Reference
The Collaborative Cross (CC) is a large (~1,000 line) panel of recombinant inbred (RI) mouse strains being developed through a community effort (Churchill et al. 2004). The CC combines the genomes of eight genetically diverse founder strains - A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ - to capture nearly 90% of the known variation present in laboratory mice. CC strains are derived using a unique funnel breeding scheme. Once inbred, the RI CC lines can be used to generate thousands of potential 'outbred' but completely reproducible genomes through the generation of recombinant inbred crosses (RIX). The designation 'PreCC' is used to describe a mapping population of CC mice that is still at incipient stages of inbreeding. CTC (2004), Churchill, G. A., et al.. The Collaborative Cross, a community resource for the genetic analysis of complex traits. Nat Genet. 36, 1133-7.
Experiment
The goal of the current study was to identify new genetic determinants of bone microarchitecture and metabolism associated with bone strength in a population of Collaborative Cross (CC) mice.
PreCC#/Tau mice aged 10 to 13 weeks (male n=86; female n=74) from 31 CC lines (average of 5 mice per line) were used to perform genome-wide haplotype mapping across 77,808 single-nucleotide polymorphisms (SNPs).
The microstructure of trabecular traits were obtained following microtomographic analysis, performed on 10-m resolution scans of the femoral distal metaphysis. In each CC animal the trabecular bone volume fraction (BV/TV), trabecular bone number (Tb.N), trabecular thickness (Tb.Th), and trabecular bone connectivity density (Conn.D) were measured in the distal femoral metaphysis. While phenotypic heterogeneity was observed in both males and females, female variability among the lines was greater (Fig S1). Irrespective of sex effects, the data indicated that body weight did not affect any of the analyzed bone parameters.
All QTL were above the genome-wide 99th percentile threshold; they were obtained empirically by 200 permutation simulations, with an experiment-wide false discovery rate (FDR) under 1 % for each scan.
After adjusting for sex and age and taking the mean for each line, haplotype association analysis was performed. For BV/TV and Tb.Th, two proximal QTL for each trait were identified on chromosome 2 and 7, respectively. Tb.N and Conn.D yielded one QTL on chromosome 8 and another on chromosome 14, respectively.
QTL Tbvq1 (trabecular bone volume QTL 1) mapped with an 95% confidence interval on Chromosome 2 that spanned from 126.2 to 135.6 Mb with a logP value of 6.67.
QTL Tbvq2 (trabecular bone volume QTL 2) mapped with an 95% confidence interval on Chromosome 2 that spanned from 115.6 to 124.9 Mb with a logP value of 6.31.
QTL Tbnq1 (trabecular bone number QTL 1) mapped with an 95% confidence interval on Chromosome 8 that spanned from 30.8 to 46.9 Mb with a logP value of 5.63.
QTL Tbthq1 (trabecular bone thickness QTL 1) mapped with an 95% confidence interval on Chromosome 7 that spanned from 62.7 to 68.1 Mb with a logP value of 7.87.
QTL Tbthq2 (trabecular bone thickness QTL 2) mapped with an 95% confidence interval on Chromosome 7 that spanned from 66.01 to 77.9 Mb with a logP value of 5.36.
QTL Tbconq1 (trabecular bone connectivity QTL 1) mapped with an 95% confidence interval on Chromosome 14 that spanned from 64.7 to 77.1 Mb with a logP value of 6.36.
Subsequently, the haplotype contribution of the founder strains to each QTL was assessed; Tbvq1, Tbvq2, Tbthq1 and Tbthq2 were predominantly affected by the PWK founder strain. In contrast, Tbnq1 and Tbconq1 had a more varied founder contribution.
Based on the literature, among the candidate genes that ranked particularly high in the analyses (P < 10-6) and which had a validated causal role in skeletal biology, were Avp, Oxt, B2m (associated with Tbvq1 and Tbvq2), Cnot7 (with Tbnq1), Pcsk6, Rgma (with Tbthq1 and Ththq2), Rb1, and Cpb2 (with Tbconq1). Other candidate genes strongly suggested by the analyses were Sgcz, Fgf20 (associated with Tbnq1), and Chd2 (with Tbthq1 and Ththq2).
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