Genetic loci affecting bone structure and strength in inbred COP and DA rats

Abstract

Previous studies have shown that the Copenhagen 2331 (COP) and Dark Agouti (DA) rats have significant differences in bone structure and strength despite their similar body mass. Thus, these inbred rat strains may provide a unique resource to identify the genetics underlying the phenotypic variation in bone fragility. A sample of 828 (405 males and 423 females) COP × DA F2 progeny had extensive phenotyping for bone structure measures including cortical bone area and polar moment of inertia at the femur midshaft and total, cortical and trabecular bone areas, for the lumbar vertebra 5 (L5). Bone strength phenotypes included ultimate force, stiffness and work to failure of femur and L5. These skeletal phenotypes were measured using peripheral quantitative computed tomography (pQCT) and mechanical testing. A whole-genome screen was conducted in the F2 rats, using microsatellite markers spaced at approximately 20 cM intervals. Genetic marker maps were generated from the F2 data and used for genome-wide linkage analyses to detect linkage to the bone structure and strength phenotypes. Permutation testing was employed to obtain the thresholds for genomewide significance (p<0.01). Significant QTL for femur structure and strength were identified on chromosome (Chr) 1 with a maximum LOD score of 33.5; evidence of linkage was found in both the male and female rats. In addition, Chrs 6, 7, 10, 13, 15 and 18 were linked to femur midshaft structure. QTL linked to femur strength were identified on Chrs 5 and 10. For L5 vertebrae, Chrs 2, 16, and 18 harbored QTL for cortical structure and trabecular structure for L5 was linked to Chrs 1, 7, 12, and 18. One female-specific QTL for femur ultimate force was identified on Chr 5, and two male-specific QTL for L5 cortical area were found on Chrs 2 and 18. Our study demonstrates strong evidence of linkage for bone structure and strength to multiple rat chromosomes.

Linkage Screen for BMD Phenotypes in Male and Female COP and DA Rat Strains

“…The study of animal models also permits the study of phenotypes that would be impractical to study in human subjects, such as bone strength and structure measures that we have also mapped in the COP/DA cross. (29) In addition to the novel sex-specific QTLs detected in the COP/DA rat cross, we also found confirmatory evidence for QTLs detected in the F344/LEW rat cross on chromosomes 1, 2, and 15. These three common BMD QTLs were detected along with 13 other QTLs in the COP/DA cross and 9 other QTLs in the F344/LEW F 2 females.…”

Differentially expressed genes strongly correlated with femur strength in rats

“…1) [19]. In addition, significant evidence of linkage of femur Fu was observed in F344×LEW F2 rats with a LOD score of 4.8, in a broad region that overlapped the COP×DA peak (Fig.…”

“…The spleens were immediately frozen in liquid nitrogen before transferring them to −80 °C. The right femurs were tested in three-point bending by positioning them on the lower supports of a three-point bending fixture and applying load at the midpoint using a material testing machine (Alliance RT/5, MTS Systems Corp., Eden Prairie, MN, USA) and ultimate force (Fu; N) was obtained from the force vs. displacement curves in TestWorks software, version 4.06 as described previously [19,20]. …”

“…Multipoint quantitative linkage analysis was then performed to identify chromosomal regions that harbor QTL for femur Fu using the programs R/qtl and QTL/Cartographer. Permutation testing was employed to obtain appropriate genome-wide significance levels for the linkage results and ANOVA was used for the most significant marker in each QTL region to further characterize significant genotypic group differences as described previously [19,20]. …”

“…We demonstrated that despite similar body size, substantial variation exists in bone geometry and biomechanical properties between adult Copenhagen 2331 (COP), Dark Agouti (DA), Fischer 344 (F344) and Lewis (LEW) rats [22]. Subsequently, we generated a large number of second filial (F2) female progeny derived from (COP×DA) and (F344×LEW) crosses, and detected significant linkage for femur ultimate force (Fu) (a biomechanical measurement that determines the maximum load that the bone can support before failing) within the region of 1q33–q54 in both of these crosses [19,20]. Thus, 1q33–q54 region harbors QTLs for a bone phenotype directly related to femur strength.…”