Revealing myopathy spectrum: integrating transcriptional and clinical features of human skeletal muscles with varying health conditions.

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.

Extension of the DNAJB2a isoform in a dominant neuromyopathy family.

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.

Long-term favorable prognosis in late onset dominant distal titinopathy: Tibial muscular dystrophy.

BACKGROUND AND PURPOSE: Tibial muscular dystrophy (TMD) is a dominant late onset distal titinopathy. It was first described in Finnish patients 3 decades ago. TMD patients with several other TTN mutations occur in many European populations. In this retrospective study, we were able to obtain longitudinal follow-up data of the disease progression over 15 years in 137 TMD patients. METHODS: We retrieved clinical data retrospectively from three examinations spanning a period of 15 years. The data were analyzed in R. Frequencies, percentages, and median values were used to describe data. Probability values were determined with the chi-squared test. RESULTS: In the cohort, the first symptoms were walking difficulties (97.8%) and weakness in distal lower limbs (98.5%). The progression of the weakness in distal lower limbs was moderate, and in the proximal lower limbs and proximal upper limbs it was mild. The distal upper limbs were not affected. Magnetic resonance imaging results indicated fatty degeneration preferentially in lower leg anterior muscles, gluteus minimus, and hamstring muscles. Serum creatine kinase values in the cohort were mostly normal (40.7%) or mildly elevated (53.7%). The data suggest that 50% of patients need walking aids by the age of 88 years. CONCLUSIONS: Despite individual variability of severity, the overall disability due to walking difficulties and upper limb weakness remained moderate even at very advanced ages, and cardiomyopathy did not develop due to the titin defect alone. The acquired results promote the correct identification of TMD, and the obtained trajectories of disease evolution can be used as natural history data for any therapeutic intervention.

Mutation update for the ACTN2 gene.

ACTN2 encodes alpha-actinin-2, a protein expressed in human cardiac and skeletal muscle. The protein, located in the sarcomere Z-disk, functions as a link between the anti-parallel actin filaments. This important structural protein also binds N-terminal titins, and thus contributes to sarcomere stability. Previously, ACTN2 mutations have been solely associated with cardiomyopathy, without skeletal muscle disease. Recently, however, ACTN2 mutations have been associated with novel congenital and distal myopathy. Previously reported variants are in varying locations across the gene, but the potential clustering effect of pathogenic locations is not clearly understood. Further, the genotype-phenotype correlations of these variants remain unclear. Here we review the previously reported ACTN2-related molecular and clinical findings and present an additional variant, c.1840-2A>T, that further expands the mutation and phenotypic spectrum. Our results show a growing body of clinical, genetic, and functional evidence, which underlines the central role of ACTN2 in the muscle tissue and myopathy. However, limited segregation and functional data are available to support the pathogenicity of most previously reported missense variants and clear-cut genotype-phenotype correlations are currently only demonstrated for some ACTN2-related myopathies.

Adult-onset dominant muscular dystrophy in Greek families caused by Annexin A11.

OBJECTIVE: Mutations in the prion-like domain of RNA binding proteins cause dysfunctional stress responses and associated aggregate pathology in patients with neurogenic and myopathic phenotypes. Recently, mutations in ANXA11 have been reported in patients with amyotrophic lateral sclerosis and multisystem proteinopathy. Here we studied families with an autosomal dominant muscle disease caused by ANXA11:c.118G > T;p.D40Y. METHODS: We performed deep phenotyping and exome sequencing of patients from four large Greek families, including seven affected individuals with progressive muscle disease but no family history of multi-organ involvement or ALS. RESULTS: In our study, all patients presented with an autosomal dominant muscular dystrophy without any Paget disease of bone nor signs of frontotemporal dementia or Parkinson's disease. Histopathological analysis showed rimmed vacuoles with annexin A11 accumulations. Electron microscopy analysis showed myofibrillar abnormalities with disorganization of the sarcomeric structure and Z-disc dissolution, and subsarcolemmal autophagic material with myeloid formations. Molecular genetic analysis revealed ANXA11:c.118G > T;p.D40Y segregating with the phenotype. INTERPRETATION: Although the pathogenic mechanisms associated with p.D40Y mutation in the prion-like domain of Annexin A11 need to be further clarified, our study provides robust and clear genetic evidence to support the expansion of the phenotypic spectrum of ANXA11.

Comprehensive transcriptomic analysis shows disturbed calcium homeostasis and deregulation of T lymphocyte apoptosis in inclusion body myositis.

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 Ca2+-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.

Dominant Distal Myopathy 3 (MPD3) Caused by a Deletion in the HNRNPA1 Gene.

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.

Out-of-Frame Mutations in ACTN2 Last Exon Cause a Dominant Distal Myopathy With Facial Weakness.

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.

Congenital asymmetric distal myopathy with hemifacial weakness caused by a heterozygous large de novo mosaic deletion in nebulin.

We report the first mosaic mutation, a deletion of exons 11-107, identified in the nebulin gene in a Finnish patient presenting with a predominantly distal congenital myopathy and asymmetric muscle weakness. The female patient is ambulant and currently 26 years old. Muscle biopsies showed myopathic features with type 1 fibre predominance, strikingly hypotrophic type 2 fibres and central nuclei, but no nemaline bodies. The deletion was detected in a copy number variation analysis based on next-generation sequencing data. The parents of the patient did not carry the deletion. Mosaicism was detected using a custom, targeted comparative genomic hybridisation array. Expression of the truncated allele, less than half the size of full-length nebulin, was confirmed by Western blotting. The clinical and histological picture resembled that of a family with a slightly smaller deletion, and that in patients with recessively inherited distal forms of nebulin-caused myopathy. Asymmetry, however, was a novel feature.

Missense mutations in small muscle protein X-linked (SMPX) cause distal myopathy with protein inclusions.

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.

Genotype-phenotype correlations in recessive titinopathies.

PURPOSE: High throughput sequencing analysis has facilitated the rapid analysis of the entire titin (TTN) coding sequence. This has resulted in the identification of a growing number of recessive titinopathy patients. The aim of this study was to (1) characterize the causative genetic variants and clinical features of the largest cohort of recessive titinopathy patients reported to date and (2) to evaluate genotype-phenotype correlations in this cohort. METHODS: We analyzed clinical and genetic data in a cohort of patients with biallelic pathogenic or likely pathogenic TTN variants. The cohort included both previously reported cases (100 patients from 81 unrelated families) and unreported cases (23 patients from 20 unrelated families). RESULTS: Overall, 132 causative variants were identified in cohort members. More than half of the cases had hypotonia at birth or muscle weakness and a delayed motor development within the first 12 months of life (congenital myopathy) with causative variants located along the entire gene. The remaining patients had a distal or proximal phenotype and a childhood or later (noncongenital) onset. All noncongenital cases had at least one pathogenic variant in one of the final three TTN exons (362-364). CONCLUSION: Our findings suggest a novel association between the location of nonsense variants and the clinical severity of the disease.

Identification and Characterization of Splicing Defects by Single-Molecule Real-Time Sequencing Technology (PacBio).

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.

Is Gene-Size an Issue for the Diagnosis of Skeletal Muscle Disorders?

Human genes have a variable length. Those having a coding sequence of extraordinary length and a high number of exons were almost impossible to sequence using the traditional Sanger-based gene-by-gene approach. High-throughput sequencing has partly overcome the size-related technical issues, enabling a straightforward, rapid and relatively inexpensive analysis of large genes.Several large genes (e.g. TTN, NEB, RYR1, DMD) are recognized as disease-causing in patients with skeletal muscle diseases. However, because of their sheer size, the clinical interpretation of variants in these genes is probably the most challenging aspect of the high-throughput genetic investigation in the field of skeletal muscle diseases.The main aim of this review is to discuss the technical and interpretative issues related to the diagnostic investigation of large genes and to reflect upon the current state of the art and the future advancements in the field.

Improved Criteria for the Classification of Titin Variants in Inherited Skeletal Myopathies.

BACKGROUND: Extensive genetic screening results in the identification of thousands of rare variants that are difficult to interpret. Because of its sheer size, rare variants in the titin gene (TTN) are detected frequently in any individual. Unambiguous interpretation of molecular findings is almost impossible in many patients with myopathies or cardiomyopathies. OBJECTIVE: To refine the current classification framework for TTN-associated skeletal muscle disorders and standardize the interpretation of TTN variants. METHODS: We used the guidelines issued by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) to re-analyze TTN genetic findings from our patient cohort. RESULTS: We identified in the classification guidelines three rules that are not applicable to titin-related skeletal muscle disorders; six rules that require disease-/gene-specific adjustments and four rules requiring quantitative thresholds for a proper use. In three cases, the rule strength need to be modified. CONCLUSIONS: We suggest adjustments are made to the guidelines. We provide frequency thresholds to facilitate filtering of candidate causative variants and guidance for the use and interpretation of functional data and co-segregation evidence. We expect that the variant classification framework for TTN-related skeletal muscle disorders will be further improved along with a better understanding of these diseases.

Mutations in the J domain of DNAJB6 cause dominant distal myopathy.

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.

Improving Copy Number Variant Detection from Sequencing Data with a Combination of Programs and a Predictive Model.

Bioinformatics tools for analyzing copy number variants (CNVs) from massively parallel sequencing (MPS) data are less well developed compared with other variant types. We present an efficient bioinformatics pipeline for CNV detection from gene panel MPS data in neuromuscular disorders. CNVs were generated in silico into samples sequenced with a previously published MPS gene panel. The in silico CNVs from these samples were analyzed with four programs having complementary CNV detection ranges: CoNIFER, XHMM, ExomeDepth, and CODEX. A logistic regression model was trained with the obtained set of in silico CNV detections to predict true-positive CNV detections among all CNV detections from samples. This model was validated using 66 control samples with a verified true-positive (n = 58) or false-positive (n = 8) CNV detection. Applying all four programs together provided more sensitive detection results with in silico CNVs than other program combinations or any program alone. Furthermore, a model with CNV detection-specific scores from all four programs as variables performed overall best in the validation. No single program could detect all CNV sizes and types equally or with enough accuracy. Therefore, a combination of carefully selected programs should be used to maximize detection accuracy. In addition, the detected CNVs should be reviewed with a statistical model to streamline and standardize the filtering of the detections for annotation.

Panorama of the distal myopathies.

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.

Actininopathy: A new muscular dystrophy caused by ACTN2 dominant mutations.

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.

Dominantly inherited distal nemaline/cap myopathy caused by a large deletion in the nebulin gene.

We report the first family with a dominantly inherited mutation of the nebulin gene (NEB). This ∼100 kb in-frame deletion encompasses NEB exons 14-89, causing distal nemaline/cap myopathy in a three-generation family. It is the largest deletion characterized in NEB hitherto. The mutated allele was shown to be expressed at the mRNA level and furthermore, for the first time, a deletion was shown to cause the production of a smaller mutant nebulin protein. Thus, we suggest that this novel mutant nebulin protein has a dominant-negative effect, explaining the first documented dominant inheritance of nebulin-caused myopathy. The index patient, a young man, was more severely affected than his mother and grandmother. His first symptom was foot drop at the age of three, followed by distal muscle atrophy, slight hypomimia, high-arched palate, and weakness of the neck and elbow flexors, hands, tibialis anterior and toe extensors. Muscle biopsies showed myopathic features with type 1 fibre predominance in the index patient and nemaline bodies and cap-like structures in biopsies from his mother and grandmother. The muscle biopsy findings constitute a further example of nemaline bodies and cap-like structures being part of the same spectrum of pathological changes.

Expanding the importance of HMERF titinopathy: new mutations and clinical aspects.

OBJECTIVE: Hereditary myopathy with early respiratory failure (HMERF) is caused by titin A-band mutations in exon 344 and considered quite rare. Respiratory insufficiency is an early symptom. A collection of families and patients with muscle disease suggestive of HMERF was clinically and genetically studied. METHODS: Altogether 12 new families with 19 affected patients and diverse nationalities were studied. Most of the patients were investigated using targeted next-generation sequencing; Sanger sequencing was applied in some of the patients and available family members. Histological data and muscle MRI findings were evaluated. RESULTS: Three families had several family members studied while the rest were single patients. Most patients had distal and proximal muscle weakness together with respiratory insufficiency. Five heterozygous TTN A-band mutations were identified of which two were novel. Also with the novel mutations the muscle pathology and imaging findings were compatible with the previous reports of HMERF. CONCLUSIONS: Our collection of 12 new families expands mutational spectrum with two new mutations identified. HMERF is not that rare and can be found worldwide, but maybe underdiagnosed. Diagnostic process seems to be complex as this study shows with mostly single patients without clear dominant family history.