Background and ObjectivesTwinkle, encoded by the TWNK gene, is a mitochondrial DNA helicase that unwinds the double helix of DNA during replication, playing a pivotal role in mitochondrial function. Twinkle-related disorders encompass a variety of genetic disorders characterized by mitochondrial dysfunction. Although several phenotypes have been described, the full clinical and molecular spectrum remains poorly defined. The aim of this study was to characterize the phenotypic and genotypic variability among multinational patients diagnosed with Twinkle-related disorders.MethodsA retrospective cohort study was conducted in patients with Twinkle-related disorders at several specialized centers in Italy, France, Germany, Spain, Denmark, Hungary, and the United States, establishing the Twinkle-Related Disorders International Consortium for Trial Readiness (TReDIC). Data were collected from medical records, including clinical features, age at onset, disease progression, and results from genetic testing. Phenotypic categories included infantile-onset cerebellar ataxia, parkinsonism, primary mitochondrial myopathy (PMM), multisystem involvement, asymptomatic carriers, undetermined phenotypes, and other phenotypes. All patients' diagnoses were confirmed by genetic analysis, and their genetic variants were noted. Outcomes included prevalence of phenotypes, symptom chronology, and mutational patterns.ResultsThe study included a total of 189 patients (116 female), with a mean age at symptom onset of 40.3 years. At the time of analysis, 70.4% were alive. PMM was the predominant syndrome (85.2%), and most common features were progressive external ophthalmoplegia (84.7%) and skeletal myopathy (55.6%), followed by hearing loss (17.5%) and psychiatric symptoms (15.3%). Most patients (76.8%) presented with neuromuscular symptoms, with fewer showing CNS (19.6%) or multiorgan (3.6%) features at onset; by more than 8 years from onset, these proportions shifted to 54.4%, 23.3%, and 23.3%, respectively. A total of 73 TWNK variants (16 novel) were found, mostly missense, clustered in functionally critical regions.DiscussionThis large multinational cohort analysis advances our understanding of Twinkle-related disorders by identifying mutational hotspots with clinical relevance and illustrating the broad phenotypic spectrum and progression patterns. In the context of such rare diseases, the formation of international collaborations, such as TReDIC, can enhance our understanding and support the design of upcoming clinical trials.

BACKGROUND AND OBJECTIVES:Twinkle, encoded by the TWNK gene, is a mitochondrial DNA helicase that unwinds the double helix of DNA during replication, playing a pivotal role in mitochondrial function. Twinkle-related disorders encompass a variety of genetic disorders characterized by mitochondrial dysfunction. Although several phenotypes have been described, the full clinical and molecular spectrum remains poorly defined. The aim of this study was to characterize the phenotypic and genotypic variability among multinational patients diagnosed with Twinkle-related disorders. METHODS:A retrospective cohort study was conducted in patients with Twinkle-related disorders at several specialized centers in Italy, France, Germany, Spain, Denmark, Hungary, and the United States, establishing the Twinkle-Related Disorders International Consortium for Trial Readiness (TReDIC). Data were collected from medical records, including clinical features, age at onset, disease progression, and results from genetic testing. Phenotypic categories included infantile-onset cerebellar ataxia, parkinsonism, primary mitochondrial myopathy (PMM), multisystem involvement, asymptomatic carriers, undetermined phenotypes, and other phenotypes. All patients' diagnoses were confirmed by genetic analysis, and their genetic variants were noted. Outcomes included prevalence of phenotypes, symptom chronology, and mutational patterns. RESULTS:The study included a total of 189 patients (116 female), with a mean age at symptom onset of 40.3 years. At the time of analysis, 70.4% were alive. PMM was the predominant syndrome (85.2%), and most common features were progressive external ophthalmoplegia (84.7%) and skeletal myopathy (55.6%), followed by hearing loss (17.5%) and psychiatric symptoms (15.3%). Most patients (76.8%) presented with neuromuscular symptoms, with fewer showing CNS (19.6%) or multiorgan (3.6%) features at onset; by more than 8 years from onset, these proportions shifted to 54.4%, 23.3%, and 23.3%, respectively. A total of 73 TWNK variants (16 novel) were found, mostly missense, clustered in functionally critical regions. DISCUSSION:This large multinational cohort analysis advances our understanding of Twinkle-related disorders by identifying mutational hotspots with clinical relevance and illustrating the broad phenotypic spectrum and progression patterns. In the context of such rare diseases, the formation of international collaborations, such as TReDIC, can enhance our understanding and support the design of upcoming clinical trials.

Background: The TWNK gene encodes a protein that colocalizes with mitochondrial DNA (mtDNA) in mitochondrial nucleoids. It acts as mtDNA helicase during replication, thus playing a pivotal role in the replication and maintenance of mtDNA stability. TWNK mutations are associated with a wide spectrum of clinical phenotypes and a marked heterogeneity. However, heterozygous nonsense variants in the gene have never been described in association with disease. Methods: We analyzed a next-generation sequencing (NGS) targeted gene panel in a cohort including 40 patients with high clinical suspicion of mitochondrial disorders. Selected patients underwent a complete neurological examination, electrophysiology tests, and muscle biopsy. Segregation analysis was performed in available family members. The 3D structure of twinkle was visualized and analyzed using Swiss Model and Pymol version 3.1.6.1. Results: We found four TWNK-mutated subjects from two unrelated families. They exhibited a variable clinical spectrum, ranging from asymptomatic individuals to subjects with psychiatric disorder, chronic progressive external ophthalmoplegia (CPEO), and CPEO-plus. All the subjects shared the heterozygous TWNK p.Glu665Ter variant. Discussion and Conclusions: We describe the clinical phenotype and muscle biopsy findings associated with the first reported heterozygous nonsense TWNK variant, thus expanding the current knowledge of Twinkle-related disorders. Our findings are in line with the high intrafamilial clinical variability associated with TWNK mutations. Although PEO and skeletal muscle involvement remain hallmarks of the disease, extra-muscular features should be carefully assessed.

Malignant Hyperthermia Susceptibility (MHS) is a pharmacogenetic disorder triggered by volatile anesthetics and muscle relaxants, leading to a hypermetabolic reaction in skeletal muscle that can be fatal if untreated. Diagnosis relies on an in vitro contracture test (IVCT) on muscle biopsy and/or genetic testing for pathogenic variants in the RYR1 gene, which accounts for most cases; less frequently, variants are found in CACNA1S and STAC3. However, unlike the IVCT, genetic testing is limited by the large number of RYR1 variants classified as of uncertain significance (VUS), preventing it from fully replacing the IVCT. We report data from 250 MHS individuals followed over 20 years, in whom 100 RYR1 and 3 CACNA1S variants were identified. Among the RYR1 variants, 81 were previously reported and 19 were novel, all classified as VUS according to European Malignant Hyperthermia Group (EMHG) and Variant Curation Expert Panel (VCEP) criteria. Similarly, of the 3 CACNA1S variants, 1 was previously reported and 2 were classified as VUS. The reporting of novel variants is crucial for improving the accuracy and consistency of variant classification, thereby supporting a more precise interpretation of their clinical significance.

Limb-Girdle Muscular Dystrophies (LGMDs) are genetically heterogeneous disorders primarily affecting proximal limb muscles. The most common form, LGMDR1, results from biallelic CAPN3 mutations encoding calpain-3, a muscle-specific protease. Recently, growing evidence implicates heterozygous CAPN3 variants in autosomal dominant disease (LGMDD4), with pathogenic mechanisms still incompletely understood. In a retrospective multicenter Italian study of patients harboring monoallelic CAPN3 variants (ClinicalTrials.gov NCT05956132), the p.Asp753Asn substitution was the most frequent change, detected in eight unrelated individuals. These patients, aged 6-80 years, exhibited a spectrum of presentations ranging from asymptomatic hyperCKemia and exertional myalgia to mild proximal weakness. Muscle biopsies showed mild, nonspecific myopathic changes, while calpain-3 expression was variably reduced. Structural modeling suggested that Asp753 may stabilize the Ca2+-bound conformation, with substitution potentially disrupting inter-domain interactions. Literature review identified 31 additional reports worldwide, confirming recurrence while highlighting marked phenotypic heterogeneity and limited clinical annotation. The aggregated evidence supports a pathogenic role for p.Asp753Asn, though the precise mechanism, potentially involving a dominant-negative effect, remains to be validated. These findings emphasize diagnostic challenges posed by single CAPN3 variants and underscore the need for integrated clinical, segregation, and functional studies to clarify pathogenic mechanisms, refine counseling, and guide patient-specific rehabilitation and therapeutic strategies.

BACKGROUND AND OBJECTIVES:The mitochondrial DNA (mtDNA) genes MT-ATP6 and MT-ATP8 encode for subunits α and 8 (A6L) of the adenosine triphosphate synthase complex. Pathogenetic variants in MT-ATP6/8 cause incurable mitochondrial syndromes encompassing a wide spectrum of clinical features including ataxia, motor and language developmental delay, deafness, retinitis pigmentosa, and Leigh pattern in brain MRI. Typically, higher levels of mtDNA variants lead to more severe symptomatology although even individuals with similar mtDNA mutational loads exhibit high clinical variability. Hence, the establishment of potential therapeutics is currently challenging. In this article, we present an international multicenter study designed to provide a retrospective natural history of patients with MT-ATP6/8 deficiency and to identify primary and secondary end points for future clinical trials. METHODS:Clinical, biochemical, and molecular genetics data of patients with genetically confirmed MT-ATP6/8 defects were collected and analyzed from Italian, German, US, and Spain national reference centers through ethical committee-approved mitochondrial patients' national registries or local programs. RESULTS:A cohort of 111 patients, 98 unreported, were analyzed (55 male, 56 female). Patients had infantile-onset disease (<1 year) in 44% of cases, pediatric-onset (≥1 year and ≤12 years) in 36%, and late-onset (>12 years) in 20%. Kaplan-Meier analysis showed a significant difference (p value = 0.0349) in the survival of infantile and pediatric patients compared with adult patients, although only 8% of patients were not alive at the last follow-up. The CNS was the most frequently affected tissue (93%), followed by the muscle (75%), eye (46%), and heart (18%). Brain MRI showed isolated Leigh-like lesions (58%), Leigh-like lesions and cortical and/or cerebellar atrophy (15%), isolated cerebellar atrophy (10%), and other lesions (21%). At the last follow-up, 11% of patients were wheelchair-bound. Metabolic acidosis or acute deterioration complicated the clinical course in ≅55% of early-onset patients. Molecular genetics studies identified 26 pathogenic variants (6 of them novel). Reduced citrulline levels and increased alanine and lactate levels were reported in 56%, 49%, and 71% of patients, respectively, suggesting their role as potential biomarkers. DISCUSSION:Our results define a more accurate classification based on the age at onset for MT-ATPase deficiency and provide fundamental clinical and biochemical data for disease management.

Nesprins are a family of spectrin-repeat proteins located at the nuclear envelope which play an important role in nuclear morphology and mechano-transduction. Nesprin-1 and Nesprin-2, encoded by the synaptic nuclear envelope SYNE1 and SYNE2, are highly expressed in cardiac and skeletal muscle. Mutations in SYNE1 have been associated with the autosomal dominant Emery‐Dreifuss Muscular Dystrophy (EDMD) type 4, Congenital Muscular Dystrophy, and Arthrogryposis Multiplex Congenita while mutations in SYNE2 only with Emery‐Dreifuss muscular dystrophy type 5. In this study, we aimed at defining the clinical characteristics, histopathological features and molecular profile of 54 patients carrying single nucleotide variants in SYNE1 and SYNE2 genes. Multiple algorithms were used to predict the deleteriousness of the variants and only variants with Combined Annotation Dependent Depletion (CADD) scores ≥20 were selected. In our cohort 56% of patients were women; 60% of patients had late onset myopathy and 23% of patients presented with EDMD-like phenotype. In 80% of patients muscle biopsy showed myopathic signs, with dystrophic changes in the 28% of cases. The subcellular localization of Nesprin-1 and -2 and their co-localization with Emerin, an inner nuclear membrane protein, were evaluated by immunofluorescence in muscle tissue from 21 patients and neither reduced nuclear staining nor mislocalization of both nesprins were detected. Our data provide a further insight into the clinical phenotypes and muscle pathology associated with SYNE1 or SYNE2 mutations and suggest that immunohistochemistry does not represent a useful diagnostic tool.

Background and Objectives The diagnostic process for myofibrillar myopathies (MFM) and distal myopathies (DM) is particularly complex because of the large number of causative genes, the existence of still molecularly undefined disease entities, and the overlapping features between the 2 categories. This study aimed to characterize a large cohort of patients affected by MFM and DM and identify the most important diagnostic and prognostic aspects of these diseases. Methods Patients with either a myopathological diagnosis of MFM or a clinical diagnosis of DM were included in this retrospective multicentric national study. Demographic, genetic, clinical, and histopathologic data of anonymized patients were collected from the neuromuscular centers of the Italian Association of Myology network. Results Data regarding 132 patients with MFM (mean age 57.0 +/- 15.8 years, 49% female) and 298 patients with DM (mean age 50.7 +/- 15.9 years, 40% female) were gathered from 20 neuromuscular centers. 69 patients fulfilled the criteria for both groups (distal myopathies with myofibrillar pathology, DM-MP). Molecular confirmation was achieved in 63% of the patients. Fifty-two percent of the patients with MFM carried pathogenic variants in either DES (n = 30), MYOT (n = 20), or DNAJB6 (n = 18), which were also the most frequent disease-causing genes in DM-MP, while GNE (n = 44) and MYH7 (n = 23) were the genes most commonly carrying pathogenic variants in DM. The mean age at onset varied from <25 years in patients with causative variants in MYH7 and DYSF to 59 years in patients with myotilinopathies. Cardiac involvement was reported in 29% of patients with MFM and 16% of patients with DM, with DES and MYH7 variants significantly associated with the development of cardiomyopathy. Respiratory impairment was more prevalent in patients with TTN and DES variants and rare in other disorders such as GNE myopathy and dysferlinopathies, which were instead associated, together with DNAJB6-related and PLIN4-related myopathies, with the risk of losing ambulation during the disease course. Discussion The Italian cohort of patients with MFM and DM recapitulates the phenotypic heterogeneity and the partial overlap between the 2 groups. However, in relative contrast to the encountered phenotypic variability, only 5 genes accounted for most of the molecular diagnoses. Specific genetic entities are associated with significantly increased risk of developing cardiorespiratory complications or loss of ambulation, which has relevant prognostic implications.

Objective: To identify novel biomarkers as an alternative diagnostic tool for limb girdle muscular dystrophy (LGMD). Background: LGMD encompasses a group of muscular dystrophies characterized by proximal muscles weakness, elevated CK levels and dystrophic findings on muscle biopsy. Heterozygous CAPN3 mutations are associated with autosomal dominant LGMD-4, while biallelic mutations can cause autosomal recessive LGMD-1. Diagnosis is currently often based on invasive methods requiring muscle biopsy or blood tests. In most cases Western blotting (WB) analysis from muscle biopsy is essential for a diagnosis, as muscle samples are currently the only known tissues to express the full-length CAPN3 isoform. Methods: We analyzed CAPN3 in a cohort including 60 LGMD patients. Selected patients underwent a complete neurological examination, electromyography, muscle biopsy, and skin biopsies for primary fibroblasts isolation. The amount of CAPN3 was evaluated by WB analysis in muscle and skin tissues. The total RNA isolated from muscle, fibroblast and urine was processed, and cDNA was used for qualitative analysis. The expression of CAPN3 was investigated by qRT-PCR. The CAPN3 3D structure has been visualized and analyzed using PyMOL. Results: Among our patients, seven different CAPN3 mutations were detected, of which two were novel. After sequencing CAPN3 transcripts from fibroblast and urine, we detected different CAPN3 isoforms surprisingly including the full-length transcript. We found comparable protein levels from fibroblasts and muscle tissue; in particular, patients harboring a novel CAPN3 mutation showed a 30% reduction in protein compared to controls from both tissues. Conclusions: Our findings showed for the first time the presence of the CAPN3 full-length transcript in urine and skin samples. Moreover, we demonstrated surprisingly comparable CAPN3 protein levels between muscle and skin samples, thus allowing us to hypothesize the use of skin biopsy and probably of urine samples as an alternative less invasive method to assess the amount of CAPN3 when molecular diagnosis turns out to be inconclusive.

CCDC78 was identified as a novel candidate gene for autosomal dominant centronuclear myopathy-4 (CNM4) approximately ten years ago. However, to date, only one family has been described, and the function of CCDC78 remains unclear. Here, we analyze for the first time a family harboring a CCDC78 nonsense mutation to better understand the role of CCDC78 in muscle. Methods: We conducted a comprehensive histopathological analysis on muscle biopsies, including immunofluorescent assays to detect multiple sarcoplasmic proteins. We examined CCDC78 transcripts and protein using WB in CCDC78-mutated muscle tissue; these analyses were also performed on muscle, lymphocytes, and fibroblasts from healthy subjects. Subsequently, we conducted RT-qPCR and transcriptome profiling through RNA-seq to evaluate changes in gene expression associated with CCDC78 dysfunction in muscle. Lastly, coimmunoprecipitation (Co-Ip) assays and mass spectrometry (LC-MS/MS) studies were carried out on extracted muscle proteins from both healthy and mutated subjects. Results: The histopathological features in muscle showed novel histological hallmarks, which included areas of dilated and swollen sarcoplasmic reticulum (SR). We provided evidence of nonsense-mediated mRNA decay (NMD), identified the presence of novel CCDC78 transcripts in muscle and lymphocytes, and identified 1035 muscular differentially expressed genes, including several involved in the SR. Through the Co-Ip assays and LC-MS/MS studies, we demonstrated that CCDC78 interacts with two key SR proteins: SERCA1 and CASQ1. We also observed interactions with MYH1, ACTN2, and ACTA1. Conclusions: Our findings provide insight, for the first time, into the interactors and possible role of CCDC78 in skeletal muscle, locating the protein in the SR. Furthermore, our data expand on the phenotype previously associated with CCDC78 mutations, indicating potential histopathological hallmarks of the disease in human muscle. Based on our data, we can consider CCDC78 as the causative gene for CNM4.

Autosomal-dominant spinocerebellar ataxia (ADCA) due to intronic GAA repeat expansion in FGF14 (SCA27B) is a recent, relatively common form of late-onset ataxia. Here, we aimed to: (1) investigate the relative frequency of SCA27B in different clinically defined disease subgroups with late-onset ataxia collected among 16 tertiary Italian centers; (2) characterize phenotype and diagnostic findings of patients with SCA27B; (3) compare the Italian cohort with other cohorts reported in recent studies. We screened 396 clinically diagnosed late-onset cerebellar ataxias of unknown cause, subdivided in sporadic cerebellar ataxia, ADCA, and multisystem atrophy cerebellar type. We identified 72 new genetically defined subjects with SCA27B. Then, we analyzed the clinical, neurophysiological, and imaging features of 64 symptomatic cases. In our cohort, the prevalence of SCA27B was 13.4

Introduction NAGLU encodes N-acetyl-alpha-glucosaminidase, an enzyme that degrades heparan sulfate. Biallelic NAGLU mutations cause mucopolysaccharidosis IIIB, a severe childhood-onset neurodegenerative disease, while monoallelic mutations are associated to late-onset, dominantly inherited painful sensory neuropathy. However, to date, only one family with a dominant NAGLU -related neuropathy has been described. Case report Here we describe a patient with early-onset motor polyneuropathy harboring a novel monoallelic NAGLU mutation. We found reduced NAGLU enzymatic activity thus corroborating the pathogenic role of the new variant. Discussion Our report represents the second ever described case with dominant NAGLU- related neuropathy and the first case with early-onset motor symptoms. We underlie the importance of a thorough clinical description of this probably underestimated new clinical entity.

Thanks to advances in gene sequencing, RYR1-related myopathy (RYR1-RM) is now known to manifest itself in vastly heterogeneous forms, whose clinical interpretation is, therefore, highly challenging. We set out to develop a novel unsupervised cluster analysis method in a large patient population. The objective was to analyze the main RYR1-related characteristics to identify distinctive features of RYR1-RM and, thus, offer more precise genotype–phenotype correlations in a group of potentially life-threatening disorders. We studied 600 patients presenting with a suspicion of inherited myopathy, who were investigated using next-generation sequencing. Among them, 73 index cases harbored variants in RYR1. In an attempt to group genetic variants and fully exploit information derived from genetic, morphological, and clinical datasets, we performed unsupervised cluster analysis in 64 probands carrying monoallelic variants. Most of the 73 patients with positive molecular diagnoses were clinically asymptomatic or pauci-symptomatic. Multimodal integration of clinical and histological data, performed using a non-metric multi-dimensional scaling analysis with k-means clustering, grouped the 64 patients into 4 clusters with distinctive patterns of clinical and morphological findings. In addressing the need for more specific genotype–phenotype correlations, we found clustering to overcome the limits of the “single-dimension” paradigm traditionally used to describe genotype–phenotype relationships.

Objective: Mutations in the valosin-containing protein (VCP) gene cause autosomal dominant multisystem proteinopathy 1 (MSP1), characterized by a variable combination of inclusion body myopathy (IBM), Paget's disease of bone (PDB), and frontotemporal dementia (FTD). Here we report a novel VCP missense mutations in an Italian family with FTD as the prevalent manifestation and compare our results with those described in the literature. Methods: We described the clinical, molecular, and imaging data of the studied family. We also conducted a systematic literature search with the aim of comparing our findings with previously reported VCP-related phenotypes. Results: A novel heterozygous VCP missense mutation (c 0.473 T > C/p.Met158Thr) was found in all the affected family members. The proband is a 69-year-old man affected by progressive muscle weakness since the age of 49. Muscle MRI showed patchy fatty infiltration in most muscles, and STIR sequences revealed an unusual signal increase in distal leg muscles. At age 65, he presented a cognitive disorder suggestive of behavioral variant FTD. A bone scintigraphy also revealed PDB. The patient's mother, his maternal aunt and her daughter had died following a history of cognitive deterioration consistent with FTD; the mother also had PDB. No relatives had any muscular impairments. Reviewing the literature data, we observed a different sex distribution of VCP-related phenotypes, being FTD prevalence higher among women as compared to men (51.2 % vs 31.2 %) and IBM prevalence higher among men as compared to women (92.1 % vs 72.8 %). Discussion: This study broadened our clinical, genetic, and imaging knowledge of VCP-related disorders.

Background The development of e-health technologies for teleconsultation and exchange of knowledge is one of the core purposes of European Reference Networks (ERNs), including the ERN EURO-NMD for rare neuromuscular diseases. Within ERNs, the Clinical Patient Management System (CPMS) is a web-based platform that seeks to boost active collaboration within and across the network, implementing data sharing. Through CPMS, it is possible to both discuss patient cases and to make patients’ data available for registries and databases in a secure way. In this view, CPMS may be considered a sort of a temporary storage for patients’ data and an effective tool for data sharing; it facilitates specialists’ consultation since rare diseases (RDs) require multidisciplinary skills, specific, and outstanding clinical experience. Following European Union (EU) recommendation, and to promote the use of CPMS platform among EURO-NMD members, a twelve-month pilot project was set up to train the 15 Italian Health Care Providers (HCPs). In this paper, we report the structure, methods, and results of the teaching course, showing that tailored, ERN-oriented, training can significantly enhance the profitable use of the CPMS. Results Throughout the training course, 45 professionals learned how to use the many features of the CPMS, eventually opening 98 panels of discussion—amounting to 82% of the total panels included in the EURO-NMD. Since clinical, genetic, diagnostic, and therapeutic data of patients can be securely stored within the platform, we also highlight the importance of this platform as an effective tool to discuss and share clinical cases, in order to ease both case solving and data storing. Conclusions In this paper, we discuss how similar course could help implementing the use of the platform, highlighting strengths and weaknesses of e-health for ERNs. The expected result is the creation of a “map” of neuromuscular patients across Europe that might be improved by a wider use of CPMS.

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.