High-Resolution Genome Screen for Bone Mineral Density in Heterogeneous Stock Rat

Abstract

We previously demonstrated that skeletal mass, structure and biomechanical properties vary considerably in heterogeneous stock (HS) rat strains. In addition, we observed strong heritability for several of these skeletal phenotypes in the HS rat model, suggesting that it represents a unique genetic resource for dissecting the complex genetics underlying bone fragility. The purpose of this study was to identify and localize genes associated with bone mineral density in HS rats. We measured bone phenotypes from 1524 adult male and female HS rats between 17 to 20 weeks of age. Phenotypes included DXA measurements for bone mineral content and areal bone mineral density for femur and lumbar spine (L3-5), and volumetric BMD measurements by CT for the midshaft and distal femur, femur neck and 5th lumbar vertebra. A total of 70,000 polymorphic SNPs distributed throughout the genome were selected from genotypes obtained from the Affymetrix rat custom SNPs array for the HS rat population. These SNPs spanned the HS rat genome with a mean linkage disequilibrium coefficient between neighboring SNPs of 0.95. Haplotypes were estimated across the entire genome for each rat using a multipoint haplotype reconstruction method, which calculates the probability of descent for each genotyped locus from each of the 8 founder HS strains. The haplotypes were tested for association with each bone density phenotype via a mixed model with covariate adjustment. We identified quantitative trait loci (QTLs) for bone mineral density phenotypes on chromosomes 2, 9, 10 and 13 meeting a conservative genome-wide empiric significance threshold (FDR=5%; P<3 × 10−6). Importantly, most QTLs were localized to very small genomic regions (1-3 Mb), allowing us to identify a narrow set of potential candidate genes including both novel genes and genes previously shown to have roles in skeletal development and homeostasis.

Fine mapping of bone structure and strength QTLs in heterogeneous stock rat

“…Recently, using the sequence data from these strains and genotypes for a dense SNP marker map in the HS offspring population, we identified several QTLs and underlying genetic variants for multiple bone phenotypes [18]; however, no single genetic variants explaining associations with bone phenotypes were detected, consistent with the complex genetic architecture of skeletal phenotypes observed previously both in humans and animal models [18-19]. The purpose of this study is to identify and localize QTLs for bone structure and strength phenotypes using high-resolution mapping in the HS rat offspring at the most common skeletal fracture sites.…”

“…To reconstruct the genome of each HS rat, genotypes for over 900,000 SNPs for each rat were selected from an Affymetrix rat custom SNP array (www.affymetrix.com) as described previously [19]. We used only high quality informative (SNP call rate more than 0.99, polymorphic SNPs and no missing genotyping) markers.…”

“…Haplotypes were constructed for each rat across the genome using the multipoint haplotype reconstruction method HAPPY (http://www.well.ox.ac.uk/happy) [20] as described previously [19]. A mixed model approach was employed to test for association between each haplotype and the bone phenotype of interest.…”

Genetic regulation of bone strength: a review of animal model studies

“…Although loci for bone strength phenotypes have not yet been reported in the HS rat, QTL for bone mass have been, and the narrow confidence intervals of these QTL demonstrate the superior mapping resolution possible when using this population. 26 This highlights the superiority of the HS rat for any forward genetic mapping study.…”

“…These include the use of commercially available outbred animals or 'designer' outbred populations, which are created by breeding two or more strains over the course of multiple generations in a manner that minimizes inbreeding. [26][27][28][29] Additional study designs have been used to better define the genetic etiology of bone strength, including the use of congenic and consomic genetic reference populations. 23 A consomic, which is sometimes referred to as chromosome substitution, is a strain wherein all of the alleles for an entire chromosome from one inbred strain of mice or rats have been moved onto an otherwise pure background of a second strain.…”