data of patients with persistent hyperCKemia and normal neurologic examination were reviewed and their muscle biopsies evaluated., Results: The study group included 40 patients aged 7-67 years; the male to female ratio was 3:1. Nineteen patients were completely asymptomatic, 20 had mild non-specific myalgia, and 1 had muscle cramps. Electromyography was performed in 27 patients and showed myopathic changes in 7 (26%). Abnormal muscle biopsy findings (e.g., increased variation in fiber size, increased number of central nuclei, and occasional degenerating fibers) were detected in 22 of the 40 patients (55%). No fat or glycogen accumulation was detected. Immunohistochemistry demonstrated abnormal dystrophin staining in 3 patients (8%), resembling the pathologic changes of Becker muscular dystrophy. No abnormal findings were detected on immunohistochemical staining for merosin, dysferlin, caveolin 3, or alpha and gamma sarcoglycans. The EMG findings did not correlate with the pathologic findings., Conclusions: Abnormal muscle biopsies were found in 55% of patients with asymptomatic or minimally symptomatic hyperCKemia. Specific diagnosis of muscular dystrophy, however, was possible in only 8% of the patients.

Distal myopathy with rimmed vacuoles is an autosomal recessive muscle disease with preferential involvement of the tibialis anterior that spares the quadriceps muscles in young adulthood. In a Japanese patient with distal myopathy with rimmed vacuoles, we identified pathogenic mutations in the gene encoding the bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase, which catalyzes the initial two steps in the biosynthesis of sialic acid. In this study, we demonstrated the relationship between the genetic mutations and enzymatic activities using an in vitro expression assay system. Furthermore, we also showed that the levels of sialic acid in muscle and primary cultured cells from DMRV patients were reduced to 60-75% of control. The reactivities to lectins were also variable in some myofibers, suggesting that hyposialylation and abnormal glycosylation in muscles may contribute to the focal accumulations of autophagic vacuoles, amyloid deposits, or both in patient muscle tissue. The addition of ManNAc and NeuAc to primary cultured cells normalized sialylation levels, thus demonstrating the therapeutic potential of these compounds for this disease.

The diagnosis of limb girdle muscular dystrophy (LGMD) type 2A (due to mutations in the gene encoding for calpain-3) is currently based on protein analysis, but mutant patients with normal protein expression have also been identified. In this study we investigated 150 LGMD patients with normal calpain-3 protein expression, identified gene mutations by an allele-specific polymerase chain reaction test, and analyzed the mutant calpain-3 catalytic activity. Four different mutations were found in eight patients (5.5%): a frame-shifting deletion (550 A del) and three missense (R490Q, R489Q, R490W). Patients with normal calpain-3 protein expression on Western blot are a considerable proportion (20%) of our total LGMD2A population. While in control muscle the calpain-3 Ca(++)-dependent autocatalytic activity was evident within 5 minutes and was prevented by ethylene diaminetetraacetic acid, in all mutant patient samples the protein was not degraded, indicating that the normal autocatalytic function had been lost. By this new functional test, we show that conventional protein diagnosis fails to detect some mutant proteins, and prove the pathogenetic role of R490Q, R489Q, R490W missense mutations. We suggest that these mutations impair protein activity by affecting interdomain protein interaction, or reduce autocatalytic activity by lowering the Ca(++) sensitivity.

Limb girdle muscular dystrophy type 2A (LGMD2A) is caused by mutations in the calpain 3 gene. In a large family affected by LGMD2A with four severely affected members, three additional asymptomatic relatives had very high serum creatine kinase concentrations. All were homozygous for the R110X mutation and showed a total absence of calpain 3 in the muscle. Histological analysis of muscle in these three rare preclinical cases showed a consistent but unusual pattern, with isolated fascicles of degenerating fibres in an almost normal muscle. This pattern was also seen in one patient with early stage LGMD2A who had a P82L missense mutation and a partial deficiency of calpain 3 in the muscle, but was not seen in early stage patients affected by other forms of LGMD. These findings suggest that a peculiar pattern of focal degeneration occurs in calpainopathy, independently of the type of mutation or the amount of calpain 3 in the muscle.

Duchenne muscular dystrophy (DMD) is a congenital X-linked myopathy caused by lack of dystrophin protein expression. In DMD, the expression of many dystrophin-associated proteins (DAPs) is reduced along the sarcolemmal membrane, but the same proteins remain concentrated at the neuromuscular junction where utrophin, a dystrophin homologue, is expressed [Matsumura, K., Ervasti, J. M., Ohlendieck, K., Kahl, K. D. & Campbell, K. (1992) Nature (London) 360, 588-591]. This outcome has led to the concept that ectopic expression of a "synaptic scaffold" of DAPs and utrophin along myofibers might compensate for the molecular defects in DMD. Here we show that transgenic overexpression of the synaptic CT GalNAc transferase in the skeletal muscles of mdx animals (mdx/CT) increases the expression of utrophin and many DAPs, including dystroglycans, sarcoglycans, and dystrobrevins, along myofibers. Protein expression of utrophin and DAPs was equal to or above that of wild-type mice. In addition, alpha-dystroglycan was glycosylated with the CT carbohydrate antigen in mdx/CT but not in mdx muscles. mdx/CT mice have little or no evidence of muscular dystrophy by several standard measures; Serum creatine kinase levels, percentage of centrally located myofiber nuclei, and variance in myofiber diameter in mdx/CT muscles were dramatically reduced compared with mdx mice. These data suggest that ectopic expression of the CT GalNAc transferase creates a functional dystrophin-related complex along myofibers in the absence of dystrophin and should be considered as a target for therapeutic intervention in DMD.

Limb-girdle muscular dystrophy type 2H (LGMD2H) is a mild autosomal recessive myopathy that was first described in the Manitoba Hutterite population. Previous studies in our laboratory mapped the causative gene for this disease to a 6.5-Mb region in chromosomal region 9q31-33, flanked by D9S302 and D9S1850. We have now used additional families and a panel of 26 microsatellite markers to construct haplotypes. Twelve recombination events that reduced the size of the candidate region to 560 kb were identified or inferred. This region is flanked by D9S1126 and D9S737 and contains at least four genes. Exons of these genes were sequenced in one affected individual, and four sequence variations were identified. The families included in our study and 100 control individuals were tested for these variations. On the basis of our results, the mutation in the tripartite-motif-containing gene (TRIM32) that replaces aspartate with asparagine at position 487 appears to be the causative mutation of LGMD2H. All affected individuals were found to be homozygous for D487N, and this mutation was not found in any of the controls. This mutation occurs in an NHL (named after the proteins NCL1, HT2A, and LIN-41) domain at a position that is highly conserved. NHL domains are known to be involved in protein-protein interactions. Although the function of TRIM32 is unknown, current knowledge of the domain structure of this protein suggests that it may be an E3-ubiquitin ligase. If proven, this represents a new pathogenic mechanism leading to muscular dystrophy.

Muscle-eye-brain disease (MEB) is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities, and lissencephaly. Mammalian O-mannosyl glycosylation is a rare type of protein modification that is observed in a limited number of glycoproteins of brain, nerve, and skeletal muscle. Here we isolated a human cDNA for protein O-mannose beta-1,2-N-acetylglucosaminyltransferase (POMGnT1), which participates in O-mannosyl glycan synthesis. We also identified six independent mutations of the POMGnT1 gene in six patients with MEB. Expression of most frequent mutation revealed a great loss of the enzymatic activity. These findings suggest that interference in O-mannosyl glycosylation is a new pathomechanism for muscular dystrophy as well as neuronal migration disorder.

Limb girdle muscular dystrophies (LGMDs) are a group of clinically heterogeneous genetic diseases characterized by progressive weakness and atrophy of scapular and pelvic muscles, with either a dominant or recessive autosomic mode of inheritance. The first symptoms of the disorder appear during the first 20 years of life and progresses gradually, and a walking disability develops 10-20 years later. The gene responsible for LGMD2A has been identified and encodes calpain 3, a protease expressed mainly in skeletal muscle. Apoptotic myonuclei were recently detected in muscular biopsy specimens of LGMD2A patients, and apoptosis was found to be correlated with altered subcellular distribution of inhibitory protein kappaBalpha (IkappaBalpha) and nuclear factor kappaB (NF-kappaB), resulting in sarcoplasmic sequestration of NF-kappaB. Calpain 3 dependent IkappaBalpha degradation was reconstituted in vitro, supporting a possible in vivo sequence of events leading from calpain 3 deficiency to IkappaBkappa accumulation, prevention of nuclear translocation of NF-kappaB, and ultimately apoptosis. Therefore calpain 3, present in healthy muscle as sarcoplasmic and nuclear forms, may control IkappaBalpha turnover and indirectly regulate NF-kappaB dependent expression of survival genes. Recent data reported from a new model of LGMD2A in mice and from other muscular disorders strengthen understanding of the molecular links between calpain 3 and the Ikappaalpha/NF-kappaB pathway. Finally, in light of the lack of apoptosis observed in inflammatory myopathies, a unifying model for the control of cell survival in muscle is proposed and discussed

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder characterized by selective atrophy of the proximal limb muscles. Its occurrence is correlated, in a large number of patients, with defects in the human CAPN3 gene, a gene that encodes the skeletal muscle-specific member of the calpain family, calpain 3 (or p94). Because calpain 3 is difficult to study due to its rapid autolysis, we have developed a molecular model of calpain 3 based on the recently reported crystal structures of m-calpain and on the high-sequence homology between p94 and m-calpain (47% sequence identity). On the basis of this model, it was possible to explain many LGMD2A point mutations in terms of calpain 3 inactivation, supporting the idea that loss of calpain 3 activity is responsible for the disease. The majority of the LGMD2A mutations appear to affect domain/domain interaction, which may be critical in the assembly and the activation of the multi-domain calpain 3. In particular, we suggest that the flexibility of protease domain I in calpain 3 may play a critical role in the functionality of calpain 3. In support of the model, some clinically observed calpain 3 mutations were generated and analyzed in recombinant m-calpain. Mutations of residues forming intramolecular domain contacts caused the expected loss of activity, but mutations of some surface residues had no effect on activity, implying that these residues in calpain 3 may interact in vivo with other target molecules. These results contribute to an understanding of structure-function relationships and of pathogenesis in calpain 3.

Spinal muscular atrophy (SMA) is characterized by degeneration of motor neurons of the spinal cord associated with muscle paralysis and caused by mutations of the survival motor neuron gene (SMN). To determine whether SMN gene defect in skeletal muscle might have a role in SMA pathogenesis, deletion of murine SMN exon 7, the most frequent mutation found in SMA, has been restricted to skeletal muscle by using the Cre-loxP system. Mutant mice display ongoing muscle necrosis with a dystrophic phenotype leading to muscle paralysis and death. The dystrophic phenotype is associated with elevated levels of creatine kinase activity, Evans blue dye uptake into muscle fibers, reduced amount of dystrophin and upregulation of utrophin expression suggesting a destabilization of the sarcolemma components. The mutant mice will be a valuable model for elucidating the underlying mechanism. Moreover, our results suggest a primary involvement of skeletal muscle in human SMA, which may contribute to motor defect in addition to muscle denervation caused by the motor neuron degeneration. These data may have important implications for the development of therapeutic strategies in SMA.

Calpain 3 is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. It was previously shown that defects in the human calpain 3 gene are responsible for limb girdle muscular dystrophy type 2A (LGMD2A), an inherited disease affecting predominantly the proximal limb muscles. To better understand the function of calpain 3 and the pathophysiological mechanisms of LGMD2A and also to develop an adequate model for therapy research, we generated capn3-deficient mice by gene targeting. capn3-deficient mice are fully fertile and viable. Allele transmission in intercross progeny demonstrated a statistically significant departure from Mendel's law. capn3-deficient mice show a mild progressive muscular dystrophy that affects a specific group of muscles. The age of appearance of myopathic features varies with the genetic background, suggesting the involvement of modifier genes. Affected muscles manifest a similar apoptosis-associated perturbation of the IkappaBalpha/nuclear factor kappaB pathway as seen in LGMD2A patients. In addition, Evans blue staining of muscle fibers reveals that the pathological process due to calpain 3 deficiency is associated with membrane alterations.

In order to offer carrier detection, genetic counseling, and prenatal diagnosis to families with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) in our country, segregation analysis of highly polymorphic short tandem repeats (STR) (dC-dA)n: (dG-dT)n loci was utilized. The risks to females of 15 DMD BMD families (9 familial and 6 sporadic) were evaluated on STR, pedigree and serum creatine kinase (SCK) data. From the 36 females at risk of being carriers (not including 8 obligate carriers), results of STR analysis were compatible with carrier status in 7 and not compatible in 20. In 9 females, no information regarding carriership was derived from the STR analysis. Prenatal diagnosis is now possible on the carrier females. Previously identified deletions in the central part of the gene were confirmed by STR analysis in 3 families. Five new alleles were identified in Argentine individuals; allele frequencies differed from those of North American people. Results derived from this study are useful for carrier detection and genetic counseling in DMD/BMD. One case of probable mosaicism in an unaffected father was detected on a pedigree basis in a family with DMD patients.

Objective: To study the changes of creatine kinase MM (CK-MM) isoforms in Duchenne muscular dystrophy (DMD) patients., Methods: Serum samples from 49 DMD patients and 40 control subjects were collected for CK-MM isoforms measurement. CK-MM isoforms were separated within 30 minutes by electrophoresis on agarose gel with a discontinuous buffer system at constant current of 30 mA and low voltage of 200-300 V, then measured by fluorescence scanning., Results: Significant differences of MM2/MM1 ratio were found between DMD patients and control subjects (P < 0.05) as well as among the three different age groups of DMD patients (P < 0.05)., Conclusions: CK-MM isoforms may present useful information for the early diagnosis and evaluation of DMD and the ratio of MM2/MM1 can be considered as a specific indicator of the degree of seriousness for DMD patients.

Five male children are reported in whom incidental recognition of elevated serum alanine aminotransferase (ALT) activity initiated investigation to identity the cause of suspected hepatocellular injury. All five were later diagnosed with X chromosome-linked muscular dystrophy. The serum level of ALT, generally considered to be specific for hepatocellular injury, was increased two to 25 times above normal in all the reported cases. Paradoxically, the increase in ALT activity was greater than that of serum aspartate aminotransferase (three to 16 times normal), an enzyme whose elevation is generally recognized as being less specific and indicative of muscle, cardiac, kidney, pancreatic, red blood cell or hepatic injury. At presentation to the gastrointestinal service, one case, age 2.5 months, had no symptoms or signs of neuromuscular dysfunction, while the other four had previously unrecognized hypertrophy of the calves, proximal limb weakness, positive Gower's sign or delayed gross motor skills. All five patients had marked elevation of serum creatine kinase activity and histopathologically confirmed muscular dystrophy. The practical clinical implication of this report is that children with elevated serum ALT, in the absence of other signs and symptoms of hepatic injury, may have occult muscular disease--most frequently muscular dystrophy. Although the clinical signs of muscular dystrophy may be subtle or absent, early determination of creatine kinase will suggest the correct diagnosis and minimize extensive and invasive investigation focusing on hepatic injury.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.

Becker muscular dystrophy is an X-linked disease due to mutations of the dystrophin gene. We now show that neuronal-type nitric oxide synthase (nNOS), an identified enzyme in the dystrophin complex, is uniquely absent from skeletal muscle plasma membrane in many human Becker patients and in mouse models of dystrophinopathy. An NH2-terminal domain of nNOS directly interacts with alpha 1-syntrophin but not with other proteins in the dystrophin complex analyzed. However, nNOS does not associate with alpha 1-syntrophin on the sarcolemma in transgenic mdx mice expressing truncated dystrophin proteins. This suggests a ternary interaction of nNOS, alpha 1-syntrophin, and the central domain of dystrophin in vivo, a conclusion supported by developmental studies in muscle. These data indicate that proper assembly of the dystrophin complex is dependent upon the structure of the central rodlike domain and have implications for the design of dystrophin-containing vectors for gene therapy.

Nitric oxide (NO) is synthesized in skeletal muscle by neuronal-type NO synthase (nNOS), which is localized to sarcolemma of fast-twitch fibers. Synthesis of NO in active muscle opposes contractile force. We show that nNOS partitions with skeletal muscle membranes owing to association of nNOS with dystrophin, the protein mutated in Duchenne muscular dystrophy (DMD). The dystrophin complex interacts with an N-terminal domain of nNOS that contains a GLGF motif. mdx mice and humans with DMD evince a selective loss of nNOS protein and catalytic activity from muscle membranes, demonstrating a novel role for dystrophin in localizing a signaling enzyme to the myocyte sarcolemma. Aberrant regulation of nNOS may contribute to preferential degeneration of fast-twitch muscle fibers in DMD.

Limb-girdle muscular dystrophies (LGMDs) are a group of inherited diseases whose genetic etiology has yet to be elucidated. The autosomal recessive forms (LGMD2) constitute a genetically heterogeneous group with LGMD2A mapping to chromosome 15q15.1-q21.1. The gene encoding the muscle-specific calcium-activated neutral protease 3 (CANP3) large subunit is located in this region. This cysteine protease belongs to the family of intracellular calpains. Fifteen nonsense, splice site, frameshift, or missense calpain mutations cosegregate with the disease in LGMD2A families, six of which were found within La Réunion island patients. A digenic inheritance model is proposed to account for the unexpected presence of multiple independent mutations in this small inbred population. Finally, these results demonstrate an enzymatic rather than a structural protein defect causing a muscular dystrophy, a defect that may have regulatory consequences, perhaps in signal transduction.

We examine the effect of each variable on the following statistics: the one-sample and two-sample Hotelling's T2, Wilks' lambda for multivariate analysis of variance, and R2 in multiple regression. For T2, the net effect of each variable is an increase in the multivariate statistic, and the particular factors determining the amount of increase are (i) the multiple correlation of the variable with all other variables, and (ii) how well the variable's contribution to falsifying the hypothesis can be linearly predicted from the other variables. The effect of each predictor variable on R2 is similar to the effect of each variable on T2. For Wilks' lambda, each variable induces a decrease, due to (i) the F for that variable alone, and (ii) the change in multiple correlation from within-sample to total-sample.

The activities and contents of the lysosomal cysteine proteinases cathepsins B, H and L were examined in xenografts of biopsied muscles transplanted from age-matched normal subjects and Duchenne-muscular-dystrophy (DMD) patients into nude mice. The activity of cathepsin B increased 9-fold and that of B-plus-L increased 24-fold in the first week after transplantation in normal muscle xenografts. By the third week, the activity of cathepsin B increased a total of 20-fold and B-plus-L increased to 36-fold the original level. The activity levels of cathepsin B, B-plus-L, H and D, and acid phosphatase in normal and DMD xenografts were not significantly different when compared 2 weeks after transplantation. However, the protein content of cathepsin B in DMD muscle xenografts was more than 3-fold that of normal xenografts at 2 weeks. The profile of cathepsin H activity in normal muscle xenografts was different than those of cathepsins B and B-plus-L. In the first week, the cathepsin H diminished sharply to about one-third of the biopsied muscle level and then, by 3 weeks after transplantation, it had increased slightly to about half the original level. The amount of endogenous cysteine-proteinase inhibitor changed in parallel with the activity of cathepsins B and B-plus-L. Cathepsins B and H, but not cathepsin L, were found immunohistochemically in regenerating muscle fibres of normal and DMD xenografts 2 weeks after transplantation. Staining of cathepsin B in DMD xenografts was slightly stronger than that in normal subjects. There was no immunostaining in degenerating or necrotic muscle fibres 2 weeks after transplantation. Western-blot analysis revealed that the cathepsin B band at 29 kDa was increased in normal xenografts 2 and 3 weeks after transplantation. Also, 2 weeks after transplantation the staining intensity of this band was slightly stronger in DMD xenografts than in normal xenografts. These results suggest that cathepsin B participates in the regeneration of transplanted muscle, both normal and DMD, and in the DMD muscle fibre-wasting processes, during regeneration.

We studied 12 Japanese families of cytochrome b558 (b) negative chronic granulomatous disease (13 patients and 23 family members) and 15 controls for cytochrome b content, O2-, and H2O2 production. Cytochrome b content (nmol/10(8) cells), O2- production (nmol/10(7) cells/min), and percentage of H2O2 generating cells (%) were 1) 0.45-1.19, 64.1-174.2, and 85.8-100.0 in controls (mean +/- 2 S.D.), 2) 0.13-0.38, 22.6-50.9, and 20.0-62.4 in healthy carriers, and 3) undetectable, undetectable, and 0 in patients, respectively. These findings indicate that healthy carriers and normal homozygotes are separable by these three parameters, and that 4 of the 12 families studied represent fresh gene mutation. Pooling of data by Segal et al., Ohno et al., and ours yielded the overall incidence of a fresh gene mutation to be 19.4% (7 of 36 cases). The lower fresh mutation rate than predicted from Haldane's formula suggests a higher mutation rate in males than in females, as previously suggested in Duchenne muscular dystrophy.

Using a histochemical method, 5'-nucleotidase activity was investigated in 80 muscle biopsy specimens, including specimens from eight patients with muscular dystrophy, 18 with nonspecific type II fiber atrophy, 15 with polymyositis, and 29 histologically normal controls. An interstitial reaction for 5'-nucleotidase was associated with an inflammatory infiltrate in 19 of 21 positive cases. Of the 15 cases of polymyositis, 14 showed an extensive interstitial reaction surrounding most of the myofibers and extending well away from the areas infiltrated by inflammatory cells. The extensive nature of this reaction makes 5'-nucleotidase activity a useful adjuvant in the diagnosis of inflammatory muscle disease.

As blood cells such as platelets, lymphocytes and erythrocytes from patients with Duchenne muscular dystrophy show evidence of membrane alterations and elevation of intra-cellular calcium, one of the calcium related changes i.e., the activity of calcium activated neutral protease (CANP) was monitored and found to be elevated in erythrocytes, lymphocytes and platelets. As similar changes were observed in platelets of carriers of this disease, CANP in platelets may serve as a useful index for carrier detection.

Skeletal muscles obtained from myopathies with myofiber necrosis, including mdx dystrophic mice, plasmocid-induced myopathy in rats, and patients with Duchenne muscular dystrophy, were examined immunohistochemically with anticathepsin-peroxidase conjugates. Strong reactions for lysosomal cysteine proteinases, which can degrade myofibrillar proteins, were demonstrated in macrophages invading and surrounding the necrotic areas and some degenerative myofibers and also in intramyofibral portions of atrophic fibers of dystrophic mice and humans. Apparently normal and regenerating myofibers did not stain for lysosomal cathepsins. Abnormal increases of cathepsins L and B were seen even in the early stage of plasmocid myopathy and in a 20-day-old young mdx mouse before infiltration of macrophages, suggesting that autodigestion by intramyofibral lysosomal proteinases is an important event before digestion of the necrotic fibers by macrophage proteinases. Activation of the intramyofibral lysosomal system, as in muscular dystrophy, was also observed in distal myopathy with rimmed vacuoles without macrophageal infiltration (Am J Pathol 1986, 122:193-198). Thus, this activation seems to be an important, early response to myocellular damage.

Extracts of platelets were subjected to vertical starch gel electrophoresis followed by enzyme staining to further investigate earlier reports of a unique dihydroxyphenylalanine (DOPA) oxidase polymorphism among boys with Duchenne's muscular dystrophy (DMD) and their mothers. This was said to be distinct from those found in healthy controls. Platelets were separated from citrated venous blood by differential centrifugation and checked for purity by phase contrast microscopy. The isolated platelets were disrupted by freeze-thawing in Tris-HC1 buffer (0.02M, pH 8.2), and the resulting platelet extracts were electrophoresed for 15 hours at 130V at 4 degrees C. The starch gels were then sliced and stained with 0.2% DOPA and 0.1% MnCl2. Three electrophoretic patterns of DOPA oxidase activity were found in normal men and women: a single rapidly moving band; a single slowly moving band; and a broad band. These three patterns were also seen in boys with DMD, their mothers, and their fathers. Thus, a unique polymorphism was not found in boys with Duchenne's muscular dystrophy or their carrier mothers.

In extracts derived from whole blood, a high molecular weight fraction of the diphenoloxidase enzymes has a significantly diminished specific activity in patients and definite carriers (heterozygotes) of the X-linked, recessive (Duchenne) form of muscular dystrophy. This anomaly was studied using spots of blood which had been collected on absorbent paper and stored at 4 degrees C for variable periods of time. Fractions enriched in the enzymes were obtained by subjecting aqueous extracts of the spots to treatment with an anion exchange resin (DEAE Sephadex A 50) followed by gel filtration on Sephadex G-25. It is of interest that this anomaly was observed in some definite carriers of the mutant gene who had on several occasions a serum creatine kinase level in the normal range. The significance of these observations is discussed.

1. Procedures are given for the syntheses of alpha,omega-dinucleoside 5'-polyphosphates as inhibitors of adenylate kinases. The following order for the ability of inhibiting pig muscle adenylate kinase was observed: Ap5A greater than 1:N6-etheno-Ap5A greater than Ap6A greater than Gp5A greater than Ap4A greater than Up5A. The synthesis of adenosine tetraphosphate, the starting material for Ap5A, is also described. 2. One molecule of pig muscle adenylate kinase binds one molecule of Ap5A. The difference spectrum of Ap5A-adenylate kinase with its maximum of 5050 M-1 - cm-1 at 271 nm, as well as the fluorescence properties of 1:N6-etheno-Ap5A can be used for kinetic and binding studies. 3. The specific binding of the negatively charged Ap5A was exploited in the preparation of human muscle adenylate kinase. The enzyme was purified to homogeneity with an overall yield of 65%, the absolute value being 70 mg per kg of muscle. 4. The effect of Ap5A on adenylate kinase in extracts of various cells and cell organelles was tested. A ratio of 1:50 (mol/mol) for Ap5A to other nucleotides was used for suppressing the adenylate kinase activity in extracts of mammalian and insect skeletal muscel, of human erythrocytes and of Staphylococcus aureus. A ratio of 1:5 was found to be necessary for the adenylate kinase from tobacco leaves and spinach chloroplasts, and a ratio of 2:1 was needed for suppressing the adenylate kinase from bovine liver mitochondria, human kidney homogenate and from Escherichia coli. Ap5A appears not to be metabolized in any of the above extracts. These results indicate that Ap5A can be used for evaluating the contribution of adenylate kinase to the production of ATP fro ADP in energy-transducing systems. 5. Contaminating adenylate kinase can be inhibited by a concentration of Ap5A which does not interfere in the study of many (phospho)kinases and ATPases. The applications of Ap5A in the assay for nucleoside diphosphokinase and in the study of mechanical and biochemical properties of contractile proteins are representative examples. The use of Ap5A makes it possible to study the effect of ADP per se in such systems. 6. Sepharose-bound Ap5A was used for removing traces of adenylate kinase from samples of myosin and creatine kinase. 7. In the presence of Ap5A the activity of creatine kinase was measured in hemolytic serum of venous blood, in plasma of capillary blood and in samples of whole blood after complete hemolysis had been induced. The clinical significance of these findings are shown for cases of myocardial infarction and muscular dystrophy.

External intercostal muscle biopsies were examined histochemically and by electron microscopy. The use of this muscle allowed correlation with physiological and pharmacological studies on the same specimens. Changes observed in musclar dystrophy and motor neurone disease resembled those previously described in biopsied limb muscle and underline the particular usefulness of this preparation in the study of human neuromuscular disease.

Several lysosomal enzymes were assayed in cultured human skin fibroblasts from patients with Duchenne's muscular dystrophy (DMD) and age- and sex-matched control patients (N). The activity of four glycosidases, cathepsin B(1), and total autoproteolysis at pH 4.0 were unchanged between the groups, but dipeptidyl aminopeptidase I (DAP-I, or cathepsin C) in the DMD cells was found to be only 30% as active as in the control cells (P < 0.003). This difference is not the result of a redistribution or loss of enzyme during homogenization because the difference occurs in all homogenate fractions. DAP-I activity existing in N and DMD fibroblasts behaves identically with respect to activation by chloride ion, activation by the sulfhydryl reducing agent dithiothreitol, changes in hydrogen ion concentration (pH), changes in substrate concentration (i.e., apparent K(m) values), and changes in temperature (i.e., apparent activation energies). Mixtures of N and DMD cell sonicates display an additivity in DAP-I activity. These results support the conclusion that the catalytic function of the DAP-I molecule is equivalent between N and DMD fibroblasts, and that the decrease in tissue-specific DAP-I activity probably results from the fact that fewer enzyme molecules are present in the DMD cells. These results are also an indication that these nonmuscle cells are expressing some of the phenotypic aspects of the genetic defect in DMD. Cultured human skin fibroblasts may therefore be a useful cellular model in DMD research.

Two species of adenylate kinase isozymes (ATP:AMP phosphotransferase, EC 2.7.4.3) from human Duchenne dystrophic serum were separated by Blue Sepharose CL-6B affinity column chromatography. One of these species was the "aberrant" adenylate kinase isozyme, found specifically in the Duchenne type of this disease (Hamada, M., Okuda, H., Oka, K., Watanabe, T., Ueda, K., Nojima, M., Kuby, S.A., Manship, M., Tyler, F., and Ziter, F. (1981) Biochim. Biophys. Acta 660, 227-237). The separated aberrant form possessed a molecular size of 98,000 (+/- 1,500), whereas the normal serum species of the enzyme was 87,000 (+/- 1,600) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by gel filtration, and by sedimentation equilibrium. The sedimentation coefficient of each species was found to be 5.8 S for the aberrant form and 5.6 S for the normal form, respectively. The subunit size (Mr = 24,700) of the aberrant enzyme in 8 M urea proved to be very similar to that of the normal human liver enzyme (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S.A. (1982) J. Biol. Chem. 257, 13120-13128), and the normal species subunit (Mr = 21,700) was found to be very similar to that of the normal human muscle enzyme (Kuby, S.A., Fleming, G., Frischat, A., Cress, M.C., and Hamada, M. (1983) J. Biol. Chem. 258, 1901-1907). Both species were tetrameric enzymes in the serum. The amino acid composition for the normal species was similar to that for the muscle-type enzyme, and that for the aberrant species was similar to the liver enzyme, but with some notable exceptions in both cases. Thus, the normal species had no tryptophan and two half-cystine residues/subunit; whereas, there was 1 tryptophan and 4 half-cystine residues/subunit of the aberrant molecule. The amino acid composition of both serum isozymes when compared to their respective muscle or liver-type enzyme differed mainly in the content of Glu, Asp, His, Leu, Ile, Gly. Kinetic properties of the two forms of human serum adenylate kinase were studied at limiting concentrations of both ADP3- and MgADP- in the reverse reaction and of AMP2- and MgATP2- in the forward reaction. The type of reaction mechanism compatible with the data was a two-substrate random quasiequilibrium type of mechanism without independent binding of the substrates and with a rate-limiting step largely at the interconversion of the ternary complexes.

A report by Bullock and coworkers has emphasized the need for standardization of the CK assay in carrier detection for DMD. A collaborative study, according to a well-specified design and involving two laboratories in Paris and Lyon, indicates that the reliability of this assay can be improved provided that special attention is paid to information about participating subjects, laboratory protocol, and repeated sampling. On a random sample of young women, mean and variance of ln(CK) are 1.66 and 0.015 respectively. Within and between variance components are in a 3:4 ratio, homogeneous between populations. Additional use of a common test serum should insure good reliability of this assay among laboratories.

An extended kindred in the Sudan, where a severe muscular dystrophy has been interpreted as of autosomal recessive inheritance, has been further analysed. The within-sibship ratio, the autosomal and X-linked inbreeding coefficients, and the creatine kinase levels suggest a possible alternative interpretation: X-linked muscular dystrophy with failure of clinical expression in some males.

1. A procedure is described for the fractionation into age classes of human red cells on a Ficoll/Triosil discontinuous density gradient. The fractionated cells appear minimally affected by the procedure. 2. Individual blood samples show a normal cell distribution, but the mean cell density may vary slightly. To allow for this in comparing samples, cell ages are expressed in terms of the mean and standard deviations from the mean. 3. Reticulocyte number decays exponentially in the gradient. By back extrapolation, a standard deviation can be computed corresponding to the theoretical age at which the sample contains 100% reticulocytes. This value can be used to calculate the theoretical activity of an enzyme in the reticulocytes and the enzyme half-life. 4. Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase activities in unfractionated and age-fractionated cells were investigated in adults, children and patients with Duchenne muscular dystrophy. 5. The half-life for adult glucose 6-phosphate dehydrogenase (48 days) compares with that found by other workers. In children the half-life is 24 days, although the initial activity is essentially unaltered. 6. The half-life of 6-phosphogluconate dehydrogenase was found to be 117 days; the activity of glutathione reductase did not alter throughout the life of the cell. There was no difference between adults and children for these enzymes. The same results were obtained with dystrophic patients as for control children for all three enzymes.

A familial myopathy affecting a man, his three daughters, and one of his brothers is reported. The quadriceps muscle was predominantly involved, with aching pain as an early feature, and later prominent areas of hypertrophy projecting from patches of atrophy gave the quadriceps a most striking and unusual appearance. Presentation was in adult life, and the course was relatively benign, pelvic girdle and hand muscles becoming involved later. The evidence suggests a hereditary selective muscular dystrophy rather than polymyositis, although a hereditary form of spinal muscular atrophy could not be excluded entirely.