Loss of function variants in DNAJB4 cause a myopathy with early respiratory failure.

DNAJ/HSP40 co-chaperones are integral to the chaperone network, bind client proteins and recruit them to HSP70 for folding. We performed exome sequencing on patients with a presumed hereditary muscle disease and no genetic diagnosis. This identified four individuals from three unrelated families carrying an unreported homozygous stop gain (c.856A > T; p.Lys286Ter), or homozygous missense variants (c.74G > A; p.Arg25Gln and c.785 T > C; p.Leu262Ser) in DNAJB4. Affected patients presented with axial rigidity and early respiratory failure requiring ventilator support between the 1st and 4th decade of life. Selective involvement of the semitendinosus and biceps femoris muscles was seen on MRI scans of the thigh. On biopsy, muscle was myopathic with angular fibers, protein inclusions and occasional rimmed vacuoles. DNAJB4 normally localizes to the Z-disc and was absent from muscle and fibroblasts of affected patients supporting a loss of function. Functional studies confirmed that the p.Lys286Ter and p.Leu262Ser mutant proteins are rapidly degraded in cells. In contrast, the p.Arg25Gln mutant protein is stable but failed to complement for DNAJB function in yeast, disaggregate client proteins or protect from heat shock-induced cell death consistent with its loss of function. DNAJB4 knockout mice had muscle weakness and fiber atrophy with prominent diaphragm involvement and kyphosis. DNAJB4 knockout muscle and myotubes had myofibrillar disorganization and accumulated Z-disc proteins and protein chaperones. These data demonstrate a novel chaperonopathy associated with DNAJB4 causing a myopathy with early respiratory failure. DNAJB4 loss of function variants may lead to the accumulation of DNAJB4 client proteins resulting in muscle dysfunction and degeneration.

A cryptic intronic LAMA2 insertion in a boy with mild congenital muscular dystrophy type 1A.

Recessive mutations in the LAMA2 gene lead to congenital muscular dystrophy type 1A and limb girdle muscular dystrophy R23 with complete or partial laminin α2 chain deficiency. Complete laminin α2 chain deficiency presents with early onset of severe hypotonia and generalized weakness, whereas partial deficiency shows a milder and more variable course with limb girdle weakness. Here, we report a child with mildly delayed motor development, elevated serum creatine kinase levels (>1000 U/l) and brain white matter hypointensity, indicative of laminin α2 chain deficiency. In addition to a stop gain variant in exon 39, the patient was found to carry an intronic insertion of 72 bp in intron 38 of the LAMA2 gene in trans. RNA analysis revealed that this insertion results in abnormally spliced as well as wild type transcript, which explains the partial laminin α2 chain deficiency observed in the muscle biopsy.

INPP5K and SIL1 associated pathologies with overlapping clinical phenotypes converge through dysregulation of PHGDH.

Marinesco-Sjögren syndrome is a rare human disorder caused by biallelic mutations in SIL1 characterized by cataracts in infancy, myopathy and ataxia, symptoms which are also associated with a novel disorder caused by mutations in INPP5K. While these phenotypic similarities may suggest commonalties at a molecular level, an overlapping pathomechanism has not been established yet. In this study, we present six new INPP5K patients and expand the current mutational and phenotypical spectrum of the disease showing the clinical overlap between Marinesco-Sjögren syndrome and the INPP5K phenotype. We applied unbiased proteomic profiling on cells derived from Marinesco-Sjögren syndrome and INPP5K patients and identified alterations in d-3-PHGDH as a common molecular feature. d-3-PHGDH modulates the production of l-serine and mutations in this enzyme were previously associated with a neurological phenotype, which clinically overlaps with Marinesco-Sjögren syndrome and INPP5K disease. As l-serine administration represents a promising therapeutic strategy for d-3-PHGDH patients, we tested the effect of l-serine in generated sil1, phgdh and inpp5k a+b zebrafish models, which showed an improvement in their neuronal phenotype. Thus, our study defines a core phenotypical feature underpinning a key common molecular mechanism in three rare diseases and reveals a common and novel therapeutic target for these patients.

MEGF10 related myopathies: A new case with adult onset disease with prominent respiratory failure and review of reported phenotypes.

Recessive mutations in MEGF10 (multiple epidermal growth factor 10) have been reported in a severe early onset disorder named Early Myopathy, Areflexia, Respiratory Distress and Dysphagia, and a milder form with cores in the muscle biopsy; and a possible genotype-phenotype correlation determining the clinical presentation has been suggested. We undertook exome sequencing in a 66 year old male with a 20 year history of progressive proximal and distal weakness of upper and lower limbs, facial weakness and dysphagia, who developed respiratory failure requiring ventilation while still ambulant in his 50s. Muscle biopsy demonstrated myopathic changes with aggregation of myofibrillar proteins. Mutations in MEGF10 were identified: a novel essential splice site (c.1426+1G>T) and a novel missense variant (c.352T>C, p.(Cys118Arg)). We performed a detailed review of all reported MEGF10 cases (n = 20), and confirmed the presence of a genotype-phenotype correlation, namely that with ≥1 null mutation onset of respiratory dysfunction occurs in the first year of life, whereas with 2 missense mutations, respiratory dysfunction occurs at 10 years old or much later, as in the patient reported here. Our findings expand the phenotype of MEGF10 mutations to include onset in the 5th decade, and discuss the spectrum of MEGF10 related disease.

Mutations in INPP5K, Encoding a Phosphoinositide 5-Phosphatase, Cause Congenital Muscular Dystrophy with Cataracts and Mild Cognitive Impairment.

Phosphoinositides are small phospholipids that control diverse cellular downstream signaling events. Their spatial and temporal availability is tightly regulated by a set of specific lipid kinases and phosphatases. Congenital muscular dystrophies are hereditary disorders characterized by hypotonia and weakness from birth with variable eye and central nervous system involvement. In individuals exhibiting congenital muscular dystrophy, early-onset cataracts, and mild intellectual disability but normal cranial magnetic resonance imaging, we identified bi-allelic mutations in INPP5K, encoding inositol polyphosphate-5-phosphatase K. Mutations impaired phosphatase activity toward the phosphoinositide phosphatidylinositol (4,5)-bisphosphate or altered the subcellular localization of INPP5K. Downregulation of INPP5K orthologs in zebrafish embryos disrupted muscle fiber morphology and resulted in abnormal eye development. These data link congenital muscular dystrophies to defective phosphoinositide 5-phosphatase activity that is becoming increasingly recognized for its role in mediating pivotal cellular mechanisms contributing to disease.

Recessive mutations in the kinase ZAK cause a congenital myopathy with fibre type disproportion.

Congenital myopathies define a heterogeneous group of neuromuscular diseases with neonatal or childhood hypotonia and muscle weakness. The genetic cause is still unknown in many patients, precluding genetic counselling and better understanding of the physiopathology. To identify novel genetic causes of congenital myopathies, exome sequencing was performed in three consanguineous families. We identified two homozygous frameshift mutations and a homozygous nonsense mutation in the mitogen-activated protein triple kinase ZAK. In total, six affected patients carry these mutations. Reverse transcription polymerase chain reaction and transcriptome analyses suggested nonsense mRNA decay as a main impact of mutations. The patients demonstrated a generalized slowly progressive muscle weakness accompanied by decreased vital capacities. A combination of proximal contractures with distal joint hyperlaxity is a distinct feature in one family. The low endurance and compound muscle action potential amplitude were strongly ameliorated on treatment with anticholinesterase inhibitor in another patient. Common histopathological features encompassed fibre size variation, predominance of type 1 fibre and centralized nuclei. A peculiar subsarcolemmal accumulation of mitochondria pointing towards the centre of the fibre was a novel histological hallmark in one family. These findings will improve the molecular diagnosis of congenital myopathies and implicate the mitogen-activated protein kinase (MAPK) signalling as a novel pathway altered in these rare myopathies.

Factors associated with low fitness in adolescents--a mixed methods study.

BACKGROUND: Fitness and physical activity are important for cardiovascular and mental health but activity and fitness levels are declining especially in adolescents and among girls. This study examines clustering of factors associated with low fitness in adolescents in order to best target public health interventions for young people. METHODS: 1147 children were assessed for fitness, had blood samples, anthropometric measures and all data were linked with routine electronic data to examine educational achievement, deprivation and health service usage. Factors associated with fitness were examined using logistic regression, conditional trees and data mining cluster analysis. Focus groups were conducted with children in a deprived school to examine barriers and facilitators to activity for children in a deprived community. RESULTS: Unfit adolescents are more likely to be deprived, female, have obesity in the family and not achieve in education. There were 3 main clusters for risk of future heart disease/diabetes (high cholesterol/insulin); children at low risk (not obese, fit, achieving in education), children 'visibly at risk' (overweight, unfit, many hospital/GP visits) and 'invisibly at risk' (unfit but not overweight, failing in academic achievement). Qualitative findings show barriers to physical activity include cost, poor access to activity, lack of core physical literacy skills and limited family support. CONCLUSIONS: Low fitness in the non-obese child can reveal a hidden group who have high risk factors for heart disease and diabetes but may not be identified as they are normal weight. In deprived communities low fitness is associated with non-achievement in education but in non-deprived communities low fitness is associated with female gender. Interventions need to target deprived families and schools in deprived areas with community wide campaigns.

Phenotypic heterogeneity in British patients with a founder mutation in the FHL1 gene.

Mutations in the four-and-a-half LIM domain 1 (FHL1) gene, which encodes a 280-amino-acid protein containing four LIM domains and a single zinc-finger domain in the N-terminal region, have been associated with a broad clinical spectrum of X-linked muscle diseases encompassing a variety of different phenotypes. Patients might present with a scapuloperoneal myopathy, a myopathy with postural muscle atrophy and generalized hypertrophy, an Emery-Dreifuss muscular dystrophy, or an early onset myopathy with reducing bodies. It has been proposed that the phenotypic variability is related to the position of the mutation within the FHL1 gene. Here, we report on three British families with a heterogeneous clinical presentation segregating a single FHL1 gene mutation and haplotype, suggesting that this represents a founder mutation. The underlying FHL1 gene mutation was detected by direct sequencing and the founder effect was verified by haplotype analysis of the FHL1 gene locus. A 3-bp insertion mutation (p.Phe127_Thr128insIle) within the second LIM domain of the FHL1 gene was identified in all available affected family members of the three families. Haplotype analysis of the FHL1 region on Xq26 revealed that the families shared a common haplotype. The p.Phe127_Thr128insIle mutation in the FHL1 gene therefore appears to be a British founder mutation and FHL1 gene screening, in particular of exon 6, should therefore be indicated in British patients with a broad phenotypic spectrum of X-linked muscle diseases.

Progressive dysphagia in limb-girdle muscular dystrophy type 2B.

Dysphagia has not been reported in genetically confirmed limb-girdle muscular dystrophy type 2B (LGMD2B). A 40-year-old woman reported exercise-induced calf pain at age 34, followed by progressive lower and upper limb weakness. At age 38, progressive dysphagia for solids, and subsequently liquids, ensued. Endoscopic and videofluoroscopic-radiological findings indicated a myopathic swallowing disorder. Molecular genetic analysis confirmed two dysferlin gene mutations consistent with a compound heterozygote state. Progressive dysphagia should be considered as part of the expanding dysferlinopathy phenotype.

The relationship between addiction and reward bundling: an experiment comparing smokers and non-smokers.

AIMS: Previous studies indicate that addicts show reduced preference for more delayed versus more immediate rewards compared to non-addicts. This may reflect a lower propensity to view such decisions in terms of the larger sequences to which they typically belong (e.g. smoking is a frequently repeated act). Therefore, this study aims to test whether, in a sequence of decisions involving smaller, sooner (SS) versus larger, later (LL) rewards, suggesting or forcing people with a propensity to addiction to make the decision for the series as a whole would increase LL preference. It is hypothesized that people without a propensity to addiction should benefit less from being encouraged to think of reward sequences because they already tend to take that view. DESIGN: Thirty regular smokers (as exemplars of addicted individuals) and 30 non-smokers chose between small short-term and larger long-term monetary rewards over a sequence of four decisions spaced 2 weeks apart. Subjects were divided into three groups: one who made each decision independently with no suggestion that they be considered as a series ('free'), a group to whom it was suggested from the start that they consider each decision as part of the series ('suggested') and a group who were told that their very first choice in the series would be used for the remaining decisions ('forced'). All subjects were paid the amounts they had chosen. SETTING: A laboratory room at the University of Cape Town (UCT). PARTICIPANTS: UCT undergraduate volunteers. ANALYSES: The proportion of LL choices in each subgroup was evaluated by χ(2) tests and a probit model. FINDINGS: Smokers increased their preference for LL rewards when 'bundling' of individual decisions into a sequence was either suggested or forced. This preference increased with repeated experience. Non-smokers showed neither pattern. CONCLUSIONS: The propensity of smokers to prefer small short-term rewards over larger delayed rewards may be mitigated, over a sequence of decisions of this kind, by encouraging or forcing them to think of the sequence as a whole. If replicated, this finding may form the basis of an intervention that could attenuate the choice patterns characteristic of addiction.

Dysferlin associates with the developing T-tubule system in rodent and human skeletal muscle.

Mutations in the dysferlin gene cause limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal anterior compartment myopathy. Dysferlin mainly localizes to the sarcolemma in mature skeletal muscle where it is implicated in membrane fusion and repair. In different forms of muscular dystrophy, a predominantly cytoplasmic localization of dysferlin can be observed in regenerating myofibers, but the subcellular compartment responsible for this labeling pattern is not yet known. We have previously demonstrated an association of dysferlin with the developing T-tubule system in vitro. To investigate the role of dysferlin in adult skeletal muscle regeneration, we studied dysferlin localization at high resolution in a rat model of regeneration and found that the subcellular labeling of dysferlin colocalizes with the developing T-tubule system. Furthermore, ultrastructural analysis of dysferlin-deficient muscle revealed primary T-tubule anomalies similar to those seen in caveolin-3-deficient muscle. These findings indicate that dysferlin is necessary for correct T-tubule formation, and dysferlin-deficient skeletal muscle is characterized by abnormally configured T-tubules.

New aspects on patients affected by dysferlin deficient muscular dystrophy.

Mutations in the dysferlin gene lead to limb girdle muscular dystrophy 2B, Miyoshi myopathy and distal anterior compartment myopathy. A cohort of 36 patients affected by dysferlinopathy is described, in the first UK study of clinical, genetic, pathological and biochemical data. The diagnosis was established by reduction of dysferlin in the muscle biopsy and subsequent mutational analysis of the dysferlin gene. Seventeen mutations were novel; the majority of mutations were small deletions/insertions, and no mutational hotspots were identified. Sixty-one per cent of patients (22 patients) initially presented with limb girdle muscular dystrophy 2B, 31% (11 patients) with a Miyoshi phenotype, one patient with proximodistal mode of onset, one patient with muscle stiffness after exercise and one patient as a symptomatic carrier. A wider range of age of onset was noted than previously reported, with 25% of patients having first symptoms before the age of 13 years. Independent of the initial mode of presentation, in our cohort of patients the gastrocnemius muscle was the most severely affected muscle leading to an inability to stand on tiptoes, and lower limbs were affected more severely than upper limbs. As previous anecdotal evidence on patients affected by dysferlinopathy suggests good muscle prowess before onset of symptoms, we also investigated pre-symptomatic fitness levels of the patients. Fifty-three per cent of the patients were very active and sporty before the onset of symptoms which makes the clinical course of dysferlinopathy unusual within the different forms of muscular dystrophy and provides a challenge to understanding the underlying pathomechanisms in this disease.

Immunohistochemical analysis of calpain 3: advantages and limitations in diagnosing LGMD2A.

Immunoblot is currently the preferred laboratory test to assist the diagnosis of limb-girdle muscular dystrophy (LGMD) 2A (calpainopathy). To assess whether immunohistochemistry may offer a reliable alternative screening we used two antibodies, Calp3-2C4 (exon 1) and Calp3-12A2 (exon 8), to label blots and sections of skeletal muscle from controls and patients with LGMD2A and other muscle diseases. In LGMD2A muscle biopsies a high degree of concordance was found with Calp3-2C4: labelling on sections was absent in patients with no bands on immunoblot and detected in those where CAPN3 bands were seen. Calp3-12A2 results were less consistent, with most samples retaining labelling. Interestingly, CAPN3 was found in all muscle sections from disease control patients irrespective of its detection on immunoblot. Our results show that immunohistochemistry with Calp3-2C4 has a similar pickup rate of LGMD2A as immunoblot and it may therefore be useful for distinguishing the majority of genuine CAPN3 defects from secondary protein reduction. However immunoblot is still needed when CAPN3 is present on sections to show secondary CAPN3 reduction and to identify LGMD2A with variable reduction of CAPN3 bands.

Attenuated muscle regeneration is a key factor in dysferlin-deficient muscular dystrophy.

Skeletal muscle requires an efficient and active membrane repair system to overcome the rigours of frequent contraction. Dysferlin is a component of that system and absence of dysferlin causes muscular dystrophy (dysferlinopathy) characterized by adult onset muscle weakness, high serum creatine kinase levels and a prominent inflammatory infiltrate. We have observed that dysferlinopathy patient biopsies show an excess of immature fibres and therefore investigated the role of dysferlin in muscle regeneration. Using notexin-induced muscle damage, we have shown that regeneration is attenuated in a mouse model of dysferlinopathy, with delayed removal of necrotic fibres, an extended inflammatory phase and delayed functional recovery. Satellite cell activation and myoblast fusion appear normal, but there is a reduction in early neutrophil recruitment in regenerating and also needle wounded muscle in dysferlin-deficient mice. Primary mouse dysferlinopathy myoblast cultures show reduced cytokine release upon stimulation, indicating that the secretion of chemotactic molecules is impaired. We suggest an extension to the muscle membrane repair model, where in addition to fusing patch repair vesicles with the sarcolemma dysferlin is also involved in the release of chemotactic agents. Reduced neutrophil recruitment results in incomplete cycles of regeneration in dysferlinopathy which combines with the membrane repair deficit to ultimately trigger dystrophic pathology. This study reveals a novel pathomechanism affecting muscle regeneration and maintenance in dysferlinopathy and highlights enhancement of the neutrophil response as a potential therapeutic avenue in these disorders.

Sarcoglycanopathies: can muscle immunoanalysis predict the genotype?

Muscle immunoanalysis of the sarcoglycan complex is an important part of the diagnostic evaluation of muscle biopsies in patients with autosomal recessive limb-girdle muscular dystrophy. Reduced or absent sarcolemmal expression of one or all of the four sarcoglycans (alpha-, beta-, gamma-, delta-sarcoglycan) can be found in patients with limb-girdle muscular dystrophy 2C-F (LGMD2C-F) and also in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). It has previously been suggested that different patterns of sarcoglycan expression could predict the primary genetic defect, and that genetic analysis could be directed by these patterns. In this first UK study we studied 24 genetically characterized patients with sarcoglycan deficient LGMD, in 22 of whom muscle immunoanalysis data were available. Thirteen patients showed alpha-sarcoglycan deficient LGMD2D, 7 patients beta-sarcoglycan deficient LGMD2E, 3 patients gamma-sarcoglycan deficient LGDM2C, and one patient delta-sarcoglycan deficient LGMD2F. Muscle biopsies were analysed in one centre without knowledge of the established genetic diagnosis. Our results demonstrated that residual sarcoglycan expression is highly variable and does not enable an accurate prediction of the genotype. Considering previous reports of sarcoglycanopathy patients with an isolated loss of one sarcoglycan we recommend to use antibodies against all four sarcoglycans for immunoanalysis of skeletal muscle sections. A concomitant reduction of dystrophin and beta-dystroglycan was observed more frequently than previously reported and illustrates the important differential diagnosis of DMD and BMD for sarcoglycan deficient LGMD.

Caveolinopathy--new mutations and additional symptoms.

Mutations in the caveolin-3 gene (CAV3) can lead to a broad spectrum of clinical phenotypes. Phenotypes that have so far been associated with primary caveolin-3 deficiency include limb girdle muscular dystrophy, rippling muscle disease, distal myopathy and hyperCKaemia. This is the first report describing the clinical, pathological and genetic features of patients with caveolinopathy from the UK. Ten patients (six families) were identified via the National Commissioning Group (NCG) service for patients with limb girdle muscle dystrophy in Newcastle. Myalgia was the most prominent symptom in our cohort of patients and for 50% it was the reason for referral. Muscle weakness was only found in 60% of the patients, whereas rippling muscle movement was present in 80%. One of the patients reported episodes of myoglobinuria and another one episodes of hypoglycaemia. Five different mutations were identified, two of which were novel and three that had previously been described. Caveolinopathy needs to be considered as a differential diagnosis in a range of clinical situations, including in patients who do not have any weakness. Indeed, rippling muscles are a more frequent symptom than weakness, and can be detected in childhood. Presentation with myalgia is common and management of it as well as of myoglobinuria and hypoglycaemia may have a major impact on the patients' quality of life.

Analysis of the UK diagnostic strategy for limb girdle muscular dystrophy 2A.

Diagnosis of limb girdle muscular dystrophy type 2A can be complex due to phenotypic variability, lack of precision of protein analysis in muscle biopsies, and absence of mutational hot spots in the CAPN3 gene. The aim of this study was to review clinical and biopsy data from a group of patients with known CAPN3 genetic status to validate and refine our current diagnostic strategy, which combines clinical information and protein analysis to direct gene testing. We analysed 85 patients in whom CAPN3 gene sequencing had been performed. Forty-two patients had two mutations, 15 a single mutation and in 28 no mutation was found. We identified clinical features that clearly discriminated the LGMD2A patients. These were: presence of scapular winging, contractures and normal respiratory function. In addition, a typical pattern of muscle weakness on manual muscle testing could be confirmed. Interpretation of protein expression obtained by Western blot was complex and involved the analysis of a number of bands detected by two antibodies for calpain 3. Loss of all calpain 3 bands was 100% specific for LGMD2A, but this pattern was found in only 23%. Absence or reduction of the approximately 60 kDa bands was also highly specific for LGMD2A, while increased abundance was highly predictive of no mutations being found even where other bands were reduced, suggesting that this is the most sensitive marker of artefactual protein degradation. Twenty-three percent of the patients with two mutations had normal full-sized calpain 3 protein, consistent with the finding of mutations localized in parts of the gene likely or proven to be involved in autolytic activity. Clinical and biochemical findings in patients with only one mutation were similar to patients with two mutations, indicating that other gene analysis techniques should be used before excluding the diagnosis. Our analysis confirms that our strategy is still valid to prioritize genetic testing in this complex group of patients, provided patients with normal protein but a suggestive clinical phenotype are not excluded from genetic testing.

Mouse model for hereditary hemorrhagic telangiectasia has a generalized vascular abnormality.

BACKGROUND: Mutations in endoglin or activin like kinase-1, both involved in the endothelial transforming growth factor-beta signaling pathway, cause the autosomal dominant bleeding disorder hereditary hemorrhagic telangiectasia. We and others have reported mouse models for this disease that share the characteristic phenotype of dilated vessels and sporadic hemorrhage. The reasons for the variable phenotype in hereditary hemorrhagic telangiectasia are not understood. METHODS AND RESULTS: After a detailed immunohistochemical analysis of 129/Ola mice, which are heterozygous for a targeted deletion in the endoglin gene, we observed intrinsic abnormalities in the vascular walls throughout the cutaneous vasculature. Postcapillary venules were dilated, and up to 70% of the vascular wall had no smooth muscle cells. The supporting layers of collagens and elastin were irregular, with thin areas, adding to the fragility of these vessels. A variable hemorrhagic phenotype was observed in which local bleeding is associated not only with fragile vessels but also with regions of inflammation. CONCLUSIONS: These findings have relevance to our understanding of the molecular basis of vascular integrity in a wide range of diseases.