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.

TRAPPC11-CDG muscular dystrophy: Review of 54 cases including a novel patient.

The trafficking protein particle (TRAPP) complex is a multisubunit protein complex that functions as a tethering factor involved in intracellular trafficking. TRAPPC11, a crucial subunit of this complex, is associated with pathogenic variants that cause a spectrum of disease, which can range from a limb girdle muscular dystrophy (LGMD) to developmental disability with muscle disease, movement disorder and global developmental delay (GDD)/intellectual disability (ID), or even a congenital muscular dystrophy (CMD). We reviewed the phenotype of all reported individuals with TRAPPC11-opathies, including an additional Mexican patient with novel compound heterozygous missense variants in TRAPPC11 (c.751 T > C and c.1058C > G), restricted to the Latino population. In these 54 patients muscular dystrophy signs are common (early onset muscle weakness, increased serum creatine kinase levels, and dystrophic changes in muscle biopsy). They present two main phenotypes, one with a slowly progressive LGMD with or without GDD/ID (n = 12), and another with systemic involvement characterized by short stature, GDD/ID, microcephaly, hypotonia, poor speech, seizures, cerebral atrophy, cerebellar abnormalities, movement disorder, scoliosis, liver disease, and cataracts (n = 42). In 6 of them CMD was identified. Obstructive hydrocephaly, retrocerebellar cyst, and talipes equinovarus found in the individual reported here has not been described in TRAPPC11 deficiency. As in previous patients, membrane trafficking assays in our patient showed defective abnormal endoplasmic reticulum-Golgi transport as well as decreased expression of LAMP2, and ICAM-1 glycoproteins. This supports previous statements that TRAPPC11-opathies are in fact a congenital disorder of glycosylation (CDG) with muscular dystrophy.

A knock down strategy for rapid, generic, and versatile modelling of muscular dystrophies in 3D-tissue-engineered-skeletal muscle.

BACKGROUND: Human iPSC-derived 3D-tissue-engineered-skeletal muscles (3D-TESMs) offer advanced technology for disease modelling. However, due to the inherent genetic heterogeneity among human individuals, it is often difficult to distinguish disease-related readouts from random variability. The generation of genetically matched isogenic controls using gene editing can reduce variability, but the generation of isogenic hiPSC-derived 3D-TESMs can take up to 6 months, thereby reducing throughput. METHODS: Here, by combining 3D-TESM and shRNA technologies, we developed a disease modelling strategy to induce distinct genetic deficiencies in a single hiPSC-derived myogenic progenitor cell line within 1 week. RESULTS: As proof of principle, we recapitulated disease-associated pathology of Duchenne muscular dystrophy and limb-girdle muscular dystrophy type 2A caused by loss of function of DMD and CAPN3, respectively. shRNA-mediated knock down of DMD or CAPN3 induced a loss of contractile function, disruption of tissue architecture, and disease-specific proteomes. Pathology in DMD-deficient 3D-TESMs was partially rescued by a candidate gene therapy treatment using micro-dystrophin, with similar efficacy compared to animal models. CONCLUSIONS: These results show that isogenic shRNA-based humanized 3D-TESM models provide a fast, cheap, and efficient tool to model muscular dystrophies and are useful for the preclinical evaluation of novel therapies.

Novel Biomarkers for Limb Girdle Muscular Dystrophy (LGMD).

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.

The CAPN3 p.Lys 254del variant is not always associated with dominant CAPN3-related muscular dystrophy.

INTRODUCTION/AIMS: Limb-girdle muscular dystrophy R1 (LGMDR1) calpain 3-related usually presents as a recessively transmitted weakness of proximal limb-girdle muscles due to pathogenic variants in the CAPN3 gene. Pathogenic variants in this gene have also been found in patients with an autosomal dominantly inherited transmission pattern (LGMDD4). The mechanism underlying this difference in transmission patterns has not yet been elucidated. Camptocormia, progressive limb weakness, myalgia, back pain, and increased CK levels are common clinical features associated with dominant forms. The p.Lys254del pathogenic variant was associated with camptocormia in two LGMDD4 families. This study aimed to present carriers found in recessively transmitted LGMDR1 families bearing the p.Lys254del variant that do not show muscle weakness. METHODS: DNA sequencing was performed on exon 5 of CAPN3 in family members to establish the carrier status of the pathogenic variant. They were evaluated clinically and MRI was performed when available. RESULTS: Two families presented with the p.Lys254del pathogenic variant in a homozygous or compound heterozygous state. Family members carrying only the pathogenic variant in the heterozygous state did not demonstrate the myopathic characteristics described in dominant patients. Camptocormia and other severe clinical symptoms were not observed. DISCUSSION: We conclude that the p.Lys254del pathogenic variant per se cannot be solely responsible for camptocormia in dominant patients. Other undisclosed factors may regulate the phenotype associated with the dominant inheritance pattern in CAPN3 pathogenic variant carriers.

Differential histological features and myogenic protein levels in distinct muscles of d-sarcoglycan null muscular dystrophy mouse model.

Skeletal muscle (SkM) comprises slow and fast-twitch fibers, which differ in molecular composition, function, and systemic energy consumption. In addition, muscular dystrophies (DM), a group of diverse hereditary diseases, present different patterns of muscle involvement, progression, and severity, suggesting that the regeneration-degeneration process may differ depending on the muscle type. Therefore, the study aimed to explore the expression of proteins involved in the repair process in different muscles at an early stage of muscular dystrophy in the δ-sarcoglycan null mice (Sgcd-null), a limb-girdle muscular dystrophy 2 F model. Hematoxylin & Eosin (H&E) Staining showed a high number of central nuclei in soleus (Sol), tibialis (Ta), gastrocnemius (Gas), and extensor digitorum longus (Edl) from four months Sgcd-null mice. However, fibrosis, determined by trichrome of Gomori modified staining, was only observed in Sgcd-null Sol. In addition, the number of Type I and II fibers variated differentially in the Sgcd-null muscles vs. wild-type muscles. Besides, the protein expression level of ß-catenin, myomaker, MyoD, and myogenin also presented different expression levels in all the Sgcd-null muscles studied. In summary, our study reveals that muscles with different metabolic characteristics showed distinct expression patterns of proteins involved in the muscle regeneration process. These results could be relevant in designing therapies for genetic and acquired myopathy.

The clinical features and TCAP mutation spectrum in a Chinese cohort of patients with limb-girdle muscular dystrophy R7.

Limb-girdle muscular dystrophy R7 (LGMDR7) is an autosomal recessive hereditary muscular dystrophy caused by mutations in titin-cap (TCAP). Here, we summarized the clinical characteristics and TCAP mutations in a Chinese cohort of 30 patients with LGMDR7. The onset age of Chinese patients was 19.89 ± 6.70 years old, which is later than European and South Asian patients (P < 0.05). Clinically speaking, 20.0% of patients presented with predominant distal weakness, and 73.3% of patients presented with predominant pelvic girdle weakness. Radiological study revealed semitendinosus and magnus adductor were severely involved in Chinese LGMDR7 patients. Rectus femoris, vastus lateralis, vastus intermedius, soleus and tibialis anterior were moderately to severely involved. The most prevalent mutation in this cohort is c.26_33dupAGGTGTCG, while c.165dupG and c.110 + 5G > A are unique in Chinese population as two of the common mutations. Besides, variant c.26_33dupAGGGTGTCG might be a founder mutation in Asian patients. Internal nuclei, lobulated fibers, and scattered rimmed vacuoles were typical morphological changes in Chinese LGMDR7 patients. This is the largest LGMDR7 cohort in the Chinese population and in the world. This article also expands the clinical, pathological, mutational and radiological spectrum of patients with LGMDR7 in China and in the world.

Histopathological correlations and fat replacement imaging patterns in recessive limb-girdle muscular dystrophy type 12.

BACKGROUND: Despite the widespread use of proton density fat fraction (PDFF) measurements with magnetic resonance imaging (MRI) to track disease progression in muscle disorders, it is still unclear how these findings relate to histopathological changes in muscle biopsies of patients with limb-girdle muscular dystrophy autosomal recessive type 12 (LGMDR12). Furthermore, although it is known that LGMDR12 leads to a selective muscle involvement distinct from other muscular dystrophies, the spatial distribution of fat replacement within these muscles is unknown. METHODS: We included 27 adult patients with LGMDR12 and 27 age-matched and sex-matched healthy controls and acquired 6-point Dixon images of the thighs and T1 and short tau inversion recovery (STIR) MR images of the whole body. In 16 patients and 15 controls, we performed three muscle biopsies, one in the semimembranosus, vastus lateralis, and rectus femoris muscles, which are severely, intermediately, and mildly affected in LGMDR12, respectively. We correlated the PDFF to the fat percentage measured on biopsies of the corresponding muscles, as well as to the Rochester histopathology grading scale. RESULTS: In patients, we demonstrated a strong correlation of PDFF on MRI and muscle biopsy fat percentage for the semimembranosus (r = 0.85, P < 0.001) and vastus lateralis (r = 0.68, P = 0.005). We found similar results for the correlation between PDFF and the Rochester histopathology grading scale. Out of the five patients with inflammatory changes on muscle biopsy, three showed STIR hyperintensities in the corresponding muscle on MRI. By modelling the PDFF on MRI for 18 thigh muscles from origin to insertion, we observed a significantly inhomogeneous proximo-distal distribution of fat replacement in all thigh muscles of patients with LGMDR12 (P < 0.001), and different patterns of fat replacement within each of the muscles. CONCLUSIONS: We showed a strong correlation of fat fraction on MRI and fat percentage on muscle biopsy for diseased muscles and validated the use of Dixon fat fraction imaging as an outcome measure in LGMDR12. The inhomogeneous fat replacement within thigh muscles on imaging underlines the risk of analysing only samples of muscles instead of the entire muscles, which has important implications for clinical trials.

A homozygous loss of function variant in POPDC3: From invalidating exercise intolerance to a limb-girdle muscular dystrophy phenotype.

Recessive pathogenic variants in POPDC3 have recently been associated with the rare limb-girdle muscular dystrophy (LGMD) subtype LGMDR26. We studied three siblings and a distantly related individual with a skeletal muscle disorder, harboring the c.486-6T>A splice site variant in POPDC3 in homozygosity. Immunohistochemistry, western blot, and mRNA experiments on patients' skeletal muscle tissue as well as on patients' myoblasts were performed to study the pathogenicity of the predicted loss of function mechanism of the variant. Patients mainly presented with invalidating myalgia and exercise intolerance and limited to no segmentary muscle weakness. CK levels were markedly elevated in all patients. A loss of function mechanism at the RNA level was shown (r.485_486insauag, p.Ile163*). Muscle biopsies performed in three out of four patients showed non-specific myopathic features with a marked type 2 fiber predominance and the presence of a large number of severely atrophic fibers with pyknotic nuclear clumps. We show that skeletal muscle symptoms in LGMDR26 may range from an overt late juvenile to young adult-onset limb-girdle muscular dystrophy phenotype to severe exercise intolerance and myalgia, with consistently highly elevated CK levels. We further prove a clear LOF mechanism of POPDC3 in this rare disorder.

Minimal expression of dysferlin prevents development of dysferlinopathy in dysferlin exon 40a knockout mice.

Dysferlin is a Ca2+-activated lipid binding protein implicated in muscle membrane repair. Recessive variants in DYSF result in dysferlinopathy, a progressive muscular dystrophy. We showed previously that calpain cleavage within a motif encoded by alternatively spliced exon 40a releases a 72 kDa C-terminal minidysferlin recruited to injured sarcolemma. Herein we use CRISPR/Cas9 gene editing to knock out murine Dysf exon 40a, to specifically assess its role in membrane repair and development of dysferlinopathy. We created three Dysf exon 40a knockout (40aKO) mouse lines that each express different levels of dysferlin protein ranging from ~ 90%, ~ 50% and ~ 10-20% levels of wild-type. Histopathological analysis of skeletal muscles from all 12-month-old 40aKO lines showed virtual absence of dystrophic features and normal membrane repair capacity for all three 40aKO lines, as compared with dysferlin-null BLAJ mice. Further, lipidomic and proteomic analyses on 18wk old quadriceps show all three 40aKO lines are spared the profound lipidomic/proteomic imbalance that characterises dysferlin-deficient BLAJ muscles. Collective results indicate that membrane repair does not depend upon calpain cleavage within exon 40a and that ~ 10-20% of WT dysferlin protein expression is sufficient to maintain the muscle lipidome, proteome and membrane repair capacity to crucially prevent development of dysferlinopathy.

Effects of Glucocorticoids in Murine Models of Duchenne and Limb-Girdle Muscular Dystrophy.

In vivo testing of glucocorticoid steroids in dystrophic mice offers important insights in benefits and risks of those drugs in the pathological context of muscular dystrophy. Frequency of dosing changes the spectrum of glucocorticoid effects on muscle and metabolic homeostasis. Here, we describe a combination of non-invasive and invasive methods to quantitatively discriminate the specific effects of intermittent (once-weekly) versus mainstay (once-daily) regimens on muscle fibrosis, muscle function, and metabolic homeostasis in murine models of Duchenne and limb-girdle muscular dystrophies.

Genotype-phenotype correlation and natural history study of dysferlinopathy: a single-centre experience from India.

Dysferlinopathies are a group of limb-girdle muscular dystrophies causing significant disability in the young population. There is a need for studies on large cohorts to describe the clinical, genotypic and natural history in our subcontinent. To describe and correlate the clinical, genetic profile and natural history of genetically confirmed dysferlinopathies. We analysed a retrospective cohort of patients with dysferlinopathy from a single quaternary care centre in India. A total of 124 patients with dysferlinopathy were included (40 females). Median age at onset and duration of illness were 21 years (range, 13-50) and 48 months (range, 8-288), respectively. The average follow-up period was 60 months (range, 12-288). Fifty-one percent had LGMD pattern of weakness at onset; 23.4% each had Miyoshi and proximo-distal type while isolated hyperCKemia was noted in 1.6%. About 60% were born to consanguineous parents and 26.6% had family history of similar illness. Twenty-three patients (18.6%) lost ambulation at follow-up; the median time to loss of independent ambulation was 120 months (range, 72-264). Single-nucleotide variants (SNVs) constituted 78.2% of patients; INDELs 14.5% and 7.3% had both SNVs and INDELs. Earlier age at onset was noted with SNVs. There was no correlation between the other clinical parameters and ambulatory status with the genotype. Thirty-seven (45.7%) novel pathogenic/likely pathogenic (P/LP) variants were identified out of a total of 81 variations. The c.3191G > A variant was the most recurrent mutation. Our cohort constitutes a clinically and genetically heterogeneous group of dysferlinopathies. There is no significant correlation between the clinico-genetic profile and the ambulatory status.

Clinical exome sequencing identifies novel compound heterozygous mutations of the POMT2 gene in patients with limb-girdle muscular dystrophy.

OBJECTIVE: Mutations in protein O-mannosyltransferase 2 (POMT2) (MIM#607439) have been identified in severe congenital muscular dystrophy such as Walker-Warburg syndrome (WWS) and milder limb-girdle muscular dystrophy type 2N (LGMD2N). The aim of this study is to investigate the genetic causes in patients with LGMD2N. METHODS: Three patients diagnosed with mild limb-girdle muscular dystrophy were recruited. The genetically pathogenic variant was identified by clinical exome sequencing, and healthy controls were verified by Sanger sequencing. RESULTS: Novel compound heterozygous mutations c.800A > G and c.1074_1075delinsAT of POMT2 were revealed in one affected individual by clinical exome sequencing. There was no report of these two variants and predicted to be highly damaging to the function of the POMT2. CONCLUSION: The novel variants extend the spectrum of POMT2 mutations, which promotes the prognostic value of testing for POMT2 mutations in patients with LGMD2N.

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OBJECTIVES: To summarize the skeletal muscle magnetic resonance imaging (MRI) features of the lower limbs in common subtypes of muscular dystrophy (MD) and the experience in the application of MRI in the diagnosis of MD. METHODS: A total of 48 children with MD who were diagnosed by genetic testing were enrolled as subjects. The muscle MRI features of the lower limbs were analyzed. Cumulative fatty infiltration score was calculated for each subtype, and the correlation of cumulative fatty infiltration score with clinical indices was analyzed for Duchenne muscular dystrophy (DMD). RESULTS: DMD was characterized by the involvement of the gluteus maximus and the adductor magnus. Becker muscular dystrophy was characterized by the involvement of the vastus lateralis muscle. Limb-girdle muscular dystrophy was characterized by the involvement of the adductor magnus, the vastus intermedius, the vastus medialis, and the vastus lateralis muscle. For DMD, the cumulative fatty infiltration score of the lower limb muscles was significantly correlated with age, course of the disease, muscle strength, and motor function (P<0.05), while it was not significantly correlated with the serum creatine kinase level (P>0.05). CONCLUSIONS: Different subtypes of MD have different MRI manifestations, and MRI may help with the diagnosis and assessment of MD.

Inhibition of the immunoproteasome modulates innate immunity to ameliorate muscle pathology of dysferlin-deficient BlAJ mice.

Muscle repair in dysferlinopathies is defective. Although macrophage (Mø)-rich infiltrates are prominent in damaged skeletal muscles of patients with dysferlinopathy, the contribution of the immune system to the disease pathology remains to be fully explored. Numbers of both pro-inflammatory M1 Mø and effector T cells are increased in muscle of dysferlin-deficient BlAJ mice. In addition, symptomatic BlAJ mice have increased muscle production of immunoproteasome. In vitro analyses using bone marrow-derived Mø of BlAJ mice show that immunoproteasome inhibition results in C3aR1 and C5aR1 downregulation and upregulation of M2-associated signaling. Administration of immunoproteasome inhibitor ONX-0914 to BlAJ mice rescues muscle function by reducing muscle infiltrates and fibro-adipogenesis. These findings reveal an important role of immunoproteasome in the progression of muscular dystrophy in BlAJ mouse and suggest that inhibition of immunoproteasome may produce therapeutic benefit in dysferlinopathy.

Tripartite motif-containing protein 32 regulates Ca2+ movement in skeletal muscle.

Mutations in tripartite motif-containing protein 32 (TRIM32), especially in NHL repeats, have been found in skeletal muscle in patients with type 2H limb-girdle muscular dystrophy (LGMD2H). However, the roles of the NHL repeats of TRIM32 in skeletal muscle functions have not been well addressed. In the present study, to examine the functional role(s) of the TRIM32 NHL repeats in skeletal muscle, TRIM32-binding proteins in skeletal muscle were first searched using a binding assay and MALDI-TOF/TOF. Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) was found to be a TRIM32-binding protein. Next, a deletion mutant of TRIM32 missing the NHL repeats (NHL-Del) was expressed in mouse primary skeletal myotubes during myoblast differentiation into myotubes. Ca2+ movement in the myotubes was examined using single-cell Ca2+ imaging. Unlike wild-type (WT) TRIM32, NHL-Del did not enhance the amount of Ca2+ release from the sarcoplasmic reticulum (SR), Ca2+ release for excitation-contraction (EC) coupling, or extracellular Ca2+ entry via store-operated Ca2+ entry (SOCE). In addition, even compared with the vector control, NHL-Del resulted in reduced SOCE due to reduced expression of extracellular Ca2+ entry channels. Transmission electron microscopy (TEM) observation of the myotubes revealed that NHL-Del induced the formation of abnormal vacuoles and tubular structures in the cytosol. Therefore, by binding to SERCA1a via its NHL repeats, TRIM32 may participate in the regulation of Ca2+ movement for skeletal muscle contraction and the formation of cellular vacuoles and tubular structures in skeletal muscle. Functional defects in TRIM32 due to mutations in NHL repeats may be pathogenic toward LGMD2H.

Water T2 could predict functional decline in patients with dysferlinopathy.

BACKGROUND: Water T2 (T2H2O ) mapping is increasingly being used in muscular dystrophies to assess active muscle damage. It has been suggested as a surrogate outcome measure for clinical trials. Here, we investigated the prognostic utility of T2H2O to identify changes in muscle function over time in limb girdle muscular dystrophies. METHODS: Patients with genetically confirmed dysferlinopathy were assessed as part of the Jain Foundation Clinical Outcomes Study in dysferlinopathy. The cohort included 18 patients from two sites, both equipped with 3-tesla magnetic resonance imaging (MRI) systems from the same vendor. T2H2O value was defined as higher or lower than the median in each muscle bilaterally. The degree of deterioration on four functional tests over 3 years was assessed in a linear model against covariates of high or low T2H2O at baseline, age, disease duration, and baseline function. RESULTS: A higher T2H2O at baseline significantly correlated with a greater decline on functional tests in 21 out of 35 muscles and was never associated with slower decline. Higher baseline T2H2O in adductor magnus, vastus intermedius, vastus lateralis, and vastus medialis were the most sensitive, being associated bilaterally with greater decline in multiple timed tests. Patients with a higher than median baseline T2H2O (>40.6 ms) in the right vastus medialis deteriorated 11 points more on the North Star Ambulatory Assessment for Dysferlinopathy and lost an additional 86 m on the 6-min walk than those with a lower T2H2O (<40.6 ms). Optimum sensitivity and specificity thresholds for predicting decline were 39.0 ms in adductor magnus and vastus intermedius, 40.0 ms in vastus medialis, and 40.5 ms in vastus lateralis from different sites equipped with different MRI systems. CONCLUSIONS: In dysferlinopathy, T2H2O did not correlate with current functional ability. However, T2H2O at baseline was higher in patients who worsened more rapidly on functional tests. This suggests that inter-patient differences in functional decline over time may be, in part, explained by different severities of the active muscle damage, assessed by T2H2O measure at baseline. Significant challenges remain in standardizing T2H2O values across sites to allow determining globally applicable thresholds. The results from the present work are encouraging and suggest that T2H2O could be used to improve prognostication, patient selection, and disease modelling for clinical trials.

Antisense Morpholino-Based In Vitro Correction of a Pseudoexon-Generating Variant in the SGCB Gene.

Limb-girdle muscular dystrophies (LGMD) are clinically and genetically heterogenous presentations displaying predominantly proximal muscle weakness due to the loss of skeletal muscle fibers. Beta-sarcoglycanopathy (LGMDR4) results from biallelic molecular defects in SGCB and features pediatric onset with limb-girdle involvement, often complicated by respiratory and heart dysfunction. Here we describe a patient who presented at the age of 12 years reporting high creatine kinase levels and onset of cramps after strenuous exercise. Instrumental investigations, including a muscle biopsy, pointed towards a diagnosis of beta-sarcoglycanopathy. NGS panel sequencing identified two variants in the SGCB gene, one of which (c.243+1548T>C) was found to promote the inclusion of a pseudoexon between exons 2 and 3 in the SGCB transcript. Interestingly, we detected the same genotype in a previously reported LGMDR4 patient, deceased more than twenty years ago, who had escaped molecular diagnosis so far. After the delivery of morpholino oligomers targeting the pseudoexon in patient-specific induced pluripotent stem cells, we observed the correction of the physiological splicing and partial restoration of protein levels. Our findings prompt the analysis of the c.243+1548T>C variant in suspected LGMDR4 patients, especially those harbouring monoallelic SGCB variants, and provide a further example of the efficacy of antisense technology for the correction of molecular defects resulting in splicing abnormalities.

Recurrent, non-traumatic, non-exertional rhabdomyolysis after immunologic stimuli in a healthy adolescent female: a case report.

BACKGROUND: Dysferlinopathy refers to a heterogenous group of autosomal recessive disorders that affect a skeletal muscle protein called dysferlin. These mutations are associated with limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, asymptomatic hyperCKemia, and distal myopathy with anterior tibial onset. CASE PRESENTATION: A 16 year old female presented with myalgia, weakness and dark urine one week after her second BNT162b2 mRNA (Pfizer) vaccine. Initial serum creatine kinase (CK) was measured at 153,000 IU/L, eventually up-trending to over 200,000 IU/L. However, stable renal function precluded hemodialysis allowing discharge after 10 days of intravenous (IV) hydration and alkaline diuresis. Just two years prior to the current presentation, the patient was hospitalized following Group A Streptococcal pharyngitis infection complicated by rhabdomyolysis. She presented with fatigue, lower extremity weakness, and dark oliguria with CK measuring 984,800 IU/L. IV hydration was attempted however hemodialysis was ultimately required throughout her 24-day hospital stay. Her episode was presumed to be idiopathic and no further work-up was performed at that time. During the patient's current hospitalization, she reported similar symptomology (myalgias and weakness) following her first quadrivalent Gardasil vaccine at age 11. No hospitalization was required at that time. A comprehensive workup was now initiated while the patient was being treated for her suspected second or third non-exertional, non-traumatic rhabdomyolysis. Rheumatologic, metabolic, infectious, and endocrinologic workup were all unremarkable. Patient eventually had whole exome sequencing performed which revealed a heterozygous pathogenic variant in the DYSF gene (DYSF c.2643 + 1G > A) encoding dysferlin. No clinically significant sequelae occurred thus far. CONCLUSIONS: While there have been reports of symptomatic heterozygote carriers of dysferlinopathies, to our knowledge none have been associated with recurrent rhabdomyolysis after immunogenic stimuli. This unique case presentation highlights the importance of a multi-disciplinary care team, the utility of modern whole-exome gene sequencing, and the future challenges of balancing vaccine risk vs benefit.

A Novel Homozygous Variant in DYSF Gene Is Associated with Autosomal Recessive Limb Girdle Muscular Dystrophy R2/2B.

Mutations in the DYSF gene, encoding dysferlin, are responsible for Limb Girdle Muscular Dystrophy type R2/2B (LGMDR2/2B), Miyoshi myopathy (MM), and Distal Myopathy with Anterior Tibialis onset (MDAT). The size of the gene and the reported inter and intra familial phenotypic variability make early diagnosis difficult. Genetic analysis was conducted using Next Gene Sequencing (NGS), with a panel of 40 Muscular Dystrophies associated genes we designed. In the present study, we report a new missense variant c.5033G>A, p.Cys1678Tyr (NM_003494) in the exon 45 of DYSF gene related to Limb Girdle Muscular Dystrophy type R2/2B in a 57-year-old patient affected with LGMD from a consanguineous family of south Italy. Both healthy parents carried this variant in heterozygosity. Genetic analysis extended to two moderately affected sisters of the proband, showed the presence of the variant c.5033G>A in both in homozygosity. These data indicate a probable pathological role of the variant c.5033G>A never reported before in the onset of LGMDR2/2B, pointing at the NGS as powerful tool for identifying LGMD subtypes. Moreover, the collection and the networking of genetic data will increase power of genetic-molecular investigation, the management of at-risk individuals, the development of new therapeutic targets and a personalized medicine.