Xiomara Q. Rosales

Sustained alpha‐sarcoglycan gene expression after gene transfer in limb‐girdle muscular dystrophy, type 2D

The aim of this study was to attain long‐lasting alpha‐sarcoglycan gene expression in limb‐girdle muscular dystrophy, type 2D (LGMD2D) subjects mediated by adeno‐associated virus (AAV) gene transfer under control of a muscle specific promoter (tMCK).

A Phase 1/2a Follistatin Gene Therapy Trial for Becker Muscular Dystrophy

Becker muscular dystrophy (BMD) is a variant of dystrophin deficiency resulting from DMD gene mutations. Phenotype is variable with loss of ambulation in late teenage or late mid-life years. There is currently no treatment for this condition. In this BMD proof-of-principle clinical trial, a potent myostatin antagonist, follistatin (FS), was used to inhibit the myostatin pathway. Extensive preclinical studies, using adeno-associated virus (AAV) to deliver follistatin, demonstrated an increase in strength. For this trial, we used the alternatively spliced FS344 to avoid potential binding to off target sites. AAV1.CMV.FS344 was delivered to six BMD patients by direct bilateral intramuscular quadriceps injections. Cohort 1 included three subjects receiving 3u2009×u200910(11) vg/kg/leg. The distance walked on the 6MWT was the primary outcome measure. Patients 01 and 02 improved 58 meters (m) and 125 m, respectively. Patient 03 showed no change. In Cohort 2, Patients 05 and 06 received 6u2009×u200910(11) vg/kg/leg with improved 6MWT by 108 m and 29 m, whereas, Patient 04 showed no improvement. No adverse effects were encountered. Histological changes corroborated benefit showing reduced endomysial fibrosis, reduced central nucleation, more normal fiber size distribution with muscle hypertrophy, especially at high dose. The results are encouraging for treatment of dystrophin-deficient muscle diseases.

Novel diagnostic features of dysferlinopathies

Reports of dysferlinopathy have suggested a clinically heterogeneous group of patients. We identified specific novel molecular and phenotypic features that help distinguish dysferlinopathies from other forms of limb‐girdle muscular dystrophy (LGMD). A detailed history, physical exam, and protein and mutation analysis of genomic DNA was done for all subjects. Five of 21 confirmed DYSF gene mutations were not previously reported. A distinct “bulge” of the deltoid muscle in combination with other findings was a striking feature in all patients. Six subjects had atypical calf enlargement, and 3 of these exhibited a paradoxical pattern of dysferlin expression: severely reduced by direct immunofluorescence with overexpression on Western blots. Six patients showed amyloid deposits in muscle that extended these findings to new domains of the dysferlin gene, including the C2G domain. Correlative studies showed colocalization of amyloid with deposition of dysferlin. The present data further serve to guide clinicians facing the expensive task of molecular characterization of patients with an LGMD phenotype. Muscle Nerve 42: 14–21, 2010

Pharmacokinetics and safety of single doses of drisapersen in non-ambulant subjects with Duchenne muscular dystrophy: Results of a double-blind randomized clinical trial

Duchenne muscular dystrophy (DMD) is a progressive, lethal neuromuscular disorder caused by the absence of dystrophin protein due to mutations of the dystrophin gene. Drisapersen is a 2′-O-methyl-phosphorothioate oligonucleotide designed to skip exon 51 in dystrophin pre-mRNA to restore the reading frame of the mRNA. This study assessed safety, tolerability, and pharmacokinetics of drisapersen after a single subcutaneous administration in non-ambulatory subjects. Eligible subjects were non-ambulant boys aged ≥9 years, in wheelchairs for ≥1 to ≤4 years, with a diagnosis of DMD resulting from a mutation correctable by drisapersen treatment. Four dose cohorts were planned (3, 6, 9 and 12 mg/kg), but study objectives were met with the 9 mg/kg dose. Less than proportional increase in exposure was demonstrated over the 3–9 mg/kg dose range, though post hoc analysis showed dose proportionality was more feasible over the 3–6 mg/kg range. Single doses of drisapersen at 3 and 6 mg/kg did not result in significant safety or tolerability concerns; however, at the 9 mg/kg dose, pyrexia and transient elevations in inflammatory parameters were seen. The maximum tolerated dose of 6 mg/kg drisapersen was identified for further characterization in multiple dose studies in the non-ambulant DMD population.

Cardiovascular magnetic resonance of cardiomyopathy in limb girdle muscular dystrophy 2B and 2I

BackgroundLimb girdle muscular dystrophies (LGMD) are inclusive of 7 autosomal dominant and 14 autosomal recessive disorders featuring progressive muscle weakness and atrophy. Studies of cardiac function have not yet been well-defined in deficiencies of dysferlin (LGMD2B) and fukutin related protein (LGMD2I). In this study of patients with these two forms of limb girdle muscular dystrophy, cardiovascular magnetic resonance (CMR) was used to more specifically define markers of cardiomyopathy including systolic dysfunction, myocardial fibrosis, and diastolic dysfunction.MethodsConsecutive patients with genetically-proven LGMD types 2I (n = 7) and 2B (n = 9) and 8 control subjects were enrolled. All subjects underwent cardiac magnetic resonance (CMR) on a standard 1.5 Tesla clinical scanner with cine imaging for left ventricular (LV) volume and ejection fraction (EF) measurement, vector velocity analysis of cine data to calculate myocardial strain, and late post-gadolinium enhancement imaging (LGE) to assess for myocardial fibrosis.ResultsSixteen LGMD patients (7 LGMD2I, 9 LGMD2B), and 8 control subjects completed CMR. All but one patient had normal LV size and systolic function; one (type 2I) had severe dilated cardiomyopathy. Of 15 LGMD patients with normal systolic function, LGE imaging revealed focal myocardial fibrosis in 7 (47%). Peak systolic circumferential strain rates were similar in patients vs. controls: εendo was -23.8 ± 8.5vs. -23.9 ± 4.2%, εepi was -11.5 ± 1.7% vs. -10.1 ± 4.2% (p = NS for all). Five of 7 LGE-positive patients had grade I diastolic dysfunction [2I (n = 2), 2B (n = 3)]. that was not present in any LGE-negative patients or controls.ConclusionsLGMD2I and LGMD2B generally result in mild structural and functional cardiac abnormalities, though severe dilated cardiomyopathy may occur. Long-term studies are warranted to evaluate the prognostic significance of subclinical fibrosis detected by CMR in these patients.

KNEE EXTENSOR STRENGTH EXHIBITS POTENTIAL TO PREDICT FUNCTION IN SPORADIC INCLUSION-BODY MYOSITIS

Introduction: In this study we address the challenging issue of potential use of muscle strength to predict function in clinical trials. This has immediate relevance to translational studies that attempt to improve quadriceps strength in sporadic inclusion‐body myositis (sIBM). Methods: Maximum voluntary isometric contraction testing as a measure of muscle strength and a battery of functional outcomes were tested in 85 ambulatory subjects with sIBM. Results: Marked quadriceps weakness was noted in all patients. Strength was correlated with distance walked at 2 and 6 minutes. Additional correlations were found with time to get up from a chair, climb stairs, and step up on curbs. Conclusions: Quadriceps (knee extensor) strength correlated with performance in this large cohort of sIBM subjects, which demonstrated its potential to predict function in this disease. These data provide initial support for use of muscle strength as a surrogate for function, although validation in a clinical trial is required. Muscle Nerve, 2012

Impaired regeneration in LGMD2A supported by increased Pax7 positive satellite cell content and muscle specific microRNA dysregulation

Introduction: Recent in vitro studies suggest that CAPN3 deficiency leads initially to accelerated myofiber formation followed by depletion of satellite cells (SC). In normal muscle, up‐regulation of miR‐1 and miR‐206 facilitates transition from proliferating SCs to differentiating myogenic progenitors. Methods: We examined the histopathological stages, Pax7 SC content, and muscle‐specific microRNA expression in biopsy specimens from well‐characterized LGMD 2A patients to gain insight into disease pathogenesis. Results: Three distinct stages of pathological changes were identified that represented the continuum of the dystrophic process from prominent inflammation with necrosis and regeneration to prominent fibrosis, which correlated with age and disease duration. Pax7‐positive SCs were highest in the fibrotic group and correlated with down‐regulation of miR‐1, miR‐133a, and miR‐206. Conclusions: These observations, and other published reports, are consistent with microRNA dysregulation leading to inability of Pax7‐positive SCs to transit from proliferation to differentiation. This results in impaired regeneration and fibrosis. Muscle Nerve 47: 731–739, 2013

A slowly progressive form of limb-girdle muscular dystrophy type 2C associated with founder mutation in the SGCG gene in Puerto Rican Hispanics.

Limb‐girdle muscular dystrophy type 2C (LGMD2C) is considered one of the severe forms of childhood‐onset muscular dystrophy. The geographical distribution of founder mutations in the SGCG gene has a prominent effect on the prevalence of LGMD2C in certain populations. The aim of this study was to confirm the hypothesis that the c.787G>A (p.E263K) mutation in the SGCG gene is a founder mutation among Puerto Rican Hispanics and to characterize the associated clinical and immunohistochemical phenotype. Genotyping of six polymorphic microsatellite markers internal to (D13S232) and flanking (D13S175, D13S292, D13S787, D13S1243, D13S283) the SGCG gene was performed on four unrelated Puerto Rican patients with LGMD2C. Preserved ambulation to the second decade of life was observed in at least two subjects. Immunostaining of skeletal muscle demonstrated absence of γ‐sarcoglycan in all affected subjects. Two markers, D13S232 and D13S292, were highly informative and confirmed that all four families share the haplotype of the mutant allele. Our findings confirm that the E263K missense mutation in the SGCG gene is a founder mutation in Puerto Rican Hispanics. A slowly progressive disease course with prolonged preservation of ambulation can be seen in association with this mutation, providing evidence for phenotypic variability.

D.P.4 Comparison of commercially-available exome capture kits in the diagnosis of neuromuscular disorders

Abstract Next generation sequencing technologies present opportunities for improved clinical molecular diagnosis of neuromuscular disorders (NMD). We sought to determine the feasibility of using whole exome sequencing to detect mutations in 347 isoforms of 143 NMD genes listed in the World Muscle Society Muscle Gene Table ( www.musclegenetable.org ). We initially tested DNA samples from eight subjects with limb-girdle muscular dystrophies (LGMD) and known mutations, using exome capture with one of three commercially available kits. We detected the same mutations as gene-specific Sanger sequencing had previously detected in seven of eight, missing 1 MYOT mutation due to a lack of representation of this specific exon in the capture kit. This led us to compare commercially available exome capture kits for coverage of NMD genes, by both predicted and by empiric coverage, in normal control genomic DNA. Based on manufacturer targeted region files, the three kits each targeted 92–94% of the known NMD exons; however, our actual capture results demonstrated that at best ∼60% of these exons obtained 100% coverage. In our hands the best performing kit, Agilent SureSelect v3, captures an average of 92.7% of the bases in the NMD gene exons, but only 58% of the NMD isoforms had all exons captured at 100% (e.g., 42% of the NMD genes had at least one exon with one base insufficiently captured to make a genotyping call). Illumina TruSeq captured 89.2% of exons but only 36.5% of genes had 100% coverage, and Nimblegen v2 captured 90.5% of genes but only 46.3% genes had 100% sequence coverage. We predict that by utilizing all three kits, ∼70% of the known NMD isoforms would have sufficient sequence coverage to make a genotyping call for 100% of all exons. When considering adoption of this approach clinically, this is an important limitation to be aware of but continued improvements in commercially available capture kits should allow economical and rapid NMD gene sequence analysis.

G.O.25

Becker muscular dystrophy (BMD), a dystrophinopathy with milder skeletal muscle manifestations compared to Duchenne muscular dystrophy, characteristically demonstrates weakness of knee extensors (KE), accompanied by muscle atrophy and fibrosis. The weakness predisposes to gait impairment and loss of ambulation. Myostatin, a member of the TGF-β superfamily of signal peptides, is expressed in skeletal muscle, acting through the activin type IIB (ACTRIIB) receptor. Follistatin, a myostatin antagonist, inhibits this pathway resulting in muscle hypertrophy, and improved muscle strength. FS315 and FS288 are two isoforms of follistatin generated by alternative splicing. FS315 has no cell-surface affinity providing a safety margin avoiding pituitary–gonadal axis complications. FS344 is the precursor cDNA to FS315. When FS344 is delivered to mdx mice in adeno-associated virus (AAV1. CMV. FS344), muscle mass and strength increase. Similar findings are seen in non-human primates without safety concerns. Six BMD patients, 18xa0years and older with proven in-frame DMD gene mutations were enrolled in a clinical trial. The first 3 subjects received 12 intramuscular (IM) injections of AAV1. CMV. FS344 at a total dose of 6e11 vg/kg in the quadriceps muscles bilaterally. A second cohort received the same number of IM injections at higher total dose (1.2e12 vg/kg). Studies extending beyond one year in Cohort 1 and for six months in Cohort 2 showed no adverse events. The distance walked (meters) on the 6-min walk test (6MWT) was the primary functional outcome measure. At one year the distance walked by two subjects in Cohort 1 increased by 125xa0m and 58xa0m; subject 3 improved modestly by 9xa0m. In Cohort 2, one patient reaching the 6-month time point increased by 108 meters. For the entire group of BMD patients only a single patient declined in distance. Preliminary analysis of muscle biopsies favor increased muscle regeneration contributing to improved function.