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.

Moderate intrinsic phenotypic alterations in C9orf72 ALS/FTD iPSC-microglia despite the presence of C9orf72 pathological features.

While motor and cortical neurons are affected in C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), it remains largely unknown if and how non-neuronal cells induce or exacerbate neuronal damage. We differentiated C9orf72 ALS/FTD patient-derived induced pluripotent stem cells into microglia (iPSC-MG) and examined their intrinsic phenotypes. Similar to iPSC motor neurons, C9orf72 ALS/FTD iPSC-MG mono-cultures form G4C2 repeat RNA foci, exhibit reduced C9orf72 protein levels, and generate dipeptide repeat proteins. Healthy control and C9orf72 ALS/FTD iPSC-MG equally express microglial specific genes and perform microglial functions, including inflammatory cytokine release and phagocytosis of extracellular cargos, such as synthetic amyloid beta peptides and healthy human brain synaptoneurosomes. RNA sequencing analysis revealed select transcriptional changes of genes associated with neuroinflammation or neurodegeneration in diseased microglia yet no significant differentially expressed microglial-enriched genes. Moderate molecular and functional differences were observed in C9orf72 iPSC-MG mono-cultures despite the presence of C9orf72 pathological features suggesting that a diseased microenvironment may be required to induce phenotypic changes in microglial cells and the associated neuronal dysfunction seen in C9orf72 ALS/FTD neurodegeneration.

Focal reactive periostitis ossificans in a long bone: radiologic and pathologic findings.

Florid reactive periostitis ossificans (FRPO) is a benign juxta-cortical lesion of unknown etiology which most commonly occurs in the hands and feet. We report the radiographic, CT, and MR features of a pathologically confirmed FRPO in the distal femur, a location in which only a handful of cases has been reported. A 26-year-old male who presented with distal thigh pain initially underwent radiograph and CT, which illustrated a well-circumscribed, ossified lesion associated with the cortex of the femur without contiguity with the medullary canal. A subsequent MRI demonstrated heterogeneous signal intensity corresponding to the ossified portion of the lesion with a T2 hyperintense cartilaginous cap and surrounding edema. The lesion was surgically excised and pathologic diagnosis of FRPO, a mixture of osteoid, mature bone, cartilage and fibrous tissue, with associated inflammatory cells, was confirmed. Follow up four months after surgery revealed significant improvement in the patient's pain.

Evaluating the Impact of a Call Triage Assistant on Resident Efficiency, Errors, and Stress.

OBJECTIVE: The aim of this study was to evaluate whether a call triage assistant, who answered telephone calls to the main reading room during the busiest hours of weekend call, would impact resident workflow efficiency, diagnostic errors, and stress level. METHODS: The call triage assistant answered all telephone calls to the main reading room from 12 pm to 7 pm on 6 weekend days over a 3-month period. We compared report turnaround times and resident discrepancy rates on these days with control days, when the same residents were on call without the assistant. We also surveyed residents to determine whether the assistants relieved anxiety associated with the call shift. RESULTS: We recorded 168 telephone calls over the study period. We found the majority of telephone calls could be handled by the assistant without disturbing the on-call resident, resulting in a 71% reduction in interruptions. The mean turnaround time for studies read on the days the assistant was on duty was 44.3 min, compared with 75.2 min on the control days (P < .01). Resident major discrepancy rates (0.4% on the intervention days compared to 0.2% on the control days) were similar (P = .58), as were minor discrepancy rates (7.5% on the intervention days compared with 6.7% on the control days; P = .61). Residents reported fewer distractions, improved workflow efficiency, and decreased call-related stress when the assistant was on duty. CONCLUSIONS: A call triage assistant effectively improved workflow efficiency and reduced resident stress on call. Resident error rates were unaffected by the presence of the assistant.

Correction to: ADAR2 mislocalization and widespread RNA editing aberrations in C9orf72-mediated ALS/FTD.

The original article was published erroneously without mentioning the support of the U.S.

ADAR2 mislocalization and widespread RNA editing aberrations in C9orf72-mediated ALS/FTD.

The hexanucleotide repeat expansion GGGGCC (G4C2)n in the C9orf72 gene is the most common genetic abnormality associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent findings suggest that dysfunction of nuclear-cytoplasmic trafficking could affect the transport of RNA binding proteins in C9orf72 ALS/FTD. Here, we provide evidence that the RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) is mislocalized in C9orf72 repeat expansion mediated ALS/FTD. ADAR2 is responsible for adenosine (A) to inosine (I) editing of double-stranded RNA, and its function has been shown to be essential for survival. Here we show the mislocalization of ADAR2 in human induced pluripotent stem cell-derived motor neurons (hiPSC-MNs) from C9orf72 patients, in mice expressing (G4C2)149, and in C9orf72 ALS/FTD patient postmortem tissue. As a consequence of this mislocalization we observe alterations in RNA editing in our model systems and across multiple brain regions. Analysis of editing at 408,580 known RNA editing sites indicates that there are vast RNA A to I editing aberrations in C9orf72-mediated ALS/FTD. These RNA editing aberrations are found in many cellular pathways, such as the ALS pathway and the crucial EIF2 signaling pathway. Our findings suggest that the mislocalization of ADAR2 in C9orf72 mediated ALS/FTD is responsible for the alteration of RNA processing events that may impact vast cellular functions, including the integrated stress response (ISR) and protein translation.

A Census of Midsize to Large Supermarkets in Toronto: A Cross-Sectional Analysis of the Consumer Nutrition Environment.

OBJECTIVE: Assess the consumer nutrition environment in midsize to large supermarkets by supermarket type and area-level socioeconomic variables. DESIGN: Cross-sectional census of 257 supermarkets using the Toronto Nutrition Environment Measures Survey in Stores. SETTING: Toronto, Canada. VARIABLES MEASURED: Availability; price and linear shelf space of fruits and vegetables vs energy-dense snack foods by supermarket type; after-tax, low-income measure; and neighborhood improvement area. ANALYSIS: Multivariate linear regression. RESULTS: There was a high availability of fruits (7.7 of 8) and vegetables (9.5 of 11). There was similar linear shelf space for fruits and vegetables vs energy-dense snack foods (ratio, 1.1 m). Adjusted fruit prices were lowest in quintiles 1 (ß = -$1.30; P = .008), 2 (ß = -$1.41; P = .005), and 3 (ß = -$1.89; P < .001) vs quintile 5 (lowest percentage of people living with low income) and in ethnic (ß = -$3.47; P < .001) and discount stores (ß = -$5.64; P < .001) vs conventional. Adjusted vegetable prices were lowest in quintiles 2 (ß = -$1.87; P = .04), 3 (ß = -$1.78; P = .03), and 4 (ß = -$2.65; P = .001) vs quintile 5 and in ethnic (ß = -$7.10; P < .001) and discount (ß = -$5.49; P < .001) stores. They were highest in other (ß = + $3.08; P = .003) vs conventional stores. Adjusted soda and chips prices were lower in discount (ß = -$1.16; P < .001) and higher in other stores (ß = + $0.67; P < .001) vs conventional. CONCLUSIONS AND IMPLICATIONS: Findings do not indicate inequities in shelf space, availability, or price across diverse neighborhoods. Practitioners can use findings to help consumers navigate supermarkets to make healthy choices.

Stenosis of gastric sleeve after laparoscopic sleeve gastrectomy: clinical, radiographic and endoscopic findings.

OBJECTIVE: To determine the clinical, radiographic, and endoscopic findings of sleeve stenosis after sleeve gastrectomy and to correlate treatment with outcomes. METHODS: We identified 43 patients who underwent barium studies to evaluate upper GI symptoms after laparoscopic sleeve gastrectomy. The clinical, radiographic, and endoscopic findings were reviewed and correlated with treatment and outcomes. RESULTS: 26 patients (60%) had sleeve stenoses. All stenoses appeared as short segments of smooth, tapered narrowing, with a mean length of 8.0 mm and mean width of 7.5 mm, and 24 (92%) were located in the proximal or distal third of the sleeve. 23 patients (88%) had upstream dilation, and 1 (4%) had retained food proximal to the stenosis. 23 (70%) of 33 patients with obstructive symptoms and 3 (30%) of 10 without obstructive symptoms had sleeve stenoses. Endoscopy revealed sleeve stenosis in 8 (67%) of 12 patients with radiographic stenosis. Endoscopic dilation resulted in improvement/resolution of symptoms in seven (88%) of 8 patients. CONCLUSION: Sleeve stenosis after sleeve gastrectomy was characterized radiographically by a short segment of smooth, tapered narrowing, typically in the proximal or distal third of the sleeve. Approximately, 70% of patients with obstructive symptoms and 30% with non-obstructive symptoms had sleeve stenosis. One-third of radiographically diagnosed stenoses were not seen at endoscopy. The barium study, therefore, is a useful test for sleeve stenosis in patients with obstructive or nonobstructive symptoms after sleeve gastrectomy. Advances in knowledge: This article describes the appearance and location of sleeve stenoses after laparoscopic sleeve gastrectomy and the clinical presentation and treatment options for these patients.

Post-transcriptional Inhibition of Hsc70-4/HSPA8 Expression Leads to Synaptic Vesicle Cycling Defects in Multiple Models of ALS.

Amyotrophic lateral sclerosis (ALS) is a synaptopathy accompanied by the presence of cytoplasmic aggregates containing TDP-43, an RNA-binding protein linked to ∼97% of ALS cases. Using a Drosophila model of ALS, we show that TDP-43 overexpression (OE) in motor neurons results in decreased expression of the Hsc70-4 chaperone at the neuromuscular junction (NMJ). Mechanistically, mutant TDP-43 sequesters hsc70-4 mRNA and impairs its translation. Expression of the Hsc70-4 ortholog, HSPA8, is also reduced in primary motor neurons and NMJs of mice expressing mutant TDP-43. Electrophysiology, imaging, and genetic interaction experiments reveal TDP-43-dependent defects in synaptic vesicle endocytosis. These deficits can be partially restored by OE of Hsc70-4, cysteine-string protein (Csp), or dynamin. This suggests that TDP-43 toxicity results in part from impaired activity of the synaptic CSP/Hsc70 chaperone complex impacting dynamin function. Finally, Hsc70-4/HSPA8 expression is also post-transcriptionally reduced in fly and human induced pluripotent stem cell (iPSC) C9orf72 models, suggesting a common disease pathomechanism.

Exome sequencing reveals independent SGCD deletions causing limb girdle muscular dystrophy in Boston terriers.

BACKGROUND: Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of inherited autosomal myopathies that preferentially affect voluntary muscles of the shoulders and hips. LGMD has been clinically described in several breeds of dogs, but the responsible mutations are unknown. The clinical presentation in dogs is characterized by marked muscle weakness and atrophy in the shoulder and hips during puppyhood. METHODS: Following clinical evaluation, the identification of the dystrophic histological phenotype on muscle histology, and demonstration of the absence of sarcoglycan-sarcospan complex by immunostaining, whole exome sequencing was performed on five Boston terriers: one affected dog and its three family members and one unrelated affected dog. RESULTS: Within sarcoglycan-δ (SGCD), a two base pair deletion segregating with LGMD in the family was discovered, and a deletion encompassing exons 7 and 8 was found in the unrelated dog. Both mutations are predicted to cause an absence of SGCD protein, confirmed by immunohistochemistry. The mutations are private to each family. CONCLUSIONS: Here, we describe the first cases of canine LGMD characterized at the molecular level with the classification of LGMD2F.

Bringing Menu Labelling to Independent Restaurants: Findings from a Voluntary Pilot Project in Toronto.

Toronto Public Health conducted a pilot project to assess the feasibility of menu labelling by independent restaurants. The pilot project was informed by consultations with the industry and other jurisdictions that have implemented a similar initiative. Public Health Dietitians worked closely with these restaurants to help them work toward posting calories and sodium on their menus. This paper reports on the findings of a feasibility assessment that took a mixed-methods approach resulting in a comprehensive process evaluation. Results showed that having highly motivated restaurants and early adopters of menu labelling is a necessary starting point. However, this alone is not sufficient to make voluntary menu labelling successful. It may be feasible only for select independent restaurants who: (i) are highly motivated and ready to make a substantial time commitment; (ii) value offering healthy food choices; (iii) have fairly standardized recipes to begin with; (iv) receive extensive specialized, individualized support; and (v) receive incentives, cost offsetting, and recognition. Full-scale implementation of a menu labelling program with Toronto independent restaurants was not justified given the current level of interest and capacity.

Impact of hands-on care on infant sleep in the neonatal intensive care unit.

STUDY OBJECTIVES: Sleep disruption is increasingly recognized in hospitalized patients. Impaired sleep is associated with measureable alterations in neurodevelopment. The neonatal intensive care unit (NICU) environment has the potential to affect sleep quality and quantity. We aimed: (i) to determine the frequency and duration of hands-on care, and its impact on sleep, for NICU patients; and (ii) to assess the incidence of respiratory events associated with handling for a cohort of sick neonates. METHODS: Term and near-term neonates admitted to the NICU and at risk for cerebral dysfunction due to severity of illness or clinical suspicion for seizures underwent attended, bedside polysomnography. Continuous polysomnogram segments were analyzed and data on handling, infant behavioral state, and associated respiratory events were recorded. RESULTS: Video and polysomnography data were evaluated for 25 infants (gestational age 39.4 ± 1.6 weeks). The maximum duration between handling episodes for each infant was 50.9 ± 26.2 min, with a median of 2.3 min between contacts. Handling occurred across all behavioral states (active sleep 29.5%; quiet sleep 23.1%; awake 29.9%; indeterminate 17.4%; P = 0.99). Arousals or awakenings occurred in 57% of contacts with a sleeping infant. Hypopnea, apnea, and oxygen desaturation occurred with 16%, 8%, and 19.5% of contacts, respectively. Hypopnea was most likely to occur following contact with infants in active sleep (28%; P < 0.001). CONCLUSIONS: Infants in the NICU experience frequent hands-on care, associated with disturbances of sleep and respiration. The potential health and developmental impact of these disturbances merits study, as strategies to monitor sleep and minimize sleep-disordered breathing might then improve NICU outcomes. Pediatr Pulmonol. 2017;52:84-90 © 2016 Wiley Periodicals, Inc.

Findings of Esophagography for 25 Patients After Peroral Endoscopic Myotomy for Achalasia.

OBJECTIVE: The purpose of this study is to better characterize the findings of esophagography after peroral endoscopic myotomy for achalasia. MATERIALS AND METHODS: We evaluated 25 patients who underwent peroral endoscopic myotomy for achalasia. The findings noted on pre- and postprocedural esophagrams were reviewed retrospectively and were correlated with clinical outcomes. RESULTS: None of the patients had esophageal perforation noted on esophagrams obtained after myotomy, and all but two patients had a hospital stay that lasted 1 day only. Esophagrams obtained on postoperative day 1 revealed endoscopic clips in 25 patients (100%), pneumoperitoneum in 18 (72%), retroperitoneal gas in 10 (40%), gastric pneumatosis in nine (36%), intramural dissections in seven (28%), and pneumomediastinum in four (16%). Repeat esophagrams obtained 3 weeks later for 22 of the patients revealed endoscopic clips in 16 patients (73%) and intramural dissections in five patients (23%), but the remaining findings had resolved. Eighteen patients (72%) had a successful myotomy and seven (28%) had suboptimal results on the basis of clinical outcomes. Observation of a distal esophageal width of 5 mm or less on postprocedural esophagrams was often associated with suboptimal results. CONCLUSION: Peroral endoscopic myotomy is a novel procedure that is less invasive than is laparoscopic Heller myotomy for the treatment of achalasia, with fewer complications and shorter recovery times. Radiologists should be aware of the findings expected on esophagography (including pneumoperitoneum, retroperitoneal gas, gastric pneumatosis, intramural dissections, and pneumomediastinum) and should also know that fluoroscopic studies may be helpful for predicting patient outcomes on the basis of the width of the distal esophagus after myotomy.

Percutaneous biliary drainage effectively lowers serum bilirubin to permit chemotherapy treatment.

PURPOSE: For digestive tract cancers, the bilirubin threshold for administration of systemic chemotherapy can be 5 or 2 mg/dL (85.5 or 34.2 µmol/L) depending upon the regimen. We examined the ability of percutaneous biliary drainage (PBD) in patients with malignant biliary obstruction to achieve these clinically relevant endpoints. METHODS: 106 consecutive patients with malignant biliary obstruction and a baseline serum bilirubin >2 mg/dL underwent PBD. Time to achieve a bilirubin of 5 mg/dL (85.5 µmol/L), 2 mg/dL (34.2 µmol/L), and survival was estimated by Kaplan-Meier analysis. Potential technical and clinical prognostic factors were subjected to univariate and multivariate analysis. Categorical variables were analyzed by the log rank test. Hazard ratios were calculated for continuous variables. RESULTS: Median survival was 100 days (range 1-3771 days). Among 88 patients with a pre-drainage bilirubin >5 mg/dL, 62% achieved a serum bilirubin ≤5 mg/dL within 30 days and 84% within 60 days, median 21 days. Among 106 patients with a pre-drainage bilirubin >2 mg/dL, 37% achieved a serum bilirubin ≤2 mg/dL by 30 days and 70% within 60 days, median 43 days. None of the technical or clinical factors evaluated, including pre-drainage bilirubin, were significant predictors of time to achieve a bilirubin ≤2 mg/dL (p = 0.51). Size and type of biliary device were the only technical variables found to affect time to bilirubin of 5 mg/dL (p = 0.016). CONCLUSION: PBD of malignant obstruction achieves clinically relevant reduction in serum bilirubin in the majority of patients within 1-2 months, irrespective of the pre-drainage serum bilirubin, sufficient to allow administration of systemic chemotherapy. However, the decision to undergo this procedure for this indication alone must be considered in the context of patients' prognosis and treatment goals.

Collagen VI deficiency reduces muscle pathology, but does not improve muscle function, in the γ-sarcoglycan-null mouse.

Muscular dystrophy is characterized by progressive skeletal muscle weakness and dystrophic muscle exhibits degeneration and regeneration of muscle cells, inflammation and fibrosis. Skeletal muscle fibrosis is an excessive deposition of components of the extracellular matrix including an accumulation of Collagen VI. We hypothesized that a reduction of Collagen VI in a muscular dystrophy model that presents with fibrosis would result in reduced muscle pathology and improved muscle function. To test this hypothesis, we crossed γ-sarcoglycan-null mice, a model of limb-girdle muscular dystrophy type 2C, with a Col6a2-deficient mouse model. We found that the resulting γ-sarcoglycan-null/Col6a2Δex5 mice indeed exhibit reduced muscle pathology compared with γ-sarcoglycan-null mice. Specifically, fewer muscle fibers are degenerating, fiber size varies less, Evans blue dye uptake is reduced and serum creatine kinase levels are lower. Surprisingly, in spite of this reduction in muscle pathology, muscle function is not significantly improved. In fact, grip strength and maximum isometric tetanic force are even lower in γ-sarcoglycan-null/Col6a2Δex5 mice than in γ-sarcoglycan-null mice. In conclusion, our results reveal that Collagen VI-mediated fibrosis contributes to skeletal muscle pathology in γ-sarcoglycan-null mice. Importantly, however, our data also demonstrate that a reduction in skeletal muscle pathology does not necessarily lead to an improvement of skeletal muscle function, and this should be considered in future translational studies.

Effects of manual task complexity on gait parameters in school-aged children and adults.

This study examined the dual-task interference effects of complexity (simple vs. complex), type of task (carrying a pitcher vs. tray), and age (young adults vs. 7-10 year old children) on temporal-spatial and variability measures of gait. All participants first walked on the GAITRite walkway without any concurrent task, followed by four dual-task gait conditions. The group of children had a more variable step length and step time than adults across all walking conditions. They also slowed down, took fewer, smaller steps and spent more time in double limb support than adults in the complex dual task conditions. Gait in healthy young adults and school aged children was relatively unaffected by concurrent performance of simple versions of the manual tasks. Our overall analysis suggests that dual-task gait in school aged children is still developing and has not yet reached adult capacity. This study also highlights the critical role of task demand and complexity in dual-task interference.

MG53's new identity.

Mitsugumin 53 (MG53) is a relatively newly identified tripartite motif-containing (TRIM) family muscle-specific E3 ubiquitin ligase that is expressed in skeletal muscle and the heart. It has been postulated to facilitate repair by targeting the site of an injury, and acting as a scaffold for assembly of a repair complex made up of dysferlin, annexin V, caveolin-3, and polymerase I and transcript release factor (PTRF). A recent letter published in Nature by Song et al. proposes an alternate function for MG53: as an E3 ligase that targets the insulin receptor and insulin receptor substrate 1 (IRS1) for degradation, therefore regulating muscle insulin signaling. This work is exciting, as it not only presents a novel role for MG53, but also suggests that muscle insulin signaling has a systemic influence on insulin resistance and the metabolic syndrome.

Ready for policy? Stakeholder attitudes toward menu labelling in Toronto, Canada.

OBJECTIVES: The purpose of this research was to assess key stakeholder attitudes regarding menu labelling in Toronto, the largest municipality in Canada. Menu labelling is a population health intervention where food-labelling principles are applied to the eating-out environment through disclosure of nutrient content of food items on restaurant menus at the point of sale. Menu-labelling legislation has been implemented in the United States, but has yet to be adopted in Canada. As provincial voluntary programs and federal analyses progress, municipal jurisdictions will need to assess the feasibility of moving forward with parallel interventions. METHODS: Data were collected and analyzed in late 2011 to early 2012, including: a consumer eating-out module incorporated into a public health surveillance telephone survey (n=1,699); an online survey of independent restaurant operators (n=256); in-depth key informant interviews with executives and decision makers at chain restaurants (n=9); and a policy consultation with local restaurant associations. RESULTS: Toronto residents, particularly men, younger adults, and those with higher income or education, frequently eat out. A majority indicated that nutrition information is important to them; 69% note that they currently use it and 78% reported they would use it if it were readily available. Resistance to menu-labelling requirements at the municipal level was articulated by franchise/chain restaurant executives and industry associations. Despite overall low interest among independent restaurant operators, 57% reported feeling some responsibility to provide nutrition information and 50% believed it could be good for business. CONCLUSIONS: This research supports earlier literature that indicates strong public support for menu labelling alongside perceived barriers among the restaurant and foodservices sector. Leverage points for effective operator engagement for menu-labelling adoption were identified, nonetheless, highlighting the need for public health support.

Illuminating regeneration: noninvasive imaging of disease progression in muscular dystrophy.

Muscular dystrophies are characterized by progressive muscle weakness and wasting. Among the key obstacles to the development of therapies is the absence of an assay to monitor disease progression in live animals. In this issue of the JCI, Maguire and colleagues use noninvasive bioluminescence imaging to monitor luciferase activity in mice expressing an inducible luciferase reporter gene in satellite cells. These cells proliferate in response to degeneration, therefore increasing the level of luciferase expression in dystrophic muscle.

Dystrophin deficiency exacerbates skeletal muscle pathology in dysferlin-null mice.

BACKGROUND: Mutations in the genes coding for either dystrophin or dysferlin cause distinct forms of muscular dystrophy. Dystrophin links the cytoskeleton to the sarcolemma through direct interaction with ß-dystroglycan. This link extends to the extracellular matrix by ß-dystroglycan's interaction with α-dystroglycan, which binds extracellular matrix proteins, including laminin α2, agrin and perlecan, that possess laminin globular domains. The absence of dystrophin disrupts this link, leading to compromised muscle sarcolemmal integrity. Dysferlin, on the other hand, plays an important role in the Ca2+-dependent membrane repair of damaged sarcolemma in skeletal muscle. Because dysferlin and dystrophin play different roles in maintaining muscle cell integrity, we hypothesized that disrupting sarcolemmal integrity with dystrophin deficiency would exacerbate the pathology in dysferlin-null mice and allow further characterization of the role of dysferlin in skeletal muscle. METHODS: To test our hypothesis, we generated dystrophin/dysferlin double-knockout (DKO) mice by breeding mdx mice with dysferlin-null mice and analyzed the effects of a combined deficiency of dysferlin and dystrophin on muscle pathology and sarcolemmal integrity. RESULTS: The DKO mice exhibited more severe muscle pathology than either mdx mice or dysferlin-null mice, and, importantly, the onset of the muscle pathology occurred much earlier than it did in dysferlin-deficient mice. The DKO mice showed muscle pathology of various skeletal muscles, including the mandible muscles, as well as a greater number of regenerating muscle fibers, higher serum creatine kinase levels and elevated Evans blue dye uptake into skeletal muscles. Lengthening contractions caused similar force deficits, regardless of dysferlin expression. However, the rate of force recovery within 45 minutes following lengthening contractions was hampered in DKO muscles compared to mdx muscles or dysferlin-null muscles, suggesting that dysferlin is required for the initial recovery from lengthening contraction-induced muscle injury of the dystrophin-glycoprotein complex-compromised muscles. CONCLUSIONS: The results of our study suggest that dysferlin-mediated membrane repair helps to limit the dystrophic changes in dystrophin-deficient skeletal muscle. Dystrophin deficiency unmasks the function of dysferlin in membrane repair during lengthening contractions. Dystrophin/dysferlin-deficient mice provide a very useful model with which to evaluate the effectiveness of therapies designed to treat dysferlin deficiency.