title: Next-generation sequencing methylation profiling of subjects with obesity identifies novel gene changes.
abstract: BACKGROUND
  Obesity is a metabolic disease caused by environmental and genetic factors. However, the epigenetic mechanisms of obesity are incompletely understood. The aim of our study was to investigate the role of skeletal muscle DNA methylation in combination with transcriptomic changes in obesity.
  
  
  RESULTS
  Muscle biopsies were obtained basally from lean (n = 12; BMI = 23.4 ± 0.7 kg/m(2)) and obese (n = 10; BMI = 32.9 ± 0.7 kg/m(2)) participants in combination with euglycemic-hyperinsulinemic clamps to assess insulin sensitivity. We performed reduced representation bisulfite sequencing (RRBS) next-generation methylation and microarray analyses on DNA and RNA isolated from vastus lateralis muscle biopsies. There were 13,130 differentially methylated cytosines (DMC; uncorrected P < 0.05) that were altered in the promoter and untranslated (5' and 3'UTR) regions in the obese versus lean analysis. Microarray analysis revealed 99 probes that were significantly (corrected P < 0.05) altered. Of these, 12 genes (encompassing 22 methylation sites) demonstrated a negative relationship between gene expression and DNA methylation. Specifically, sorbin and SH3 domain containing 3 (SORBS3) which codes for the adapter protein vinexin was significantly decreased in gene expression (fold change -1.9) and had nine DMCs that were significantly increased in methylation in obesity (methylation differences ranged from 5.0 to 24.4 %). Moreover, differentially methylated region (DMR) analysis identified a region in the 5'UTR (Chr.8:22,423,530-22,423,569) of SORBS3 that was increased in methylation by 11.2 % in the obese group. The negative relationship observed between DNA methylation and gene expression for SORBS3 was validated by a site-specific sequencing approach, pyrosequencing, and qRT-PCR. Additionally, we performed transcription factor binding analysis and identified a number of transcription factors whose binding to the differentially methylated sites or region may contribute to obesity.
  
  
  CONCLUSIONS
  These results demonstrate that obesity alters the epigenome through DNA methylation and highlights novel transcriptomic changes in SORBS3 in skeletal muscle.
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, AZ USA.
authors.forename: Samantha E
authors.initials: SE
authors.lastname: Day
authors.affiliation: School for the Science of Health Care Delivery, Arizona State University, Phoenix, AZ USA.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Coletta
authors.affiliation: Division of Weight Management and Wellness Children's Hospital of Pittsburgh, Pittsburgh, PA USA.
authors.forename: Joon Young
authors.initials: JY
authors.lastname: Kim
authors.affiliation: School of Life Sciences, Arizona State University, Tempe, AZ USA.
authors.forename: Latoya E
authors.initials: LE
authors.lastname: Campbell
authors.affiliation: Endocrinology Department, Mayo Clinic in Arizona, Scottsdale, AZ USA.
authors.forename: Tonya R
authors.initials: TR
authors.lastname: Benjamin
authors.affiliation: Endocrinology Department, Mayo Clinic in Arizona, Scottsdale, AZ USA.
authors.forename: Lori R
authors.initials: LR
authors.lastname: Roust
authors.affiliation: Endocrinology Department, Mayo Clinic in Arizona, Scottsdale, AZ USA.
authors.forename: Elena A
authors.initials: EA
authors.lastname: De Filippis
authors.affiliation: The Department of Biomedical Informatics, Arizona State University, Phoenix, AZ USA.
authors.forename: Valentin
authors.initials: V
authors.lastname: Dinu
authors.affiliation: College of Nursing and Health Innovation Arizona State University, Phoenix, AZ USA ; Mayo/ASU Center for Metabolic and Vascular Biology, Mayo Clinic in Arizona, Scottsdale, AZ USA.
authors.forename: Gabriel Q
authors.initials: GQ
authors.lastname: Shaibi
authors.affiliation: Division of Endocrinology, Diabetes and Metabolism in the Department of Medicine at the UA College of Medicine, University of Arizona, Tucson, AZ USA.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Mandarino
authors.affiliation: Mayo/ASU Center for Metabolic and Vascular Biology, Mayo Clinic in Arizona, Scottsdale, AZ USA ; School of Nutrition and Health Promotion, College of Health Solutions, Arizona State University, 550 N. 3rd Street, Phoenix, AZ 85004 USA ; Department of Basic Medical Sciences, The University of Arizona College of Medicine, Phoenix, AZ USA.
authors.forename: Dawn K
authors.initials: DK
authors.lastname: Coletta
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D000328:Adult; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D009124:Muscle Proteins; D009765:Obesity / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods; D017423:Sequence Analysis, RNA / Q000379:methods
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D009124:Muscle Proteins; C486523:SORBS3 protein, human
keywords: Methylation; Next-generation sequencing; Obesity; Skeletal muscle