Loss of Calpain 3 dysregulates store-operated calcium entry and its exercise response in mice.

Limb-Girdle Muscular Dystrophy R1/2A (LGMD R1/2A) is caused by mutations in the CAPN3 gene encoding Calpain 3, a skeletal-muscle specific, Ca2+-dependent protease. Localization of Calpain 3 within the triad suggests it contributes to Ca2+ homeostasis. Through live-cell Ca2+ measurements, muscle mechanics, immunofluorescence, and electron microscopy (EM) in Capn3 deficient (C3KO) and wild-type (WT) mice, we determined whether loss of Calpain 3 altered Store-Operated Calcium Entry (SOCE) activity. Direct Ca2+ influx measurements revealed loss of Capn3 elicits elevated resting SOCE and increased resting cytosolic Ca2+, supported by high incidence of calcium entry units (CEUs) observed by EM. C3KO and WT mice were subjected to a single bout of treadmill running to elicit SOCE. Within 1HR post-treadmill running, C3KO mice exhibited diminished force production in extensor digitorum longus muscles and a greater decay of Ca2+ transients in flexor digitorum brevis muscle fibers during repetitive stimulation. Striking evidence for impaired exercise-induced SOCE activation in C3KO mice included poor colocalization of key SOCE proteins, stromal-interacting molecule 1 (STIM1) and ORAI1, combined with disappearance of CEUs in C3KO muscles. These results demonstrate that Calpain 3 is a key regulator of SOCE in skeletal muscle and identify SOCE dysregulation as a contributing factor to LGMD R1/2A pathology.

Engineered mischarged transfer RNAs for correcting pathogenic missense mutations.

Missense mutations account for approximately 50% of pathogenic mutations in human genetic diseases, and most lack effective treatments. Gene therapies, gene editing, and RNA therapies, including transfer RNA (tRNA) modalities, are common strategies for genetic disease treatments. However, reported tRNA therapies are for nonsense mutations only. It has not been explored how tRNAs can be engineered to correct missense mutations. Here, we describe missense-correcting tRNAs (mc-tRNAs) as a potential therapeutic for correcting pathogenic missense mutations. Mc-tRNAs are engineered tRNAs charged with one amino acid, but read codons of another in translation. We first developed a series of fluorescent protein-based reporters that indicate the successful correction of missense mutations via restoration of fluorescence. We engineered mc-tRNAs that effectively corrected serine and arginine missense mutations in the reporters and confirmed the amino acid substitution by mass spectrometry and mc-tRNA expression by sequencing. We examined the transcriptome response to mc-tRNA expression and found some mc-tRNAs induced minimum transcriptomic changes. Furthermore, we applied an mc-tRNA to rescue a pathogenic CAPN3 Arg-to-Gln mutant involved in LGMD2A. These results establish a versatile pipeline for mc-tRNA engineering and demonstrate the potential of mc-tRNA as an alternative therapeutic platform for the treatment of genetic disorders.

Two Novel Variants of the CAPN3 Gene in Chinese Patients with Limb-Girdle Muscular Dystrophy Recessive 1.

INTRODUCTION: Recessive mutations in the CAPN3 gene can lead to limb-girdle muscular dystrophy recessive 1 (LGMD R1). Targeted next-generation sequencing facilitates the discovery of new mutations linked with disease, owing to its ability to selectively enrich specific genomic regions. METHODS: We performed targeted next-generation sequencing of all exons of the CAPN3 gene in 4 patients with sporadic limb-girdle muscular dystrophy (LGMD) and further analyzed the effects of the novel identified variant using various software tools. RESULTS: We found 5 variants in CAPN3 gene in 4 patients, c.82_83insC (insertion mutation) and c.1115+2T>C (splicing mutation) are reported for the first time in CAPN3 (NM_000070.2). The bioinformatics analysis indicated that these two novel variants affected CAPN3 transcription as well as translation. DISCUSSION: Our findings reveal previously unreported splicing mutation and insertion mutation in CAPN3 gene, further expanding the pathogenic gene profile of LGMD.

Clinical and molecular characterization of limb-girdle muscular dystrophy 2G/R7 in a large cohort of Brazilian patients.

Limb-girdle muscular dystrophy type 2G/R7 (LGMD2G/R7) is an ultra-rare condition initially identified within the Brazilian population. We aimed to expand clinical and genetic information about this disease, including its worldwide distribution. A multicenter historical cohort study was performed at 13 centers in Brazil in which data from index cases and their affected relatives from consecutive families with LGMD2G/R7 were reviewed from July 2017 to August 2023. Additionally, a systematic literature review was conducted to identify case reports and series of the disease worldwide. Forty-one LGMD2G/R7 cases were described in the Brazilian cohort, being all subjects homozygous for the c.157C>T/(p.Gln53*) variant in TCAP. Survival curves showed that the median disease duration before individuals required walking aids was 21 years. Notably, women exhibited a slower disease progression, requiring walking aids 13 years later than men. LGMD2G/R7 was frequently reported not only in Brazil but also in China and Bulgaria, with 119 cases identified globally, with possible founder effects in the Brazilian, Eastern European, and Asian populations. These findings are pivotal in raising awareness of LGMD2G/R7, understanding its progression, and identifying potential modifiers. This can significantly contribute to the development of future natural history studies and clinical trials for this disease.

Quantitative muscle magnetic resonance imaging in limb-girdle muscular dystrophy type R1 (LGMDR1): A prospective longitudinal cohort study.

Limb-girdle muscular dystrophy (LGMD) type R1 (LGMDR1) is the most common subtype of LGMD in Europe. Prospective longitudinal data, including clinical assessments and new biomarkers such as quantitative magnetic resonance imaging (qMRI), are needed to evaluate the natural course of the disease and therapeutic options. We evaluated eight thigh and seven leg muscles of 13 LGMDR1 patients (seven females, mean age 36.7 years, body mass index 23.9 kg/m2) and 13 healthy age- and gender-matched controls in a prospective longitudinal design over 1 year. Clinical assessment included testing for muscle strength with quick motor function measure (QMFM), gait analysis and patient questionnaires (neuromuscular symptom score, activity limitation [ACTIVLIM]). MRI scans were performed on a 3-T MRI scanner, including a Dixon-based sequence, T2 mapping and diffusion tensor imaging. The qMRI values of fat fraction (FF), water T2 relaxation time (T2), fractional anisotropy, mean diffusivity, axial diffusivity and radial diffusivity were analysed. Within the clinical outcome measures, significant deterioration between baseline and follow-up was found for ACTIVLIM (p = 0.029), QMFM (p = 0.012). Analysis of qMRI parameters of the patient group revealed differences between time points for both FF and T2 when analysing all muscles (FF: p < 0.001; T2: p = 0.016). The highest increase of fat replacement was found in muscles with an FF of between 10% and 50% at baseline. T2 in muscles with low-fat replacement increased significantly. No significant differences were found for the diffusion metrics. Significant correlations between qMRI metrics and clinical assessments were found at baseline and follow-up, while only T2 changes in thigh muscles correlated with changes in ACTIVLIM over time (ρ = -0.621, p < 0.05). Clinical assessments can show deterioration of the general condition of LGMDR1 patients. qMRI measures can give additional information about underlying pathophysiology. Further research is needed to establish qMRI outcome measures for clinical trials.

Validation of a novel western blot assay to monitor patterns and levels of alpha dystroglycan in skeletal muscle of patients with limb girdle muscular dystrophies.

The cell membrane protein, dystroglycan, plays a crucial role in connecting the cytoskeleton of a variety of mammalian cells to the extracellular matrix. The α-subunit of dystroglycan (αDG) is characterized by a high level of glycosylation, including a unique O-mannosyl matriglycan. This specific glycosylation is essential for binding of αDG to extracellular matrix ligands effectively. A subset of muscular dystrophies, called dystroglycanopathies, are associated with aberrant, dysfunctional glycosylation of αDG. This defect prevents myocytes from attaching to the basal membrane, leading to contraction-induced injury. Here, we describe a novel Western blot (WB) assay for determining levels of αDG glycosylation in skeletal muscle tissue. The assay described involves extracting proteins from fine needle tibialis anterior (TA) biopsies and separation using SDS-PAGE followed by WB. Glycosylated and core αDG are then detected in a multiplexed format using fluorescent antibodies. A practical application of this assay is demonstrated with samples from normal donors and patients diagnosed with LGMD2I/R9. Quantitative analysis of the WB, which employed the use of a normal TA derived calibration curve, revealed significantly reduced levels of αDG in patient biopsies relative to unaffected TA. Importantly, the assay was able to distinguish between the L276I homozygous patients and a more severe form of clinical disease observed with other FKRP variants. Data demonstrating the accuracy and reliability of the assay are also presented, which further supports the potential utility of this novel assay to monitor changes in ⍺DG of TA muscle biopsies in the evaluation of potential therapeutics.

Defining clinical endpoints in limb girdle muscular dystrophy: a GRASP-LGMD study.

BACKGROUND: The Limb Girdle Muscular Dystrophies (LGMDs) are characterized by progressive weakness of the shoulder and hip girdle muscles as a result of over 30 different genetic mutations. This study is designed to develop clinical outcome assessments across the group of disorders. METHODS/DESIGN: The primary goal of this study is to evaluate the utility of a set of outcome measures on a wide range of LGMD phenotypes and ability levels to determine if it would be possible to use similar outcomes between individuals with different phenotypes. We will perform a multi-center, 12-month study of 188 LGMD patients within the established Genetic Resolution and Assessments Solving Phenotypes in LGMD (GRASP-LGMD) Research Consortium, which is comprised of 11 sites in the United States and 2 sites in Europe. Enrolled patients will be clinically affected and have mutations in CAPN3 (LGMDR1), ANO5 (LGMDR12), DYSF (LGMDR2), DNAJB6 (LGMDD1), SGCA (LGMDR3), SGCB (LGMDR4), SGCD (LGMDR6), or SGCG (LGMDR5, or FKRP-related (LGMDR9). DISCUSSION: To the best of our knowledge, this will be the largest consortium organized to prospectively validate clinical outcome assessments (COAs) in LGMD at its completion. These assessments will help clinical trial readiness by identifying reliable, valid, and responsive outcome measures as well as providing data driven clinical trial decision making for future clinical trials on therapeutic agents for LGMD. The results of this study will permit more efficient clinical trial design. All relevant data will be made available for investigators or companies involved in LGMD therapeutic development upon conclusion of this study as applicable. TRIAL REGISTRATION: Clinicaltrials.gov NCT03981289; Date of registration: 6/10/2019.

Exome sequencing revealed variants in SGCA and SIL1 genes underlying limb girdle muscular dystrophy and Marinesco-Sjögren syndrome patients.

BACKGROUND: Inherited neuromuscular (NMD) and neurodegenerative diseases (NDD) belong to two distinct categories that disturb different components of the nervous system, leading to a variety of different symptoms and clinical manifestations. Both NMD and NDD are a heterogeneous group of genetic conditions. Genetic variations in the SGCA and SIL1 genes have been implicated in causing Limb Girdle Muscular Dystrophy (LGMD), a type of neuromuscular disorder, and Marinesco-Sjögren Syndrome (MSS) which is a neurodegenerative disorder. METHODS: In the present study, we have investigated four patients presenting LGMD and five patients with MSS features. After collecting detailed clinical and family history, necessary laboratory investigations, including estimation of a skeletal muscle marker enzyme serum creatine kinase (CK), nerve conduction study (NCS), electromyography (EMG), echocardiography (Echo), Magnetic resonance imaging (MRI -brain), CT-brain and X-rays were performed. Whole exome followed by Sanger sequencing was employed to search for the disease-causing variants. RESULTS: Physical examination in LGMD patients revealed poor muscle tone and facing difficulty in straightening up from the floor. Clinical history revealed frequent falls and strenuousness in climbing stairs. They started toe-walking in early childhood. Laboratory investigations confirmed elevated CK levels and abnormal NCS and EMG. The MSS patients showed abnormalities in gate and jerking movement, abnormal speech, and strabismus with cataract. MRI-brain showed cerebral atrophy in some MSS patients with elevated CK levels. Whole exome sequencing revealed a nonsense variant [c.C574T, p.(Arg192*)] in the SGCA gene and a frameshift [c.936dupG, p.(Leu313AlaFs*39)] in the SIL1 gene in LGMD and MSS patients, respectively. CONCLUSION: Our study emphasizes the significance of integrating clinical and genetic analyses for precise diagnosis and tailored management strategies in inherited NMD and NDD disorders. To the best of our knowledge, this is the first study documenting SGCA and SIL1 recurrent variants in subcontinent populations with few rare clinical features. The recurrent mutations expanding the global understanding of the mutation's geographic and ethnic distribution and contributing valuable epidemiological data. The study will facilitate genetic counseling for families experiencing similar clinical features, both within Pakistani populations and in other regions.

A novel homozygous variant (c.5876T > C: p. Leu1959Pro) in DYSF segregates with limb-girdle muscular dystrophy: a case report.

BACKGROUND: Limb girdle muscular dystrophies (LGMDs) constitute a heterogeneous group of neuromuscular disorders with a very variable clinical presentation and overlapping traits. The clinical symptoms of LGMD typically appear in adolescence or early adulthood. Genetic variation in the dysferlin gene (DYSF) has been associated with LGMD. METHODS: We characterized a recessive LGMD in a young adult from consanguineous Irani families using whole-exome sequencing (WES) technology. Sanger sequencing was performed to verify the identified variant. Computational modeling and protein-protein docking were used to investigate the impact of the variant on the structure and function of the DYSF protein. RESULTS: By WES, we identified a novel homozygous missense variant in DYSF (NM_003494.4: c.5876T > C: p. Leu1959Pro) previously been associated with LGMD phenotypes. CONCLUSIONS: The identification and validation of new pathogenic DYSF variant in the present study further highlight the importance of this gene in LGMD.

The Influence of a Genetic Variant in CCDC78 on LMNA-Associated Skeletal Muscle Disease.

Mutations in the LMNA gene-encoding A-type lamins can cause Limb-Girdle muscular dystrophy Type 1B (LGMD1B). This disease presents with weakness and wasting of the proximal skeletal muscles and has a variable age of onset and disease severity. This variability has been attributed to genetic background differences among individuals; however, such variants have not been well characterized. To identify such variants, we investigated a multigeneration family in which affected individuals are diagnosed with LGMD1B. The primary genetic cause of LGMD1B in this family is a dominant mutation that activates a cryptic splice site, leading to a five-nucleotide deletion in the mature mRNA. This results in a frame shift and a premature stop in translation. Skeletal muscle biopsies from the family members showed dystrophic features of variable severity, with the muscle fibers of some family members possessing cores, regions of sarcomeric disruption, and a paucity of mitochondria, not commonly associated with LGMD1B. Using whole genome sequencing (WGS), we identified 21 DNA sequence variants that segregate with the family members possessing more profound dystrophic features and muscle cores. These include a relatively common variant in coiled-coil domain containing protein 78 (CCDC78). This variant was given priority because another mutation in CCDC78 causes autosomal dominant centronuclear myopathy-4, which causes cores in addition to centrally positioned nuclei. Therefore, we analyzed muscle biopsies from family members and discovered that those with both the LMNA mutation and the CCDC78 variant contain muscle cores that accumulated both CCDC78 and RyR1. Muscle cores containing mislocalized CCDC78 and RyR1 were absent in the less profoundly affected family members possessing only the LMNA mutation. Taken together, our findings suggest that a relatively common variant in CCDC78 can impart profound muscle pathology in combination with a LMNA mutation and accounts for variability in skeletal muscle disease phenotypes.

High Prevalence of a c.5979dupA Variant in the Dysferlin Gene (DYSF) in Individuals from a Semiarid Region of Brazil.

Background: Dysferlinopathies represent a group of limb girdle or distal muscular dystrophies with an autosomal-recessive inheritance pattern resulting from the presence of pathogenic variants in the dysferlin gene (DYSF). Objective: In this work, we describe a population from a small city in Brazil carrying the c.5979dupA pathogenic variant of DYSF responsible for limb girdle muscular dystrophy type 2R and distal muscular dystrophy. Methods: Genotyping analyses were performed by qPCR using customized probe complementary to the region with the duplication under analysis in the DYSF. Results: A total of 104 individuals were examined. c.5979dupA was identified in 48 (46.15%) individuals. Twenty-three (22%) were homozygotes, among whom 13 (56.5%) were female. A total of 91.3% (21) of homozygous individuals had a positive family history, and seven (30.4%) reported consanguineous marriages. Twenty-five (24%) individuals were heterozygous (25.8±16 years) for the same variant, among whom 15 (60%) were female. The mean CK level was 697 IU for homozygotes, 140.5 IU for heterozygotes and 176 IU for wild-type homo-zygotes. The weakness distribution pattern showed 17.3% of individuals with a proximal pattern, 13% with a distal pattern and 69.6% with a mixed pattern. Fatigue was present in 15 homozygotes and one heterozygote. Conclusion: The high prevalence of this variant in individuals from this small community can be explained by a possible founder effect associated with historical, geographical and cultural aspects.

Co-Occurrence of Myotonic Dystrophy Type 1 and Limb-Girdle Muscular Dystrophy Type 2B: A Case Report.

Introduction: Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disease whose pattern of weakness is predominantly distal. Limb-girdle muscular dystrophy type 2B/R2-dysferlin-related (LGMD2B/R2) is another neuromuscular disease, which presents an autosomal recessive inheritance and is marked by proximal muscle weakness. Even if uncommon, comorbid inherited pathologies must be considered in cases of atypical presentations, especially in those with family history of consanguinity. Case Presentation: Herein, we report the unique case of a patient diagnosed with both DM1 and LGMD2B/R2: a 38-year-old woman in follow-up of DM1 in a neuromuscular disease service presenting prominent proximal weakness. The patient's parents were consanguineous, and creatine kinase levels were elevated. A multi-gene panel test was performed and revealed the diagnosis of LGMD2B/R2. Conclusion: Genetic diseases with atypical presentations should raise the possibility of a second disorder, prompting an appropriate investigation. Overlooking a second diagnosis can implicate in not offering adequate genetic counseling, support, or specific treatment.

Three novel missense variants in two families with JAG2-associated limb-girdle muscular dystrophy.

Limb-girdle muscular dystrophy recessive 27 is associated with biallelic variants in JAG2, encoding the JAG2 notch ligand. Twenty-four affected individuals from multiple families have been described in two reports. We present two Australian families with three novel JAG2 missense variants: (c.1021G>T, p.(Gly341Cys)) homozygous in two siblings of Pakistani origin, and compound heterozygous variants (c.703T>C, p.(Trp235Arg); c.2350C>T, p.(Arg784Cys)) in a proband of European ancestry. Patients presented with childhood-onset limb-girdle-like myopathy with difficulty or inability walking. MRI revealed widespread torso and limb muscle involvement. Muscle pathology showed myopathic changes with fatty infiltration. Muscle RNA sequencing revealed significant downregulation of myogenesis genes PAX7, MYF5, and MEGF10 similar to previous JAG2-related muscular dystrophy cases or Jag2-knockdown cells. In absence of functional assays to characterise JAG2 variants, clinical, MRI and transcriptomic profiling collectively may help discern JAG2-related muscular dystrophy, diagnosis of which is essential for patients and families given the severity of disease and reoccurrence risk.

Assessment of the quality of life in patients with LGMD. The case of transportinopathy.

The Quality of Life (QOL) is influenced by several disease-related factors, support, resources, expectations, and aspirations, within the disease-related concepts. The Individualized Neuromuscular Quality of Life (INQoL) is a validated muscle disease-specific measure of the QoL developed from the experiences of patients with muscle disease and can be used for people or large cohorts. This review of QoL in transportinopathy cases reports adjustments in an autosomal dominant (AD) LGMD, and a comparison is made with autosomal recessive (AR) LGMD evaluated by INQoL. The locus for this form of LGMD with AD inheritance was found on chromosome 7, and then identification of the gene and its encoded protein (transportin-3) was obtained in 2013. A large three-generation family with several branches in Spain and Italy was previously reported and described in detail. Some patients had an early onset weakness, but others had an adult onset of the disease, as late as 58 years. The severity of the appearance of the phenotype is correlated with QoL and progresses with age. Assessing the impact on their QoL is particularly relevant to know whether the treatment is reducing their suffering.

A Case of a Patient with Calpainopathy Carrying Compound Heterozygous Mutations of a de novo Pathogenic Variant of c.1333G>A and a Novel Variant of c.1331C>T in CAPN3.

Calpainopathy is primarily an autosomal recessive inherited myopathy; however, dominantly inherited cases with a pathogenic variant of c.1333G>A have been reported. A 13-year-old Japanese girl presented with toe walking and elevated serum creatine kinase levels. Genetic panel testing revealed compound heterozygosity for c.1333G>A and a novel variant of c.1331C>T in CAPN3, leading to a diagnosis of calpainopathy. A genetic analysis of her parents revealed the possibility that c.1333G>A was de novo. In this patient, the onset age was earlier than that of the reported autosomal dominant cases, suggesting the influence of the novel variant in the contralateral allele.

Identification and functional characterization of a novel heterozygous splice­site mutation in the calpain 3 gene causes rare autosomal dominant limb­girdle muscular dystrophy.

Limb-girdle muscular dystrophies are a group of extremely heterogenous neuromuscular disorders that manifest with gradual and progressive weakness of both proximal and distal muscles. Autosomal dominant limb-girdle muscular dystrophy (LGMDD4) or calpainopathy is a very rare form of myopathy characterized by weakness and atrophy of both proximal and distal muscles with a variable age of onset. LGMDD4 is caused by germline heterozygous mutations of the calpain 3 (CAPN3) gene. Patients with LGMDD4 often show extreme phenotypic heterogeneity; however, most patients present with gait difficulties, increased levels of serum creatine kinase, myalgia and back pain. In the present study, a 16-year-old male patient, clinically diagnosed with LGMDD4, was investigated. The proband had been suffering from weakness and atrophy of both of their proximal and distal muscles, and had difficulty walking and standing independently. The serum creatine kinase levels (4,754 IU/l; normal, 35-232 IU/l) of the patient were markedly elevated. The younger sister and mother of the proband were also clinically diagnosed with LGMDD4, while the father was phenotypically normal. Whole exome sequencing identified a heterozygous novel splice-site (c.2440-1G>A) mutation in intron 23 of the CAPN3 gene in the proband. Sanger sequencing confirmed that this mutation was also present in both the younger sister and mother of the proband, but the father was not a carrier of this mutation. This splice-site (c.2440-1G>A) mutation causes aberrant splicing of CAPN3 mRNA, leading to the skipping of the last exon (exon 24) of CAPN3 mRNA and resulting in the removal of eight amino acids from the C-terminal of domain IV of the CAPN3 protein. Hence, this splice site mutation causes the formation of a truncated CAPN3 protein (p.Trp814*) of 813 amino acids instead of the wild-type CAPN3 protein that consists of 821 amino acids. This mutation causes partial loss of domain IV (PEF domain) in the CAPN3 protein, which is involved in calcium binding and homodimerization; therefore, this is a loss-of-function mutation. Relative expression of the mutated CAPN3 mRNA was reduced in comparison with the wild-type CAPN3 mRNA in the proband, and their younger sister and mother. This mutation was also not present in 100 normal healthy control individuals of the same ethnicity. The present study reported the first case of CAPN3 gene-associated LGMDD4 in the Chinese population.

Molecular Diagnosis of Limb-Girdle Muscular Dystrophy Using Next-Generation Sequencing Panels.

Introduction: Limb-girdle muscular dystrophies (LGMDs) are clinically and genetically heterogeneous muscle disorders. We aimed to share the diagnostic yield of an NGS gene panel containing LGMD-related genes and our experience with LGMD. Methods: Between February 2019 and October 2022, patients with a suspicion of LGMD and their relatives were reviewed in terms of demographic, clinical, and individual genetic data, age of symptom onset, sex, clinical features, LGMD types, cardiac involvement, muscle biopsy results, family history, and consanguinity. Our NGS gene panel consisted of ANO5, CAPN3, CAV3, DAG1, DES, DNAJB6, DYSF, FKTN, FLNC, FRKP, GAA, GMPPB, HNRNPDL, ISPD, LIMS2, LMNA, MYOT, PLEC, POMGNT1, POMK, POMT1, POMT2, SGCA, SGCB, SGCD, SGCG, TCAP, TNPO3, TRAPPC11, TRIM32, and TTN genes. Results: The diagnosis rate was 61.1% (11/18). Twelve (80%) patients with LGMD were male and three (20%) were female. The median age was 15.9 (range, 1.5-39) years. Our patient collective was drawn up out of patients with the following variants: LGMDR1 (n = 6; 40%), LGMDR2 (n = 4; 26.6%), LGMDR3 (n = 4; 26.6%), and LGMDR12 (n = 1; 6.7%). Conclusion: The present study showed that the NGS panel has a high success rate in the diagnosis of LGMD and contributes to early diagnosis.

Pilot investigations into the mechanistic basis for adverse effects of glucocorticoids in dysferlinopathy.

BACKGROUND: Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by gene mutations resulting in deficiency of the membrane-associated protein dysferlin. They manifest post-growth and are characterised by muscle wasting (primarily in the limb and limb-gridle muscles), inflammation, and replacement of myofibres with adipose tissue. The precise pathomechanism for dysferlinopathy is currently unclear; as such there are no treatments currently available. Glucocorticoids (GCs) are widely used to reduce inflammation and treat muscular dystrophies, but when administered to patients with dysferlinopathy, they have unexpected adverse effects, with accelerated loss of muscle strength. METHODS: To investigate the mechanistic basis for the adverse effects of GCs in dysferlinopathy, the potent GC dexamethasone (Dex) was administered for 4-5 weeks (0.5-0.75 µg/mL in drinking water) to dysferlin-deficient BLA/J and normal wild-type (WT) male mice, sampled at 5 (Study 1) or 10 months (Study 2) of age. A wide range of analyses were conducted. Metabolism- and immune-related gene expression was assessed in psoas muscles at both ages and in quadriceps at 10 months of age. For the 10-month-old mice, quadriceps and psoas muscle histology was assessed. Additionally, we investigated the impact of Dex on the predominantly slow and fast-twitch soleus and extensor digitorum longus (EDL) muscles (respectively) in terms of contractile function, myofibre-type composition, and levels of proteins related to contractile function and metabolism, plus glycogen. RESULTS: At both ages, many complement-related genes were highly expressed in BLA/J muscles, and WT mice were generally more responsive to Dex than BLA/J. The effects of Dex on BLA/J mice included (i) increased expression of inflammasome-related genes in muscles (at 5 months) and (ii) exacerbated histopathology of quadriceps and psoas muscles at 10 months. A novel observation was pronounced staining for glycogen in many myofibres of the damaged quadriceps muscles, with large pale vacuolated myofibres, suggesting possible myofibre death by oncosis. CONCLUSION: These pilot studies provide a new focus for further investigation into the adverse effects of GCs on dysferlinopathic muscles.

Evaluation of Neuromuscular Diseases and Complaints by Quantitative Muscle MRI.

Background: Quantitative muscle MRI (qMRI) is a promising tool for evaluating and monitoring neuromuscular disorders (NMD). However, the application of different imaging protocols and processing pipelines restricts comparison between patient cohorts and disorders. In this qMRI study, we aim to compare dystrophic (limb-girdle muscular dystrophy), inflammatory (inclusion body myositis), and metabolic myopathy (Pompe disease) as well as patients with post-COVID-19 conditions suffering from myalgia to healthy controls. Methods: Ten subjects of each group underwent a 3T lower extremity muscle MRI, including a multi-echo, gradient-echo, Dixon-based sequence, a multi-echo, spin-echo (MESE) T2 mapping sequence, and a spin-echo EPI diffusion-weighted sequence. Furthermore, the following clinical assessments were performed: Quick Motor Function Measure, patient questionnaires for daily life activities, and 6-min walking distance. Results: Different involvement patterns of conspicuous qMRI parameters for different NMDs were observed. qMRI metrics correlated significantly with clinical assessments. Conclusions: qMRI metrics are suitable for evaluating patients with NMD since they show differences in muscular involvement in different NMDs and correlate with clinical assessments. Still, standardisation of acquisition and processing is needed for broad clinical use.

Identification of LAMA2 compound heterozygous variants: a case report.

Background: Laminin-α2 (LAMA2) chain-deficient muscular dystrophy (LAMA2-MD) is the most common congenital muscular dystrophy (CMD) in the world. Its main manifestations are muscle weakness and hypotonia that occur after birth or at early infancy. Case Description: We reported a case of a 3-year-old and 6-month-old boy presented with delayed motor development, elevated creatine kinase (CK) levels, and abnormal white matter in the brain. Whole exome sequencing (WES) showed compound heterozygous variants of the LAMA2 gene. This case reports for the first time the compound heterozygous LAMA2 variants c.5476C>T (p.R1826*) (paternal inheritance) with c.2749 + 2dup (maternal inheritance), as both variants are interpreted as pathogenic/potentially pathogenic variants. Conclusions: This study reports a novel heterozygous variant, including two pathogenic variants in the LAMA2 gene, and highlights the effectiveness of highly efficient exome sequencing applying in patients with undefined CMDs.