Expert Panel Curation of 31 Genes in Relation to Limb Girdle Muscular Dystrophy.

Introduction: Limb girdle muscular dystrophies (LGMDs) are a group of genetically heterogeneous autosomal conditions with some degree of phenotypic homogeneity. LGMD is defined as having onset >2 years of age with progressive proximal weakness, elevated serum creatine kinase levels and dystrophic features on muscle biopsy. Advances in massively parallel sequencing have led to a surge in genes linked to LGMD. Methods: The ClinGen Muscular Dystrophies and Myopathies gene curation expert panel (MDM GCEP, formerly Limb Girdle Muscular Dystrophy GCEP) convened to evaluate the strength of evidence supporting gene-disease relationships (GDR) using the ClinGen gene-disease clinical validity framework to evaluate 31 genes implicated in LGMD. Results: The GDR was exclusively LGMD for 17 genes, whereas an additional 14 genes were related to a broader phenotype encompassing congenital weakness. Four genes (CAPN3, COL6A1, COL6A2, COL6A3) were split into two separate disease entities, based on each displaying both dominant and recessive inheritance patterns, resulting in curation of 35 GDRs. Of these, 30 (86%) were classified as Definitive, 4 (11%) as Moderate and 1 (3%) as Limited. Two genes, POMGNT1 and DAG1, though definitively related to myopathy, currently have insufficient evidence to support a relationship specifically with LGMD. Conclusions: The expert-reviewed assertions on the clinical validity of genes implicated in LGMDs form an invaluable resource for clinicians and molecular geneticists. We encourage the global neuromuscular community to publish case-level data that help clarify disputed or novel LGMD associations.

Utilization of Targeted RNA-Seq for the Resolution of Variant Pathogenicity and Enhancement of Diagnostic Yield in Dysferlinopathy.

For inherited diseases, obtaining a definitive diagnosis is critical for proper disease management, family planning, and participation in clinical trials. This can be challenging for dysferlinopathy due to the significant clinical overlap between the 30+ subtypes of limb-girdle muscular dystrophy (LGMD) and the large number of variants of unknown significance (VUSs) that are identified in the dysferlin gene, DYSF. We performed targeted RNA-Seq using a custom gene-panel in 77 individuals with a clinical/genetic suspicion of dysferlinopathy and evaluated all 111 identified DYSF variants according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. This evaluation identified 11 novel DYSF variants and allowed for the classification of 87 DYSF variants as pathogenic/likely pathogenic, 8 likely benign, while 16 variants remained VUSs. By the end of the study, 60 of the 77 cases had a definitive diagnosis of dysferlinopathy, which was a 47% increase in diagnostic yield over the rate at study onset. This data shows the ability of RNA-Seq to assist in variant pathogenicity classification and diagnosis of dysferlinopathy and is, therefore, a type of analysis that should be considered when DNA-based genetic analysis is not sufficient to provide a definitive diagnosis.

Expanding the muscle imaging spectrum in dysferlinopathy: description of an outlier population from the classical MRI pattern.

Dysferlinopathy is a muscle disease characterized by a variable clinical presentation and is caused by mutations in the DYSF gene. The Jain Clinical Outcome Study for Dysferlinopathy (COS) followed the largest cohort of patients (n=187) with genetically confirmed dysferlinopathy throughout a three-year natural history study, in which the patients underwent muscle function tests and muscle magnetic resonance imaging (MRI). We previously described the pattern of muscle pathology in this population and established a series of imaging criteria for diagnosis. In this paper, we describe the muscle imaging and clinical features of a subgroup of COS participants whose muscle imaging results did not completely meet the diagnostic criteria. We reviewed 184 T1-weighted (T1w) muscle MRI scans obtained at the baseline visit of the COS study, of which 106 were pelvic and lower limb only and 78 were whole-body scans. We identified 116 of the 184 patients (63%) who did not meet at least one of the established imaging criteria. The highest number found of unmet criteria was four per patient. We identified 24 patients (13%) who did not meet three or more of the nine established criteria and considered them as "outliers". The most common unmet criterion (27.3% of cases) was the adductor magnus being equally or more affected than the adductor longus. We compared the genetic, demographic, clinical and muscle function data of the outlier patients with those who met the established criteria and observed that the outlier patients had an age of disease onset that was significantly older than the whole group (29.3 vs 20.5 years, p=0.0001). This study expands the phenotypic muscle imaging spectrum of patients with dysferlinopathy and can help to guide the diagnostic process in patients with limb girdle weakness of unknown origin.

Patient reported pregnancy and birth outcomes in genetic neuromuscular diseases.

Pregnancy and birth in women with neuromuscular conditions has been associated with more rapid disease progression and obstetric complications. This study assessed the impact of functional status and specific diagnosis on patient reported pregnancy and birth outcomes in 26 genetic neuromuscular diseases. Pregnancy and birth outcomes were collected through electronic patient questionnaires and analysed by mobility group and diagnosis. Free text responses were grouped into themes. 721 pregnancies were reported by 305 women. Miscarriage (21% of pregnancies), caesarean delivery (38% of births) and instrumental vaginal delivery (19% of births) were all more frequent in respondents than in the general population (p<0.05), and were more common in those who were non-ambulant at conception than other mobility levels (p <0.05). Falls occurred during 42% of pregnancies and a deterioration in muscle strength during 43%. There was not an increased incidence of maternal complications, apart from maternal hypertension which was more common in limb girdle muscular dystrophy 2A/R1 (35%) and myotonic dystrophy (24%). Patients offered specific practical advice to prospective mothers. Women with neuromuscular conditions have a more complex antenatal and perinatal course than unaffected women. Prenatal counselling, specialist obstetric review and additional occupational therapy support should be considered.

Myostatin and follistatin as monitoring and prognostic biomarkers in dysferlinopathy.

Myostatin is a myokine which acts upon skeletal muscle to inhibit growth and regeneration. Myostatin is endogenously antagonised by follistatin. This study assessed serum myostatin and follistatin concentrations as monitoring or prognostic biomarkers in dysferlinopathy, an autosomal recessively inherited muscular dystrophy. Myostatin was quantified twice with a three-year interval in 76 patients with dysferlinopathy and 38 controls. Follistatin was quantified in 62 of these patients at the same timepoints, and in 31 controls. Correlations with motor function, muscle fat fraction and contractile cross-sectional area were performed. A regression model was used to account for confounding variables. Baseline myostatin, but not follistatin, correlated with baseline function and MRI measures. However, in individual patients, three-year change in myostatin did not correlate with functional or MRI changes. Linear modelling demonstrated that function, serum creatine kinase and C-reactive protein, but not age, were independently related to myostatin concentration. Baseline myostatin concentration predicted loss of ambulation but not rate of change of functional or MRI measures, even when relative inhibition with follistatin was considered. With adjustment for extra-muscular causes of variation, myostatin could form a surrogate measure of functional ability or muscle mass, however myostatin inhibition does not form a promising treatment target in dysferlinopathy.

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.

Comparison of strength testing modalities in dysferlinopathy.

INTRODUCTION/AIMS: Dysferlinopathy demonstrates heterogeneity in muscle weakness between patients, which can progress at different rates over time. Changing muscle strength due to disease progression or from an investigational product is associated with changing functional ability. The purpose of this study was to compare three methods of strength testing used in the Clinical Outcome Study (COS) for dysferlinopathy to understand which method and which muscle groups were most sensitive to change over time. METHODS: Patients were evaluated at each study visit using functional scales, manual muscle testing, and handheld dynamometry (HHD) at all 15 sites. A fixed-frame system (Fixed) was used at a subset of seven sites. Screening and baseline visits were evaluated for reliability. Data over a 1-year period were analyzed to determine sensitivity to change among strength modalities and individual muscle groups. RESULTS: HHD and Fixed captured significant change across 1 year in summed muscle strength score of four muscle groups (P < .01). Strength summed scores were significantly correlated with functional scales (rho = 0.68-0.92, P < .001). Individual muscle groups, however, showed high levels of variability between visits. DISCUSSION: Although both HHD and Fixed demonstrate change over 12 months, HHD is a less expensive option that provides data on a continuous scale and may be easier to implement. Due to variability in strength measures, researchers should carefully consider use of strength testing as an outcome and may wish to select functional measures with less variability as clinical trial endpoints.

Three-year quantitative magnetic resonance imaging and phosphorus magnetic resonance spectroscopy study in lower limb muscle in dysferlinopathy.

BACKGROUND: Natural history studies in neuromuscular disorders are vital to understand the disease evolution and to find sensitive outcome measures. We performed a longitudinal assessment of quantitative magnetic resonance imaging (MRI) and phosphorus magnetic resonance spectroscopy (31 P MRS) outcome measures and evaluated their relationship with function in lower limb skeletal muscle of dysferlinopathy patients. METHODS: Quantitative MRI/31 P MRS data were obtained at 3 T in two different sites in 54 patients and 12 controls, at baseline, and three annual follow-up visits. Fat fraction (FF), contractile cross-sectional area (cCSA), and muscle water T2 in both global leg and thigh segments and individual muscles and 31 P MRS indices in the anterior leg compartment were assessed. Analysis included comparisons between patients and controls, assessments of annual changes using a linear mixed model, standardized response means (SRM), and correlations between MRI and 31 P MRS markers and functional markers. RESULTS: Posterior muscles in thigh and leg showed the highest FF values. FF at baseline was highly heterogeneous across patients. In ambulant patients, median annual increases in global thigh and leg segment FF values were 4.1% and 3.0%, respectively (P < 0.001). After 3 years, global thigh and leg FF increases were 9.6% and 8.4%, respectively (P < 0.001). SRM values for global thigh FF were over 0.8 for all years. Vastus lateralis muscle showed the highest SRM values across all time points. cCSA decreased significantly after 3 years with median values of 11.0% and 12.8% in global thigh and global leg, respectively (P < 0.001). Water T2 values in ambulant patients were significantly increased, as compared with control values (P < 0.001). The highest water T2 values were found in the anterior part of thigh and leg. Almost all 31 P MRS indices were significantly different in patients as compared with controls (P < 0.006), except for pHw , and remained, similar as to water T2 , abnormal for the whole study duration. Global thigh water T2 at baseline was significantly correlated to the change in FF after 3 years (ρ = 0.52, P < 0.001). There was also a significant relationship between the change in functional score and change in FF after 3 years in ambulant patients (ρ = -0.55, P = 0.010). CONCLUSIONS: This multi-centre study has shown that quantitative MRI/31 P MRS measurements in a heterogeneous group of dysferlinopathy patients can measure significant changes over the course of 3 years. These data can be used as reference values in view of future clinical trials in dysferlinopathy or comparisons with quantitative MRI/S data obtained in other limb-girdle muscular dystrophy subtypes.

Assessing the Relationship of Patient Reported Outcome Measures With Functional Status in Dysferlinopathy: A Rasch Analysis Approach.

Dysferlinopathy is a muscular dystrophy with a highly variable functional disease progression in which the relationship of function to some patient reported outcome measures (PROMs) has not been previously reported. This analysis aims to identify the suitability of PROMs and their association with motor performance.Two-hundred and four patients with dysferlinopathy were identified in the Jain Foundation's Clinical Outcome Study in Dysferlinopathy from 14 sites in 8 countries. All patients completed the following PROMs: Individualized Neuromuscular Quality of Life Questionnaire (INQoL), International Physical Activity Questionnaire (IPAQ), and activity limitations for patients with upper and/or lower limb impairments (ACTIVLIMs). In addition, nonambulant patients completed the Egen Klassifikation Scale (EK). Assessments were conducted annually at baseline, years 1, 2, 3, and 4. Data were also collected on the North Star Assessment for Limb Girdle Type Muscular Dystrophies (NSAD) and Performance of Upper Limb (PUL) at these time points from year 2. Data were analyzed using descriptive statistics and Rasch analysis was conducted on ACTIVLIM, EK, INQoL. For associations, graphs (NSAD with ACTIVLIM, IPAQ and INQoL and EK with PUL) were generated from generalized estimating equations (GEE). The ACTIVLIM appeared robust psychometrically and was strongly associated with the NSAD total score (Pseudo R 2 0.68). The INQoL performed less well and was poorly associated with the NSAD total score (Pseudo R 2 0.18). EK scores were strongly associated with PUL (Pseudo R 2 0.69). IPAQ was poorly associated with NSAD scores (Pseudo R 2 0.09). This study showed that several of the chosen PROMs demonstrated change over time and a good association with functional outcomes. An alternative quality of life measure and method of collecting data on physical activity may need to be selected for assessing dysferlinopathy.

Cardiac and pulmonary findings in dysferlinopathy: A 3-year, longitudinal study.

INTRODUCTION/AIMS: There is debate about whether and to what extent either respiratory or cardiac dysfunction occurs in patients with dysferlinopathy. This study aimed to establish definitively whether dysfunction in either system is part of the dysferlinopathy phenotype. METHODS: As part of the Jain Foundation's International Clinical Outcome Study (COS) for dysferlinopathy, objective measures of respiratory and cardiac function were collected twice, with a 3-y interval between tests, in 188 genetically confirmed patients aged 11-86 y (53% female). Measures included forced vital capacity (FVC), electrocardiogram (ECG), and echocardiogram (echo). RESULTS: Mean FVC was 90% predicted at baseline, decreasing to 88% at year 3. FVC was less than 80% predicted in 44 patients (24%) at baseline and 48 patients (30%) by year 3, including ambulant participants. ECGs showed P-wave abnormalities indicative of delayed trans-atrial conduction in 58% of patients at baseline, representing a risk for developing atrial flutter or fibrillation. The prevalence of impaired left ventricular function or hypertrophy was comparable to that in the general population. DISCUSSION: These results demonstrate clinically significant respiratory impairment and abnormal atrial conduction in some patients with dysferlinopathy. Therefore, we recommend that annual or biannual follow-up should include FVC measurement, enquiry about arrhythmia symptoms and peripheral pulse palpation to assess cardiac rhythm. However, periodic specialist cardiac review is probably not warranted unless prompted by symptoms or abnormal pulse findings.

4-Phenylbutyrate restores localization and membrane repair to human dysferlin mutations.

Dysferlinopathies are muscular dystrophies caused by recessive loss-of-function mutations in dysferlin (DYSF), a membrane protein involved in skeletal muscle membrane repair. We describe a cell-based assay in which human DYSF proteins bearing missense mutations are quantitatively assayed for membrane localization by flow cytometry and identified 64 localization-defective DYSF mutations. Using this platform, we show that the clinically approved drug 4-phenylbutryric acid (4-PBA) partially restores membrane localization to 25 mutations, as well as membrane repair to cultured myotubes expressing 2 different mutations. Two-day oral administration of 4-PBA to mice homozygous for one of these mutations restored myofiber membrane repair. 4-PBA may hold therapeutic potential for treating a subset of humans with muscular dystrophy due to dysferlin deficiency.

Assessing Dysferlinopathy Patients Over Three Years With a New Motor Scale.

OBJECTIVE: Dysferlinopathy is a muscular dystrophy with a highly variable clinical presentation and currently unpredictable progression. This variability and unpredictability presents difficulties for prognostication and clinical trial design. The Jain Clinical Outcomes Study of Dysferlinopathy aims to establish the validity of the North Star Assessment for Limb Girdle Type Muscular Dystrophies (NSAD) scale and identify factors that influence the rate of disease progression using NSAD. METHODS: We collected a longitudinal series of functional assessments from 187 patients with dysferlinopathy over 3 years. Rasch analysis was used to develop the NSAD, a motor performance scale suitable for ambulant and nonambulant patients. Generalized estimating equations were used to evaluate the impact of patient factors on outcome trajectories. RESULTS: The NSAD detected significant change in clinical progression over 1 year. The steepest functional decline occurred during the first 10 years after symptom onset, with more rapid decline noted in patients who developed symptoms at a younger age (p = 0.04). The most rapidly deteriorating group over the study was patients 3 to 8 years post symptom onset at baseline. INTERPRETATION: The NSAD is the first validated limb girdle specific scale of motor performance, suitable for use in clinical practice and clinical trials. Longitudinal analysis showed it may be possible to identify patient factors associated with greater functional decline both across the disease course and in the short-term for clinical trial preparation. Through further work and validation in this cohort, we anticipate that a disease model incorporating functional performance will allow for more accurate prognosis for patients with dysferlinopathy. ANN NEUROL 2021;89:967-978.

Miyoshi myopathy and limb girdle muscular dystrophy R2 are the same disease.

This study aims to determine clinically relevant phenotypic differences between the two most common phenotypic classifications in dysferlinopathy, limb girdle muscular dystrophy R2 (LGMDR2) and Miyoshi myopathy (MMD1). LGMDR2 and MMD1 are reported to involve different muscles, with LGMDR2 showing predominant limb girdle weakness and MMD1 showing predominant distal lower limb weakness. We used heatmaps, regression analysis and principle component analysis of functional and Magnetic Resonance Imaging data to perform a cross-sectional review of the pattern of muscle involvement in 168 patients from the Jain Foundation's international Clinical Outcomes Study for Dysferlinopathy. We demonstrated that there is no clinically relevant difference in proximal vs distal involvement between diagnosis. There is a continuum of distal involvement at any given degree of proximal involvement and patients do not fall into discrete distally or proximally affected groups. There appeared to be geographical preference for a particular diagnosis, with MMD1 being more common in Japan and LGMDR2 in Europe and the USA. We conclude that the dysferlinopathies do not form two distinct phenotypic groups and therefore should not be split into separate cohorts of LGMDR2 and MM for the purposes of clinical management, enrolment in clinical trials or access to subsequent treatments.

Clinical and Genomic Evaluation of 207 Genetic Myopathies in the Indian Subcontinent.

Objective: Inherited myopathies comprise more than 200 different individually rare disease-subtypes, but when combined together they have a high prevalence of 1 in 6,000 individuals across the world. Our goal was to determine for the first time the clinical- and gene-variant spectrum of genetic myopathies in a substantial cohort study of the Indian subcontinent. Methods: In this cohort study, we performed the first large clinical exome sequencing (ES) study with phenotype correlation on 207 clinically well-characterized inherited myopathy-suspected patients from the Indian subcontinent with diverse ethnicities. Results: Clinical-correlation driven definitive molecular diagnosis was established in 49% (101 cases; 95% CI, 42-56%) of patients with the major contributing pathogenicity in either of three genes, GNE (28%; GNE-myopathy), DYSF (25%; Dysferlinopathy), and CAPN3 (19%; Calpainopathy). We identified 65 variant alleles comprising 37 unique variants in these three major genes. Seventy-eight percent of the DYSF patients were homozygous for the detected pathogenic variant, suggesting the need for carrier-testing for autosomal-recessive disorders like Dysferlinopathy that are common in India. We describe the observed clinical spectrum of myopathies including uncommon and rare subtypes in India: Sarcoglycanopathies (SGCA/B/D/G), Collagenopathy (COL6A1/2/3), Anoctaminopathy (ANO5), telethoninopathy (TCAP), Pompe-disease (GAA), Myoadenylate-deaminase-deficiency-myopathy (AMPD1), myotilinopathy (MYOT), laminopathy (LMNA), HSP40-proteinopathy (DNAJB6), Emery-Dreifuss-muscular-dystrophy (EMD), Filaminopathy (FLNC), TRIM32-proteinopathy (TRIM32), POMT1-proteinopathy (POMT1), and Merosin-deficiency-congenital-muscular-dystrophy-type-1 (LAMA2). Thirteen patients harbored pathogenic variants in >1 gene and had unusual clinical features suggesting a possible role of synergistic-heterozygosity/digenic-contribution to disease presentation and progression. Conclusions: Application of clinically correlated ES to myopathy diagnosis has improved our understanding of the clinical and genetic spectrum of different subtypes and their overlaps in Indian patients. This, in turn, will enhance the global gene-variant-disease databases by including data from developing countries/continents for more efficient clinically driven molecular diagnostics.

Intensive Teenage Activity Is Associated With Greater Muscle Hyperintensity on T1W Magnetic Resonance Imaging in Adults With Dysferlinopathy.

Practice of sports during childhood or adolescence correlates with an earlier onset and more rapidly progressing phenotype in dysferlinopathies. To determine if this correlation relates to greater muscle pathology that persists into adulthood, we investigated the effect of exercise on the degree of muscle fatty replacement measured using muscle MRI. We reviewed pelvic, thigh and leg T1W MRI scans from 160 patients with genetically confirmed dysferlinopathy from the Jain Foundation International clinical outcomes study in dysferlinopathy. Two independent assessors used the Lamminen-Mercuri visual scale to score degree of fat replacement in each muscle. Exercise intensity for each individual was defined as no activity, minimal, moderate, or intensive activity by using metabolic equivalents and patient reported frequency of sports undertaken between the ages of 10 and 18. We used ANCOVA and linear modeling to compare the mean Lamminen-Mercuri score for the pelvis, thigh, and leg between exercise groups, controlling for age at assessment and symptom duration. Intensive exercisers showed greater fatty replacement in the muscles of the pelvis than moderate exercisers, but no significant differences of the thigh or leg. Within the pelvis, Psoas was the muscle most strongly associated with this exercise effect. In patients with a short symptom duration of <15 years there was a trend toward greater fatty replacement in the muscles of the thigh. These findings define key muscles involved in the exercise-phenotype effect that has previously been observed only clinically in dysferlinopathy and support recommendations that pre-symptomatic patients should avoid very intensive exercise.

Estimating prevalence for limb-girdle muscular dystrophy based on public sequencing databases.

PURPOSE: Limb-girdle muscular dystrophies (LGMD) are a genetically heterogeneous category of autosomal inherited muscle diseases. Many genes causing LGMD have been identified, and clinical trials are beginning for treatment of some genetic subtypes. However, even with the gene-level mechanisms known, it is still difficult to get a robust and generalizable prevalence estimation for each subtype due to the limited amount of epidemiology data and the low incidence of LGMDs. METHODS: Taking advantage of recently published exome and genome sequencing data from the general population, we used a Bayesian method to develop a robust disease prevalence estimator. RESULTS: This method was applied to nine recessive LGMD subtypes. The estimated disease prevalence calculated by this method was largely comparable with published estimates from epidemiological studies; however, it highlighted instances of possible underdiagnosis for LGMD2B and 2L. CONCLUSION: The increasing size of aggregated population variant databases will allow for robust and reproducible prevalence estimates of recessive disease, which is critical for the strategic design and prioritization of clinical trials.

Correction of pseudoexon splicing caused by a novel intronic dysferlin mutation.

OBJECTIVE: Dysferlin is a large transmembrane protein that functions in critical processes of membrane repair and vesicle fusion. Dysferlin-deficiency due to mutations in the dysferlin gene leads to muscular dystrophy (Miyoshi myopathy (MM), limb girdle muscular dystrophy type 2B (LGMD2B), distal myopathy with anterior tibial onset (DMAT)), typically with early adult onset. At least 416 pathogenic dysferlin mutations are known, but for approximately 17% of patients, one or both of their pathogenic variants remain undefined following standard exon sequencing methods that interrogate exons and nearby flanking intronic regions but not the majority of intronic regions. METHODS: We sequenced RNA from myogenic cells to identify a novel dysferlin pathogenic variant in two affected siblings that previously had only one disease-causing variant identified. We designed antisense oligonucleotides (AONs) to bypass the effects of this mutation on RNA splicing. RESULTS: We identified a new pathogenic point mutation deep within dysferlin intron 50i. This intronic variant causes aberrant mRNA splicing and inclusion of an additional pseudoexon (PE, we term PE50.1) within the mature dysferlin mRNA. PE50.1 inclusion alters the protein sequence, causing premature translation termination. We identified this mutation in 23 dysferlinopathy patients (seventeen families), revealing it to be one of the more prevalent dysferlin mutations. We used AON-mediated exon skipping to correct the aberrant PE50.1 splicing events in vitro, which increased normal mRNA production and significantly restored dysferlin protein expression. INTERPRETATION: Deep intronic mutations can be a common underlying cause of dysferlinopathy, and importantly, could be treatable with AON-based exon-skipping strategies.

Assessment of disease progression in dysferlinopathy: A 1-year cohort study.

OBJECTIVE: To assess the ability of functional measures to detect disease progression in dysferlinopathy over 6 months and 1 year. METHODS: One hundred ninety-three patients with dysferlinopathy were recruited to the Jain Foundation's International Clinical Outcome Study for Dysferlinopathy. Baseline, 6-month, and 1-year assessments included adapted North Star Ambulatory Assessment (a-NSAA), Motor Function Measure (MFM-20), timed function tests, 6-minute walk test (6MWT), Brooke scale, Jebsen test, manual muscle testing, and hand-held dynamometry. Patients also completed the ACTIVLIM questionnaire. Change in each measure over 6 months and 1 year was calculated and compared between disease severity (ambulant [mild, moderate, or severe based on a-NSAA score] or nonambulant [unable to complete a 10-meter walk]) and clinical diagnosis. RESULTS: The functional a-NSAA test was the most sensitive to deterioration for ambulant patients overall. The a-NSAA score was the most sensitive test in the mild and moderate groups, while the 6MWT was most sensitive in the severe group. The 10-meter walk test was the only test showing significant change across all ambulant severity groups. In nonambulant patients, the MFM domain 3, wrist flexion strength, and pinch grip were most sensitive. Progression rates did not differ by clinical diagnosis. Power calculations determined that 46 moderately affected patients are required to determine clinical effectiveness for a hypothetical 1-year clinical trial based on the a-NSAA as a clinical endpoint. CONCLUSION: Certain functional outcome measures can detect changes over 6 months and 1 year in dysferlinopathy and potentially be useful in monitoring progression in clinical trials. CLINICALTRIALSGOV IDENTIFIER: NCT01676077.

Genetic landscape and novel disease mechanisms from a large LGMD cohort of 4656 patients.

OBJECTIVE: Limb-girdle muscular dystrophies (LGMDs), one of the most heterogeneous neuromuscular disorders (NMDs), involves predominantly proximal-muscle weakness with >30 genes associated with different subtypes. The clinical-genetic overlap among subtypes and with other NMDs complicate disease-subtype identification lengthening diagnostic process, increases overall costs hindering treatment/clinical-trial recruitment. Currently seven LGMD clinical trials are active but still no gene-therapy-related treatment is available. Till-date no nation-wide large-scale LGMD sequencing program was performed. Our objectives were to understand LGMD genetic basis, different subtypes' relative prevalence across US and investigate underlying disease mechanisms. METHODS: A total of 4656 patients with clinically suspected-LGMD across US were recruited to conduct next-generation sequencing (NGS)-based gene-panel testing during June-2015 to June-2017 in CLIA-CAP-certified Emory-Genetics-Laboratory. Thirty-five LGMD-subtypes-associated or LGMD-like other NMD-associated genes were investigated. Main outcomes were diagnostic yield, gene-variant spectrum, and LGMD subtypes' prevalence in a large US LGMD-suspected population. RESULTS: Molecular diagnosis was established in 27% (1259 cases; 95% CI, 26-29%) of the patients with major contributing genes to LGMD phenotypes being: CAPN3(17%), DYSF(16%), FKRP(9%) and ANO5(7%). We observed an increased prevalence of genetically confirmed late-onset Pompe disease, DNAJB6-associated LGMD subtype1E and CAPN3-associated autosomal-dominant LGMDs. Interestingly, we identified a high prevalence of patients with pathogenic variants in more than one LGMD gene suggesting possible synergistic heterozygosity/digenic/multigenic contribution to disease presentation/progression that needs consideration as a part of diagnostic modality. INTERPRETATION: Overall, this study has improved our understanding of the relative prevalence of different LGMD subtypes, their respective genetic etiology, and the changing paradigm of their inheritance modes and novel mechanisms that will allow for improved timely treatment, management, and enrolment of molecularly diagnosed individuals in clinical trials.

Muscle MRI in patients with dysferlinopathy: pattern recognition and implications for clinical trials.

BACKGROUND AND OBJECTIVE: Dysferlinopathies are a group of muscle disorders caused by mutations in the DYSF gene. Previous muscle imaging studies describe a selective pattern of muscle involvement in smaller patient cohorts, but a large imaging study across the entire spectrum of the dysferlinopathies had not been performed and previous imaging findings were not correlated with functional tests. METHODS: We present cross-sectional T1-weighted muscle MRI data from 182 patients with genetically confirmed dysferlinopathies. We have analysed the pattern of muscles involved in the disease using hierarchical analysis and presented it as heatmaps. Results of the MRI scans have been correlated with relevant functional tests for each region of the body analysed. RESULTS: In 181 of the 182 patients scanned, we observed muscle pathology on T1-weighted images, with the gastrocnemius medialis and the soleus being the most commonly affected muscles. A similar pattern of involvement was identified in most patients regardless of their clinical presentation. Increased muscle pathology on MRI correlated positively with disease duration and functional impairment. CONCLUSIONS: The information generated by this study is of high diagnostic value and important for clinical trial development. We have been able to describe a pattern that can be considered as characteristic of dysferlinopathy. We have defined the natural history of the disease from a radiological point of view. These results enabled the identification of the most relevant regions of interest for quantitative MRI in longitudinal studies, such as clinical trials. CLINICAL TRIAL REGISTRATION: NCT01676077.