Search

OBJECTIVE Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disease caused by loss-of-function dystrophin (DMD) mutations in boys, who typically suffer loss of ambulation by age 12. Previously, we reported that coding variants in latent transforming growth factor beta (TGFβ)-binding protein 4 (LTBP4) were associated with reduced TGFβ signaling and prolonged ambulation (p = 1.0 × 10-3 ) in DMD patients; this result was subsequently replicated by other groups. In this study, we evaluated whether additional DMD modifier genes are observed using whole-genome association in the original cohort. METHODS We performed a genome-wide association study (GWAS) for single-nucleotide polymorphisms (SNPs) influencing loss of ambulation (LOA) in the same cohort of 253 DMD patients used to detect the candidate association with LTBP4 coding variants. Gene expression and chromatin interaction databases were used to fine-map association signals above the threshold for genome-wide significance. RESULTS Despite the small sample size, two loci associated with prolonged ambulation met genome-wide significance and were tagged by rs2725797 (chr15, p = 6.6 × 10-9 ) and rs710160 (chr19, p = 4.7 × 10-8 ). Gene expression and chromatin interaction data indicated that the latter SNP tags regulatory variants of LTBP4, whereas the former SNP tags regulatory variants of thrombospondin-1 (THBS1): an activator of TGFβ signaling by direct binding to LTBP4 and an inhibitor of proangiogenic nitric oxide signaling. INTERPRETATION Together with previous evidence implicating LTBP4, the THBS1 modifier locus emphasizes the role that common regulatory variants in gene interaction networks can play in mitigating disease progression in muscular dystrophy. Ann Neurol 2018;84:234-245.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is a severe X-linked recessive disease caused by loss-of-function dystrophin (DMD) mutations in boys, who typically suffer loss of ambulation by age 12. Previously we reported that coding variants in latent transforming growth factor beta-binding protein 4 (LTBP4) were associated with reduced TGFβ signaling and prolonged ambulation (p-value = 1.0 × 10(−3)) in DMD patients; this result was subsequently replicated by other groups. In this study, we evaluated whether additional DMD modifier genes are observed using whole genome association in the original cohort. METHODS: We performed a genome-wide association study (GWAS) for SNPs influencing loss of ambulation (LOA) in the same cohort of 253 DMD patients used to detect the candidate association with LTBP4 coding variants. Gene expression and chromatin interaction databases were used to fine map association signals above the threshold for genome-wide significance. RESULTS: Despite the small sample size, two loci associated with prolonged ambulation met genome-wide significance and were tagged by rs2725797 (chr15, p-value = 6.6 × 10(−9)) and rs710160 (chr19, p-value = 4.7 × 10(−8)). Gene expression and chromatin interaction data indicated that the latter SNP tags regulatory variants of LTBP4, while the former SNP tags regulatory variants of thrombospondin-1 (THBS1): an activator of TGFβ signaling via direct binding to LTBP4 and an inhibitor of pro-angiogenic nitric oxide signaling. INTERPRETATION: Together with prior evidence implicating LTBP4, the THBS1 modifier locus emphasizes the role that common regulatory variants in gene interaction networks can play in mitigating disease progression in muscular dystrophy.

Exon duplication mutations account for up to 11% of all cases of Duchenne muscular dystrophy (DMD), and a duplication of exon 2 is the most common duplication in patients. For use as a platform for testing of duplication-specific therapies, we developed a mouse model that carries a Dmd exon 2 duplication. By using homologous recombination we duplicated exon 2 within intron 2 at a location consistent with a human duplication hotspot. mRNA analysis confirms the inclusion of a duplicated exon 2 in mouse muscle. Dystrophin expression is essentially absent by immunofluorescent and immunoblot analysis, although some muscle specimens show very low-level trace dystrophin expression. Phenotypically, the mouse shows similarities to mdx, the standard laboratory model of DMD. In skeletal muscle, areas of necrosis and phagocytosis are seen at 3 weeks, with central nucleation prominent by four weeks, recapitulating the "crisis" period in mdx. Marked diaphragm fibrosis is noted by 6 months, and remains unchanged at 12 months. Our results show that the Dup2 mouse is both pathologically (in degree and distribution) and physiologically similar to mdx. As it recapitulates the most common single exon duplication found in DMD patients, this new model will be a useful tool to assess the potential of duplicated exon skipping.

Objective Exon-skipping therapies aim to convert Duchenne muscular dystrophy (DMD) into less severe Becker muscular dystrophy (BMD) by altering pre-mRNA splicing to restore an open reading frame, allowing translation of an internally deleted and partially functional dystrophin protein. The most common single exon deletion—exon 45 (Δ45)—may theoretically be treated by skipping of either flanking exon (44 or 46). We sought to predict the impact of these by assessing the clinical severity in dystrophinopathy patients. Methods Phenotypic data including clinical diagnosis, age at wheelchair use, age at loss of ambulation, and presence of cardiomyopathy were analyzed from 41 dystrophinopathy patients containing equivalent in-frame deletions. Results As expected, deletions of either exons 45 to 47 (Δ45–47) or exons 45 to 48 (Δ45–48) result in BMD in 97% (36 of 37) of subjects. Unexpectedly, deletion of exons 45 to 46 (Δ45–46) is associated with the more severe DMD phenotype in 4 of 4 subjects despite an in-frame transcript. Notably, no patients with a deletion of exons 44 to 45 (Δ44–45) were found within the United Dystrophinopathy Project database, and this mutation has only been reported twice before, which suggests an ascertainment bias attributable to a very mild phenotype. Interpretation The observation that Δ45–46 patients have typical DMD suggests that the conformation of the resultant protein may result in protein instability or altered binding of critical partners. We conclude that in DMD patients with Δ45, skipping of exon 44 and multiexon skipping of exons 46 and 47 (or exons 46–48) are better potential therapies than skipping of exon 46 alone. Ann Neurol 2015 Ann Neurol 2015;77:668–674

Duchenne muscular dystrophy (DMD) is associated with the loss of dystrophin, which plays an important role in myofiber integrity via interactions with β-dystroglycan and other members of the transmembrane dystrophin-associated protein complex. The ZZ domain, a cysteine-rich zinc-finger domain near the dystrophin C-terminus, is implicated in forming a stable interaction between dystrophin and β-dystroglycan, but the mechanism of pathogenesis of ZZ missense mutations has remained unclear because not all such mutations have been shown to alter β-dystroglycan binding in previous experimental systems. We engineered three ZZ mutations (p.Cys3313Phe, p.Asp3335His, and p.Cys3340Tyr) into a short construct similar to the Dp71 dystrophin isoform for in vitro and in vivo studies and delineated their effect on protein expression, folding properties, and binding partners. Our results demonstrate two distinct pathogenic mechanisms for ZZ missense mutations. The cysteine mutations result in diminished or absent subsarcolemmal expression because of protein instability, likely due to misfolding. In contrast, the aspartic acid mutation disrupts binding with β-dystroglycan despite an almost normal expression at the membrane, confirming a role for the ZZ domain in β-dystroglycan binding but surprisingly demonstrating that such binding is not required for subsarcolemmal localization of dystrophin, even in the absence of actin binding domains.

A spectrum of dystrophin mutations causes Duchenne muscular dystrophy (DMD). The extreme genomic size of the DMD gene (2.2 Mb) leads to kilobase size deletions or duplications in approximately 70% of DMD patients, and the remaining 30% arise from point mutations that introduce premature stop codons or disrupt splicing1. DMD mutations typically completely ablate dystrophin protein expression, while the milder Becker muscular dystrophy (BMD) mutations are most often in-frame deletions that produce truncated, but partially functional dystrophin. DMD patients are clinically defined by loss of ambulation before the age of 12, whereas BMD patients often remain ambulatory beyond age 15, a distinction that generally correlates with the degree of protein expression. Those who lose ambulation between 12 and 15 years are described as intermediate muscular dystrophy (IMD). Many DMD boys now ambulate beyond the age of 12 reflecting their treatment with prednisone or deflazacort, which remain the only medications that alter the natural history of DMD, prolonging ambulation for up to 1 to more than 3 years2-5. Despite the complete absence of dystrophin, the DMD phenotype is associated with a range in rates of progression, supporting that genetic or environmental modifiers help determine clinical course. Animal models, particularly mice, can be used to map genetic modifiers since inbred strains and standardized environmental conditions reduce variation. Like DMD patients, muscular dystrophy patients with a founder mutation in the dystrophin associated protein, γ-sarcoglycan, (SGCG) lose ambulation over a wide age range, consistent with the presence of genetic modifiers6, 7. The Sgcg null mouse was used in a genomewide screen for modifiers of muscular dystrophy and identified the Ltbp4 gene encoding the latent TGFβ binding protein (LTBP) 4 as a modifier of muscular dystrophy8. Given the shared pathological mechanisms between loss of dystrophin and its associated proteins, the sarcoglycans, we reasoned that LTBP4 may modify DMD. LTBP4, a member of the fibrillin superfamily, binds to transforming growth factor (TGF)-β in the extracellular matrix where it regulates TGFβ activity9. We now examined human LTBP4 polymorphisms in 254 nonambulatory patients from the United Dystrophinopathy Project cohort. This United Dystrophinopathy cohort includes 900 DMD patients, most who still ambulate, identified from seven sites representing by far the largest uniformly characterized DMD population. We assessed the effect of LTBP4 genotype on the age at ambulatory loss, since it reflects disease progression, is not influenced by ascertainment bias, and is a milestone for which the month or age is recalled by most patients and/or families. A protective haplotype in LTBP4 was identified.

L. E. Taylor, Y. J. Kaminoh, C. K. Rodesch and K. M. Flanigan (2012) Neuropathology and Applied Neurobiology38, 591–601 Quantification of dystrophin immunofluorescence in dystrophinopathy muscle specimens Aims: Duchenne muscular dystrophy (DMD) is usually associated with absent or nearly absent dystrophin expression at the sarcolemmal membrane. Quantification of very low levels of dystrophin signal in immunofluorescent studies of muscle biopsy sections presents a technical challenge. This is particularly true in the setting of proof-of-principle drug trials, in which the detection and quantification of what may be significant changes in levels of expression is important, even if absolute dystrophin levels remain low. Methods: We have developed a method of image analysis that allows reliable and semi-automated immunofluorescent quantification of low-level dystrophin expression in sections co-stained for spectrin. Using a custom Metamorph script to create a contiguous region spectrin mask, we quantify dystrophin signal intensity only at pixels within the spectrin mask that presumably represent the sarcolemmal membrane. Using this method, we analysed muscle biopsy tissue from a series of patients with DMD, Becker muscular dystrophy, intermediate muscular dystrophy and normal control tissue. Results: Analysis of serial sections on multiple days confirms reproducibility, and normalized dystrophin : spectrin intensity ratios (expressed as a percentage of normal control tissue) correlate well with the dystrophin expression levels as determined by Western blot analysis. Conclusion: This method offers a robust and reliable method of biomarker detection for trials of DMD therapies.

Single nucleotide polymorphism (SNP), rs11200638, in the promoter of HTRA1 has recently been shown to increase the risk for AMD. In order to investigate the association of this HTRA1 polymorphism and the bilaterality of AMD, we genotyped rs11200638 in control, unilateral, and bilateral advanced AMD patients. The A allele for SNP rs11200638 in HTRA1 , was significantly more prevalent in bilateral wet AMD and GA patients than in unilateral groups ( p = .02 and p = .03, respectively). The homozygote odds ratios of bilateral wet AMD and GA are significantly greater than those seen in unilateral groups (twofold and threefold increase, respectively). This finding is consistent with the role of HTRA1 in AMD pathogenesis and will help aid in the clinical management and prognosis of AMD patients.

Objective: We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials. Methods: Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels. Results: Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable. Conclusions: Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval.

Duchenne muscular dystrophy (DMD) displays a clinical range that is not fully explained by the primary DMD mutations. Ltbp4, encoding latent transforming growth factor-β binding protein 4, was previously discovered in a genome-wide scan as a modifier of murine muscular dystrophy. We sought to determine whether LTBP4 genotype influenced DMD severity in a large patient cohort.We analyzed nonsynonymous single nucleotide polymorphisms (SNPs) from human LTBP4 in 254 nonambulatory subjects with known DMD mutations. These SNPs, V194I, T787A, T820A, and T1140M, form the VTTT and IAAM LTBP4 haplotypes.Individuals homozygous for the IAAM LTBP4 haplotype remained ambulatory significantly longer than those heterozygous or homozygous for the VTTT haplotype. Glucocorticoid-treated patients who were IAAM homozygotes lost ambulation at 12.5 ± 3.3 years compared to 10.7 ± 2.1 years for treated VTTT heterozygotes or homozygotes. IAAM fibroblasts exposed to transforming growth factor (TGF) β displayed reduced phospho-SMAD signaling compared to VTTT fibroblasts, consistent with LTBP4' role as a regulator of TGFβ.LTBP4 haplotype influences age at loss of ambulation, and should be considered in the management of DMD patients.

The innate immune system provides a first line of defense against invading pathogens by releasing multiple inflammatory cytokines, such as interleukin-1beta and tumor necrosis factor-alpha, which directly combat the infectious agent and recruit additional immune responses. This exuberant cytokine release paradoxically injures the host by triggering leakage from capillaries, tissue edema, organ failure, and shock. Current medical therapies target individual pathogens with antimicrobial agents or directly either blunt or boost the host's immune system. We explored a third approach: activating with the soluble ligand Slit an endothelium-specific, Robo4-dependent signaling pathway that strengthens the vascular barrier, diminishing deleterious aspects of the host's response to the pathogen-induced cytokine storm. This approach reduced vascular permeability in the lung and other organs and increased survival in animal models of bacterial endotoxin exposure, polymicrobial sepsis, and H5N1 influenza. Thus, enhancing the resilience of the host vascular system to the host's innate immune response may provide a therapeutic strategy for treating multiple infectious agents.

Familial macular degeneration is a clinically and genetically heterogeneous group of disorders characterized by progressive central vision loss. Here we show that an R373C missense mutation in the prominin 1 gene (PROM1) causes 3 forms of autosomal-dominant macular degeneration. In transgenic mice expressing R373C mutant human PROM1, both mutant and endogenous PROM1 were found throughout the layers of the photoreceptors, rather than at the base of the photoreceptor outer segments, where PROM1 is normally localized. Moreover, the outer segment disk membranes were greatly overgrown and misoriented, indicating defective disk morphogenesis. Immunoprecipitation studies showed that PROM1 interacted with protocadherin 21 (PCDH21), a photoreceptor-specific cadherin, and with actin filaments, both of which play critical roles in disk membrane morphogenesis. Collectively, our results identify what we believe to be a novel complex involved in photoreceptor disk morphogenesis and indicate a possible role for PROM1 and PCDH21 in macular degeneration.

Significant morbidity and mortality among patients with diabetes mellitus result largely from a greatly increased incidence of microvascular complications. Proliferative diabetic retinopathy (PDR) and end stage renal disease (ESRD) are two of the most common and severe microvascular complications of diabetes. A high concordance exists in the development of PDR and ESRD in diabetic patients, as well as strong familial aggregation of these complications, suggesting a common underlying genetic mechanism. However, the precise gene(s) and genetic variant(s) involved remain largely unknown. Erythropoietin (EPO) is a potent angiogenic factor observed in the diabetic human and mouse eye. By a combination of case–control association and functional studies, we demonstrate that the T allele of SNP rs1617640 in the promoter of the EPO gene is significantly associated with PDR and ESRD in three European-American cohorts [Utah: P = 1.91 × 10 −3 ; Genetics of Kidneys in Diabetes (GoKinD) Study: P = 2.66 × 10 −8 ; and Boston: P = 2.1 × 10 −2 ]. The EPO concentration in human vitreous body was 7.5-fold higher in normal subjects with the TT risk genotype than in those with the GG genotype. Computational analysis suggests that the risk allele (T) of rs1617640 creates a matrix match with the EVI1/MEL1 or AP1 binding site, accounting for an observed 25-fold enhancement of luciferase reporter expression as compared with the G allele. These results suggest that rs1617640 in the EPO promoter is significantly associated with PDR and ESRD. This study identifies a disease risk-associated gene and potential pathway mediating severe diabetic microvascular complications.

Age-related macular degeneration (AMD) is the most common cause of irreversible visual impairment in the developed world. The two forms of advanced AMD, geographic atrophy (GA) and choroidal neovascularization (wet AMD), represent two types of degenerative processes in the macula that lead to loss of central vision. Soft confluent drusen, characterized by deposits in macula without visual loss are considered a precursor of advanced AMD. A single nucleotide polymorphism, rs11200638, in the promoter of HTRA1 has been shown to increases the risk for wet AMD. However, its impact on soft confluent drusen and GA or the relationship between them is unclear. To better understand the role the HTRA1 polymorphism plays in AMD subtypes, we genotyped an expanded Utah population with 658 patients having advanced AMD or soft confluent drusen and 294 normal controls and found that the rs11200638 was significantly associated with GA. This association remains significant conditional on LOC387715 rs10490924. In addition, rs11200638 was significantly associated with soft confluent drusen, which are strongly immunolabeled with HTRA1 antibody in an AMD eye with GA similar to wet AMD. Two-locus analyses were performed for CFH Y402H variant at 1q31 and the HTRA1 polymorphism. Together CFH and HTRA1 risk variants increase the odds of having AMD by more than 40 times. These findings expand the role of HTRA1 in AMD. Understanding the underlying molecular mechanism will provide an important insight in pathogenesis of AMD.

Nonsense mutations within exon 1 of DMD do not result in severe DMD but instead lead to very mild BMD, due to an alternative initiation of translation at AUG codons within in exon 6. This leads to translation of a nearly full-length but N-terminal truncated dystrophin lacking the first half of the canonical actin binding domain 1 (ABD1). We have identified the motif encoded in exon 5 that recruits ribosomes for alternate translational initiation within exon 6, and using a dual luciferase reporter system we have determined that this motif is selectively activated in muscle cell lines but not fibroblasts or HEK cells. Our data suggest that this motif is an IRES (internal ribosome entry site) as complementary experiments have ruled out any promoter activity or aberrant splicing. The exceedingly mild clinical features of patients with an exon 1 DMD founder allele (p.Trp3X) suggest that the product of this IRES initiation is a highly functional protein. We are therefore exploring different strategies to induce IRES utilisation for therapeutic purposes in patients with mutations in exons 1 through 4. One of them is based on forced synthesis of this IRES protein isoform that can be induced by exon 2 skipping. This leads to a frameshift and premature stop codon in exon 3, which thereby force the use of the IRES. We have developed four different antisense sequences for efficient skipping of exon 2 that are incorporated into a short U7 RNA derivative that was previously used in other studies to induce efficient exon skipping in DMD . We are currently testing these antisense constructs in patient cells carrying mutations in the first exons to evaluate the potential positive benefit of this out of frame skipping strategy.

Objective: Establish safety of rAAV.CMV.FS gene transfer by intramuscular (i.m.) injection for improving quadriceps strength in preparation for phase I clinical trial. Background FS is a potent myostatin inhibitor that increases muscle size and strength in mice and non-human primates when secreted by muscle following delivery by AAV. Safety concerns have included impeded release of pituitary-gonadal axis hormones and general organ toxicity. In this study we used a non-tissue binding FS isoform (FS344) to address these safety concerns in mice at viral doses 10-fold greater than those planned for clinical use. Design/Methods: Mice were randomized to three experimental arms with bilateral i.m. injections to quadriceps muscle of rAAV1.CMV.FS344 equal to highest clinical dose (2.0e12 vg/kg) or 10-fold higher (2.0e13 vg/kg). Animals were followed to sacrifice time points at 6-,12-,24-, and 36-weeks (5 animals/sex/cohort/timepoint) post-injection (p.i.). Outcomes included: bodyweight, hematology, chemistry panels, pituitary-gonadal hormones; immune studies, and histopathology of 24 organs from each animal. Results: No treatment-related adverse clinical or toxicology signs were observed and viral DNA in non-injected tissues dissipated by 24 weeks. Within 6 weeks, high dose animals showed scattered focal infiltrates of inflammatory cells corresponding to T-cell immunity to AAV1 identified by IFN-γ ELISpot, but no other pathology was observed. Wet weights of quadriceps increased by 3.5-fold at 36 weeks p.i. in high dose animals. Muscle fiber hypertrophy was observed at 6 weeks and fiber diameter increased by 50% compared to controls by 36 weeks. Hypertrophic-multinucleated fibers were observed at 12 weeks p.i. with increased density of PAX7 positive nuclei. Conclusions: This study shows rAAV1.CMV.FS344 is safe and well tolerated at the proposed clinical doses and causes satellite cell proliferation and significant muscle hypertrophy by fusion with differentiated satellite cells. Early transient inflammation in muscle is related to immune response to AAV1. These results support this approach for clinical trial use. Supported by: Grants from The Myositis Association and Parent Project Muscular Dystrophy, Inc; and support from the Foundation at Nationwide Children9s Hospital. Disclosure: Dr. Kota has nothing to disclose. Dr. Shilling has nothing to disclose. Dr. Montgomery has nothing to disclose. Dr. Lewis has nothing to disclose. Dr. Bevan has nothing to disclose. Dr. Shontz has nothing to disclose. Dr. Kaminoh has nothing to disclose. Dr. Rosales has nothing to disclose. Dr. Viollet has nothing to disclose. Dr. Flanigan has received personal compensaiton for activities with AVI Therapeutics, Prosensa Therapeutics, and PTC, Inc. Dr. Flanigan has received research support from PTC Therapeutics. Dr. Clark has nothing to disclose. Dr. Kaspar has nothing to disclose. Dr. Sahenk has nothing to disclose. Dr. Mendell has received research support from AVI BioPharmaceuticals Inc.

We examined familial aggregation and risk of age-related macular degeneration in the Utah population using a population-based case-control study. Over one million unique patient records were searched within the University of Utah Health Sciences Center and the Utah Population Database (UPDB), identifying 4764 patients with AMD. Specialized kinship analysis software was used to test for familial aggregation of disease, estimate the magnitude of familial risks, and identify families at high risk for disease. The population-attributable risk (PAR) for AMD was calculated to be 0.34. Recurrence risks in relatives indicate increased relative risks in siblings (2.95), first cousins (1.29), second cousins (1.13), and parents (5.66) of affected cases. There were 16 extended large families with AMD identified for potential use in genetic studies. Each family had five or more living affected members. The familial aggregation of AMD shown in this study exemplifies the merit of the UPDB and supports recent research demonstrating significant genetic contribution to disease development and progression.