Kevin M. Flanigan

Distinctive patterns of microRNA expression in primary muscular disorders

The primary muscle disorders are a diverse group of diseases caused by various defective structural proteins, abnormal signaling molecules, enzymes and proteins involved in posttranslational modifications, and other mechanisms. Although there is increasing clarification of the primary aberrant cellular processes responsible for these conditions, the decisive factors involved in the secondary pathogenic cascades are still mainly obscure. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, we searched for miRNAs regulated during the degenerative process of muscle to gain insight into the specific regulation of genes that are disrupted in pathological muscle conditions. We describe 185 miRNAs that are up- or down-regulated in 10 major muscular disorders in humans [Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophies types 2A and 2B, Miyoshi myopathy, nemaline myopathy, polymyositis, dermatomyositis, and inclusion body myositis]. Although five miRNAs were found to be consistently regulated in almost all samples analyzed, pointing to possible involvement of a common regulatory mechanism, others were dysregulated only in one disease and not at all in the other disorders. Functional correlation between the predicted targets of these miRNAs and mRNA expression demonstrated tight posttranscriptional regulation at the mRNA level in DMD and Miyoshi myopathy. Together with direct mRNA–miRNA predicted interactions demonstrated in DMD, some of which are involved in known secondary response functions and others that are involved in muscle regeneration, these findings suggest an important role of miRNAs in specific physiological pathways underlying the disease pathology.

A Phase I/II trial of MYO-029 in Adult Subjects with Muscular Dystrophy

Myostatin is an endogenous negative regulator of muscle growth and a novel target for muscle diseases. We conducted a safety trial of a neutralizing antibody to myostatin, MYO‐029, in adult muscular dystrophies (Becker muscular dystrophy, facioscapulohumeral dystrophy, and limb‐girdle muscular dystrophy).

Eteplirsen for the treatment of Duchenne muscular dystrophy

In prior open‐label studies, eteplirsen, a phosphorodiamidate morpholino oligomer, enabled dystrophin production in Duchenne muscular dystrophy (DMD) with genetic mutations amenable to skipping exon 51. The present study used a double‐blind placebo‐controlled protocol to test eteplirsens ability to induce dystrophin production and improve distance walked on the 6‐minute walk test (6MWT).

Evidence‐based path to newborn screening for duchenne muscular dystrophy

Creatine kinase (CK) levels are increased on dried blood spots in newborns related to the birthing process. As a marker for newborn screening, CK in Duchenne muscular dystrophy (DMD) results in false‐positive testing. In this report, we introduce a 2‐tier system using the dried blood spot to first assess CK with follow‐up DMD gene testing.

Sequence specificity of aminoglycoside-induced stop codon readthrough: Potential implications for treatment of Duchenne muscular dystrophy

As a result of their ability to induce translational readthrough of stop codons, the aminoglycoside antibiotics are currently being tested for efficacy in the treatment of Duchenne muscular dystrophy patients carrying a nonsense mutation in the dystrophin gene. We have undertaken a systematic analysis of aminoglycoside‐induced readthrough of each stop codon in human tissue culture cells using a dual luciferase reporter system. Significant differences in the efficiency of aminoglycoside‐induced readthrough were observed, with UGA showing greater translational readthrough than UAG or UAA. Additionally, the nucleotide in the position immediately downstream from the stop codon had a significant impact on the efficiency of aminoglycoside‐induced readthrough in the order C > U > A ≥ G. Our studies show that the efficiency of stop codon readthrough in the presence of aminoglycosides is inversely proportional to the efficiency of translational termination in the absence of these compounds. Using the same assay, we analyzed a 33–base pair fragment of the mouse dystrophin gene containing the mdx premature stop codon mutation UAA (A), which is also the most efficient translational terminator. The additional flanking sequences from the dystrophin gene do not significantly change the relatively low‐level aminoglycoside‐induced stop codon readthrough of this stop codon. The implications of these results for drug efficacy in the treatment of individual patients with Duchenne muscular dystrophy or other genetic diseases caused by nonsense mutations are discussed. Ann Neurol 2000;48:164–169

Gentamicin-induced readthrough of stop codons in duchenne muscular dystrophy

The objective of this study was to establish the feasibility of long‐term gentamicin dosing to achieve stop codon readthrough and produce full‐length dystrophin. Mutation suppression of stop codons, successfully achieved in the mdx mouse using gentamicin, represents an important evolving treatment strategy in Duchenne muscular dystrophy (DMD).

Ataluren treatment of patients with nonsense mutation dystrophinopathy

Introduction: Dystrophinopathy is a rare, severe muscle disorder, and nonsense mutations are found in 13% of cases. Ataluren was developed to enable ribosomal readthrough of premature stop codons in nonsense mutation (nm) genetic disorders. Methods: Randomized, double‐blind, placebo‐controlled study; males ≥5 years with nm‐dystrophinopathy received study drug orally 3 times daily, ataluren 10, 10, 20 mg/kg (N = 57); ataluren 20, 20, 40 mg/kg (N = 60); or placebo (N = 57) for 48 weeks. The primary endpoint was change in 6‐Minute Walk Distance (6MWD) at Week 48. Results: Ataluren was generally well tolerated. The primary endpoint favored ataluren 10, 10, 20 mg/kg versus placebo; the week 48 6MWD Δ = 31.3 meters, post hoc P = 0.056. Secondary endpoints (timed function tests) showed meaningful differences between ataluren 10, 10, 20 mg/kg, and placebo. Conclusions: As the first investigational new drug targeting the underlying cause of nm‐dystrophinopathy, ataluren offers promise as a treatment for this orphan genetic disorder with high unmet medical need. Muscle Nerve 50: 477–487, 2014

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).

Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort

Mutations in the DMD gene, encoding the dystrophin protein, are responsible for the dystrophinopathies Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), and X‐linked Dilated Cardiomyopathy (XLDC). Mutation analysis has traditionally been challenging, due to the large gene size (79 exons over 2.2 Mb of genomic DNA). We report a very large aggregate data set comprised of DMD mutations detected in samples from patients enrolled in the United Dystrophinopathy Project, a multicenter research consortium, and in referral samples submitted for mutation analysis with a diagnosis of dystrophinopathy. We report 1,111 mutations in the DMD gene, including 891 mutations with associated phenotypes. These results encompass 506 point mutations (including 294 nonsense mutations) and significantly expand the number of mutations associated with the dystrophinopathies, highlighting the utility of modern diagnostic techniques. Our data supports the uniform hypermutability of CGA>TGA mutations, establishes the frequency of polymorphic muscle (Dp427m) protein isoforms and reveals unique genomic haplotypes associated with “private” mutations. We note that 60% of these patients would be predicted to benefit from skipping of a single DMD exon using antisense oligonucleotide therapy, and 62% would be predicted to benefit from an inclusive multiexonskipping approach directed toward exons 45 through 55. Hum Mutat 30:1657–1666, 2009.

Clinical and electrophysiologic features of CMT2A with mutations in the mitofusin 2 gene.

Background: Axonal neuropathy linked to the CMT2A locus was originally associated with a mutation in the KIF1B gene. However, mutations in this gene have not been described associated with any other CMT2A families. Recently, mutations in the MFN2 gene, encoding the mitochondrial GTPase mitofusin 2 (Mfn2), have been identified as causative of CMT2A in seven families. The authors report three additional CMT2A families associated with novel mutations in highly conserved regions of the Mfn2 GTPase domain. Methods: The authors performed a standardized neuromuscular and nerve conduction examination, genotyped known CMT loci, and analyzed the MFN2 gene by direct sequencing in three pedigrees and 10 additional probands affected by axonal CMT. Results: Sequencing of the MFN2 gene revealed a novel mutation in each family (c.818T>G, c.638T>C, and c.314C>T). The largest family demonstrated an age-independent variable expression such that approximately one quarter of individuals with the mutation presented with features mild enough as to remain occult even with electrophysiologic evaluation. Conclusion: These results confirm that the majority of cases of CMT linked to the CMT2A locus are due to MFN2 mutations. The phenotype is largely indistinguishable from KIF1B-related CMT and from CMT2E and CMT2F. At least in some families, as many as 25% of individuals with MFN2 mutations may be asymptomatic and have a normal electrophysiologic examination, although a detailed neuromuscular examination may suggest the trait. Given the frequency of MFN2 mutations among CMT2 probands (3/13, or 23%), genetic testing of CMT2 patients should begin with a screen of the MFN2 gene.