Jagged 1 Rescues the Duchenne Muscular Dystrophy Phenotype

Abstract

Duchenne muscular dystrophy (DMD), caused by mutations at the dystrophin gene, is the most common form of muscular dystrophy. There is no cure for DMD and current therapeutic approaches to restore dystrophin expression are only partially effective. The absence of dystrophin in muscle results in dysregulation of signaling pathways, which could be targets for disease therapy and drug discovery. Previously, we identified two exceptional Golden Retriever muscular dystrophy (GRMD) dogs that are mildly affected, have functional muscle, and normal lifespan despite the complete absence of dystrophin. Now, our data on linkage, whole-genome sequencing, and transcriptome analyses of these dogs compared to severely affected GRMD and control animals reveals that increased expression of Jagged1 gene, a known regulator of the Notch signaling pathway, is a hallmark of the mild phenotype. Functional analyses demonstrate that Jagged1 overexpression ameliorates the dystrophic phenotype, suggesting that Jagged1 may represent a target for DMD therapy in a dystrophin-independent manner. PAPERCLIP.

Keywords: DMD; Jagged1; dystrophin; genetic modifier; muscle.

Figure 1. Combining association, linkage and identity-by-descent analysis identifies a 30Mb candidate region on chromosome 24

(a) A QQ plot of 129,908 SNPs tested for association identified 27 SNPs outside the 95% confidence intervals (dashed lines) and minimal stratification relative to the expected distribution (red line), suggesting the mixed model approach corrected for close relatedness among the two escapers and 31 severely affected GRMD dogs. (b) Only the association on chromosome 24 also falls in a region where the two escapers (sire and offspring) share a long haplotype likely to be identical-by-descent (IBD, red). Other peaks on chromosomes 24, 33 and 37 show no evidence of IBD (grey) and are most likely false positives due to the small sample size. (c) The mapped region extends 27Mb from the start of chromosome 24. Linkage analysis with Merlin (solid black line) detected a significant linkage peak (dominant parametric LOD > 3) overlapping the IBD/association peak which includes the putative driver gene jagged1 (blue line) identified through gene expression profiling. See also Figures S1 and S2.

Figure 2. Altered Jagged1 expression in escaper GRMD dogs

(a) mRNA microarray comparing muscle gene expression of escaper GRMD dogs with related severely affected and wild-type littermates. (b) mRNA expression of escaper dogs confirming the expression array findings. Relative Jagged1 gene expression in muscle samples of escaper GRMD dogs as compared to related severely affected and wild-type dogs; bars indicate standard deviation from the mean. (c) Jagged1 protein levels in the muscle of escaper GRMD dogs (E) as compared to severely affected (A) and wild-type dog muscle (N); Beta-actin is the loading control. See also Table S1.

Figure 3. Variant located in the Jagged1 promoter of escaper GRMD dogs

(a) Dog and Human Jagged1 locus. Box: variant at dog chr24:11,644,709. (b) Conservation of the variant position. (c) Predicted transcription factor binding site at the region with the base pair change. (d) Consensus sequence of myogenin binding site, demonstrating the high information content of the T allele. (e) Electromobility shift assay (EMSA) showing myogenin binding to mutated probe (E) and not to the wild type probe (Wt). (f) Luciferase reporter assay showing activity of wild-type (Wt) and escaper (E) genotype vectors in muscle cells (C2C12) and embryonic kidney cells (293T) with Myogenin or MyoD overexpression, as compared to empty vectors controls (V). Error bars indicate SEM (n = 3 replicates). See also Figure S3.

Figure 4. Functional analysis of jagged1 expression

(a) Percent affected sapje fish as determined by birefringence assay at 4 dpf. Note fewer affected fish in the jagged1 injected sapje cohort. Four separate injection experiments were performed. (b) Genotype of sapje injected fish with jagged1a and jagged1b as compared to non-injected sapje fish. In red are dystrophin null fish with a wild-type phenotype, recovered by jagged1 overexpression. (c) Immunofluorescence of jagged1a and jagged1b overexpression in the sapje fish. Wild-type, phenotypically affected homozygous fish for the dystrophin mutation and jagged1a and jagged1b injected with normal birefringence (recovered) were stained for myosin heavy chain (MCH) and dystrophin antibodies. Note the organization of the muscle fibers in the recovered fish muscle comparable to the wild-type fish (n=10) even without dystrophin. Photographs were taken at 20× magnification. (d) Jagged1 protein levels in the muscle of cardiotoxin injured mice 1, 4 and 7 days after injury. (e) Jagged1 protein levels in muscle cells during in vitro muscle differentiation. (f) Muscle cell proliferation rate, as measured by MTT, of two wild-type, two escaper and two affected GRMD dogs. Error bars indicate SEM (n=2, three replicates).