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Genes influencing spinal bone mineral density in inbred F344, LEW, COP, and DA rats.

ABSTRACT: Previously, we identified the regions of chromosomes 10q12-q31 and 15p16-q21 harbor quantitative trait loci (QTLs) for lumbar volumetric bone mineral density (vBMD) in female F2 rats derived from Fischer 344 (F344) x Lewis (LEW) and Copenhagen 2331 (COP) x Dark Agouti (DA) crosses. The purpose of this study is to identify the candidate genes within these QTL regions contributing to the variation in lumbar vBMD. RNA was extracted from bone tissue of F344, LEW, COP, and DA rats. Microarray analysis was performed using Affymetrix Rat Genome 230 2.0 Arrays. Genes differentially expressed among the rat strains were then ranked based on the strength of the correlation with lumbar vBMD in F2 animals derived from these rats. Quantitative PCR (qPCR) analysis was performed to confirm the prioritized candidate genes. A total of 285 genes were differentially expressed among all strains of rats with a false discovery rate less than 10%. Among these genes, 18 candidate genes were prioritized based on their strong correlation (r (2) > 0.90) with lumbar vBMD. Of these, 14 genes (Akap1, Asgr2, Esd, Fam101b, Irf1, Lcp1, Ltc4s, Mdp-1, Pdhb, Plxdc1, Rabep1, Rhot1, Slc2a4, Xpo4) were confirmed by qPCR. We identified several novel candidate genes influencing spinal vBMD in rats.

SUBMITTER: Alam I

PROVIDER: S-EPMC2835802 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Genes influencing spinal bone mineral density in inbred F344, LEW, COP, and DA rats.

Alam Imranul I Sun Qiwei Q Koller Daniel L DL Liu Lixiang L Liu Yunlong Y Edenberg Howard J HJ Foroud Tatiana T Turner Charles H CH

Functional & integrative genomics 20091015 1

Previously, we identified the regions of chromosomes 10q12-q31 and 15p16-q21 harbor quantitative trait loci (QTLs) for lumbar volumetric bone mineral density (vBMD) in female F2 rats derived from Fischer 344 (F344) x Lewis (LEW) and Copenhagen 2331 (COP) x Dark Agouti (DA) crosses. The purpose of this study is to identify the candidate genes within these QTL regions contributing to the variation in lumbar vBMD. RNA was extracted from bone tissue of F344, LEW, COP, and DA rats. Microarray analysi ...[more]

PMID: 19841953

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Project description:Hip fracture is the most devastating osteoporotic fracture type with significant morbidity and mortality. Several studies in humans identified chromosomal regions linked to hip size and bone mass. Animal models, particularly the inbred rat, serve as complementary approaches for studying the genetic influence on hip fragility. The purpose of this study is to identify sex-independent and sex-specific quantitative trait loci (QTLs) for femoral neck density, structure, and strength in inbred Copenhagen 2331 (COP) and Dark Agouti (DA) rats.A total of 828 (405 males and 423 females) F(2) progeny derived from the inbred COP and DA strains of rats were phenotyped for femoral neck volumetric BMD (vBMD), cross-sectional area, polar moment of inertia (Ip), neck width, ultimate force, and energy to break. A whole genome screen was performed using 93 microsatellite markers with an average intermarker distance of 20 cM. Recombination-based marker maps were generated using MAPMAKER/EXP from the COP x DA F(2) data and compared with published Rat Genome Database (RGD) maps. These maps were used for genome-wide linkage analyses to detect sex-independent and sex-specific QTLs.Significant evidence of linkage (p < 0.01) for sex-independent QTLs were detected for (1) femoral neck vBMD on chromosomes (Chrs) 1, 6, 10, and 12, (2) femoral neck structure on Chrs 5, 7, 10, and 18, and (3) biomechanical properties on Chrs 1 and 4. Male-specific QTLs were discovered on Chrs 2, 9, and 18 for total vBMD, on Chr 17 for trabecular vBMD, on Chr 9 for total bone area, and on Chr 15 for ultimate force. A female-specific QTL was discovered on Chr 2 for ultimate force. The effect size of the individual QTL varied between 1% and 4%.We detected evidence that sex-independent and sex-specific QTLs contribute to hip fragility in the inbred rat. Several QTLs regions identified in this study are homologous to human chromosomal regions previously linked to QTLs contributing to femoral neck and related phenotypes.

Project description:We used high density oligonucleotide arrays to measure the relative expression levels of ~27,000 probe sets in the left ventricles of inbred rat strains; DA (high performing), Copenhagen (COP, low performing), as well as F1(COPxDA) rats bred from these two strains whose performance was intermediate relative to both parental strains. DA and COP rats differ for both intrinsic aerobic running capacity (assessed with a treadmill exercise test to exhaustion) and intrinsic cardiac performance (as assessed by the Langendorff-Neeley working heart model). Previous work also showed a strong correlation between intrinsic aerobic running capacity and intrinsic cardiac performance. The inbred rats were maintained under specific pathogen free conditions and had access to pelleted rat chow (diet #5001, Purina Mills, Richmond, IN) and water ad libitim. The rats were housed with a target room temperature and humidity of 22oC and 40%, respectively, and 12:12 hour light dark cycle,with the light cycle occurring in the daytime (6 A.M. – 6 P.M.). The aerobic running capacity of the rats used in this study was assessed using a single week of treadmill endurance running tests, occurring at 11 weeks of age, and the rats were allowed 4 weeks to recover. Rats were killed at 15 weeks of age and their left ventricles were dissected. RNA was isolated by homogenizing the left ventricles of the heart in an ice cold guanidine thiocyanate/phenol solution (Ultraspec, Biotecx Laboratories, Houston, TX), extracted with chloroform and isolated using RNA Tack Resin (Biotecx Laboratories). Total RNA was further purified by ethanol precipitation and absorption to a column (RNeasy, Qiagen, Valencia, CA). Double-stranded cDNA was synthesized from 15 micrograms total RNA from each rat (four rats per strain) using reverse transcriptase (SuperScript II, InVitrogen, Carlsbad, CA) and T7-(dT)24 as the oligonucleotide primer. Biotinylated cRNA was synthesized using a kit (Enzo Bioarray High Yield RNA transcript Labeling Kit, Enzo Diagnostics, Farmingdale, NY) according to the manufacturer’s instruction and cRNAs were purified on columns (RNeasy mini kit, Qiagen). Each cRNA was fragmented according to the protocol in the Affymetrix GeneChip Expression Analysis manual and their quality was assessed by hybridization of a 5 microgram aliquot to a test chip (TestChip3, Affymetrix). 15 micrograms of the probe was hybridized to each of a set of three rat GeneChips (U34A, B, and C; Affymetrix). Hybridization, washing, staining with streptavidin-phycoerythrin, and scanning were performed at the Medical College of Ohio Core Facility according to the manufacturer's (Affymetrix) instructions. Complete transcription and hybridization were validated with bacterial sequences as an external control and several housekeeping genes as an internal control. Absolute analysis of data were conducted using the default settings of the Affymetrix software (GeneChip Software MAS-5.0). Microarray images were scaled to an average hybridization intensity of 150 to normalize the signals between individual chips. Keywords = quantitative real-time RT-PCR Keywords = insulin receptor substrate 2 (Irs2) Keywords = phosphatidylinositol 3-kinase Keywords = regulatory subunit 1 Keywords = (Pik3r1) Keywords = endurance running Keywords = treadmill running Keywords: parallel sample

Project description:IntroductionObservational studies have yielded inconsistent findings regarding the correlation between bone mineral density (BMD) and various spinal disorders. To explore the relationship between total-body BMD and various spinal disorders further, we conducted a Mendelian randomization analysis to assess this association.MethodsTwo-sample bidirectional Mendelian randomization (MR) analysis was employed to investigate the association between total-body BMD and various spinal disorders. The inverse-variance weighted (IVW) method was used as the primary effect estimate, and additional methods, including weighted median, MR-Egger, simple mode, and weighted mode, were used to assess the reliability of the results. To examine the robustness of the data further, we conducted a sensitivity analysis using alternative bone-density databases, validating the outcome data.ResultsMR revealed a significant positive association between total-body BMD and the prevalence of spondylosis and spinal stenosis. When total-body BMD was considered as the exposure factor, the analysis demonstrated an increased risk of spinal stenosis (IVW odds ratio [OR] 1.23; 95% confidence interval [CI], 1.14-1.32; P < 0.001) and spondylosis (IVW: OR 1.24; 95%CI, 1.16-1.33; P < 0.001). Similarly, when focusing solely on heel BMD as the exposure factor, we found a positive correlation with the development of both spinal stenosis (IVW OR 1.13, 95%CI, 1.05-1.21; P < 0.001) and spondylosis (IVW OR 1.10, 95%CI, 1.03-1.18; P = 0.0048). However, no significant associations were found between total-body BMD and other spinal disorders, including spinal instability, spondylolisthesis/spondylolysis, and scoliosis (P > 0.05).ConclusionThis study verified an association of total-body BMD with spinal stenosis and with spondylosis. Our results imply that when an increasing trend in BMD is detected during patient examinations and if the patient complains of numbness and pain, the potential occurrence of conditions such as spondylosis or spinal stenosis should be investigated and treated appropriately.

Dataset Information

Genes influencing spinal bone mineral density in inbred F344, LEW, COP, and DA rats.

Publications

Genes influencing spinal bone mineral density in inbred F344, LEW, COP, and DA rats.

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