Standards of Care (SoC) are important for individuals with dysferlinopathy to proactively maximise function and quality of life within a multidisciplinary model of care, and for the robustness of clinical trials by ensuring patients are receiving a similar care standard across trial sites and international boundaries. With potential therapies for dysferlinopathy entering clinical trials, the development and implementation of internationally agreed SoC has become urgent. Clinicians with significant experience in managing patients with dysferlinopathy were invited to join working groups to develop consensus SoC guidelines. Working groups covered diagnosis; outcome measures; cardiac and respiratory function; and endocrine, pregnancy and nutritional management. A series of online workshops were used to present, review and discuss evidence, before a face to face meeting agreed recommendations in each area. The majority of evidence used came from the International Clinical Outcomes study for Dysferlinopathy (COS1) (NCT01676077) and other cohort studies and case reports. Guidelines were proposed for diagnosis and disease management at different stages of the disease. This collaboration created the first SoC in limb girdle muscular dystrophy and forms a framework which may be useful for other conditions. This document provides an important reference for clinicians and patients to ensure accurate diagnosis and proactive management of dysferlinopathy. The second phase of COS includes a care discussion with patients. This will inform a future iteration of the SoC. Further studies are required to understand the disease progression in ethnically diverse populations or the impact of exercise. Ongoing work to evaluate the impact of new therapies will be needed to ensure these SoC remain up to date. This work was completed with support of the ENMC and Jain Foundation and is submitted on behalf of all ENMC Dysferlin Standards of Care Workshop participants. Standards of Care (SoC) are important for individuals with dysferlinopathy to proactively maximise function and quality of life within a multidisciplinary model of care, and for the robustness of clinical trials by ensuring patients are receiving a similar care standard across trial sites and international boundaries. With potential therapies for dysferlinopathy entering clinical trials, the development and implementation of internationally agreed SoC has become urgent. Clinicians with significant experience in managing patients with dysferlinopathy were invited to join working groups to develop consensus SoC guidelines. Working groups covered diagnosis; outcome measures; cardiac and respiratory function; and endocrine, pregnancy and nutritional management. A series of online workshops were used to present, review and discuss evidence, before a face to face meeting agreed recommendations in each area. The majority of evidence used came from the International Clinical Outcomes study for Dysferlinopathy (COS1) (NCT01676077) and other cohort studies and case reports. Guidelines were proposed for diagnosis and disease management at different stages of the disease. This collaboration created the first SoC in limb girdle muscular dystrophy and forms a framework which may be useful for other conditions. This document provides an important reference for clinicians and patients to ensure accurate diagnosis and proactive management of dysferlinopathy. The second phase of COS includes a care discussion with patients. This will inform a future iteration of the SoC. Further studies are required to understand the disease progression in ethnically diverse populations or the impact of exercise. Ongoing work to evaluate the impact of new therapies will be needed to ensure these SoC remain up to date. This work was completed with support of the ENMC and Jain Foundation and is submitted on behalf of all ENMC Dysferlin Standards of Care Workshop participants.

Fat replacement in muscles measured by Dixon MRI provides an excellent marker for disease progression in dysferlinopathy patients. Although no specific therapies exist for dysferlinopathies, these disorders entail multiple pathways to muscle cell death, each of which is potentially a target for intervention; therefore, developing methods to measure therapeutic outcomes of novel drug trials is extremely important. In this study, we looked at a cohort of 11 patients from the JAIN COS natural history study who had been scanned at least 6 times over a 10-year period. Fat fraction analysis to measure the quantity of fat in muscles was carried out for seven muscles in the lower leg and nine muscles in the thigh. In both lower legs and thighs, percentage fat fraction showed a steady increase over the years with lower legs regressing slightly faster (slope = 3.24 % per year, SD = 0.85, range 1.62-4.74) than thighs (slope = 2.16 % per year, SD = 1.21, range 0.42-4.57), this difference was significant (p=0.015). The R2 fit was almost linear with a mean of 0.94 (range 0.81 to 0.99); there was no sign of sigmoidal behaviour which is commonly seen in other muscular dystrophies. Furthermore, certain muscles undergo disease progression early with gastrocnemius medialis and soleus in the lower leg, semimembranosus and adductor magnus in the thighs being most likely to show fatty transformation before other muscles. Linear regression analysis may predict the clinical direction a patient may take and time to lose ambulation as well as mapping the disease progression of individual muscles. In conclusion, Dixon MRI provides a valuable tool to capture any changes in disease progression following therapeutic drug intervention by measuring deviations in the slope of fat fraction percentage progression. Fat replacement in muscles measured by Dixon MRI provides an excellent marker for disease progression in dysferlinopathy patients. Although no specific therapies exist for dysferlinopathies, these disorders entail multiple pathways to muscle cell death, each of which is potentially a target for intervention; therefore, developing methods to measure therapeutic outcomes of novel drug trials is extremely important. In this study, we looked at a cohort of 11 patients from the JAIN COS natural history study who had been scanned at least 6 times over a 10-year period. Fat fraction analysis to measure the quantity of fat in muscles was carried out for seven muscles in the lower leg and nine muscles in the thigh. In both lower legs and thighs, percentage fat fraction showed a steady increase over the years with lower legs regressing slightly faster (slope = 3.24 % per year, SD = 0.85, range 1.62-4.74) than thighs (slope = 2.16 % per year, SD = 1.21, range 0.42-4.57), this difference was significant (p=0.015). The R2 fit was almost linear with a mean of 0.94 (range 0.81 to 0.99); there was no sign of sigmoidal behaviour which is commonly seen in other muscular dystrophies. Furthermore, certain muscles undergo disease progression early with gastrocnemius medialis and soleus in the lower leg, semimembranosus and adductor magnus in the thighs being most likely to show fatty transformation before other muscles. Linear regression analysis may predict the clinical direction a patient may take and time to lose ambulation as well as mapping the disease progression of individual muscles. In conclusion, Dixon MRI provides a valuable tool to capture any changes in disease progression following therapeutic drug intervention by measuring deviations in the slope of fat fraction percentage progression.

Aims Dysferlinopathy is an autosomal recessive muscular dystrophy, caused by bi-allelic variants in the gene encoding dysferlin (DYSF). Onset typically occurs in the second to third decade and is characterised by slowly progressive skeletal muscle weakness and atrophy of the proximal and/or distal muscles of the four limbs. There are rare cases of symptomatic DYSF variant carriers. Here, we report a large family with a dominantly inherited hyperCKaemia and late-onset muscular dystrophy. Methods and Results Genetic analysis identified a co-segregating novel DYSF variant [NM_003494.4:c.6207del p.(Tyr2070Metfs*4)]. No secondary variants in DYSF or other dystrophy-related genes were identified on whole genome sequencing and analysis of the proband's DNA. Skeletal muscle involvement was milder and later onset than typical dysferlinopathy presentations; these clinical signs manifested in four individuals, all between the fourth and sixth decades of life. All individuals heterozygous for the c.6207del variant had hyperCKaemia. Histological analysis of skeletal muscle biopsies across three generations showed clear dystrophic signs, including inflammatory infiltrates, regenerating myofibres, increased variability in myofibre size and internal nuclei. Muscle magnetic resonance imaging revealed fatty replacement of muscle in two individuals. Western blot and immunohistochemical analysis of muscle biopsy demonstrated consistent reduction of dysferlin staining. Allele-specific quantitative PCR analysis of DYSF mRNA from patient muscle found that the variant, localised to the extreme C-terminus of dysferlin, does not activate post-transcriptional mRNA decay. Conclusions We propose that this inheritance pattern may be underappreciated and that other late-onset muscular dystrophy cases with mono-allelic DYSF variants, particularly C-terminal premature truncation variants, may represent dominant forms of disease.

The Jain clinical outcome study (COS) is an international study-evaluating patients with genetically confirmed dysferlinopathy. COS aims to develop understanding of disease progression and support future scientific research 197 patients were recruited across 15 sites and in 8 countries. All sites completed year 3 visits. Further to the COS data collection allowing the characterization of the disease (results reported elsewhere), patients also had the option to donate biobank samples to the MRC biobank for rare and neuromuscular diseases curated at Newcastle. Standardised protocols for collection, processing and shipment were provided to each site. Blood for DNA and RNA was collected at one time point only. Plasma and serum were collected annually. Patients could opt to provide a skin sample to generate fibroblast cell lines. Samples have been shipped to the Newcastle MRC Centre Biobank for rare and neuromuscular disease. 12 sites of 15 sites provided samples. 97% patients opted to donate blood samples. 43% patients opted to donate skin samples for fibroblasts. Samples currently at Newcastle are as follows: blood for RNA n164; DNA n161; baseline plasma n147; year 1 plasma n151; year 2 plasma n134; year 3 plasma n132 (pending final shipments); baseline serum n145; year 1 serum n150; year 2 serum n133; year 3 serum n87 (pending final shipments); limited year 4 and year 5 samples are also available. This is a valuable resource for scientists worldwide to develop understanding of potential biomarkers for dysferlinopathies necessary to support design of future clinical trials. Samples can be requested from the Newcastle MRC center for rare and neuromuscular disease. The Jain COS steering group will consider requests for study data to support the analysis of results generated by research using these samples. The COS study has been extended for two further years. COS2 will also collect urine across all participating sites.

The clinical outcome study of dysferlinopathy (COS) is an international natural history study that recruited 197 patients with genetically confirmed dysferlinopathy. In the study 11 women reported a pregnancy in the last 12 months prior to or during the study. A total of 16 pregnancies with 10 live deliveries and 6 miscarriages were recorded at study visits. Delivery was by C-section with regional anaesthesia in half of cases. There were no major delivery problems or medical concerns with the neonates. Baseline data from COS shows that respiratory and cardiac involvement is not a prominent feature of dysferlinopathy. Patient concerns focused on the impact of pregnancy on muscle function and ability to care for the infant. Difficulties carrying and feeding the baby were reported. During 7 of the 10 term pregnancies, women perceived a worsening of muscle symptoms, with 4 describing further deterioration immediately after delivery. 7 of the live deliveries occurred after the baseline visit therefore functional data before and after delivery is available. The mean progression for ambulant participants over one year as measured by the North Star for Dysferlin (NSAD) is -2.13 NSAD points while for non-ambulant participants it is -0.84 NSAD points. Two participants experienced significantly greater than mean decline. (-7.87 and -5 NSAD points over one year), but the remaining five demonstrated less than the mean decline. Muscle MRI was acquired pre and post-natally for 5 participants and will be reviewed to determine the impact of pregnancy on individual muscles. The numbers of pregnancies observed are small in even a large-scale natural history study. Further longitudinal work is required in collaboration with patient registries to develop understanding of the impact of pregnancy on disease progression.

Vitamin D is known to be important for musculoskeletal health and as per Standards of care for Duchenne muscular dystrophy it is recommended that Vitamin D supplements should be considered in all patients where levels cannot be maintained. Public Health England recommends children (1 to 4 years) should be given daily supplements of vitamin D and that all people should consider taking vitamin D supplements during winter because of lack to sunlight exposure. We aimed to understand the impact of low vitamin D levels in neuromuscular patients complaining of low mood, myalgia and fatigue. We retrospectively reviewed Vitamin D and calcium blood levels in 100 consecutive patients attending their annual follow up appointments in the specialist muscle clinics. Vitamin D levels were categorised in deficient (<25nmol/l), insufficient (25-50nmol/l) and sufficient (>50nmol/l). Symptoms relating to low levels of Vitamin D and list of medications were also collected. We collected 100 blood samples for vitamin D and calcium levels, 49/100 samples were paediatric patients. Ten subjects were Vitamin D deficient; thirty-six patients were Vitamin D insufficient. 27% of the deficient/insufficient samples were children. Calcium levels were within normal range for all subjects. Out of the 46 patients with deficient or insufficient levels of vitamin D, 17 patients reported pain, 3 patients reported low mood, one patient reported fatigue, 2 patients reported pain and low mood, 1 patient reported pain and fatigue. Six patients with sufficient levels of vitamin D reported symptoms of low mood, fatigue and/or pain. Ten patients were already on vitamin D supplements. The remaining was advised to start vitamin D supplements following the blood test. Testing of Vitamin D levels should be considered in all neuromuscular patients complaining of myalgia, low mood or fatigue irrespectively of being prescribed vitamin D.

An aim of the COS study is to establish robust functional rating scales for use in ambulant and non-ambulant patients with dysferlinopathy. Functional outcome measures were reviewed for suitability and robustness. Functional ability, as measured by the motor function measure (MFM) and North Star Ambulatory Assessment for Dysferlinopathy (NSAA), was reviewed using modern psychometric methods (Rumm 2030). Analysis of 156 patients, with between one and four visits (n = 199), investigated the ability of the individual items of both scales to measure the underlying construct of the scale.