In vitro models of patient-derived muscle allow for more efficient development of genetic medicin... more In vitro models of patient-derived muscle allow for more efficient development of genetic medicines for the muscular dystrophies, which often present mutation-specific pathologies. One popular strategy to generate patient-specific myotubes involves reprogramming dermal fibroblasts to a muscle lineage through MyoD induction. However, creating physiologically relevant, reproducible tissues exhibiting multinucleated, aligned myotubes with organized striations is dependent on the introduction of physicochemical cues that mimic the native muscle microenvironment. Here, we engineered patient-specific control and dystrophic muscle tissues in vitro by culturing and differentiating MyoD–directly reprogrammed fibroblasts isolated from one healthy control subject, three patients with Duchenne muscular dystrophy (DMD), and two Limb Girdle 2A/R1 (LGMD2A/R1) patients on micromolded gelatin hydrogels. Engineered DMD and LGMD2A/R1 tissues demonstrated varying levels of defects in α-actinin expressi...
ObjectiveDescription of a new variant of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1... more ObjectiveDescription of a new variant of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) gene causing congenital myasthenic syndrome (CMS) in 3 children from 2 unrelated families.MethodsMuscle biopsies, EMG, and whole-exome sequencing were performed.ResultsAll 3 patients presented with congenital hypotonia, muscle weakness, respiratory insufficiency, head lag, areflexia, and gastrointestinal dysfunction. Genetic analysis identified a homozygous frameshift insertion in the GFPT1 gene (NM_001244710.1: c.686dupC; p.Arg230Ter) that was shared by all 3 patients. In one of the patients, inheritance of the variant was through uniparental disomy (UPD) with maternal origin. Repetitive nerve stimulation and single-fiber EMG was consistent with the clinical diagnosis of CMS with a postjunctional defect. Ultrastructural evaluation of the muscle biopsy from one of the patients showed extremely attenuated postsynaptic folds at neuromuscular junctions and extensive autophagic vacuolar pa...
Background Human muscle biopsies are increasingly important for diagnosis, research, and to monit... more Background Human muscle biopsies are increasingly important for diagnosis, research, and to monitor therapeutic trials. We examined the use of a self-contained, vacuum-assisted biopsy system and a novel muscle freezing technique to improve, simplify, and standardize human muscle biopsy collection and cryopreservation in older adults. Methods The Vacora vacuum-assisted biopsy system was deployed in muscle biopsies of 12 individuals ranging in age from 57 to 80 years. This office-based approach was well tolerated as it is minimally invasive, uses only local anesthetic, and has a quick recovery. To maximize biopsy sample quality and reproducibility, we developed a novel muscle sample freezing protocol. Fresh human and mouse muscle biopsy samples were placed into readily available tissue cassettes followed by direct freezing in liquid nitrogen. After this modified snap freezing protocol, frozen muscle samples were enrobed in OCT for cryosectioning. We examined the effect of this freezin...
Duchenne muscular dystrophy (DMD) is a fatal inherited genetic disorder that results in progressi... more Duchenne muscular dystrophy (DMD) is a fatal inherited genetic disorder that results in progressive muscle weakness and ultimately loss of ambulation, respiratory failure and heart failure. Cardiac MRI (MRI) plays an increasingly important role in the diagnosis and clinical care of boys with DMD and associated cardiomyopathies. Conventional cardiac MRI biomarkers permit measurements of global cardiac function and presence of fibrosis, but changes in these measures are late manifestations. Emerging MRI biomarkers of myocardial function and structure include the estimation of rotational mechanics and regional strain using MRI tagging; T1-mapping; and T2-mapping, a marker of inflammation, edema and fat. These emerging biomarkers provide earlier insights into cardiac involvement in DMD, improving patient care and aiding the evaluation of emerging therapies.
Like other single gene disorders, muscular dystrophy displays a range of phenotypic heterogeneity... more Like other single gene disorders, muscular dystrophy displays a range of phenotypic heterogeneity even with the same primary mutation. Identifying genetic modifiers capable of altering the course of muscular dystrophy is one approach to deciphering gene-gene interactions that can be exploited for therapy development. To this end, we used an intercross strategy in mice to map modifiers of muscular dystrophy. We interrogated genes of interest in an interval on mouse chromosome 10 associated with body mass in muscular dystrophy as skeletal muscle contributes significantly to total body mass. Using whole genome sequencing of the two parental mouse strains combined with deep RNA sequencing, we identified the Met62Ile substitution in the Dusp6 gene from the DBA/2J mouse strain. DUSP6 is a broadly expressed dual specificity phosphatase protein, which binds and dephosphorylates ERK, leading to decreased ERK activity. We found that the Met62Ile substitution reduced the interaction between DU...
Genetics in medicine : official journal of the American College of Medical Genetics, Jan 24, 2018
Pathogenic variants in KAT6A have recently been identified as a cause of syndromic developmental ... more Pathogenic variants in KAT6A have recently been identified as a cause of syndromic developmental delay. Within 2 years, the number of patients identified with pathogenic KAT6A variants has rapidly expanded and the full extent and variability of the clinical phenotype has not been reported. We obtained data for patients with KAT6A pathogenic variants through three sources: treating clinicians, an online family survey distributed through social media, and a literature review. We identified 52 unreported cases, bringing the total number of published cases to 76. Our results expand the genotypic spectrum of pathogenic variants to include missense and splicing mutations. We functionally validated a pathogenic splice-site variant and identified a likely hotspot location for de novo missense variants. The majority of clinical features in KAT6A syndrome have highly variable penetrance. For core features such as intellectual disability, speech delay, microcephaly, cardiac anomalies, and gast...
Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, resulting in loss of dystrophin,... more Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, resulting in loss of dystrophin, which is essential to muscle health. DMD "exon skipping" uses anti-sense oligo-nucleotides (AONs) to force specific exon exclusion during mRNA processing to restore reading frame and rescue of partially functional dystrophin protein. Although exon-skipping drugs in humans show promise, levels of rescued dystrophin protein remain suboptimal. We previously identified dantrolene as a skip booster when combined with AON in human DMD cultures and short-term mdx dystrophic mouse studies. Here, we assess the effect of dantrolene/AON combination on DMD exon-23 skipping over long-term mdx treatment under conditions that better approximate potential human dosing. To evaluate the dantrolene/AON combination treatment effect on dystrophin induction, we assayed three AON doses, with and without oral dantrolene, to assess multiple outcomes across different muscles. Meta-analyses of the resul...
Antisense oligonucleotide (AON) mediated exon skipping is an emerging therapeutic for individuals... more Antisense oligonucleotide (AON) mediated exon skipping is an emerging therapeutic for individuals with Duchenne muscular dystrophy (DMD). Skipping of exons adjacent to common exon deletions in DMD using AONs can produce in-frame transcripts and functional protein. Targeted skipping of DMD exons 8, 44, 45, 50, 51, 52, 53, and 55 are predicted to benefit 47% of affected individuals. We observed a correlation between mutation subgroups and age at loss of ambulation in DuchenneConnect, a large database of phenotypic and genetic data for DMD (N = 765). Males amenable to exon 44 (N = 74) and exon 8 skipping (N = 18) showed prolonged ambulation compared to other exon skip groups and nonsense mutations (P = 0.035 and P < 0.01, respectively). In particular, exon 45 deletions were associated with prolonged age at LOA relative to the rest of the exon 44 skip amenable cohort and other DMD mutations. Exon 3-7 deletions also showed prolonged ambulation relative to all other exon 8 skippable mu...
Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progen... more Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progenitor cells (SMPCs). However, the myogenicity of hPSC-SMPCs relative to human fetal or adult satellite cells remains unclear. We observed that hPSC-SMPCs derived by directed differentiation are less functional in vitro and in vivo compared to human satellite cells. Using RNA sequencing, we found that the cell surface receptors ERBB3 and NGFR demarcate myogenic populations, including PAX7 progenitors in human fetal development and hPSC-SMPCs. We demonstrated that hPSC skeletal muscle is immature, but inhibition of transforming growth factor-β signalling during differentiation improved fusion efficiency, ultrastructural organization and the expression of adult myosins. This enrichment and maturation strategy restored dystrophin in hundreds of dystrophin-deficient myofibres after engraftment of CRISPR-Cas9-corrected Duchenne muscular dystrophy human induced pluripotent stem cell-SMPCs. The w...
Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A. Calpain 3 (CAPN3)... more Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A. Calpain 3 (CAPN3) is a calcium dependent protease residing in the myofibrillar, cytosolic and triad fractions of skeletal muscle. At the triad, it colocalizes with calcium calmodulin kinase IIβ (CaMKIIβ). CAPN3 knock out mice (C3KO) show reduced triad integrity and blunted CaMKIIβ signaling, which correlates with impaired transcriptional activation of myofibrillar and oxidative metabolism genes in response to running exercise. These data suggest a role for CAPN3 and CaMKIIβ in gene regulation that takes place during adaptation to endurance exercise. To assess whether CAPN3- CaMKIIβ signaling influences skeletal muscle remodeling in other contexts, we subjected C3KO and WT mice to hindlimb unloading and reloading and assessed CaMKIIβ signaling and gene expression by RNA sequencing. After induced atrophy followed by 4 days of reloading, both CaMKIIβ activation and expression of inflammatory and cellular st...
Multiple glycosyltransferases are essential for the proper modification of alpha-dystroglycan, as... more Multiple glycosyltransferases are essential for the proper modification of alpha-dystroglycan, as mutations in the encoding genes cause congenital/limb-girdle muscular dystrophies. Here we elucidate further the structure of an O-mannose-initiated glycan on alpha-dystroglycan that is required to generate its extracellular matrix-binding polysaccharide. This functional glycan contains a novel ribitol structure that links a phosphotrisaccharide to xylose. ISPD is a CDP-ribitol (ribose) pyrophosphorylase that generates the reduced sugar nucleotide for the insertion of ribitol in a phosphodiester linkage to the glycoprotein. TMEM5 is a UDP-xylosyl transferase that elaborates the structure. We demonstrate in a zebrafish model as well as in a human patient that defects in TMEM5 result in muscular dystrophy in combination with abnormal brain development. Thus, we propose a novel structure - a ribitol in a phosphodiester linkage - for the moiety on which TMEM5, B4GAT1, and LARGE act to gener...
Limb girdle muscular dystrophy 2A is due to loss-of-function mutations in theCalpain 3 (Capn3)gen... more Limb girdle muscular dystrophy 2A is due to loss-of-function mutations in theCalpain 3 (Capn3)gene. Our previous data suggest that CAPN3 helps to maintain the integrity of the triad complex in skeletal muscle. InCapn3knock-out mice (C3KO), Ca(2+)release and Ca(2+)/calmodulin kinase II (CaMKII) signaling are attenuated. We hypothesized that calpainopathy may result from a failure to transmit loading-induced Ca(2+)-mediated signals, necessary to up-regulate expression of muscle adaptation genes. To test this hypothesis, we compared transcriptomes of muscles from wild-type and C3KO mice subjected to endurance exercise. In wild-type mice, exercise induces a gene signature that includes myofibrillar, mitochondrial and oxidative lipid metabolism genes, necessary for muscle adaptation. C3KO muscles fail to activate the same gene signature. Furthermore, in agreement with the aberrant transcriptional profile, we observe a commensurate functional defect in lipid metabolism whereby C3KO muscle...
This review aims to describe the benefits and limitations of using the Duchenne Connect patient r... more This review aims to describe the benefits and limitations of using the Duchenne Connect patient registry to provide information particularly in regard to active treatment choices in Duchenne muscular dystrophy and their impact on disease progression. Clinical trials and natural history studies are difficult for rare diseases like Duchenne muscular dystrophy. Using an online patient self-report survey model, Duchenne Connect provides relevant data that are difficult to gather in other ways. Validation of the overall dataset is supported by comparable mutational spectrum relative to other cohorts and demonstrated beneficial effect of corticosteroid use in prolonging ambulation. These types of analyses are provocative and allow multivariate analyses across the breadth of patient and physician medication and supplement practices. Because the data are self-reported and online, the barrier to participation is low and great potential exists for novel directions of further research in a hig...
In vitro models of patient-derived muscle allow for more efficient development of genetic medicin... more In vitro models of patient-derived muscle allow for more efficient development of genetic medicines for the muscular dystrophies, which often present mutation-specific pathologies. One popular strategy to generate patient-specific myotubes involves reprogramming dermal fibroblasts to a muscle lineage through MyoD induction. However, creating physiologically relevant, reproducible tissues exhibiting multinucleated, aligned myotubes with organized striations is dependent on the introduction of physicochemical cues that mimic the native muscle microenvironment. Here, we engineered patient-specific control and dystrophic muscle tissues in vitro by culturing and differentiating MyoD–directly reprogrammed fibroblasts isolated from one healthy control subject, three patients with Duchenne muscular dystrophy (DMD), and two Limb Girdle 2A/R1 (LGMD2A/R1) patients on micromolded gelatin hydrogels. Engineered DMD and LGMD2A/R1 tissues demonstrated varying levels of defects in α-actinin expressi...
ObjectiveDescription of a new variant of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1... more ObjectiveDescription of a new variant of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) gene causing congenital myasthenic syndrome (CMS) in 3 children from 2 unrelated families.MethodsMuscle biopsies, EMG, and whole-exome sequencing were performed.ResultsAll 3 patients presented with congenital hypotonia, muscle weakness, respiratory insufficiency, head lag, areflexia, and gastrointestinal dysfunction. Genetic analysis identified a homozygous frameshift insertion in the GFPT1 gene (NM_001244710.1: c.686dupC; p.Arg230Ter) that was shared by all 3 patients. In one of the patients, inheritance of the variant was through uniparental disomy (UPD) with maternal origin. Repetitive nerve stimulation and single-fiber EMG was consistent with the clinical diagnosis of CMS with a postjunctional defect. Ultrastructural evaluation of the muscle biopsy from one of the patients showed extremely attenuated postsynaptic folds at neuromuscular junctions and extensive autophagic vacuolar pa...
Background Human muscle biopsies are increasingly important for diagnosis, research, and to monit... more Background Human muscle biopsies are increasingly important for diagnosis, research, and to monitor therapeutic trials. We examined the use of a self-contained, vacuum-assisted biopsy system and a novel muscle freezing technique to improve, simplify, and standardize human muscle biopsy collection and cryopreservation in older adults. Methods The Vacora vacuum-assisted biopsy system was deployed in muscle biopsies of 12 individuals ranging in age from 57 to 80 years. This office-based approach was well tolerated as it is minimally invasive, uses only local anesthetic, and has a quick recovery. To maximize biopsy sample quality and reproducibility, we developed a novel muscle sample freezing protocol. Fresh human and mouse muscle biopsy samples were placed into readily available tissue cassettes followed by direct freezing in liquid nitrogen. After this modified snap freezing protocol, frozen muscle samples were enrobed in OCT for cryosectioning. We examined the effect of this freezin...
Duchenne muscular dystrophy (DMD) is a fatal inherited genetic disorder that results in progressi... more Duchenne muscular dystrophy (DMD) is a fatal inherited genetic disorder that results in progressive muscle weakness and ultimately loss of ambulation, respiratory failure and heart failure. Cardiac MRI (MRI) plays an increasingly important role in the diagnosis and clinical care of boys with DMD and associated cardiomyopathies. Conventional cardiac MRI biomarkers permit measurements of global cardiac function and presence of fibrosis, but changes in these measures are late manifestations. Emerging MRI biomarkers of myocardial function and structure include the estimation of rotational mechanics and regional strain using MRI tagging; T1-mapping; and T2-mapping, a marker of inflammation, edema and fat. These emerging biomarkers provide earlier insights into cardiac involvement in DMD, improving patient care and aiding the evaluation of emerging therapies.
Like other single gene disorders, muscular dystrophy displays a range of phenotypic heterogeneity... more Like other single gene disorders, muscular dystrophy displays a range of phenotypic heterogeneity even with the same primary mutation. Identifying genetic modifiers capable of altering the course of muscular dystrophy is one approach to deciphering gene-gene interactions that can be exploited for therapy development. To this end, we used an intercross strategy in mice to map modifiers of muscular dystrophy. We interrogated genes of interest in an interval on mouse chromosome 10 associated with body mass in muscular dystrophy as skeletal muscle contributes significantly to total body mass. Using whole genome sequencing of the two parental mouse strains combined with deep RNA sequencing, we identified the Met62Ile substitution in the Dusp6 gene from the DBA/2J mouse strain. DUSP6 is a broadly expressed dual specificity phosphatase protein, which binds and dephosphorylates ERK, leading to decreased ERK activity. We found that the Met62Ile substitution reduced the interaction between DU...
Genetics in medicine : official journal of the American College of Medical Genetics, Jan 24, 2018
Pathogenic variants in KAT6A have recently been identified as a cause of syndromic developmental ... more Pathogenic variants in KAT6A have recently been identified as a cause of syndromic developmental delay. Within 2 years, the number of patients identified with pathogenic KAT6A variants has rapidly expanded and the full extent and variability of the clinical phenotype has not been reported. We obtained data for patients with KAT6A pathogenic variants through three sources: treating clinicians, an online family survey distributed through social media, and a literature review. We identified 52 unreported cases, bringing the total number of published cases to 76. Our results expand the genotypic spectrum of pathogenic variants to include missense and splicing mutations. We functionally validated a pathogenic splice-site variant and identified a likely hotspot location for de novo missense variants. The majority of clinical features in KAT6A syndrome have highly variable penetrance. For core features such as intellectual disability, speech delay, microcephaly, cardiac anomalies, and gast...
Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, resulting in loss of dystrophin,... more Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, resulting in loss of dystrophin, which is essential to muscle health. DMD "exon skipping" uses anti-sense oligo-nucleotides (AONs) to force specific exon exclusion during mRNA processing to restore reading frame and rescue of partially functional dystrophin protein. Although exon-skipping drugs in humans show promise, levels of rescued dystrophin protein remain suboptimal. We previously identified dantrolene as a skip booster when combined with AON in human DMD cultures and short-term mdx dystrophic mouse studies. Here, we assess the effect of dantrolene/AON combination on DMD exon-23 skipping over long-term mdx treatment under conditions that better approximate potential human dosing. To evaluate the dantrolene/AON combination treatment effect on dystrophin induction, we assayed three AON doses, with and without oral dantrolene, to assess multiple outcomes across different muscles. Meta-analyses of the resul...
Antisense oligonucleotide (AON) mediated exon skipping is an emerging therapeutic for individuals... more Antisense oligonucleotide (AON) mediated exon skipping is an emerging therapeutic for individuals with Duchenne muscular dystrophy (DMD). Skipping of exons adjacent to common exon deletions in DMD using AONs can produce in-frame transcripts and functional protein. Targeted skipping of DMD exons 8, 44, 45, 50, 51, 52, 53, and 55 are predicted to benefit 47% of affected individuals. We observed a correlation between mutation subgroups and age at loss of ambulation in DuchenneConnect, a large database of phenotypic and genetic data for DMD (N = 765). Males amenable to exon 44 (N = 74) and exon 8 skipping (N = 18) showed prolonged ambulation compared to other exon skip groups and nonsense mutations (P = 0.035 and P < 0.01, respectively). In particular, exon 45 deletions were associated with prolonged age at LOA relative to the rest of the exon 44 skip amenable cohort and other DMD mutations. Exon 3-7 deletions also showed prolonged ambulation relative to all other exon 8 skippable mu...
Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progen... more Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progenitor cells (SMPCs). However, the myogenicity of hPSC-SMPCs relative to human fetal or adult satellite cells remains unclear. We observed that hPSC-SMPCs derived by directed differentiation are less functional in vitro and in vivo compared to human satellite cells. Using RNA sequencing, we found that the cell surface receptors ERBB3 and NGFR demarcate myogenic populations, including PAX7 progenitors in human fetal development and hPSC-SMPCs. We demonstrated that hPSC skeletal muscle is immature, but inhibition of transforming growth factor-β signalling during differentiation improved fusion efficiency, ultrastructural organization and the expression of adult myosins. This enrichment and maturation strategy restored dystrophin in hundreds of dystrophin-deficient myofibres after engraftment of CRISPR-Cas9-corrected Duchenne muscular dystrophy human induced pluripotent stem cell-SMPCs. The w...
Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A. Calpain 3 (CAPN3)... more Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A. Calpain 3 (CAPN3) is a calcium dependent protease residing in the myofibrillar, cytosolic and triad fractions of skeletal muscle. At the triad, it colocalizes with calcium calmodulin kinase IIβ (CaMKIIβ). CAPN3 knock out mice (C3KO) show reduced triad integrity and blunted CaMKIIβ signaling, which correlates with impaired transcriptional activation of myofibrillar and oxidative metabolism genes in response to running exercise. These data suggest a role for CAPN3 and CaMKIIβ in gene regulation that takes place during adaptation to endurance exercise. To assess whether CAPN3- CaMKIIβ signaling influences skeletal muscle remodeling in other contexts, we subjected C3KO and WT mice to hindlimb unloading and reloading and assessed CaMKIIβ signaling and gene expression by RNA sequencing. After induced atrophy followed by 4 days of reloading, both CaMKIIβ activation and expression of inflammatory and cellular st...
Multiple glycosyltransferases are essential for the proper modification of alpha-dystroglycan, as... more Multiple glycosyltransferases are essential for the proper modification of alpha-dystroglycan, as mutations in the encoding genes cause congenital/limb-girdle muscular dystrophies. Here we elucidate further the structure of an O-mannose-initiated glycan on alpha-dystroglycan that is required to generate its extracellular matrix-binding polysaccharide. This functional glycan contains a novel ribitol structure that links a phosphotrisaccharide to xylose. ISPD is a CDP-ribitol (ribose) pyrophosphorylase that generates the reduced sugar nucleotide for the insertion of ribitol in a phosphodiester linkage to the glycoprotein. TMEM5 is a UDP-xylosyl transferase that elaborates the structure. We demonstrate in a zebrafish model as well as in a human patient that defects in TMEM5 result in muscular dystrophy in combination with abnormal brain development. Thus, we propose a novel structure - a ribitol in a phosphodiester linkage - for the moiety on which TMEM5, B4GAT1, and LARGE act to gener...
Limb girdle muscular dystrophy 2A is due to loss-of-function mutations in theCalpain 3 (Capn3)gen... more Limb girdle muscular dystrophy 2A is due to loss-of-function mutations in theCalpain 3 (Capn3)gene. Our previous data suggest that CAPN3 helps to maintain the integrity of the triad complex in skeletal muscle. InCapn3knock-out mice (C3KO), Ca(2+)release and Ca(2+)/calmodulin kinase II (CaMKII) signaling are attenuated. We hypothesized that calpainopathy may result from a failure to transmit loading-induced Ca(2+)-mediated signals, necessary to up-regulate expression of muscle adaptation genes. To test this hypothesis, we compared transcriptomes of muscles from wild-type and C3KO mice subjected to endurance exercise. In wild-type mice, exercise induces a gene signature that includes myofibrillar, mitochondrial and oxidative lipid metabolism genes, necessary for muscle adaptation. C3KO muscles fail to activate the same gene signature. Furthermore, in agreement with the aberrant transcriptional profile, we observe a commensurate functional defect in lipid metabolism whereby C3KO muscle...
This review aims to describe the benefits and limitations of using the Duchenne Connect patient r... more This review aims to describe the benefits and limitations of using the Duchenne Connect patient registry to provide information particularly in regard to active treatment choices in Duchenne muscular dystrophy and their impact on disease progression. Clinical trials and natural history studies are difficult for rare diseases like Duchenne muscular dystrophy. Using an online patient self-report survey model, Duchenne Connect provides relevant data that are difficult to gather in other ways. Validation of the overall dataset is supported by comparable mutational spectrum relative to other cohorts and demonstrated beneficial effect of corticosteroid use in prolonging ambulation. These types of analyses are provocative and allow multivariate analyses across the breadth of patient and physician medication and supplement practices. Because the data are self-reported and online, the barrier to participation is low and great potential exists for novel directions of further research in a hig...
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Papers by Stanley Nelson