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.

In this study, we set out to answer the following question: how do these escaper dogs have a fully functional muscle without dystrophin? Skeletal muscle of DMD patients undergoes waves or cycles of degeneration followed by regeneration. Muscle repair is a regulated process that comprises different cell types and signaling molecules, but additional factors and genetic modifiers involved in DMD pathogenesis remain poorly understood, representing new potential therapeutic targets. Genetic modifiers have been reported in DMD patients with a slower progression, but none were associated with a nearly normal phenotype (). Here, through three independent approaches, we identified a modifier gene, Jagged1, which can modulate the GRMD phenotype. Using a mixed model association and linkage analysis, we identified a chromosomal region associated with the escaper phenotype. One gene within this region showed altered expression when comparing muscle tissue of escaper and affected dogs. By whole-genome sequencing, we found a variant present only in escaper GRMD dogs that creates a novel myogenin binding site in the Jagged1 promoter. Overexpression of jagged1 in dystrophin deficient zebrafish rescues the dystrophic phenotype in this zebrafish model. This suggests that Jagged1, when increased in expression in muscle, can rescue dystrophin-deficient phenotypes in two different animal models, pointing to a new potential therapeutic target.

These two exceptional, related GRMD dogs (here called “escapers”) remained fully ambulatory with normal lifespans, a phenotype never reported before for GRMD. They fall outside the known GRMD phenotypic range of variability, differing significantly from typically affected dogs despite their dystrophic muscle, absence of muscle dystrophin, elevated serum CK levels, and lack of evidence of utrophin upregulation (). Most importantly, these GRMD dogs show that it is possible to have a functional muscle in a mid-size dystrophin-deficient animal.

To explore the efficiency of the different therapeutic approaches for DMD, there is a need for animal models that mimic the human condition. However, animal models of dystrophin-deficiency show differences in skeletal muscle pathology in response to dystrophin-deficiency (). The dystrophin-deficient fish model sapje shows some phenotypic variability, but nearly all fish die during the first weeks of life and all show abnormal muscle structure as measured by birefringence under polarized light (). The mdx mouse is the most widely used animal model for DMD, even though its mild phenotype does not mimic severe human DMD symptoms (). The most similar to the human condition is the golden retriever muscular dystrophy (GRMD) dog (). These animals carry a point mutation on a splicing site that causes the skipping of exon 7 and a premature stop codon, resulting in the absence of dystrophin. GRMD dogs and DMD patients share many similarities in disease pathogenesis, including early progressive muscle degeneration and atrophy, fibrosis, contractures, and grossly elevated serum creatine kinase (CK) levels (). Early death may occur within the first weeks of life but usually occurs around 1–2 years of age as a result of respiratory failure or cardiomyopathy. The great majority of GRMD dogs do not survive beyond age two. In the Brazilian GRMD colony at Biosciences Institute at the University of São Paulo, we have described two exceptional dogs presenting a very mild phenotype clearly distinguishable from other affected dogs despite the absence of muscle dystrophin. Histopathological and immunohistochemistry analysis of their muscle showed typical features of a dystrophic process with variability in fiber size, splitting, degeneration, and infiltrating connective tissue ().

DMD therapeutic approaches currently under development aim to rescue dystrophin expression in the muscle (). Pre-clinical and clinical studies include exon-skipping (), AAV-delivery of μ-dystrophin (), and nonsense suppression to induce “readthrough” of nonsense mutations (). While AAV-delivery led to μ-dystrophin expression in skeletal muscle, T cell immunity against dystrophin epitopes was reported (). Also, the success of the dystrophin-based therapies relies on the quality of the recipient muscle. This requires the development of dystrophin-independent therapies to improve the muscle condition targeting the altered signaling pathways.

Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by mutations in dystrophin (), which affects 1 in 3,500 to 5,000 boys (). Deficiency of muscle dystrophin causes progressive myofiber degeneration and muscle wasting (). The first symptoms are usually evident at 3–5 years of age, with loss of ambulation between 9 and 12 years. Death occurs in the second or third decade due to respiratory or cardiac failure. While there are several treatments under development or currently in use—particularly corticotherapy, which aims to ameliorate symptoms and slow down the disease progression—there is still no cure for DMD (). Allelic to DMD, Becker muscular dystrophy (BMD) is caused by mutations that do not affect the reading frame of the dystrophin transcript; the result is a semi-functional, truncated dystrophin protein (). DMD muscle shows a complete absence of dystrophin, whereas in the BMD muscle there is a variable amount of partially functional dystrophin (). Differently from DMD, where most boys carrying null mutations show a severe phenotype, BMD patients show a variable clinical course. Genotype/phenotype correlation studies suggest that the severity of the phenotype is dependent on the amount of muscle dystrophin or the site of the mutation/deletion in the dystrophin gene (

Half the dystrophin gene is apparently enough for a mild clinical course: confirmation of its potential use for gene therapy.

When examining the effect of Jagged1 on muscle regeneration in normal mice, we found that Jagged1 expression is upregulated at day 4 after cardiotoxin-induced injury in mouse tibialis anterior muscle ( Figure 4 D). We also determined that Jagged1 is elevated during myoblast muscle differentiation in vitro ( Figure 4 E). To examine whether muscle cells from escaper dogs proliferate faster than cells from severely affected dogs, we performed a proliferation assay using myogenic cells from biopsies of age-matched dogs. Escaper dogs’ muscle showed typical dystrophic features () as evidenced by cycles of degeneration and regeneration, which is not seen in normal muscle. Because of these cycles and consistent activation, myogenic cells from affected GRMD dogs are expected to divide less frequently. We show that muscle cells from escaper dogs divide significantly faster than those from affected dogs ( Figure 4 F). These results are consistent with previous findings that show that overexpression of the Notch intracellular domain (NICD) expands the proliferative capacity of activated muscle satellite cells in vitro and in vivo ().

To evaluate if the overexpression of Jagged1 can ameliorate the dystrophic muscle phenotype in other species, we used the severely affected dystrophic sapje zebrafish DMD model. Muscle phenotype was assayed using birefringence, where fish are placed under a polarized light and dystrophin-negative fish show a decrease in the amount of light, indicative of muscle tearing or muscle fiber disorganization. In four separate experiments, we injected approximately 200 fertilized one-cell stage eggs from sapje heterozygous fish matings with mRNA of either one of the zebrafish jagged1 genetic copies of the mammalian Jagged1 gene: jagged1a or jagged1b. In all experiments, an average of 24% of the non-injected sapje fish exhibited a typical affected dystrophic, patchy birefringence phenotype. This proportion is within the 21%–27% expected range of affected fish of a heterozygous sapje mating. In contrast, fish injected with either jagged1a or jagged1b showed a significantly lower percentage of fish with poor birefringence (p = 1.31×10for jagged1a, p = 4.4×10for jagged1b, Figure 4 A). Genotypic analysis revealed that about 75% of dystrophin-null fish injected with jagged1a and 60% of of those injected with jagged1b had normal birefringence, which demonstrated a common rescue from the muscle lethality phenotype ( Figure 4 B). These results indicate that increasing jagged1 expression rescues most dystrophin-null fish from developing the abnormalities typically seen in dystrophin-null muscle. To further evaluate the jagged1a and jagged1b overexpression sapje fish, we performed immunostaining on individual fish bodies using a myosin heavy chain (MHC) antibody to evaluate muscle structure. In WT fish, MHC was clearly expressed and showed that muscle fibers were normal. Interestingly, MHC staining of jagged1 mRNA-injected dystrophin-null rescued fish showed normal myofiber structure similar to that of WT fish, whereas affected, non-injected dystrophin-null fish demonstrated clear muscle abnormalities ( Figure 4 C).

(F) Muscle cell proliferation rate, as measured by MTT, of two WT, two escaper, and two affected GRMD dogs. Error bars indicate SEM (n = 2, three replicates).

(D) Jagged1 protein levels in the muscle of cardiotoxin injured mice one, four, and seven days after injury.

(C) Immunofluorescence of jagged1a and jagged1b overexpression in the sapje fish. WT, 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 WT fish (n = 10) even without dystrophin. Photographs were taken at 20x magnification.

(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 WT phenotype, recovered by jagged1 overexpression.

To understand the effects of the escaper variant, we performed different functional analyses. This candidate variant was found to be conserved across 29 eutherian mammals, suggesting a regulatory potential for this region ( Figures 3 A and 3B). Transcription factor binding site analysis, using TRAP () and TRANSFAC (), revealed that this G>T change creates a novel myogenin binding site ( Figure 3 C) with a high information content for the mutant allele (T) in the myogenin consensus binding motif ( Figure 3 D). Myogenin is a muscle-specific transcription factor involved in muscle differentiation and repair (). To determine whether the variant affects DNA binding by myogenin, we carried out electrophoretic mobility shift assays (EMSAs) using muscle cell nuclear extracts and biotin-labeled oligonucleotide probes containing either the wild-type (WT) or escaper (E) genotype. The oligonucleotide probe containing the escaper T allele robustly bound the myogenin protein, whereas an oligonucleotide probe containing the WT G allele did not bind at all ( Figure 3 E). A competition assay showed that an unlabeled escaper probe efficiently competed with the binding of the labeled escaper probe. In contrast, the unlabeled WT probe had no effect on the binding activity of the labeled escaper probe, indicating a specific interaction between the escaper allele and myogenin ( Figure 3 E). To evaluate whether the novel myogenin binding site found in the escaper dogs was driving the increased expression of Jagged1, we performed a luciferase reporter assay using Jagged1 upstream promoter sequences containing either the WT sequence or the escaper variant fused to a luciferase reporter. Luciferase vectors containing either WT or escaper sequence were transfected into muscle cells (myoblasts) and human embryonic kidney cells (HEK293T) along with constructs that overexpress either myogenin or another E-box myogenic factor (MyoD) as control. On HEK293K cells, overexpression of myogenin was able to activate the expression of the escaper Jagged1 reporter 3-fold, but showed no activation of the WT reporter ( Figure 3 F). As predicted, the overexpression of MyoD did not activate either the WT or escaper Jagged1 luciferase reporter ( Figure 3 F). Similarly, myoblasts (that endogenously express myogenin) transfected with the escaper vector showed a similar luciferase activation that was three times higher than the WT vector, notwithstanding the presence of overexpression vectors ( Figure 3 F). These results demonstrate that the creation of the novel myogenin binding site in the escaper Jagged1 promoter is essential for driving the increase of Jagged1 expression in the escaper dog skeletal muscles.

To identify potentially causative variants behind the differential gene expression pattern observed in the escaper dogs, we performed whole-genome sequencing on three dogs (the two escapers and one severely affected related dog). We hypothesized that the compensatory variation would be novel, as the escaper phenotype had not previously been seen in GRMD dogs worldwide. We looked for variants located under the association peak on chromosome 24 and focused on the Jagged1 locus (including 3 KB upstream and downstream of the gene) in search for a variant present only in the escapers and not in the affected GRMD dogs. A total of ∼1,300 variants were detected within the escaper-associated region on chromosome 24. All variants were lifted over to the human genome, and those present in muscle enhancer regions near the promoters of the two isoforms of Jagged1 expressed in skeletal muscle ( Figure 3 A) () were further analyzed. Since the escaper variant was hypothesized to be novel, all variants detected in previous extensive canine sequencing efforts () were excluded. After this filtering, only a single point variant was found to follow the escaper haplotype: a heterozygote G>T change in the promoter region of Jagged1 (cfa24:11655709, Figure 3 A). Sanger sequencing of the Jagged1 candidate escaper variant was performed in the escaper extended pedigree, including the first escaper (M1M4), his offspring, and a sibling’s offspring (M1M5) ( Figure S3 ). We also sequenced key breeders of the kennel and found that the variant is specific to the escapers’ pedigree and was introduced in a single outcross (B1F3 mate). All affected dogs lacked the Jagged1 variant, while both escapers were heterozygous. Thus, the novel Jagged1 mutation segregates with the escaper phenotype in this family. Four additional individuals carried the candidate variant: three were stillborn puppies and the fourth was a GRMD puppy that died at 6 months of age from an accidental ingestion of a foreign object. This puppy (K2M11) was fully ambulatory with a similar phenotype to the two escaper dogs, but he was classified as affected in the mapping analysis since we cannot predict his adult phenotype with confidence.

All dogs were genotyped for the GRMD mutation and for the jagged1 variant (G>T at Chr24: 11,655,709). Escaper dogs are: M1M4 and H3M10.

(F) Luciferase reporter assay showing activity of WT and E genotype vectors in both 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

We then performed a genome-wide analysis for genes differentially expressed in muscle between the escapers and affected dogs. Using Agilent mRNA SurePrint Canine arrays, we compared muscle gene expression of the two escapers, four affected, and four wild-type dogs at two years of age. We found very similar muscle gene expression patterns in the two escaper GRMD dogs, which were more similar to muscle from wild-type dogs than from the affected dogs. In total, 114 genes were found to be differentially expressed between escapers and affected GRMD dogs, as shown by unsupervised hierarchical clustering of all ten samples ( Figure 2 A). Of these, 65 genes were also differentially expressed between escapers and wild-type dogs ( Table S1 ), implicating them in a possible compensatory mechanism active in only the escaper dogs. Only one of these 65 genes, Jagged1, is located under the association peak on chromosome 24. Jagged1 mRNA levels were two times higher in the escapers when compared to both wild-type and severely affected dogs ( Figure 2 B). Further protein level analysis confirmed the mRNA findings ( Figure 2 C).

(C) Jagged1 protein levels in the muscle of escaper GRMD dogs (E) as compared to severely affected (A) and WT dog muscle (N); Beta-actin is the loading control. See also Table S1

(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 WT dogs; bars indicate SD from the mean.

To understand the genetic basis behind the escaper phenotype in GRMD dogs, we performed a genome-wide mapping analysis comparing two related escaper GRMD dogs—the only two GRMD escapers reported to date—to 31 severely affected GRMD dogs from the same breeding population. All GRMD dogs were confirmed to carry the originally described point mutation (a change from adenine to guanine transition) in the intron 6 of the dystrophin gene. This mutation ablates a splicing site and exon 7 is skipped from the mature mRNA. The absence of exon 7 causes a premature stop codon at exon 8 (). Based on survival age and functional capacity, they were classified as escaper or affected (binary). All the dogs showing the standard range of phenotypic variability seen in GRMD dogs were classified as affected in this study. Our aim was to identify a single gene responsible for the milder phenotype seen in the two escaper dogs. We performed a two-step mapping analysis. First, we carried out an association study, utilizing the power of the many severely affected dogs expected to lack the modifier locus. This was followed by segregation analysis, taking advantage of the fact that the two escapers came from a well-defined pedigree in which a transmission-based test could be used. All dogs were genotyped using the Illumina CanineHD 170K SNP array. We tested for association genome wide using the mixed model approach implemented in EMMAX () to correct for population structure ( Figure 1 A) and identified strongly associated SNPs (p < 1x10) on chromosomes 24, 33, and 37 ( Figure 1 B). We then measured identity by descent (IBD) across the genome between the two escapers using Beagle (). Only the associated SNPs on chromosome 24 also overlapped a segment of IBD in the two escapers, consistent with a single origin of the causative mutation ( Figure 1 B). The 27 Mb segment showing both IBD and association with the escaper phenotype (CanFam2, cfa24:3,073,196-30,066,497) contains approximately 350 protein-coding genes. Linkage analysis using Merlin () strongly confirmed this region, with a maximal parametric LOD score of 3.31 (dominant inheritance model with complete penetrance, Figure S1 ). No other genomic regions showed any signs of linkage ( Figure S2 ). Thus, convergent IBD, association, and linkage analyses all pointed to the same 27 Mb region on chromosome 24 ( Figure 1 C).

(A) Linkage analysis of the escapers’ pedigree identified a 27Mb linkage peak on chromosome 24 with a maximal parametric dominant LOD score of 3.31. A smoothed linkage curve (red) is shown on top of the single SNP LOD (gray). (B) Non-dog related Jagged1 homologs all reside under this linkage peak. Shown are alignments of the human, bovine, rat, mouse, and frog Jagged1 refseq sequences, as well as the two zebrafish homologs used in this study.

(C) The mapped region extends 27 Mb 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 and association peak that includes the putative driver gene jagged1 (blue line) identified through gene expression profiling. See also Figures S1 and S2

(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 (gray) and are most likely false positives due to the small sample size.

(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 2 escapers and 31 severely affected GRMD dogs.

Discussion

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McNally E.M. The superhealing MRL background improves muscular dystrophy. Animal models for DMD are important tools for developing new therapeutic approaches. Among the different animal models for muscular dystrophy, the GRMD dog is the closest to the human condition. Both GRMD dogs and DMD patients have a severe phenotype as well as many phenotypic and biochemical similarities, including early progressive muscle degeneration and atrophy, fibrosis, contractures, and elevated serum creatine kinase levels. We identified two dogs that escaped from the typical severe phenotype associated with dystrophin deficiency. Using a combined approach of mapping and identity by descent, we identified a candidate region of association with the escaper phenotype. Only one gene within this region showed altered expression in escaper and affected dogs: Jagged1. We found a candidate variant at an upstream, conserved position creating a new muscle-specific transcription factor binding site that drives Jagged1 overexpression. Jagged1 is also in the region associated to the mild phenotype observed in a muscular dystrophy mouse model on the MRL (Murphy Roths Large) “superhealing” background. These mice show enhanced muscle regeneration and reduced dystrophic pathology. This healing phenotype was mapped to a region containing 49 genes that includes the Jagged1 locus ().

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Wang W. Soluble JAGGED1 inhibits pulmonary hypertension by attenuating notch signaling. Although the great majority of DMD patients show a severe course, exceptional cases of dystrophin-deficient patients with a milder phenotype have been identified. We have previously reported two patients carrying null mutations, with no skeletal muscle dystrophin present via immunofluorescent staining or western blot analysis, and a milder course including the maintenance of ambulation well into their second decade of life (). More recently, a dystrophin-negative patient who remained ambulant until age 30 was also reported (). Several other genetic modifiers are known to affect the severity of the clinical symptoms of Duchenne muscular dystrophy (LTBP4, SPP1, TGFBR2). However, none of these genetic variants have been shown to fully restore or delay substantially the symptoms of dystrophin-deficiency in DMD boys (). Furthermore, it would be of great interest to examine the genomes of DMD boys with varying clinical symptoms and determine if variants in Jagged1 or other Notch signaling factors exist and are causative for any variation of the dystrophic disease progression. The Notch signaling pathway, specifically Jagged1 overexpression, represents a novel therapeutic entry point for the treatment of DMD. Full restoration of Notch signaling must be achieved in the muscle satellite cell if one expects to correct the dysregulated Notch-dependent signaling that is affected in dystrophin-deficiency (). Direct injection of exogenous, soluble Jagged-1 ligand is not a viable therapeutic option, as external Jagged1 weakens Notch signaling even more than dystrophin-deficiency (). Thus, one might envision finding a small molecule or transcription factor that could increase expression of Jagged1 in all of the skeletal muscles of DMD patient.

There is currently no cure for DMD, and existing therapies aiming to rescue dystrophin expression are only partially effective. Here, we show that the overexpression of Jagged1 is likely to modulate the dystrophic phenotype in dystrophin-deficient GRMD dogs. We also show that overexpression of jagged1 rescues the dystrophic phenotype in a severe DMD model: the sapje zebrafish. Our study highlights the possibilities of across-species analysis to identify and validate disease-modifying genes and associated pathways. These results suggest that Jagged1 may be a new target for DMD therapeutic efforts in a dystrophin-independent manner, which will complement existing approaches. In addition, further investigation on the gene target Jagged1 will contribute to a better understanding of the disease pathogenesis and molecular physiology.