Objective The aim of this study was to attain long-lasting alpha-sarcoglycan gene expression in LGMD2D subjects mediated by adeno-associated virus (AAV) gene transfer under control of a muscle specific promoter (tMCK) Methods rAAV1.tMCK.hSGCA (3.25 × 1011 vg) was delivered to the extensor digitorum brevis (EDB) muscle of three subjects with documented SGCA mutations via a double-blind, randomized, placebo controlled trial. Control sides received saline. The blind was not broken until the study was completed at 6 months and all results reported to the oversight committee. Results Persistent alpha-sarcoglycan gene expression was achieved for six months in two of three LGMD2D subjects. Markers for muscle fiber transduction other than alpha-sarcoglycan included expression of major histocompatability complex I (MHC I), increase in muscle fiber size, and restoration of the full sarcoglycan complex. Mononuclear inflammatory cells recruited to the site of gene transfer appeared to undergo programmed cell death demonstrated by TUNEL and caspase-3 staining. A patient failing gene transfer demonstrated an early rise in neutralizing antibody titers and T cell immunity to AAV validated by enzyme-linked immunospot (ELISpot) on the second day post gene injection. This was in clear distinction to other participants with satisfactory gene expression. Interpretation The findings of this gene replacement study in LGMD2D subjects have important implications not previously demonstrated in muscular dystrophy. Long-term, sustainable gene expression of alpha-sarcoglycan was observed following gene transfer mediated by AAV. The merit of a muscle specific tMCK promoter, not previously used in clinical trial was evident, and the potential for reversal of disease was displayed.

Manifesting carriers of DMD gene mutations may present diagnostic challenges, particularly in the absence of a family history of dystrophinopathy. We review the clinical and genetic features in fifteen manifesting carriers identified among 860 subjects within the United Dystrophinopathy Project, a large clinical dystrophinopathy cohort whose members undergo comprehensive DMD mutation analysis. We defined manifesting carriers as females with significant weakness, excluding those with only myalgias/cramps. DNA extracted from peripheral blood was used to study X chromosome inactivation patterns. Among these manifesting carriers, age at symptom onset ranged from 2 to 47 years. Seven had no family history and eight had male relatives with Duchene muscular dystrophy (DMD). Clinical severity among the manifesting carriers varied from a DMD-like progression to a very mild Becker muscular dystrophy-like phenotype. Eight had exonic deletions or duplications and six had point mutations. One patient had two mutations (an exonic deletion and a splice site mutation), consistent with a heterozygous compound state. The X chromosome inactivation pattern was skewed toward nonrandom in four out of seven informative deletions or duplications but was random in all cases with nonsense mutations. We present the results of DMD mutation analysis in this manifesting carrier cohort, including the first example of a presumably compound heterozygous DMD mutation. Our results demonstrate that improved molecular diagnostic methods facilitate the identification of DMD mutations in manifesting carriers, and confirm the heterogeneity of mutational mechanisms as well as the wide spectrum of phenotypes.

Objective:We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials.Methods:Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels.Results:Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable.Conclusions:Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval.