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1.  Pharmacologic Management of Duchenne Muscular Dystrophy: Target Identification and Preclinical Trials 
ILAR Journal  2014;55(1):119-149.
Duchenne muscular dystrophy (DMD) is an X-linked human disorder in which absence of the protein dystrophin causes degeneration of skeletal and cardiac muscle. For the sake of treatment development, over and above definitive genetic and cell-based therapies, there is considerable interest in drugs that target downstream disease mechanisms. Drug candidates have typically been chosen based on the nature of pathologic lesions and presumed underlying mechanisms and then tested in animal models. Mammalian dystrophinopathies have been characterized in mice (mdx mouse) and dogs (golden retriever muscular dystrophy [GRMD]). Despite promising results in the mdx mouse, some therapies have not shown efficacy in DMD. Although the GRMD model offers a higher hurdle for translation, dogs have primarily been used to test genetic and cellular therapies where there is greater risk. Failed translation of animal studies to DMD raises questions about the propriety of methods and models used to identify drug targets and test efficacy of pharmacologic intervention. The mdx mouse and GRMD dog are genetically homologous to DMD but not necessarily analogous. Subcellular species differences are undoubtedly magnified at the whole-body level in clinical trials. This problem is compounded by disparate cultures in clinical trials and preclinical studies, pointing to a need for greater rigor and transparency in animal experiments. Molecular assays such as mRNA arrays and genome-wide association studies allow identification of genetic drug targets more closely tied to disease pathogenesis. Genes in which polymorphisms have been directly linked to DMD disease progression, as with osteopontin, are particularly attractive targets.
PMCID: PMC4158345  PMID: 24936034
animal models; drug development; Duchenne muscular dystrophy; golden retriever muscular dystrophy; genome wide association studies; mRNA arrays; mdx mouse; preclinical studies
2.  Short Read (Next-gen) Sequencing: A Tutorial with Cardiomyopathy Diagnostics as an Exemplar 
PMCID: PMC4116683  PMID: 23852418
next-gen sequencing; titin; dilated cardiomyopathy; exome; cardiomyopathy; genetics; human
3.  A Renaissance for Antisense Oligonucleotide Drugs in Neurology 
Archives of neurology  2009;66(1):32-38.
Antisense oligonucleotides are short nucleic acid sequences designed for use as small-molecule drugs. They recognize and bind to specific messenger RNA (mRNA) or pre-mRNA sequences to create small double-stranded regions of the target mRNA that alter mRNA splicing patterns or inhibit protein translation. Antisense approaches have been actively pursued as a form of molecular medicine for more than 20 years, but only one has been translated to a marketed drug (intraocular human immunodeficiency virus treatment). Two recent advances foreshadow a change in clinical applications of antisense strategies. First is the development of synthetic DNA analogues that show outstanding stability and sequence specificity yet little or no binding to modulator proteins. Second is the publication of impressive preclinical and clinical data using antisense in an exon-skipping strategy to increase dystrophin production in Duchenne muscular dystrophy. As long-standing barriers are successfully circumvented, attention turns toward scale-up of production, long-term toxicity studies, and the challenges to traditional drug regulatory attitudes presented by tightly targeted sequence-specific drugs.
PMCID: PMC4111150  PMID: 19139297
4.  Alterations in Osteopontin Modify Muscle Size in Females in Both Humans and Mice 
An osteopontin (OPN; SPP1) gene promoter polymorphism modifies disease severity in Duchenne muscular dystrophy, and we hypothesized that it might also modify muscle phenotypes in healthy volunteers.
Gene association studies were carried out for OPN (rs28357094) in the FAMuSS cohort (n=752; age 23.7±5.7 yrs). Phenotypes studied included muscle size (MRI), strength, and response to supervised resistance training. We also studied 147 young adults that had carried out a bout of eccentric elbow exercise (age 24.0 ± 5.2 yrs). Phenotypes analyzed included strength, soreness, and serum muscle enzymes.
In the FAMuSS cohort, the G allele was associated with 17% increase in baseline upper arm muscle volume only in women (F=26.32; p=5.32 × 10−7), explaining 5% of population variance. In the eccentric damage cohort, weak associations of the G allele were seen in women with both baseline myoglobin, and elevated CK. Sexually dimorphic effects of OPN on muscle were also seen in OPN null mice. Five of seven muscle groups examined showed smaller size in OPN null female mice, whereas two were smaller in males. Query of OPN gene transcription after experimental muscle damage in mice showed rapid induction within 12 hrs (100-fold increase from baseline), followed by sustained high level expression through 16 days of regeneration before falling to back to baseline.
OPN is a sexually dimorphic modifier of muscle size in normal humans and mice, and responds to muscle damage. The OPN gene is known to be estrogen responsive, and this may explain the female-specific genotype effects in adult volunteers.
PMCID: PMC3631433  PMID: 23274598
osteopontin (OPN); secreted phosphoprotein 1 (SPP1); genetic polymorphism; estrogen; hypertrophy; MRI
5.  An analysis of DNA methylation in human adipose tissue reveals differential modification of obesity genes before and after gastric bypass and weight loss 
Genome Biology  2015;16(1):8.
Environmental factors can influence obesity by epigenetic mechanisms. Adipose tissue plays a key role in obesity-related metabolic dysfunction, and gastric bypass provides a model to investigate obesity and weight loss in humans.
Here, we investigate DNA methylation in adipose tissue from obese women before and after gastric bypass and significant weight loss. In total, 485,577 CpG sites were profiled in matched, before and after weight loss, subcutaneous and omental adipose tissue. A paired analysis revealed significant differential methylation in omental and subcutaneous adipose tissue. A greater proportion of CpGs are hypermethylated before weight loss and increased methylation is observed in the 3′ untranslated region and gene bodies relative to promoter regions. Differential methylation is found within genes associated with obesity, epigenetic regulation and development, such as CETP, FOXP2, HDAC4, DNMT3B, KCNQ1 and HOX clusters. We identify robust correlations between changes in methylation and clinical trait, including associations between fasting glucose and HDAC4, SLC37A3 and DENND1C in subcutaneous adipose. Genes investigated with differential promoter methylation all show significantly different levels of mRNA before and after gastric bypass.
This is the first study reporting global DNA methylation profiling of adipose tissue before and after gastric bypass and associated weight loss. It provides a strong basis for future work and offers additional evidence for the role of DNA methylation of adipose tissue in obesity.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0569-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4301800  PMID: 25651499
6.  Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans 
Cell metabolism  2005;2(4):251-261.
Obesity and type 2 diabetes are strongly associated with abnormal lipid metabolism and accumulation of intramyocellular triacylglycerol, but the underlying cause of these perturbations are yet unknown. Herein, we show that the lipogenic gene, stearoyl-CoA desaturase 1 (SCD1), is robustly up-regulated in skeletal muscle from extremely obese humans. High expression and activity of SCD1, an enzyme that catalyzes the synthesis of monounsaturated fatty acids, corresponded with low rates of fatty acid oxidation, increased triacylglycerol synthesis and increased monounsaturation of muscle lipids. Elevated SCD1 expression and abnormal lipid partitioning were retained in primary skeletal myocytes derived from obese compared to lean donors, implying that these traits might be driven by epigenetic and/or heritable mechanisms. Overexpression of human SCD1 in myotubes from lean subjects was sufficient to mimic the obese phenotype. These results suggest that elevated expression of SCD1 in skeletal muscle contributes to abnormal lipid metabolism and progression of obesity.
PMCID: PMC4285571  PMID: 16213227
7.  Neck and waist circumference biomarkers of cardiovascular risk in a cohort of predominantly African-American college students: A preliminary study 
PMCID: PMC4038263  PMID: 24051106
African Americans; anthropometry; blood pressure; body fat; health risk appraisal; low density lipoprotein cholesterol; students; waist-hip ratio
8.  Non-Invasive MRI and Spectroscopy of mdx Mice Reveal Temporal Changes in Dystrophic Muscle Imaging and in Energy Deficits 
PLoS ONE  2014;9(11):e112477.
In Duchenne muscular dystrophy (DMD), a genetic disruption of dystrophin protein expression results in repeated muscle injury and chronic inflammation. Magnetic resonance imaging shows promise as a surrogate outcome measure in both DMD and rehabilitation medicine that is capable of predicting clinical benefit years in advance of functional outcome measures. The mdx mouse reproduces the dystrophin deficiency that causes DMD and is routinely used for preclinical drug testing. There is a need to develop sensitive, non-invasive outcome measures in the mdx model that can be readily translatable to human clinical trials. Here we report the use of magnetic resonance imaging and spectroscopy techniques for the non-invasive monitoring of muscle damage in mdx mice. Using these techniques, we studied dystrophic mdx muscle in mice from 6 to 12 weeks of age, examining both the peak disease phase and natural recovery phase of the mdx disease course. T2 and fat-suppressed imaging revealed significant levels of tissue with elevated signal intensity in mdx hindlimb muscles at all ages; spectroscopy revealed a significant deficiency of energy metabolites in 6-week-old mdx mice. As the mdx mice progressed from the peak disease stage to the recovery stage of disease, each of these phenotypes was either eliminated or reduced, and the cross-sectional area of the mdx muscle was significantly increased when compared to that of wild-type mice. Histology indicates that hyper-intense MRI foci correspond to areas of dystrophic lesions containing inflammation as well as regenerating, degenerating and hypertrophied myofibers. Statistical sample size calculations provide several robust measures with the ability to detect intervention effects using small numbers of animals. These data establish a framework for further imaging or preclinical studies, and they support the development of MRI as a sensitive, non-invasive outcome measure for muscular dystrophy.
PMCID: PMC4229202  PMID: 25390038
9.  VBP15, a Glucocorticoid Analogue, Is Effective at Reducing Allergic Lung Inflammation in Mice 
PLoS ONE  2013;8(5):e63871.
Asthma is a chronic inflammatory condition of the lower respiratory tract associated with airway hyperreactivity and mucus obstruction in which a majority of cases are due to an allergic response to environmental allergens. Glucocorticoids such as prednisone have been standard treatment for many inflammatory diseases for the past 60 years. However, despite their effectiveness, long-term treatment is often limited by adverse side effects believed to be caused by glucocorticoid receptor-mediated gene transcription. This has led to the pursuit of compounds that retain the anti-inflammatory properties yet lack the adverse side effects associated with traditional glucocorticoids. We have developed a novel series of steroidal analogues (VBP compounds) that have been previously shown to maintain anti-inflammatory properties such as NFκB-inhibition without inducing glucocorticoid receptor-mediated gene transcription. This study was undertaken to determine the effectiveness of the lead compound, VBP15, in a mouse model of allergic lung inflammation. We show that VBP15 is as effective as the traditional glucocorticoid, prednisolone, at reducing three major hallmarks of lung inflammation—NFκB activity, leukocyte degranulation, and pro-inflammatory cytokine release from human bronchial epithelial cells obtained from patients with asthma. Moreover, we found that VBP15 is capable of reducing inflammation of the lung in vivo to an extent similar to that of prednisone. We found that prednisolone–but not VBP15 shortens the tibia in mice upon a 5 week treatment regimen suggesting effective dissociation of side effects from efficacy. These findings suggest that VBP15 may represent a potent and safer alternative to traditional glucocorticoids in the treatment of asthma and other inflammatory diseases.
PMCID: PMC3646769  PMID: 23667681
10.  Progression of volume load and muscular adaptation during resistance exercise 
European journal of applied physiology  2010;111(6):1063-1071.
Volume load (VL) is suggested to influence the adaptation of muscle to resistance exercise (RE). We sought to examine the independent association between total VL and hypertrophy and strength following a progressive RE protocol of equated sets and intensity. Total VL was calculated in 83 subjects (n = 43 males, n = 40 females; age = 25.12 ± 5.5 years) who participated in unilateral arm RE for 12 weeks. Subjects were tested for biceps muscle volume (MRI of the upper arm), isometric maximal voluntary contraction (MVC), and dynamic biceps strength (1RM), at baseline and following RE. Linear regression analysis revealed that sex was a significant predictor of hypertrophy (β = 0.06; p = 0.01) and strength (β = 0.14; p = 0.04), and that males had greater increases. Total VL was independently associated with hypertrophy only among females (β = 0.12; p < 0.01). For males, only baseline strength was (inversely) related to hypertrophy (β = −0.12; p = 0.04). VL was strongly associated with changes in 1RM strength improvement for both males (β = 0.66; p < 0.01) and females (β = 0.26; p = 0.02), but only related to MVC among females (β = 0.20; p = 0.02). Findings reveal that VL was independently associated with hypertrophy only among females. For males baseline strength was independently and inversely related to changes in muscle mass. Conversely, VL was found to be strongly associated with changes in 1RM for both males and females, controlling for age, body mass, and baseline strength.
PMCID: PMC4215195  PMID: 21113614
Strength training; Volume load; FAMuSS; Muscle mass; Periodization
11.  The 1p13.3 LDL (C)-Associated Locus Shows Large Effect Sizes in Young Populations 
Pediatric research  2011;69(6):538-543.
Genome-wide association studies (GWAS) have identified polymorphic loci associated with coronary artery disease (CAD) risk factors (i. e. serum lipids) in adult populations (42–69 yrs). We hypothesized that younger populations would show a greater relative genetic component due to fewer confounding variables. We examined the influence of 20 GWAS loci associated with serum lipids and insulin metabolism, in a university student cohort (n=548; mean age= 24 yrs), and replicated statistically associated results in a second study cohort of primary school students (n=810, mean age= 11.5 yrs). 19 loci showed no relationship with studied risk factors in young adults. However, the ancestral allele of the rs646776 (SORT1) locus was strongly associated with increased low density lipoprotein cholesterol {LDL (C)} in young adults (TT: 97.6 ± 1.0 mg/dL {n=345}, vs. CT/CC: 87.3 ± 1.0 mg/dL {n=203}; p = 3 × 10−6) and children (TT: 94.0 ± 1.3 mg/dL {n=551}, vs. CT/CC: 84.7 ± 1.4 mg/dL {n=259}; p = 4 × 10−6). This locus is responsible for 3.6% of population variance in young adults and 2.5% of population variance in children. The effect size of the SORT1 locus is considerably higher in young populations (2.5%–4.1%) compared to older subjects (1%).
PMCID: PMC3606915  PMID: 21297524
12.  Mathematical Modeling of Corticosteroid Pharmacogenomics in Rat Muscle following Acute and Chronic Methylprednisolone Dosing 
Molecular pharmaceutics  2008;5(2):328-339.
The pharmacogenomic effects of a corticosteroid (CS) were assessed in rat skeletal muscle using microarrays. Adrenalectomized (ADX) rats were treated with methylprednisolone (MPL) by either 50 mg/kg intravenous injection or 7-day 0.3 mg/kg/h infusion through subcutaneously implanted pumps. RNAs extracted from individual rat muscles were hybridized to Affymetrix Rat Genome Genechips. Data mining yielded 653 and 2316 CS-responsive probe sets following MPL bolus and infusion treatments. Of these, 196 genes were controlled by MPL under both dosing conditions. Cluster analysis revealed that 124 probe sets exhibited three typical expression dynamic profiles following acute dosing. Cluster A consisted of up-regulated probe sets which were grouped into five subclusters each exhibiting unique temporal patterns during the infusion. Cluster B comprised down-regulated probe sets which were divided into two subclusters with distinct dynamics during the infusion. Cluster C probe sets exhibited delayed down-regulation under both bolus and infusion conditions. Among those, 104 probe sets were further grouped into subclusters based on their profiles following chronic MPL dosing. Several mathematical models were proposed and adequately captured the temporal patterns for each subcluster. Multiple types of dosing regimens are needed to resolve common determinants of gene regulation as chronic exposure results in unexpected differences in gene expression compared to acute dosing. Pharmacokinetic/pharmacodynamic (PK/PD) modeling provides a quantitative tool for elucidating the complexities of CS pharmacogenomics in skeletal muscle.
PMCID: PMC4196382  PMID: 18271548
Microarray studies; pharmacokinetics; pharmacodynamics; mathematical models; computational biology
13.  Microarray analysis of the temporal response of skeletal muscle to methylprednisolone: comparative analysis of two dosing regimens 
Physiological genomics  2007;30(3):282-299.
The transcriptional response of skeletal muscle to chronic corticosteroid exposure was examined over 168 h and compared with the response profiles observed following a single dose of corticosteroid. Male adrenalectomized Wistar rats were given a constant-rate infusion of 0.3 mg•kg−1•h−1 methylprednisolone for up to 7 days via subcutaneously implanted minipumps. Four control and forty drug-treated animals were killed at ten different time points during infusion. Liver total RNAs were hybridized to 44 individual Affymetrix REA230A gene chips. Previously, we described a filtration approach for identifying genes of interest in microarray data sets developed from tissues of rats treated with methylprednisolone (MPL) following acute dosing. Here, a similar approach involving a series of three filters was applied sequentially to identify genes of interest. These filters were designed to eliminate probe sets that were not expressed in the tissue, not regulated by the drug, or did not meet defined quality control standards. Filtering eliminated 86% of probe sets, leaving a remainder of 2,316 for further consideration. In a previous study, 653 probe sets were identified as MPL regulated following administration of a single (acute) dose of the drug. Comparison of the two data sets yielded 196 genes identified as regulated by MPL in both dosing regimens. Because of receptor downregulation, it was predicted that genes regulated by receptor-glucocorticoid response element interactions would exhibit tolerance in chronic profiles. However, many genes did not exhibit steroid tolerance, indicating that present perspectives on the mechanism of glucocorticoid action cannot entirely explain all temporal profiles.
PMCID: PMC4186702  PMID: 17473217
glucocorticoids; corticosteroids; Affymetrix gene chips; gene expression; time series
15.  Application of Scaling Factors in Simultaneous Modeling of Microarray Data from Diverse Chips 
Pharmaceutical research  2007;24(4):643-649.
Microarrays have been utilized in many biological, physiological and pharmacological studies as a high-throughput genomic technique. Several generations of Affymetrix GeneChip® microarrays are widely used in gene expression studies. However, differences in intensities of signals for different probe sets that represent the same gene on various types of Affymetrix chips make comparison of datasets complicated.
Materials and Methods
A power coefficient scaling factor was applied in the pharmacokinetic/ pharmacodynamic (PK/PD) modeling to account for differences in probe set sensitivities (i.e., signal intensities). Microarray data from muscle and liver following methylprednisolone 50 mg/kg i.v. bolus and 0.3 mg/kg/h infusion regimens were taken as an exemplar.
The scaling factor applied to the pharmacodynamic output function was used to solve the problem of intensity differences between probe sets. This approach yielded consistent pharmacodynamic parameters for the applied models.
Modeling of pharmacodynamic/pharmacogenomic (PD/PG) data from diverse chips should be performed with caution due to differential probe set intensities. In such circumstances, a power scaling factor can be applied in the modeling.
PMCID: PMC4181592  PMID: 17318415
bioinformatics; computational biology; pharmacodynamics; pharmacogenomics; pharmacokinetics
16.  Time Series Proteome Profiling To Study Endoplasmic Reticulum Stress Response 
Journal of proteome research  2008;7(6):2435-2444.
Time series profiling is a powerful approach for obtaining information on protein expression dynamics and prevailing biochemical pathways. To date, such information could only be obtained at the mRNA level using mature and highly parallel technologies such as microarray gene expression profiling. The generation of time series data at the protein level has lagged due to the lack of robust and highly reproducible methodologies. Using a combination of SILAC strategy, SDS-PAGE and LC-MS/MS, we demonstrate successful monitoring of expression levels of the same set of proteins across different time points within the ER compartment of human primary fibroblast cells when exposed to ER stress inducers tunicamycin and thapsigargin. Data visualization was facilitated using GeneSpring GX analysis platform that was designed to process Affymetrix microarray data. This software also facilitated the generation of important parameters such as data normalization, calculation of statistical values to extract significant changes in protein expression, and the cross comparison of data sets.
PMCID: PMC4154506  PMID: 18435558
SILAC; Isotope Ratio; LC-MS/MS; Proteome profiling; Time series; ER stress; GRP78; Reticulocalbin
17.  Microarray Analysis Reveals Novel Features of the Muscle Aging Process in Men and Women 
To develop a global view of muscle transcriptional differences between older men and women and sex-specific aging, we obtained muscle biopsies from the biceps brachii of young and older men and women and profiled the whole-genome gene expression using microarray. A logistic regression-based method in combination with an intensity-based Bayesian moderated t test was used to identify significant sex- and aging-related gene functional groups. Our analysis revealed extensive sex differences in the muscle transcriptome of older individuals and different patterns of transcriptional changes with aging in men and women. In older women, we observed a coordinated transcriptional upregulation of immune activation, extracellular matrix remodeling, and lipids storage; and a downregulation of mitochondrial biogenesis and function and muscle regeneration. The effect of aging results in sexual dimorphic alterations in the skeletal muscle transcriptome, which may modify the risk for developing musculoskeletal and metabolic diseases in men and women.
PMCID: PMC3826860  PMID: 23418191
Aging; Sex; Sarcopenia; Transcription profile.
18.  Variants of the Ankyrin Repeat Domain 6 Gene (ANKRD6) and Muscle and Physical Activity Phenotypes Among European-Derived American Adults 
Ankyrin repeat domain 6 (ANKRD6) is a ubiquitous protein that associates with early development in mammals and is highly expressed in the brain, spinal cord, and heart of humans. We examined the role of 8 ANKRD6 single-nucleotide polymorphisms (SNPs) on muscle performance and habitual physical activity (PA). Single-nucleotide polymorphisms were 545 T>A (rs9362667), 485 M>L (rs61736690), 233 T>M (rs2273238), 128 I>L (rs3748085), 631 P>L (rs61739327), 122 Q>E (rs16881983), 197805 G>A (rs9344950), and 710 L>X (NOVEL). This study consisted of 922 healthy, untrained, European-derived American men (n = 376, 23.6 ± 0.3 years, 25.0 ± 0.2 kg·m−2) and women (n = 546, 23.2 ± 0.2 years, 24.0 ± 0.2 kg·m−2). Muscle strength (maximum voluntary contraction [MVC] and 1 repetition maximum [1RM]) and size (cross-sectional area [CSA]) were assessed before and after 12 weeks of unilateral resistance training (RT). A subsample (n = 536, 23.4 ± 0.2 years, 24.6 ± 0.2 kg·m−2) completed the Paffenbarger Physical Activity Questionnaire. Associations among ANKRD6 genotypes and muscle phenotypes were tested with repeated measure analysis of covariance (ANCOVA) and PA phenotypes with multivariate ANCOVA, with age and body mass index as covariates. ANKRD6 122 Q>E was associated with increased baseline biceps CSA. ANKRD6 545 A>T and ANKRD6 710 L>X were associated with increased 1RM and MVC in response to RT, respectively. ANKRD6 631 P>L was associated with increased biceps CSA response to RT and time spent in moderate-intensity PA among the total sample and women. ANKRD6 genetic variants were associated with the muscle size and strength response to RT and habitual PA levels. Further research is needed to validate our results and explore mechanisms for the associations we observed.
PMCID: PMC4147939  PMID: 22580979
diversin; exercise; resistance training; genetics
19.  Losartan Decreases Cardiac Muscle Fibrosis and Improves Cardiac Function in Dystrophin-Deficient Mdx Mice 
Recent studies showed that chronic administration of losartan, an angiotensin II type I receptor antagonist, improved skeletal muscle function in dystrophin-deficient mdx mice. In this study, C57BL/10ScSn-Dmdmdx/J female mice were either untreated or treated with losartan (n = 15) in the drinking water at a dose of 600 mg/L over a 6-month period. Cardiac function was assessed via in vivo high frequency echocardiography and skeletal muscle function was assessed using grip strength testing, Digiscan monitoring, Rotarod timing, and in vitro force testing. Fibrosis was assessed using picrosirius red staining and Image J analysis. Gene expression was evaluated using real-time polymerized chain reaction (RT-PCR). Percentage shortening fraction was significantly decreased in untreated (26.9% ± 3.5%) mice compared to losartan-treated (32.2% ± 4.2%; P < .01) mice. Systolic blood pressure was significantly reduced in losartan-treated mice (56 ± 6 vs 69 ± 7 mm Hg; P < .0005). Percentage cardiac fibrosis was significantly reduced in losartan-treated hearts (P < .05) along with diaphragm (P < .01), extensor digitorum longus (P < .05), and gastrocnemius (P < .05) muscles compared to untreated mdx mice. There were no significant differences in skeletal muscle function between treated and untreated groups. Chronic treatment with losartan decreases cardiac and skeletal muscle fibrosis and improves cardiac systolic function in dystrophin-deficient mdx mice.
PMCID: PMC4147941  PMID: 21304057
Duchenne muscular dystrophy; dystrophin; mice; cardiomyopathy; angiotensin; echocardiography
20.  Differences in Fat and Muscle Mass Associated With a Functional Human Polymorphism in a Post-Transcriptional BMP2 Gene Regulatory Element 
Journal of cellular biochemistry  2009;107(6):1073-1082.
A classic morphogen, bone morphogenetic protein 2 (BMP2) regulates the differentiation of pluripotent mesenchymal cells. High BMP2 levels promote osteogenesis or chondrogenesis and low levels promote adipogenesis. BMP2 inhibits myogenesis. Thus, BMP2 synthesis is tightly controlled. Several hundred nucleotides within the 3′ untranslated regions of BMP2 genes are conserved from mammals to fishes indicating that the region is under stringent selective pressure. Our analyses indicate that this region controls BMP2 synthesis by post-transcriptional mechanisms. A common A to C single nucleotide polymorphism (SNP) in the BMP2 gene (rs15705, +A1123C) disrupts a putative post-transcriptional regulatory motif within the human ultra-conserved sequence. In vitro studies indicate that RNAs bearing the A or C alleles have different protein binding characteristics in extracts from mesenchymal cells. Reporter genes with the C allele of the ultra-conserved sequence were differentially expressed in mesenchymal cells. Finally, we analyzed MRI data from the upper arm of 517 healthy individuals aged 18–41 years. Individuals with the C/C genotype were associated with lower baseline subcutaneous fat volumes (P = 0.0030) and an increased gain in skeletal muscle volume (P = 0.0060) following resistance training in a cohort of young males. The rs15705 SNP explained 2–4% of inter-individual variability in the measured parameters. The rs15705 variant is one of the first genetic markers that maybe exploited to facilitate early diagnosis, treatment, and/or prevention of diseases associated with poor fitness. Furthermore, understanding the mechanisms by which regulatory polymorphisms influence BMP2 synthesis will reveal novel pharmaceutical targets for these disabling conditions.
PMCID: PMC4147943  PMID: 19492344
Bone Morphogenetic Protein 2 (BMP2); Messenger RNA (mRNA); Post-Transcriptional Gene Regulation; Mesenchymal Cells; Single Nucleotide Polymorphism (SNP); Fitness; Population Genetics
21.  Understanding the molecular complexity of medulloblastoma 
Nature reviews. Neurology  2012;8(10):539-540.
The understanding and treatment of medulloblastoma, the most common childhood malignant brain tumour, is rapidly evolving. Three complementary deep-sequencing studies that were recently published in Nature add to our knowledge of this disease, further refine risk stratification, and identify potential druggable targets.
PMCID: PMC4147946  PMID: 22986433
22.  MC4R Variant Is Associated With BMI but Not Response to Resistance Training in Young Females 
Obesity (Silver Spring, Md.)  2010;19(3):662-666.
Recently, a genome-wide association study (GWAS) that identified eight single-nucleotide polymorphisms (SNPs) associated with BMI highlighted a possible neuronal influence on the development of obesity. We hypothesized these SNPs would govern the response of BMI and subcutaneous fat to resistance training in young individuals (age = 24 years). We genotyped the eight GWAS-identified SNPs in the article by Willer et al. in a cohort (n = 796) that undertook a 12-week resistance-training program. Females with a copy of the rare allele (C) for rs17782313 (MC4R) had significantly higher BMIs (CC/CT: n = 174; 24.70 ± 0.33 kg/m2, TT: n = 278; 23.41 ± 0.26 kg/m2, P = 0.002), and the SNP explained 1.9% of overall variation in BMI. Males with a copy of the rare allele (T) for rs6548238 (TMEM18) had lower amounts of subcutaneous fat pretraining (CT/TT: n = 65; 156,534 ± 7,415 mm3, CC: n = 136; 177,825 ± 5,139 mm3, P = 0.019) and males with a copy of the rare allele (A) for rs9939609 (FTO) lost a significant amount of subcutaneous fat with exercise (AT/AA: n = 83; −798.35 ± 2,624.30 mm3, TT: n = 47; 9,435.23 ± 3,494.44 mm3, P = 0.021). Females with a copy of the G allele for a missense variant in the SH2B1 (rs7498665) was associated with less change of subcutaneous fat volume with exercise (AG/GG: n = 191; 9,813 ± 2,250 mm3 vs. AA: n = 126; 770 ± 2,772 mm3; P = 0.011). These data support the original finding that there is an association between measures of obesity and a variant near the MC4R gene and extends these results to a younger population and implicates FTO, TMEM18, and SH2B1 polymorphisms in subcutaneous fat regulation.
PMCID: PMC4147947  PMID: 20725061
23.  Myostatin and Follistatin Polymorphisms Interact with Muscle Phenotypes and Ethnicity 
We examined associations among myostatin (MSTN) 2379 A > G and 163 G > A and follistatin (FST) −5003 A > T and −833 G > T single nucleotide polymorphisms (SNP) on the muscle size and the strength response to resistance training (RT).
Subjects (n = 645, age = 24.1 ± 0.2 yr, body mass index [BMI] = 24.2 ± 0.2 kg·m−2) self-disclosed themselves as Caucasian (78.9%), African American (3.6%), Asian (8.4%), Hispanic (5.0%), or Other (4.2%). They were genotyped for MSTN 2379 A > G (n = 645), MSTN 163 G > A (n = 639), FST −5003 A > T (n = 580), and FST −833 G > T (n = 603). We assessed dynamic (one repetition maximum [1RM]) and isometric (maximum voluntary contraction [MVC]) muscle strength and size (cross-sectional area [CSA]) of the elbow flexors before and after 12 wk of unilateral upper-arm RT. Repeated-measures ANCOVA tested associations among genetic variants and muscle phenotypes with age and BMI as covariates.
Baseline MVC was greater among African Americans who were carriers of the MSTN G2379 allele (AG/GG, n = 15) than the A2379A homozygotes (n = 8; 64.2 ± 6.8 vs 49.8 ± 8.7 kg). African Americans who were carriers of the FST T−5003 allele (n = 12) had greater baseline 1RM (11.9 ± 0.7 vs 8.8 ± 0.5 kg) and CSA (24.4 ± 1.3 vs 19.1 ± 1.2 cm2) than African Americans with the A-5003A genotype (n = 14; P < 0.05). No MSTN or FST genotype and muscle phenotype associations were found among the other ethnic groups (P ≥ 0.05).
MSTN 2379 A > G and FST −5003 A > T were associated with baseline muscle strength and size among African Americans only. These ethnic-specific associations are hypothesis generating and should be confirmed in a larger sample of African Americans.
PMCID: PMC4147954  PMID: 19346981
Muscle & nerve  2013;48(1):32-54.
Contemporary natural history data in Duchenne muscular dystrophy (DMD) is needed to assess care recommendations and aid in planning future trials.
The Cooperative International Neuromuscular Research Group (CINRG) DMD Natural History Study (DMD-NHS) enrolled 340 individuals, aged 2–28 years, with DMD in a longitudinal, observational study at 20 centers. Assessments obtained every 3 months for 1 year, at 18 months, and annually thereafter included: clinical history; anthropometrics; goniometry; manual muscle testing; quantitative muscle strength; timed function tests; pulmonary function; and patient-reported outcomes/ health-related quality-of-life instruments.
Glucocorticoid (GC) use at baseline was 62% present, 14% past, and 24% GC-naive. In those ≥6 years of age, 16% lost ambulation over the first 12 months (mean age 10.8 years).
Detailed information on the study methodology of the CINRG DMD-NHS lays the groundwork for future analyses of prospective longitudinal natural history data. These data will assist investigators in designing clinical trials of novel therapeutics.
PMCID: PMC4147958  PMID: 23677550
adolescent; adult; child/preschool; follow-up study; health status; human; locomotion; male; muscle strength/physiology; muscular dystrophies/classification; muscular dystrophies/Duchenne/physiopathology; muscular dystrophies/therapy; phenotype; quality of life/psychology; respiratory function test
25.  Evolution and comparative genomics of subcellular specializations: EST sequencing of Torpedo electric organ 
Marine genomics  2011;4(1):33-40.
Uncharacterized open reading frames (ORFs) in human genomic sequence often show a high degree of evolutionary conservation, yet have little or no tissue EST or protein data suggestive of protein product function. The encoded proteins may have highly restricted expression in specialized cells, subcellular specializations, and/or narrow windows during development. One such highly specialized and minute subcellular compartment is the neuromuscular junction (NMJ), where motorneurons contact muscle fibers. The electric Torpedo ray has evolved to expand the NMJ structure to the size of a large organ (electroplax organ), and we hypothesized that Torpedo electroplax proteins would be candidates for human ESTs expressed at the human NMJ. A total of 9,719 primary electroplax cDNA clones were sequenced. We identified 44 human ORFs showing high (>63%) amino acid identity to Torpedo electroplax transcripts with enrichment for mRNA splicing motifs (SH2 and pre-mRNA splicing domains), an observation potentially important for the strict nuclear domains maintained by myonuclei underlying the NMJ. We generated antibodies against two uncharacterized human genes (C19orf29 [Drosophila cactin] and C15orf24) and showed that these were indeed expressed at the murine NMJ. Cactin, a member of the Rel transcription factor family in Drosophila, localized to the postsynaptic cytosol of the NMJ and nuclear membrane. C15orf24 protein localized to the murine postsynaptic sarcolemma. We show a novel approach towards identifying proteins expressed at a subcellular specializations using evolutionary diversity of organ function and cross-species mapping.
PMCID: PMC3412124  PMID: 21429463
Electric organ; Neuromuscular Junction (NMJ); Proteome; Torpedo californica; Novel Gene Discovery; Open Reading Frame (ORF)

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