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Objective: The objective of the study is to characterize the pathomechanisms underlying actininopathies. Distal myopathies are a group of rare, inherited muscular disorders characterized by progressive loss of muscle fibers that begin in the distal parts of arms and legs. Recently, variants in a new disease gene, ACTN2, have been shown to cause distal myopathy. ACTN2, a gene previously only associated with cardiomyopathies, encodes alpha-actinin-2, a protein expressed in both cardiac and skeletal sarcomeres. The primary function of alpha-actinin-2 is to link actin and titin to the sarcomere Z-disk. New ACTN2 variants are continuously discovered; however, the clinical significance of many variants remains unknown. Thus, lack of clear genotype-phenotype correlations in ACTN2-related diseases, actininopathies, persists., Methods: Functional characterization in C2C12 cell model of several ACTN2 variants is conducted, including frameshift and missense variants associated with dominant and recessive actininopathies. We assess the genotype-phenotype correlations of actininopathies using clinical data from several patients carrying these variants., Results: The results show that the missense variants associated with a recessive form of actininopathy do not cause detectable alpha-actinin-2 aggregates in the cell model. Conversely, dominant frameshift variants causing a protein extension do form alpha-actinin-2 aggregates., Interpretation: The results suggest that alpha-actinin-2 aggregation is the disease mechanism underlying some dominant actininopathies, and thus, we recommend that protein-extending frameshift variants in ACTN2 should be classified as pathogenic. However, this mechanism is likely elicited by only a limited number of variants. Alternative functional characterization methods should be explored to further investigate other molecular mechanisms underlying actininopathies., (© 2024 The Author(s). Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Background: Titin truncating variants (TTNtvs) have been repeatedly reported as causative of recessive but not dominant skeletal muscle disorders., Objective: To determine whether a single heterozygous nonsense variant in TTN can be responsible for the observed dominant myopathy in a large family., Methods: In this case series, all available family members (8 affected and 6 healthy) belonging to a single family showing autosomal dominant inheritance were thoroughly examined clinically and genetically., Results: All affected family members showed a similar clinical phenotype with a combination of cardiac and skeletal muscle involvement. Muscle imaging data revealed titin-compatible hallmarks. Genetic analysis revealed in all affected patients a nonsense TTN variant c.70051C>T p.(Arg23351*), in exon 327. RNA sequencing confirmed the lack of complete nonsense-mediated decay, and protein studies convincingly revealed expression of a shortened titin fragment of the expected size., Discussion: We conclude that a single heterozygous nonsense variant in titin occasionally can cause a dominant myopathy as shown in this large family. Therefore, monoallelic titin truncating variants should be considered as possible disease-causing variants in unsolved patients with a dominant myopathy. However, large segregation studies, muscle imaging, and RNA and protein assays are needed to support the clinical and genetic interpretation., Competing Interests: The authors report no relevant disclosures. Go to Neurology.org/NG for full disclosures., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Myopathy refers to a large group of heterogeneous, rare muscle diseases. Bulk RNA-sequencing has been utilized for the diagnosis and research of these diseases for many years. However, the existing valuable sequencing data often lack integration and clinical interpretation. In this study, we integrated bulk RNA-sequencing data from 1221 human skeletal muscles (292 with myopathies, 929 controls) from both databases and our local samples. By applying a method similar to single-cell analysis, we revealed a general spectrum of muscle diseases, ranging from healthy to mild disease, moderate muscle wasting, and severe muscle disease. This spectrum was further partly validated in three specific myopathies (97 muscles) through clinical features including trinucleotide repeat expansion, magnetic resonance imaging fat fraction, pathology, and clinical severity scores. This spectrum helped us identify 234 genuinely healthy muscles as unprecedented controls, providing a new perspective for deciphering the hallmark genes and pathways among different myopathies. The newly identified featured genes of general myopathy, inclusion body myositis, and titinopathy were highly expressed in our local muscles, as validated by quantitative polymerase chain reaction., (© 2024. The Author(s).)

In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTN gene. The co-occurrence of predicted deleterious SRPK3/TTN variants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3 -/- ; ttn.1 +/- ) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3 and ttn.1 in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases., (© 2024. The Author(s).)

Recessive mutations in the DNAJB2 gene, encoding the J-domain co-chaperones DNAJB2a and DNAJB2b, have previously been reported as the genetic cause of progressive peripheral neuropathies, rarely involving pyramidal signs, parkinsonism and myopathy. We describe here a family with the first dominantly acting DNAJB2 mutation resulting in a late-onset neuromyopathy phenotype. The c.832 T > G p.(*278Glyext*83) mutation abolishes the stop codon of the DNAJB2a isoform resulting in a C-terminal extension of the protein, with no direct effect predicted on the DNAJB2b isoform of the protein. Analysis of the muscle biopsy showed reduction of both protein isoforms. In functional studies, the mutant protein mislocalized to the endoplasmic reticulum due to a transmembrane helix in the C-terminal extension. The mutant protein underwent rapid proteasomal degradation and also increased the turnover of co-expressed wild-type DNAJB2a, potentially explaining the reduced protein amount in the patient muscle tissue. In line with this dominant negative effect, both wild-type and mutant DNAJB2a were shown to form polydisperse oligomers., (© The Author(s) 2023. Published by Oxford University Press.)

Objective: Inclusion body myositis (IBM) has an unclear molecular etiology exhibiting both characteristic inflammatory T-cell activity and rimmed-vacuolar degeneration of muscle fibers. Using in-depth gene expression and splicing studies, we aimed at understanding the different components of the molecular pathomechanisms in IBM., Methods: We performed RNA-seq on RNA extracted from skeletal muscle biopsies of clinically and histopathologically defined IBM (n = 24), tibial muscular dystrophy (n = 6), and histopathologically normal group (n = 9). In a comprehensive transcriptomics analysis, we analyzed the differential gene expression, differential splicing and exon usage, downstream pathway analysis, and the interplay between coding and non-coding RNAs (micro RNAs and long non-coding RNAs)., Results: We observe dysregulation of genes involved in calcium homeostasis, particularly affecting the T-cell activity and regulation, causing disturbed Ca 2+ -induced apoptotic pathways of T cells in IBM muscles. Additionally, LCK/p56, which is an essential gene in regulating the fate of T-cell apoptosis, shows increased expression and altered splicing usage in IBM muscles., Interpretation: Our analysis provides a novel understanding of the molecular mechanisms in IBM by showing a detailed dysregulation of genes involved in calcium homeostasis and its effect on T-cell functioning in IBM muscles. Loss of T-cell regulation is hypothesized to be involved in the consistent observation of no response to immune therapies in IBM patients. Our results show that loss of apoptotic control of cytotoxic T cells could indeed be one component of their abnormal cytolytic activity in IBM muscles., (© 2022. The Author(s).)

Background and Objectives: To determine the genetic cause of the disease in the previously reported family with adult-onset autosomal dominant distal myopathy (myopathy, distal, 3; MPD3)., Methods: Continued clinical evaluation including muscle MRI and muscle pathology. A linkage analysis with single nucleotide polymorphism arrays and genome sequencing were used to identify the genetic defect, which was verified by Sanger sequencing. RNA sequencing was used to investigate the transcriptional effects of the identified genetic defect., Results: Small hand muscles (intrinsic, thenar, and hypothenar) were first involved with spread to the lower legs and later proximal muscles. Dystrophic changes with rimmed vacuoles and cytoplasmic inclusions were observed in muscle biopsies at advanced stage. A single nucleotide polymorphism array confirmed the previous microsatellite-based linkage to 8p22-q11 and 12q13-q22. Genome sequencing of three affected family members combined with structural variant calling revealed a small heterozygous deletion of 160 base pairs spanning the second last exon 10 of the heterogeneous nuclear ribonucleoprotein A1 ( HNRNPA1) gene, which is in the linked region on chromosome 12. Segregation of the mutation with the disease was confirmed by Sanger sequencing. RNA sequencing showed that the mutant allele produces a shorter mutant mRNA transcript compared with the wild-type allele. Immunofluorescence studies on muscle biopsies revealed small p62 and larger TDP-43 inclusions., Discussion: A small exon 10 deletion in the gene HNRNPA1 was identified as the cause of MPD3 in this family. The new HNRNPA1-related phenotype, upper limb presenting distal myopathy, was thus confirmed, and the family displays the complexities of gene identification., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Background and Objectives: To clinically, genetically, and histopathologically characterize patients presenting with an unusual combination of distal myopathy and facial weakness, without involvement of upper limb or shoulder girdle muscles., Methods: Two families with a novel form of actininopathy were identified. Patients had been followed up over 10 years. Their molecular genetic diagnosis was not clear after extensive investigations, including analysis of candidate genes and FSHD1-related D4Z4 repeats., Results: Patients shared a similar clinical phenotype and a common pattern of muscle involvement. They presented with a very slowly progressive myopathy involving anterior lower leg and facial muscles. Muscle MRI finding showed complete fat replacement of anterolateral compartment muscles of the lower legs with variable involvement of soleus and gastrocnemius but sparing thigh muscles. Muscle biopsy showed internalized nuclei, myofibrillar disorganization, and rimmed vacuoles. High-throughput sequencing identified in each proband a heterozygous single nucleotide deletion (c.2558del and c.2567del) in the last exon of the ACTN2 gene. The deletions are predicted to lead to a novel but unstructured slightly extended C-terminal amino acid sequence., Discussion: Our findings indicate an unusual form of actininopathy with specific molecular and clinical features. Actininopathy should be considered in the differential diagnosis of distal myopathy combined with facial weakness., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Using deep phenotyping and high-throughput sequencing, we have identified a novel type of distal myopathy caused by mutations in the Small muscle protein X-linked (SMPX) gene. Four different missense mutations were identified in ten patients from nine families in five different countries, suggesting that this disease could be prevalent in other populations as well. Haplotype analysis of patients with similar ancestry revealed two different founder mutations in Southern Europe and France, indicating that the prevalence in these populations may be higher. In our study all patients presented with highly similar clinical features: adult-onset, usually distal more than proximal limb muscle weakness, slowly progressing over decades with preserved walking. Lower limb muscle imaging showed a characteristic pattern of muscle involvement and fatty degeneration. Histopathological and electron microscopic analysis of patient muscle biopsies revealed myopathic findings with rimmed vacuoles and the presence of sarcoplasmic inclusions, some with amyloid-like characteristics. In silico predictions and subsequent cell culture studies showed that the missense mutations increase aggregation propensity of the SMPX protein. In cell culture studies, overexpressed SMPX localized to stress granules and slowed down their clearance., (© 2021. The Author(s).)

Distal myopathies are genetic primary muscle disorders with a prominent weakness at onset in hands and/or feet. The age of onset (from early childhood to adulthood), the distribution of muscle weakness (upper versus lower limbs) and the histological findings (ranging from nonspecific myopathic changes to myofibrillar disarrays and rimmed vacuoles) are extremely variable. However, despite being characterized by a wide clinical and genetic heterogeneity, the distal myopathies are a category of muscular dystrophies: genetic diseases with progressive loss of muscle fibers. Myopathic congenital arthrogryposis is also a form of distal myopathy usually caused by focal amyoplasia. Massive parallel sequencing has further expanded the long list of genes associated with a distal myopathy, and contributed identifying as distal myopathy-causative rare variants in genes more often related with other skeletal or cardiac muscle diseases. Currently, almost 20 genes (ACTN2, CAV3, CRYAB, DNAJB6, DNM2, FLNC, HNRNPA1, HSPB8, KHLH9, LDB3, MATR3, MB, MYOT, PLIN4, TIA1, VCP, NOTCH2NLC, LRP12, GIPS1) have been associated with an autosomal dominant form of distal myopathy. Pathogenic changes in four genes (ADSSL, ANO5, DYSF, GNE) cause an autosomal recessive form; and disease-causing variants in five genes (DES, MYH7, NEB, RYR1 and TTN) result either in a dominant or in a recessive distal myopathy. Finally, a digenic mechanism, underlying a Welander-like form of distal myopathy, has been recently elucidated. Rare pathogenic mutations in SQSTM1, previously identified with a bone disease (Paget disease), unexpectedly cause a distal myopathy when combined with a common polymorphism in TIA1. The present review aims at describing the genetic basis of distal myopathy and at summarizing the clinical features of the different forms described so far., Competing Interests: Conflict of interest The Authors declare no conflict of interest, (©2020 Gaetano Conte Academy - Mediterranean Society of Myology, Naples, Italy.)

Skeletal muscle and the nervous system depend on efficient protein quality control, and they express chaperones and cochaperones at high levels to maintain protein homeostasis. Mutations in many of these proteins cause neuromuscular diseases, myopathies, and hereditary motor and sensorimotor neuropathies. In this review, we cover mutations in DNAJB6, DNAJB2, αB-crystallin (CRYAB, HSPB5), HSPB1, HSPB3, HSPB8, and BAG3, and discuss the molecular mechanisms by which they cause neuromuscular disease. In addition, previously unpublished results are presented, showing downstream effects of BAG3 p.P209L on DNAJB6 turnover and localization., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Although DNA-sequencing is the most effective procedure to achieve a molecular diagnosis in genetic diseases, complementary RNA analyses are often required.Reverse-Transcription polymerase chain reaction (RT-PCR) is still a valuable option when the clinical phenotype and/or available DNA-test results address the diagnosis toward a gene of interest or when the splicing effect of a single variant needs to be assessed.We use Single-Molecule Real-Time sequencing to detect and characterize splicing defects and single nucleotide variants in well-known disease genes (DMD, NF1, TTN). After proper optimization, the procedure could be used in the diagnostic setting, simplifying the workflow of cDNA analysis.

Eight patients from five families with undiagnosed dominant distal myopathy underwent clinical, neurophysiological and muscle biopsy examinations. Molecular genetic studies were performed using targeted sequencing of all known myopathy genes followed by segregation of the identified mutations in the affected families using Sanger sequencing. Two novel mutations in DNAJB6 J domain, c.149C>T (p.A50V) and c.161A>C (p.E54A), were identified as the cause of disease. The muscle involvement with p.A50V was distal calf-predominant, and the p.E54A was more proximo-distal. Histological findings were similar to those previously reported in DNAJB6 myopathy. In line with reported pathogenic mutations in the glycine/phenylalanine (G/F) domain of DNAJB6, both the novel mutations showed reduced anti-aggregation capacity by filter trap assay and TDP-43 disaggregation assays. Modeling of the protein showed close proximity of the mutated residues with the G/F domain. Myopathy-causing mutations in DNAJB6 are not only located in the G/F domain, but also in the J domain. The identified mutations in the J domain cause dominant distal and proximo-distal myopathy, confirming that mutations in DNAJB6 should be considered in distal myopathy cases., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: To clinically and pathologically characterize a cohort of patients presenting with a novel form of distal myopathy and to identify the genetic cause of this new muscular dystrophy., Methods: We studied 4 families (3 from Spain and 1 from Sweden) suffering from an autosomal dominant distal myopathy. Affected members showed adult onset asymmetric distal muscle weakness with initial involvement of ankle dorsiflexion later progressing also to proximal limb muscles., Results: In all 3 Spanish families, we identified a unique missense variant in the ACTN2 gene cosegregating with the disease. The affected members of the Swedish family carry a different ACTN2 missense variant., Interpretation: ACTN2 encodes for alpha actinin2, which is highly expressed in the sarcomeric Z-disk with a major structural and functional role. Actininopathy is thus a new genetically determined distal myopathy. ANN NEUROL 2019;85:899-906., (© 2019 American Neurological Association.)

Objective: To identify the genetic defect causing a distal calf myopathy with cores., Methods: Families with a genetically undetermined calf-predominant myopathy underwent detailed clinical evaluation, including EMG/nerve conduction studies, muscle biopsy, laboratory investigations, and muscle MRI. Next-generation sequencing and targeted Sanger sequencing were used to identify the causative genetic defect in each family., Results: A novel deletion-insertion mutation in ryanodine receptor 1 ( RYR1 ) was found in the proband of the index family and segregated with the disease in 6 affected relatives. Subsequently, we found 2 more families with a similar calf-predominant myopathy segregating with unique RYR1 -mutated alleles. All patients showed a very slowly progressive myopathy without episodes of malignant hyperthermia or rhabdomyolysis. Muscle biopsy showed cores or core-like changes in all families., Conclusions: Our findings expand the spectrum of RYR1 -related disorders to include a calf-predominant myopathy with core pathology and autosomal dominant inheritance. Two families had unique and previously unreported RYR1 mutations, while affected persons in the third family carried 2 previously known mutations in the same dominant allele., (© 2019 American Academy of Neurology.)

Importance: Mutations in the titin gene (TTN) cause a wide spectrum of genetic diseases. The interpretation of the numerous rare variants identified in TTN is a difficult challenge given its large size., Objective: To identify genetic variants in titin in a cohort of patients with muscle disorders., Design, Setting, and Participants: In this case series, 9 patients with titinopathy and 4 other patients with possibly disease-causing variants in TTN were identified. Titin mutations were detected through targeted resequencing performed on DNA from 504 patients with muscular dystrophy, congenital myopathy, or other skeletal muscle disorders. Patients were enrolled from 10 clinical centers in April 2012 to December 2013. All of them had not received a diagnosis after undergoing an extensive investigation, including Sanger sequencing of candidate genes. The data analysis was performed between September 2013 and January 2017. Sequencing data were analyzed using an internal custom bioinformatics pipeline., Main Outcomes and Measures: The identification of novel mutations in the TTN gene and novel patients with titinopathy. We performed an evaluation of putative causative variants in the TTN gene, combining genetic, clinical, and imaging data with messenger RNA and/or protein studies., Results: Of the 9 novel patients with titinopathy, 5 (55.5%) were men and the mean (SD) age at onset was 25 (15.8) years (range, 0-46 years). Of the 4 other patients (3 men and 1 woman) with possibly disease-causing TTN variants, 2 (50%) had a congenital myopathy and 2 (50%) had a slowly progressive distal myopathy with onset in the second decade. Most of the identified mutations were previously unreported. However, all the variants, even the already described mutations, require careful clinical and molecular evaluation of probands and relatives. Heterozygous truncating variants or unique missense changes are not sufficient to make a diagnosis of titinopathy., Conclusions and Relevance: The interpretation of TTN variants often requires further analyses, including a comprehensive evaluation of the clinical phenotype (deep phenotyping) as well as messenger RNA and protein studies. We propose a specific workflow for the clinical interpretation of genetic findings in titin.

Background and Purpose: The aim was to determine the genetic background of unknown muscular dystrophy in five French families., Methods: Twelve patients with limb girdle muscular dystrophy or distal myopathy were clinically evaluated. Gene mutations were identified using targeted exon sequencing and mutated DNAJB6 was tested in vitro., Results: Five patients presented with distal lower limb weakness whilst others had proximal presentation with a variable rate of progression starting at the mean age of 38.5 years. Two novel mutations (c.284A>T, p.Asn95Ile, two families; and c.293_295delATG, p.Asp98del, one family) as well as the previously reported c.279C>G (p.Phe93Leu, two families) mutation in DNAJB6 were identified. All showed a reduced capacity to prevent protein aggregation., Conclusions: The mutational and phenotypical spectrum of DNAJB6-caused muscle disease is larger than previously reported, including also dysphagia. The originally reported c.279C>G (p.Phe93Leu) mutation is now identified in four different populations and appears to be a mutational hotspot. Our report confirms that some DNAJB6 mutations cause distal-onset myopathy and hence DNAJB6 defects should be considered broadly in dominant muscular dystrophy families., (© 2018 EAN.)

Background: Mutations in the titin gene (TTN) cause a large spectrum of diseases affecting skeletal and/or cardiac muscle. TTN includes 363 coding exons, a repeated region with a high degree of complexity, isoform-specific elements, and metatranscript-only exons thought to be expressed only during fetal development. Although three main classes of isoforms have been described so far, alternative splicing events (ASEs) in different tissues or in different developmental and physiological states have been reported., Methods: To achieve a comprehensive view of titin ASEs in adult human skeletal muscles, we performed a RNA-Sequencing experiment on 42 human biopsies collected from 12 anatomically different skeletal muscles of 11 individuals without any skeletal-muscle disorders., Results: We confirmed that the skeletal muscle N2A isoforms are highly prevalent, but we found an elevated number of alternative splicing events, some at a very high level. These include previously unknown exon skipping events and alternative 5' and 3' splice sites. Our data suggests the partial inclusion in the TTN transcript of some metatranscript-only exons and the partial exclusion of canonical N2A exons., Conclusions: This study provides an extensive picture of the complex TTN splicing pattern in human adult skeletal muscle, which is crucial for a proper clinical interpretation of TTN variants.

Multisystem proteinopathy (MSP) involves disturbances of stress granule (SG) dynamics and autophagic protein degradation that underlie the pathogenesis of a spectrum of degenerative diseases that affect muscle, brain, and bone. Specifically, identical mutations in the autophagic adaptor SQSTM1 can cause varied penetrance of 4 distinct phenotypes: amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Paget's disease of the bone, and distal myopathy. It has been hypothesized that clinical pleiotropy relates to additional genetic determinants, but thus far, evidence has been lacking. Here, we provide evidence that a TIA1 (p.N357S) variant dictates a myodegenerative phenotype when inherited, along with a pathogenic SQSTM1 mutation. Experimentally, the TIA1-N357S variant significantly enhances liquid-liquid-phase separation in vitro and impairs SG dynamics in living cells. Depletion of SQSTM1 or the introduction of a mutant version of SQSTM1 similarly impairs SG dynamics. TIA1-N357S-persistent SGs have increased association with SQSTM1, accumulation of ubiquitin conjugates, and additional aggregated proteins. Synergistic expression of the TIA1-N357S variant and a SQSTM1-A390X mutation in myoblasts leads to impaired SG clearance and myotoxicity relative to control myoblasts. These findings demonstrate a pathogenic connection between SG homeostasis and ubiquitin-mediated autophagic degradation that drives the penetrance of an MSP phenotype.

Tibial muscular dystrophy (TMD) is the first described human titinopathy. It is a mild adult-onset slowly progressive myopathy causing weakness and atrophy in the anterior lower leg muscles. TMD is caused by mutations in the last two exons, Mex5 and Mex6, of the titin gene (TTN). The first reported TMD mutations were dominant, but the Finnish founder mutation FINmaj, an 11-bp insertion/deletion in Mex6, in homozygosity caused a completely different severe early-onset limb-girdle muscular dystrophy 2J (LGMD2J). Later, we reported that not all TMD mutations cause LGMD when homozygous or compound heterozygous with truncating mutation, but some of them rather cause a more severe TMD-like distal disease. We have now performed targeted next-generation sequencing of myopathy-related genes on seven families from Albania, Bosnia, Iran, Tunisia, Belgium, and Spain with juvenile or early adult onset recessive distal myopathy. Novel mutations in TTN Mex5, Mex6 and A-band exon 340 were identified in homozygosity or compound heterozygosity with a frameshift or nonsense mutation in TTN I- or A-band region. Family members having only one of these TTN mutations were healthy. Our results add yet another entity to the list of distal myopathies: juvenile or early adult onset recessive distal titinopathy.

Objective: To report novel disease and pathology due to HSPB8 mutations in 2 families with autosomal dominant distal neuromuscular disease showing both myofibrillar and rimmed vacuolar myopathy together with neurogenic changes., Methods: We performed whole-exome sequencing (WES) in tandem with linkage analysis and candidate gene approach as well as targeted next-generation sequencing (tNGS) to identify causative mutations in 2 families with dominant rimmed vacuolar myopathy and a motor neuropathy. Pathogenic variants and familial segregation were confirmed using Sanger sequencing., Results: WES and tNGS identified a heterozygous change in HSPB8 in both families: c.421A > G p.K141E in family 1 and c.151insC p.P173SfsX43 in family 2. Affected patients had a distal myopathy that showed myofibrillar aggregates and rimmed vacuoles combined with a clear neurogenic component both on biopsy and neurophysiologic studies. MRI of lower limb muscles demonstrated diffuse tissue changes early in the disease stage progressing later to fatty replacement typical of a myopathy., Conclusion: We expand the understanding of disease mechanisms, tissue involvement, and phenotypic outcome of HSPB8 mutations. HSPB8 is part of the chaperone-assisted selective autophagy (CASA) complex previously only associated with Charcot-Marie-Tooth type 2L (OMIM 60673) and distal hereditary motor neuronopathy type IIa. However, we now demonstrate that patients can develop a myopathy with histologic features of myofibrillar myopathy with aggregates and rimmed vacuoles, similar to the pathology in myopathies due to gene defects in other compounds of the CASA complex such as BAG3 and DNAJB6 after developing the early neurogenic effects., (© 2015 American Academy of Neurology.)

DNAJB6 is the causative gene for limb-girdle muscular dystrophy 1D (LGMD1D). Four different coding missense mutations, p.F89I, p.F93I, p.F93L, and p.P96R, have been reported in families from Europe, North America and Asia. The previously known mutations cause mainly adult-onset proximal muscle weakness with moderate progression and without respiratory involvement. A Finnish family and a British patient have been studied extensively due to a severe muscular dystrophy. The patients had childhood-onset LGMD, loss of ambulation in early adulthood and respiratory involvement; one patient died of respiratory failure aged 32. Two novel mutations, c.271T > A (p.F91I) and c.271T > C (p.F91L), in DNAJB6 were identified by whole exome sequencing as a cause of this severe form of LGMD1D. The results were confirmed by Sanger sequencing. The anti-aggregation effect of the mutant DNAJB6 was investigated in a filter-trap based system using transient transfection of mammalian cell lines and polyQ-huntingtin as a model for an aggregation-prone protein. Both novel mutant proteins show a significant loss of ability to prevent aggregation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Mutations in the extreme C-terminus of titin (TTN), situated in the sarcomeric M-band, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J). The mutations ultimately cause a loss of C-terminal titin, including a binding site for the protease calpain 3 (CAPN3), and lead to a secondary CAPN3 deficiency in LGMD2J muscle. CAPN3 has been previously shown to bind C-terminal titin and to use it as a substrate in vitro. Interestingly, mutations in CAPN3 underlie limb-girdle muscular dystrophy 2A (LGMD2A). Here, we aimed to clarify the relationship of CAPN3 and M-band titin in normal and pathological muscle. In vitro analyses identified several CAPN3 cleavage sites in C-terminal titin that were defined by protein sequencing. Furthermore, cleavage products were detected in normal muscle extracts by western blotting and in situ by immunofluorescence microscopy. The TMD/LGMD2J mutation FINmaj proved to alter this processing in vitro, while binding of CAPN3 to mutant titin was preserved. Unexpectedly, the pathological loss of M-band titin due to TMD/LGMD2J mutations was found to be independent of CAPN3, whereas the involvement of ubiquitous calpains is likely. We conclude that proteolytic processing of C-terminal titin by CAPN3 may have an important role in normal muscle, and that this process is disrupted in LGMD2A and in TMD/LGMD2J due to CAPN3 deficiency and to the loss of C-terminal titin, respectively., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG)n repeat expansion in intron 1 of CNBP. Transcription of the repeats causes a toxic RNA gain of function involving their accumulation in ribonuclear foci. This leads to sequestration of splicing factors and alters pre-mRNA splicing in a range of downstream effector genes, which is thought to contribute to the diverse DM2 clinical features. Hyperlipidemia is frequent in DM2 patients, but the treatment is problematic because of an increased risk of statin-induced adverse reactions. Hypothesizing that shared pathways lead to the increased risk, we compared the skeletal muscle expression profiles of DM2 patients and controls with patients with hyperlipidemia on statin therapy. Neural precursor cell expressed, developmentally downregulated-4 (NEDD4), an ubiquitin ligase, was one of the dysregulated genes identified in DM2 patients and patients with statin-treated hyperlipidemia. In DM2 muscle, NEDD4 mRNA was abnormally spliced, leading to aberrant NEDD4 proteins. NEDD4 was down-regulated in persons taking statins, and simvastatin treatment of C2C12 cells suppressed NEDD4 transcription. Phosphatase and tensin homologue (PTEN), an established NEDD4 target, was increased and accumulated in highly atrophic DM2 muscle fibers. PTEN ubiquitination was reduced in DM2 myofibers, suggesting that the NEDD4-PTEN pathway is dysregulated in DM2 skeletal muscle. Thus, this pathway may contribute to the increased risk of statin-adverse reactions in patients with DM2., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Tibial muscular dystrophy (TMD) is a late onset, autosomal dominant distal myopathy that results from mutations in the two last domains of titin. The cascade of molecular events leading from the causative Titin mutations to the preterm death of muscle cells in TMD is largely unknown. In this study we examined the mRNA and protein changes associated with the myopathology of TMD. To identify these components we performed gene expression profiling using muscle biopsies from TMD patients and healthy controls. The profiling results were confirmed through quantitative real-time PCR and protein level analysis. One of the pathways identified was activation of endoplasmic reticulum (ER) stress response. ER stress activates the unfolded protein response (UPR) pathway. UPR activation was supported by elevation of the marker genes HSPA5, ERN1 and the UPR specific XBP1 splice form. However, UPR activation appears to be insufficient to correct the protein abnormalities causing its activation because degenerative TMD muscle fibres show an increase in ubiquitinated protein inclusions. Abnormalities of VCP-associated degradation pathways are also suggested by the presence of proteolytic VCP fragments in western blotting, and VCP's accumulation within rimmed vacuoles in TMD muscle fibres together with p62 and LC3B positive autophagosomes. Thus, pathways controlling turnover and degradation, including autophagy, are distorted and lead to degeneration and loss of muscle fibres.

Objective: A study was undertaken to identify the molecular cause of Welander distal myopathy (WDM), a classic autosomal dominant distal myopathy., Methods: The genetic linkage was confirmed and defined by microsatellite and single nucleotide polymorphism haplotyping. The whole linked genomic region was sequenced with targeted high-throughput and Sanger sequencing, and coding transcripts were sequenced on the cDNA level. WDM muscle biopsies were studied by Western blotting and immunofluorescence microscopy. Splicing of TIA1 and its target genes in muscle and myoblast cultures was analyzed by reverse transcriptase polymerase chain reaction. Mutant TIA1 was characterized by cell biological studies on HeLa cells, including quantification of stress granules by high content analysis and fluorescence recovery after photobleaching (FRAP) experiments., Results: The linked haplotype at 2p13 was narrowed down to <806 kb. Sequencing by multiple methods revealed only 1 segregating coding mutation, c.1362 G>A (p.E384K) in the RNA-binding protein TIA1, a key component of stress granules. Immunofluorescence microscopy of WDM biopsies showed a focal increase of TIA1 in atrophic and vacuolated fibers. In HeLa cells, mutant TIA1 constructs caused a mild increase in stress granule abundance compared to wild type, and showed slower average fluorescence recovery in FRAP., Interpretation: WDM is caused by mutated TIA1 through a dominant pathomechanism probably involving altered stress granule dynamics., (Copyright © 2012 American Neurological Association.)

Limb-girdle muscular dystrophy type 1D (LGMD1D) was linked to chromosome 7q36 over a decade ago, but its genetic cause has remained elusive. Here we studied nine LGMD-affected families from Finland, the United States and Italy and identified four dominant missense mutations leading to p.Phe93Leu or p.Phe89Ile changes in the ubiquitously expressed co-chaperone DNAJB6. Functional testing in vivo showed that the mutations have a dominant toxic effect mediated specifically by the cytoplasmic isoform of DNAJB6. In vitro studies demonstrated that the mutations increase the half-life of DNAJB6, extending this effect to the wild-type protein, and reduce its protective anti-aggregation effect. Further, we show that DNAJB6 interacts with members of the CASA complex, including the myofibrillar myopathy-causing protein BAG3. Our data identify the genetic cause of LGMD1D, suggest that its pathogenesis is mediated by defective chaperone function and highlight how mutations in a ubiquitously expressed gene can exert effects in a tissue-, isoform- and cellular compartment-specific manner.

Deletion of the phosphoglucose isomerase gene, PGI1, in Saccharomyces cerevisiae leads to a phenotype for which glucose is toxic. This is related to overproduction of NADPH through the oxidative part of the pentose phosphate pathway and the incompetence of S. cerevisiae to deal with this overproduction. A similar deletion (rag2) in Kluyveromyces lactis does not lead to such a phenotype. We transformed a genomic library of K. lactis in a yeast vector to a S. cerevisiae strain with a pgi1 deletion and screened for growth on glucose. We found a gene (GDP1) which encodes a phosphorylating glyceraldehyde-3-phosphate dehydrogenase, NADP-GAPDH (EC 1.2.1.13), that accepts both NADP and NAD. This is the first report of a eukaryotic, nonplant, NADP-linked GAPDH. Presumably, operation of this enzyme in the reverse direction enabled the transformed S. cerevisiae pgi1 deletion mutant to reoxidize the excess NADPH produced when glucose catabolism was forced through the pentose pathway. On the other hand, transcription of the gene in K. lactis was upregulated during growth on D-xylose, which suggests that in K. lactis the enzyme is involved in regeneration of NADPH needed for xylose assimilation, but transcription was not detected in a rag2 mutant grown on glucose. The presence of an asparagine (Asn46 in NADP-GAPDH) instead of the conserved aspartate found in related but NAD-specific enzymes may explain the ability of NADP-GAPDH to work with NADP as well as NAD.

By analysing the surface composition of a set of protein 3D structures, complemented with predicted surface compositional information for homologous proteins, we have found significant evidence for a layer composition of protein structures. In the innermost and outermost parts of proteins there is a net negative charge, while the middle has a net positive charge. In addition, our findings indicate that the concept of conservative mutation needs substantial revision, e.g. very different spatial preferences were found for glutamic acid and aspartic acid. The alanine screening often used in protein engineering projects involves the substitution of residues to alanine, based on the assumption that alanine is a "neutral" residue. However, alanine has a high negative correlation with all but the non-polar residues. We therefore propose the use of, for example, serine as a substitute for the residues that are negatively correlated with alanine.

Esterases and lipases both hydrolyse ester bonds. Whereas the lipases display high activity towards the aggregated state of its substrate, the esterases typically show highest activity towards the soluble state of its substrate. We have compared the amino acid sequence, the 3D-structure as well as the pH-dependent electrostatic signature of selected members of the two families, for which 3D-structural information is publicly available. Lipases display a statistically significant enhanced occurrence of non-polar residues close to the surface, clustering around the active-site. Lid opening appears to strengthen this pattern further. As we have proposed earlier the active site of lipases displays negative potential in the pH-range associated with their maximum activity, typically at pH values above 8. The esterases show a very similar pattern, however, at pH values around 6 correlated with their usually lower pH-activity optimum.

Here we present an investigation of the contacts that cysteines make with residues in their three-dimensional environment and a comprehensive analysis of the conformational features of 351 disulphide bridges in 131 non-homologous single-chain protein structures. Upstream half-cystines preferentially have downstream neighbours, whereas downstream half-cystines have mainly upstream neighbours. Non-disulphide bridged cysteines (free cysteines) have no preference for upstream or downstream neighbours. Free cysteines have more contacts to non-polar residues and fewer contacts to polar/charged residues than half-cystines, which correlates with our observation that free cysteines are more buried than half-cystines. Free cysteines prefer to be located in alpha-helices while no clear preference is observed for half-cystines. Histidine and methionine are preferentially seen nearby free cysteines. Tryptophan is found preferentially nearby half-cystines. We have merged sequential and spatial information, and highly interesting novel patterns have been discovered. The number of cysteines per protein is typically an even number, peaking at four. The number of residues separating two half-cystines is preferentially 11 and 16. Left-handed and right-handed disulphide bridges display different conformational parameters. Here we present side chain torsion angle information based on a 5-12 times larger number of disulphide bridges than has previously been published. Considering the importance of cysteines for maintaining the 3D-structural scaffold of proteins, it is essential to have as accurate information as possible concerning the packing and conformational preferences. The present work may provide key information for engineering the protein environment around cysteines.

The protein surface is the interface through which a protein senses the external world. Its composition of charged, polar and hydrophobic residues is crucial for the stability and activity of the protein. The charge state of seven of the twenty naturally occurring amino acids is pH dependent. A total of 95% of all titratable residues are located on the surface of soluble proteins. In evolutionary related families of proteins such residues are particularly prone to substitutions, insertions and deletions. We present here an analysis of the residue composition of 4038 proteins, selected from 125 protein families with < 25% identity between core members of each family. Whereas only 16.8% of the residues were truly buried, 40.7% were > 30% exposed on the surface and the remainder were < 30% exposed. The individual residue types show distinct differences. The data presented provides an important new approach to protein engineering of protein surfaces. Guidelines for the optimization of solvent exposure for a given residue are given. The cutinase family of enzymes has been investigated. The stability of native cutinase has been studied as a function of pH, and has been compared with the cutinase activity towards tributyrin. Whereas the onset of enzymatic activity is linked with the deprotonation of the active site HIS188, destabilization of the 3D structure as determined by differential scanning calorimetry is coupled with the loss of activity at very basic pH values. A modeling investigation of the pH dependence of the electrostatic potentials reveals that the activity range is accompanied by the development of a highly significant negative potential in the active site cleft. The 3D structures of three mutants of the Fusarium solani pisi cutinase have been solved to high resolution using X-ray diffraction analysis. Preliminary X-ray data are presented.

The trypsin family of serine proteases is one of the most studied protein families, with a wealth of amino acid sequence information available in public databases. Since trypsin-like enzymes are widely distributed in living organisms in nature, likely evolutionary scenarios have been proposed. A novel methodology for Fourier transformation of biological sequences (FOTOBIS) is presented. The methodology is well suited for the identification of the size and extent of short repeats in protein sequences. In the present paper the trypsin family of enzymes is analyzed with FOTOBIS and strong evidence for tandem gene duplication is found. A likely evolutionary path for the development of present-day trypsins involved an intrinsic extensive tandem gene duplication of a small DNA fragment of 15-18 nucleotides, corresponding to five or six amino acids. This ancestral trypsin gene was subsequently duplicated, leading to the earliest version of a full-sized trypsin, from which the contemporary trypsins have developed.