Wild-Type Mouse Models to Screen Antisense Oligonucleotides for Exon-Skipping Efficacy in Duchenne Muscular Dystrophy

A readily available animal model is essential for rapidly identifying effective treatments for Duchenne muscular dystrophy (DMD), a devastating neuromuscular disorder caused by the lack of dystrophin protein, which results from frame-disrupting mutations in the DMD gene. Currently, the mdx mouse is the most commonly used model for antisense oligonucleotide (AO)-mediated exon skipping pre-clinical studies, with a mild phenotype. However, the accessibility of mdx mouse colonies particularly in developing countries can constrain research. Therefore in this study we explore the feasibility of using wild-type mice as models to establish exon-skipping efficiency of various DMD AO chemistries and their conjugates. Four different strains of wild-type mice and six different AO chemistries were investigated intramuscularly and the results indicated that the same exon-skipping efficiency was achieved for all tested AOs as that from mdx mice. Notably, levels of exon-skipping obtained in C57BL6 and C3H and mdx mice were most closely matched, followed by ICR and BALB/C mice. Systemic validation revealed that wild-type mice are less responsive to AO-mediated exon skipping than mdx mice. Our study provides evidence for the first time that wild-type mice can be appropriate models for assessing DMD AO exon-skipping efficiency with similar sensitivity to that of mdx mice and this finding can further accelerate the development of effective DMD AOs.     

Mutation-independent Proteomic Signatures of Pathological Progression in Murine Models of Duchenne Muscular Dystrophy

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Highlights

• •Proteomics analysis was performed in two murine models of Duchenne muscular dystrophy (mdx and mdx52) at three ages (8, 16 and 80 weeks) and compared with wild-type controls.
• •High-resolution isoelectric focusing liquid chromatography-tandem mass spectrometry enabled the quantification of 4974 proteins in all samples.
• •This study has revealed protein signatures of dystrophin deficiency and the progression of dystrophic pathology.
• •In contrast, the proteomes of the mdx and mdx52 mice were highly similar.
• •Pathway analysis revealed crosstalk between inflammatory, metabolic and muscle growth processes in dystrophic muscle.

     

A two-locus selection hypothesis for Duchenne Muscular Dystrophy

Three hypotheses to explain the incidence and familial structure of Duchenne Muscular Dystrophy are examined. The classical explanations of a high rate of mutation and heterozygote advantage are shown to be inconsistent with the data. The third hypothesis of two closely linked loci on the X chromosome is developed and shown to be compatible with a set of Utah families including many large Mormon families. The application of the results to other X-linked and autosomal diseases is discussed.     

Evidence for Locus Heterogeneity in Autosomal Dominant Limb-Girdle Muscular Dystrophy

Limb-girdle muscular dystrophy (LGMD) is a diagnostic classification encompassing a broad group of proximal myopathies. A gene for the dominant form of LGMD (LGMD1A) has recently been localized to a 7-cM region of chromosome 5q between D5S178 and IL9. We studied three additional dominant LGMD families for linkage to these two markers and excluded all from localization to this region, providing evidence for locus heterogeneity within the dominant form of LGMD. Although the patterns of muscle weakness were similar in all families studied, the majority of affected family members in the chromosome 5–linked pedigree have a dysarthric speech pattern, which is not present in any of the five unlinked families. The demonstration of heterogeneity within autosomal dominant LGMD is the first step in attempting to subclassify these families with similar clinical phenotypes on a molecular level.     

The Identification of Carriers in Duchenne Muscular Dystrophy

Following recurrent reports in the literature that it is possible to detect carriers of the gene for progressive muscular dystrophy (Duchenne), a survey of nine families was performed. No unequivocal results could be obtained in comparison of clinical data with circulation-time measurements or serum enzyme studies. It is concluded that carrier-labelling with respect to progressive muscular dystrophy is not yet at a satisfactory stage, although progress is being made.     

Genetic Modifiers of Duchenne Muscular Dystrophy and Dilated Cardiomyopathy

Objective

Dilated cardiomyopathy (DCM) is a major complication and leading cause of death in Duchenne muscular dystrophy (DMD). DCM onset is variable, suggesting modifier effects of genetic or environmental factors. We aimed to determine if polymorphisms previously associated with age at loss of independent ambulation (LoA) in DMD (rs28357094 in the SPP1 promoter, rs10880 and the VTTT/IAAM haplotype in LTBP4) also modify DCM onset.

Methods

A multicentric cohort of 178 DMD patients was genotyped by TaqMan assays. We performed a time-to-event analysis of DCM onset, with age as time variable, and finding of left ventricular ejection fraction < 50% and/or end diastolic volume > 70 mL/m² as event (confirmed by a previous normal exam < 12 months prior); DCM-free patients were censored at the age of last echocardiographic follow-up.

Results

Patients were followed up to an average age of 15.9 ± 6.7 years. Seventy-one/178 patients developed DCM, and median age at onset was 20.0 years. Glucocorticoid corticosteroid treatment (n = 88 untreated; n = 75 treated; n = 15 unknown) did not have a significant independent effect on DCM onset. Cardiological medications were not administered before DCM onset in this population. We observed trends towards a protective effect of the dominant G allele at SPP1 rs28357094 and recessive T allele at LTBP4 rs10880, which was statistically significant in steroid-treated patients for LTBP4 rs10880 (< 50% T/T patients developing DCM during follow-up [n = 13]; median DCM onset 17.6 years for C/C-C/T, log-rank p = 0.027).

Conclusions

We report a putative protective effect of DMD genetic modifiers on the development of cardiac complications, that might aid in risk stratification if confirmed in independent cohorts.     

Effective Classification and Gene Expression Profiling for the Facioscapulohumeral Muscular Dystrophy

The Facioscapulohumeral Muscular Dystrophy (FSHD) is an autosomal dominant neuromuscular disorder whose incidence is estimated in about one in 400,000 to one in 20,000. No effective therapeutic strategies are known to halt progression or reverse muscle weakness and atrophy. It is known that the FSHD is caused by modifications located within a D4ZA repeat array in the chromosome 4q, while recent advances have linked these modifications to the DUX4 gene. Unfortunately, the complete mechanisms responsible for the molecular pathogenesis and progressive muscle weakness still remain unknown. Although there are many studies addressing cancer databases from a machine learning perspective, there is no such precedent in the analysis of the FSHD. This study aims to fill this gap by analyzing two specific FSHD databases. A feature selection algorithm is used as the main engine to select genes promoting the highest possible classification capacity. The combination of feature selection and classification aims at obtaining simple models (in terms of very low numbers of genes) capable of good generalization, that may be associated with the disease. We show that the reported method is highly efficient in finding genes to discern between healthy cases (not affected by the FSHD) and FSHD cases, allowing the discovery of very parsimonious models that yield negligible repeated cross-validation error. These models in turn give rise to very simple decision procedures in the form of a decision tree. Current biological evidence regarding these genes shows that they are linked to skeletal muscle processes concerning specific human conditions.     

Paper Chromatographic Survey of Urinary Amino-acids in Muscular Dystrophy

A paper chromatographic analysis in 60 patients with muscular dystrophy from 49 families and in 351 of their relatives showed a high incidence of generalized hyperaminoaciduria in both patients and relatives. There was a higher incidence of threoninuria in patients and their relatives than in controls. One family was found to have excess histidinuria and another excess lysinuria.