Molecular and cellular basis of genetically inherited skeletal muscle disorders.

Neuromuscular disorders comprise a diverse group of human inborn diseases that arise from defects in the structure and/or function of the muscle tissue - encompassing the muscle cells (myofibres) themselves and their extracellular matrix - or muscle fibre innervation. Since the identification in 1987 of the first genetic lesion associated with a neuromuscular disorder - mutations in dystrophin as an underlying cause of Duchenne muscular dystrophy - the field has made tremendous progress in understanding the genetic basis of these diseases, with pathogenic variants in more than 500 genes now identified as underlying causes of neuromuscular disorders. The subset of neuromuscular disorders that affect skeletal muscle are referred to as myopathies or muscular dystrophies, and are due to variants in genes encoding muscle proteins. Many of these proteins provide structural stability to the myofibres or function in regulating sarcolemmal integrity, whereas others are involved in protein turnover, intracellular trafficking, calcium handling and electrical excitability - processes that ensure myofibre resistance to stress and their primary activity in muscle contraction. In this Review, we discuss how defects in muscle proteins give rise to muscle dysfunction, and ultimately to disease, with a focus on pathologies that are most common, best understood and that provide the most insight into muscle biology.

Therapeutic targeting of RNA for neurological and neuromuscular disease.

Neurological and neuromuscular diseases resulting from familial, sporadic, or de novo mutations have devasting personal, familial, and societal impacts. As the initial product of DNA transcription, RNA transcripts and their associated ribonucleoprotein complexes provide attractive targets for modulation by increasing wild-type or blocking mutant allele expression, thus relieving downstream pathological consequences. Therefore, it is unsurprising that many existing and under-development therapeutics have focused on targeting disease-associated RNA transcripts as a frontline drug strategy for these genetic disorders. This review focuses on the current range of RNA targeting modalities using examples of both dominant and recessive neurological and neuromuscular diseases.

Neuromuscular disorders: finding the missing genetic diagnoses.

Neuromuscular disorders (NMDs) are a wide-ranging group of diseases that seriously affect the quality of life of affected individuals. The development of next-generation sequencing revolutionized the diagnosis of NMD, enabling the discovery of hundreds of NMD genes and many more pathogenic variants. However, the diagnostic yield of genetic testing in NMD cohorts remains incomplete, indicating a large number of genetic diagnoses are not identified through current methods. Fortunately, recent advancements in sequencing technologies, analytical tools, and high-throughput functional screening provide an opportunity to circumvent current challenges. Here, we discuss reasons for missing genetic diagnoses in NMD, how emerging technologies and tools can overcome these hurdles, and examine future approaches to improving diagnostic yields in NMD.

Molecular mechanisms and therapeutic strategies for neuromuscular diseases.

Neuromuscular diseases encompass a heterogeneous array of disorders characterized by varying onset ages, clinical presentations, severity, and progression. While these conditions can stem from acquired or inherited causes, this review specifically focuses on disorders arising from genetic abnormalities, excluding metabolic conditions. The pathogenic defect may primarily affect the anterior horn cells, the axonal or myelin component of peripheral nerves, the neuromuscular junction, or skeletal and/or cardiac muscles. While inherited neuromuscular disorders have been historically deemed not treatable, the advent of gene-based and molecular therapies is reshaping the treatment landscape for this group of condition. With the caveat that many products still fail to translate the positive results obtained in pre-clinical models to humans, both the technological development (e.g., implementation of tissue-specific vectors) as well as advances on the knowledge of pathogenetic mechanisms form a collective foundation for potentially curative approaches to these debilitating conditions. This review delineates the current panorama of therapies targeting the most prevalent forms of inherited neuromuscular diseases, emphasizing approved treatments and those already undergoing human testing, offering insights into the state-of-the-art interventions.

Proteomic studies in VWA1-related neuromyopathy allowed new pathophysiological insights and the definition of blood biomarkers.

Bi-allelic variants in VWA1, encoding Von Willebrand Factor A domain containing 1 protein localized to the extracellular matrix (ECM), were linked to a neuromuscular disorder with manifestation in child- or adulthood. Clinical findings indicate a neuromyopathy presenting with muscle weakness. Given that pathophysiological processes are still incompletely understood, and biomarkers are still missing, we aimed to identify blood biomarkers of pathophysiological relevance: white blood cells (WBC) and plasma derived from six VWA1-patients were investigated by proteomics. Four proteins, BET1, HNRNPDL, NEFM and PHGDH, known to be involved in neurological diseases and dysregulated in WBC were further validated by muscle-immunostainings unravelling HNRNPDL as a protein showing differences between VWA1-patients, healthy controls and patients suffering from neurogenic muscular atrophy and BICD2-related neuromyopathy. Immunostaining studies of PHGDH indicate its involvement in apoptotic processes via co-localisation with caspase-3. NEFM showed an increase in cells within the ECM in biopsies of all patients studied. Plasma proteomics unravelled dysregulation of 15 proteins serving as biomarker candidates among which a profound proportion of increased ones (6/11) are mostly related to antioxidative processes and have even partially been described as blood biomarkers for other entities of neuromuscular disorders before. CRP elevated in plasma also showed an increase in the extracellular space of VWA1-mutant muscle. Results of our combined studies for the first time describe pathophysiologically relevant biomarkers for VWA1-related neuromyopathy and suggest that VWA1-patient derived blood might hold the potential to study disease processes of clinical relevance, an important aspect for further preclinical studies.

Next generation sequencing panel as an effective approach to genetic testing in patients with a highly variable phenotype of neuromuscular disorders.

Neuromuscular disorders (NMDs) include a wide range of diseases affecting the peripheral nervous system. The genetic diagnoses are increasingly obtained with using the next generation sequencing (NGS). We applied the custom-design targeted NGS panel including 89 genes, together with genotyping and multiplex ligation-dependent probe amplification (MLPA) to identify a genetic spectrum of NMDs in 52 Polish patients. As a result, the genetic diagnosis was determined by NGS panel in 29 patients so its diagnostic utility is estimated at 55.8%. The most pathogenic variants were found in CLCN1, followed by CAPN3, SCN4A, and SGCA genes. Genotyping of myotonic dystrophy type 1 and 2 (DM1 and DM2) as a secondary approach has been performed. The co-occurrence of CAPN3 and CNBP mutations in one patient as well as DYSF and CNBP mutations in another suggests possibly more complex inheritance as well as expression of a phenotype. In 7 individuals with single nucleotide variant found in NGS testing, the MLPA of the CAPN3 gene was performed detecting the deletion encompassing exons 2-8 in the CAPN3 gene in one patient, confirming recessive limb-girdle muscular dystrophy type 1 (LGMDR1). Thirty patients obtained a genetic diagnosis (57.7%) after using NGS testing, genotyping and MLPA analysis. The study allowed for the identification of 27 known and 4 novel pathogenic/likely pathogenic variants and variants of uncertain significance (VUS) associated with NMDs.In conclusion, the diagnostic approach with diverse molecular techniques enables to broaden the mutational spectrum and maximizes the diagnostic yield. Furthermore, the co-occurrence of DM2 and LGMD has been detected in 2 individuals.

Deubiquitinases in muscle physiology and disorders.

In vivo, muscle and neuronal cells are post-mitotic, and their function is predominantly regulated by proteostasis, a multilayer molecular process that maintains a delicate balance of protein homeostasis. The ubiquitin-proteasome system (UPS) is a key regulator of proteostasis. A dysfunctional UPS is a hallmark of muscle ageing and is often impacted in neuromuscular disorders (NMDs). Malfunction of the UPS often results in aberrant protein accumulation which can lead to protein aggregation and/or mis-localization affecting its function. Deubiquitinating enzymes (DUBs) are key players in the UPS, controlling protein turnover and maintaining the free ubiquitin pool. Several mutations in DUB encoding genes are linked to human NMDs, such as ATXN3, OTUD7A, UCHL1 and USP14, whilst other NMDs are associated with dysregulation of DUB expression. USP5, USP9X and USP14 are implicated in synaptic transmission and remodeling at the neuromuscular junction. Mice lacking USP19 show increased maintenance of lean muscle mass. In this review, we highlight the involvement of DUBs in muscle physiology and NMDs, particularly in processes affecting muscle regeneration, degeneration and inflammation following muscle injury. DUBs have recently garnered much respect as promising drug targets, and their roles in muscle maturation, regeneration and degeneration may provide the framework for novel therapeutics to treat muscular disorders including NMDs, sarcopenia and cachexia.

Neuromuscular disease genetics in under-represented populations: increasing data diversity.

Neuromuscular diseases (NMDs) affect ∼15 million people globally. In high income settings DNA-based diagnosis has transformed care pathways and led to gene-specific therapies. However, most affected families are in low-to-middle income countries (LMICs) with limited access to DNA-based diagnosis. Most (86%) published genetic data is derived from European ancestry. This marked genetic data inequality hampers understanding of genetic diversity and hinders accurate genetic diagnosis in all income settings. We developed a cloud-based transcontinental partnership to build diverse, deeply-phenotyped and genetically characterized cohorts to improve genetic architecture knowledge, and potentially advance diagnosis and clinical management. We connected 18 centres in Brazil, India, South Africa, Turkey, Zambia, Netherlands and the UK. We co-developed a cloud-based data solution and trained 17 international neurology fellows in clinical genomic data interpretation. Single gene and whole exome data were analysed via a bespoke bioinformatics pipeline and reviewed alongside clinical and phenotypic data in global webinars to inform genetic outcome decisions. We recruited 6001 participants in the first 43 months. Initial genetic analyses 'solved' or 'possibly solved' ∼56% probands overall. In-depth genetic data review of the four commonest clinical categories (limb girdle muscular dystrophy, inherited peripheral neuropathies, congenital myopathy/muscular dystrophies and Duchenne/Becker muscular dystrophy) delivered a ∼59% 'solved' and ∼13% 'possibly solved' outcome. Almost 29% of disease causing variants were novel, increasing diverse pathogenic variant knowledge. Unsolved participants represent a new discovery cohort. The dataset provides a large resource from under-represented populations for genetic and translational research. In conclusion, we established a remote transcontinental partnership to assess genetic architecture of NMDs across diverse populations. It supported DNA-based diagnosis, potentially enabling genetic counselling, care pathways and eligibility for gene-specific trials. Similar virtual partnerships could be adopted by other areas of global genomic neurological practice to reduce genetic data inequality and benefit patients globally.

Utility of exome sequencing for the diagnosis of pediatric-onset neuromuscular diseases beyond diagnostic yield: a narrative review.

Diagnosis of neuromuscular diseases (NMD) can be challenging because of the heterogeneity of this group of diseases. This review aimed to describe the diagnostic yield of whole exome sequencing (WES) for pediatric-onset neuromuscular disease diagnosis, as well as other benefits of this approach in patient management since WES can contribute to appropriate treatment selection in NMD patients. WES increases the possibility of reaching a conclusive genetic diagnosis when other technologies have failed and even exploring new genes not previously associated with a specific NMD. Moreover, this strategy can be useful when a dual diagnosis is suspected in complex congenital anomalies and undiagnosed cases.

Undetected Neuromuscular Disease in Patients after Heart Transplantation.

(1) Heart transplantation (HTX) improves the overall survival and functional status of end-stage heart failure patients with cardiomyopathies (CMPs). The majority of CMPs have genetic causes, and the overlap between CMPs and inherited myopathies is well documented. However, the long-term outcome in skeletal muscle function and possibility of an undiagnosed underlying genetic cause of both a cardiac and skeletal pathology remain unknown. (2) Thirty-nine patients were assessed using open and standardized interviews on muscle function, a quality-of-life (EuroQol EQ-5D-3L) questionnaire, and a physical examination (Medical Research Council Muscle scale). Whole-exome sequencing was completed in three stages for those with skeletal muscle weakness. (3) Seven patients (17.9%) reported new-onset muscle weakness and motor limitations. Objective muscle weakness in the upper and lower extremities was seen in four patients. In three of them, exome sequencing revealed pathogenic/likely pathogenic variants in the genes encoding nexilin, myosin heavy chain, titin, and SPG7. (4) Our findings support a positive long-term outcome of skeletal muscle function in HTX patients. However, 10% of patients showed clinical signs of myopathy due to a possible genetic cause. The integration of genetic testing and standardized neurological assessment of motor function during the peri-HTX period should be considered.

The spectrum of hereditary neuromuscular disorders in the Pakistani population.

Hereditary neuromuscular disorders (NMDs) are a broad group of clinically heterogeneous disorders with varying inheritance patterns, that are associated with over 500 implicated genes. In the context of a highly consanguineous Pakistani population, we expect that autosomal recessive NMDs may have a higher prevalence compared with patients of European descent. This is the first study to offer a detailed description of the spectrum of genes causing hereditary NMDs in the Pakistani population using NGS testing. To study the clinical and genetic profiles of patients presenting for evaluation of a hereditary neuromuscular disorder. This is a retrospective chart review of patients seen in the Neuromuscular Disorders Clinic and referred to the Genetics Clinic with a suspected hereditary neuromuscular disorder, between 2016 and 2020 at the Aga Khan University Hospital, Karachi and Mukhtiar A. Sheikh Hospital, Multan, Pakistan. The genetic testing for these patients included NGS-based single gene sequencing, NGS-based multi-gene panel and whole exome sequencing. In a total of 112 patients studied, 35 (31.3%) were female. The mean age of onset in all patients was 14.6 years (SD ±12.1 years), with the average age at presentation to the clinic of 22.4 years (SD ±14.10 years). Forty-seven (41.9%) patients had a positive genetic test result, 53 (47.3%) had one or more variants of uncertain significance (VUS), and 12 (10.7%) had a negative result. Upon further genotype-phenotype correlation and family segregation analysis, the diagnostic yield improved, with 59 (52.7%) patients reaching a diagnosis of a hereditary NMD. We also report probable founder variants in COL6A2, FKTN, GNE, and SGCB, previously reported in populations that have possible shared ancestry with the Pakistani population. Our findings reemphasizes that the rate of VUSs can be reduced by clinical correlation and family segregation studies.

Exome sequencing in the pediatric neuromuscular clinic leads to more frequent diagnosis of both neuromuscular and neurodevelopmental conditions.

INTRODUCTION/AIMS: Exome sequencing (ES) has proven to be a valuable diagnostic tool for neuromuscular disorders, which often pose a diagnostic challenge. The aims of this study were to investigate the clinical outcomes associated with utilization of ES in the pediatric neuromuscular clinic and to determine if specific phenotypic features or abnormal neurodiagnostic tests were predictive of a diagnostic result. METHODS: This was a retrospective medical record review of 76 pediatric neuromuscular clinic patients who underwent ES. Based upon clinical assessment prior to ES, patients were divided into two groups: affected by neuromuscular (n = 53) or non-neuromuscular (n = 23) syndromes. RESULTS: A diagnosis was made in 28/76 (36.8%), with 29 unique disorders identified. In the neuromuscular group, a neuromuscular condition was confirmed in 78% of those receiving a genetic diagnosis. Early age of symptom onset was associated with a significantly higher diagnostic yield. The most common reason neuromuscular diagnoses were not detected on prior testing was due to causative genes not being present on disease-specific panels. Changes to medical care were made in 57% of individuals receiving a diagnosis on ES. DISCUSSION: These data further support ES as a powerful diagnostic tool in the pediatric neuromuscular clinic and highlight the advantages of ES over gene panels, including the ability to identify diagnoses regardless of etiology, identify genes newly associated with disease, and identify multiple confounding diagnoses. Rapid and accurate diagnosis by ES can not only end the patient's diagnostic odyssey, but often impacts patients' medical management and genetic counseling of families.

Transcriptome-based variant calling and aberrant mRNA discovery enhance diagnostic efficiency for neuromuscular diseases.

BACKGROUND: Whole-exome sequencing-based diagnosis of rare diseases typically yields 40%-50% of success rate. Precise diagnosis of the patients with neuromuscular disorders (NMDs) has been hampered by locus heterogeneity or phenotypic heterogeneity. We evaluated the utility of transcriptome sequencing as an independent approach in diagnosing NMDs. METHODS: The RNA sequencing (RNA-Seq) of muscle tissues from 117 Korean patients with suspected Mendelian NMD was performed to evaluate the ability to detect pathogenic variants. Aberrant splicing and CNVs were inspected to identify additional causal genetic factors for NMD. Aberrant splicing events in Dystrophin (DMD) were investigated by using antisense oligonucleotides (ASOs). A non-negative matrix factorisation analysis of the transcriptome data followed by cell type deconvolution was performed to cluster samples by expression-based signatures and identify cluster-specific gene ontologies. RESULTS: Our pipeline called 38.1% of pathogenic variants exclusively from the muscle transcriptomes, demonstrating a higher diagnostic rate than that achieved via exome analysis (34.9%). The discovery of variants causing aberrant splicing allowed the application of ASOs to the patient-derived cells, providing a therapeutic approach tailored to individual patients. RNA-Seq data further enabled sample clustering by distinct gene expression profiles that corresponded to clinical parameters, conferring additional advantages over exome sequencing. CONCLUSION: The RNA-Seq-based diagnosis of NMDs achieves an increased diagnostic rate and provided pathogenic status information, which is not easily accessible through exome analysis.

[Molecular therapies: present and future in neuromuscular diseases]. / Molekulare Therapien: Gegenwart und Zukunft bei neuromuskulären Erkrankungen.

BACKGROUND: The possibilities in the field of molecular therapies of neuromuscular diseases have rapidly developed in recent years. First compounds are already available in clinical practice and numerous other substances are in advanced phases of clinical trials. This article gives an exemplary overview of the current state of clinical research in molecular therapies of neuromuscular diseases. It also gives a view into the near future of the clinical application, including the challenges. DISCUSSION: Using Duchenne muscular dystrophy (DMD) and myotubular myopathy as examples, the principles of gene addition in monogenetic skeletal muscle diseases, which are already manifested in childhood are described. In addition to initial successes, the challenges and setbacks hindering the approval and regular clinical application of further compounds are demonstrated. Furthermore, the state of current clinical research in Becker-Kiener muscular dystrophy (BMD) and the numerous forms of limb-girdle muscular dystrophy (LGMD) are summarized. Numerous new therapeutic approaches and a corresponding outlook are also shown for facioscapulohumeral muscular dystrophy (FSHD), Pompe disease, and myotonic dystrophy. CONCLUSION: Clinical research in the field of molecular therapy of neuromuscular diseases is one of the pacesetters of modern precision medicine; however, challenges need to be seen, jointly addressed and overcome in the future.

Renal pathology in a mouse model of severe Spinal Muscular Atrophy is associated with downregulation of Glial Cell-Line Derived Neurotrophic Factor (GDNF).

Spinal muscular atrophy (SMA) occurs as a result of cell-ubiquitous depletion of the essential survival motor neuron (SMN) protein. Characteristic disease pathology is driven by a particular vulnerability of the ventral motor neurons of the spinal cord to decreased SMN. Perhaps not surprisingly, many other organ systems are also impacted by SMN depletion. The normal kidney expresses very high levels of SMN protein, equivalent to those found in the nervous system and liver, and levels are dramatically lowered by ~90-95% in mouse models of SMA. Taken together, these data suggest that renal pathology may be present in SMA. We have addressed this using an established mouse model of severe SMA. Nephron number, as assessed by gold standard stereological techniques, was significantly reduced. In addition, morphological assessment showed decreased renal vasculature, particularly of the glomerular capillary knot, dysregulation of nephrin and collagen IV, and ultrastructural changes in the trilaminar filtration layers of the nephron. To explore the molecular drivers underpinning this process, we correlated these findings with quantitative PCR measurements and protein analyses of glial cell-line-derived neurotrophic factor, a crucial factor in ureteric bud branching and subsequent nephron development. Glial cell-line-derived neurotrophic factor levels were significantly reduced at early stages of disease in SMA mice. Collectively, these findings reveal significant renal pathology in a mouse model of severe SMA, further reinforcing the need to develop and administer systemic therapies for this neuromuscular disease.

Mimicking human riboflavin responsive neuromuscular disorders by silencing flad-1 gene in C. elegans: Alteration of vitamin transport and cholinergic transmission.

Riboflavin (Rf), or vitamin B2, is the precursor of FMN and FAD, redox cofactors of several dehydrogenases involved in energy metabolism, redox balance and other cell regulatory processes. FAD synthase, coded by FLAD1 gene in humans, is the last enzyme in the pathway converting Rf into FAD. Mutations in FLAD1 gene are responsible for neuromuscular disorders, in some cases treatable with Rf. In order to mimic these disorders, the Caenorhabditis elegans (C. elegans) gene orthologue of FLAD1 (flad-1) was silenced in a model strain hypersensitive to RNA interference in nervous system. Silencing flad-1 resulted in a significant decrease in total flavin content, paralleled by a decrease in the level of the FAD-dependent ETFDH protein and by a secondary transcriptional down-regulation of the Rf transporter 1 (rft-1) possibly responsible for the total flavin content decrease. Conversely an increased ETFDH mRNA content was found. These biochemical changes were accompanied by significant phenotypical changes, including impairments of fertility and locomotion due to altered cholinergic transmission, as indicated by the increased sensitivity to aldicarb. A proposal is made that neuronal acetylcholine production/release is affected by alteration of Rf homeostasis. Rf supplementation restored flavin content, increased rft-1 transcript levels and eliminated locomotion defects. In this aspect, C. elegans could provide a low-cost animal model to elucidate the molecular rationale for Rf therapy in human Rf responsive neuromuscular disorders and to screen other molecules with therapeutic potential.

Challenging Neuromuscular Disease Cases.

The diagnosis of neuromuscular disorders requires a thorough history including family history and examination, with the next steps broadened now beyond electromyography and neuropathology to include genetic testing. The challenge in diagnosis can often be putting all the information together. With advances in genetic testing, some diagnoses that adult patients may have received as children deserve a second look and may result in diagnoses better defined or alternative diagnoses made. Clearly defining or redefining a diagnosis can result in understanding of potential other systems involved, prognosis, or potential treatments. This article presents several cases and approach to diagnosis as well as potential treatment and prognostic concerns, including seipinopathy, congenital myasthenic syndrome, central core myopathy, and myotonic dystrophy type 2.

Bi-allelic truncating mutations in VWA1 cause neuromyopathy.

The von Willebrand Factor A domain containing 1 protein, encoded by VWA1, is an extracellular matrix protein expressed in muscle and peripheral nerve. It interacts with collagen VI and perlecan, two proteins that are affected in hereditary neuromuscular disorders. Lack of VWA1 is known to compromise peripheral nerves in a Vwa1 knock-out mouse model. Exome sequencing led us to identify bi-allelic loss of function variants in VWA1 as the molecular cause underlying a so far genetically undefined neuromuscular disorder. We detected six different truncating variants in 15 affected individuals from six families of German, Arabic, and Roma descent. Disease manifested in childhood or adulthood with proximal and distal muscle weakness predominantly of the lower limbs. Myopathological and neurophysiological findings were indicative of combined neurogenic and myopathic pathology. Early childhood foot deformity was frequent, but no sensory signs were observed. Our findings establish VWA1 as a new disease gene confidently implicated in this autosomal recessive neuromyopathic condition presenting with child-/adult-onset muscle weakness as a key clinical feature.

The diversity of hereditary neuromuscular diseases: Experiences from molecular diagnosis.

BACKGROUND: Hereditary neuromuscular diseases (NMDs) are a group of rare disorders, and the diagnosis of these diseases is a substantial burden for referral centers. Although next-generation sequencing (NGS) has identified a large number of genes associated with hereditary NMDs, the diagnostic rates still vary across centers. METHODS: Patients with a suspected hereditary NMD were referred to neuromuscular specialists at the National Taiwan University Hospital. Molecular diagnoses were performed by employing a capture panel containing 194 genes associated with NMDs. RESULTS: Among the 50 patients referred, 43 had a suspicion of myopathy, and seven had polyneuropathy. The overall diagnostic rate was 58%. Pathogenic variants in 19 genes were observed; the most frequent pathogenic variant found in this cohort (DYSF) was observed in only four patients, and 10 pathogenic variants were observed in one patient each. One case of motor neuron disease was clinically mistaken for myopathy. A positive family history increased the diagnostic rate (positive: 72.7% vs. negative: 56.3%). Fourteen patients with elevated plasma creatine kinase levels remained without a diagnosis. CONCLUSION: The application of NGS in this single-center study proves the great diversity of hereditary NMDs. A capture panel is essential, but high-quality clinical and laboratory evaluations of patients are also indispensable.

A Schematic Approach to Defining the Prevalence of COL VI Variants in Five Years of Next-Generation Sequencing.

OBJECTIVE: To define the prevalence of variants in collagen VI genes through a next-generation sequencing (NGS) approach in undiagnosed patients with suspected neuromuscular disease and to propose a diagnostic flowchart to assess the real pathogenicity of those variants. METHODS: In the past five years, we have collected clinical and molecular information on 512 patients with neuromuscular symptoms referred to our center. To pinpoint variants in COLVI genes and corroborate their real pathogenicity, we sketched a multistep flowchart, taking into consideration the bioinformatic weight of the gene variants, their correlation with clinical manifestations and possible effects on protein stability and expression. RESULTS: In Step I, we identified variants in COLVI-related genes in 48 patients, of which three were homozygous variants (Group 1). Then, we sorted variants according to their CADD score, clinical data and complementary studies (such as muscle and skin biopsy, study of expression of COLVI on fibroblast or muscle and muscle magnetic resonance). We finally assessed how potentially pathogenic variants (two biallelic and 12 monoallelic) destabilize COL6A1-A2-A3 subunits. Overall, 15 out of 512 patients were prioritized according to this pipeline. In seven of them, we confirmed reduced or absent immunocytochemical expression of collagen VI in cultured skin fibroblasts or in muscle tissue. CONCLUSIONS: In a real-world diagnostic scenario applied to heterogeneous neuromuscular conditions, a multistep integration of clinical and molecular data allowed the identification of about 3% of those patients harboring pathogenetic collagen VI variants.