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
Host genetics plays an important role in determining the outcome of Mycobacterium tuberculosis infection. The authors previously found that Collaborative Cross (CC) mouse strains differ in their susceptibility to M. tuberculosis and that the CC042/GeniUnc (CC042) strain suffered from a rapidly progressive disease and failed to produce the protective cytokine gamma interferon (IFN-) in the lung. Here, the authors used parallel genetic and immunological approaches to investigate the basis of CC042 mouse susceptibility to M. tuberculosis.
To investigate the genetic basis of CC042 mouse susceptibility, the authors created an F2 population between CC042 and CC001 mice. The CC001 strain was chosen as a partner to cross with the CC042 strain because of its relative TB resistance (which is similar to that of C57BL/6J mice) and to match the CC042 mouse major histocompatibility locus (H-2b). The authors crossed female CC001 mice with male CC042 mice to generate F1 progeny [(CC001 x CC042)F1 mice], which were then intercrossed to produce 201 F2 offspring. They infected male and female F1 and F2 progeny, along with parental strains, with M. tuberculosis (H37Rv) via low-dose aerosol. The mice were sacrificed at between 28 and 31 days postinfection, a time point that maximized phenotypic differences while minimizing morbidity. The phenotypes measured included the numbers of lung CFU, the numbers of spleen CFU, and lung IFN- levels.
In total, 170 F2 mice (86 female and 84 male mice) were genotyped with the MiniMUGA array (GRCm38/mm10).
QTL mapping was conducted on the tuberculosis-associated phenotypes, consisting of the numbers of lung CFU, numbers of spleen CFU, and IFN- levels. Genotype and phenotype data were imported into R (version 3.4.3) and reformatted for R/qtl (version 1.42-8). Genotype probabilities were calculated at a 0.25-cM spacing, and QTL mapping was carried out using the scanone function and batch and sex as additive covariates.
Five significant QTL that affect the measured tuberculosis immunophenotypes were identified:
Tip1 (tuberculosis immunophenotype 1, spleen CFU) maps to Chr 7: 3.6 - 72.9 Mb with a peak LOD score of 9.2 at 72.1 Mb (gUNC13104259). Tip1 has an additive mode of inheritance and explains 17.6% of the trait variance. CAST/EiJ alleles contribute the low haplotype at Tip1, while WSB/EiJ alleles contribute the high haplotype.
Tip2 (tuberculosis immunophenotype 2, spleen CFU) maps to Chr 7: 124.7 - 127.3 Mb with a peak LOD score of 12.3 at 125.4 Mb (gUNC13793270). Tip2 has a recessive mode of inheritance and explains 22.5% of the trait variance. NZO/HlLtJ alleles contribute the low haplotype at Tip2, while WSB/EiJ alleles contribute the high haplotype. Mice homozygous for the WSB/EiJ allele at Tip2 demonstrate a 10-fold increase in the number of spleen CFU, on average.
Tip3 (tuberculosis immunophenotype 3, IFN-g level) maps to Chr 15: 53.7 - 89.7 Mb with a peak LOD score of 5.4 at 83.1 Mb (mbackupUNC150396514). Tip3 has an additive (nonlinear) mode of inheritance and explains 13.5% of the trait variance. CAST/EiJ alleles contribute the low haplotype at Tip3, while 129S1/SvImJ alleles contribute the high haplotype.
Tip4 (tuberculosis immunophenotype 4, IFN-g level) maps to Chr 16: 4.5 - 44.7 Mb with a peak LOD score of 4.2 at 40.8 Mb (UNC26693650). Tip4 has an additive (nonlinear) mode of inheritance and explains 10.6% of the trait variance. WSB/EiJ alleles contribute the low haplotype at Tip4, while CAST/EiJ alleles contribute the high allele.
Tip5 (tuberculosis immunophenotype 5, IFN-g level) maps to Chr 7: 121.0 - 130.7 Mb with a peak LOD score of 9.1 at 125.4 Mb (gUNC13793270). Tip5 has a recessive mode of inheritance and explains 21.4% of the trait variance. NZO/HlLtJ alleles contribute the low haplotype at Tip2, while WSB/EiJ alleles contribute the high haplotype.
No QTL were associated with the numbers of lung CFU, suggesting that this trait is under more complex genetic control than the others.
The lack of CD11a expression on CC042 lymphocytes implicated Itgal variation as the basis for Tip2. To investigate this possibility, CD11a expression was assayed in the WSB and CC011 strains, which contain the susceptibility-associated WSB haplotype at Tip2 (Fig. 7A). We found that WSB and CC011 mouse splenocytes expressed CD11a levels similar to those expressed by B6 mouse splenocytes, leading us to hypothesize that CC042 mice had incurred a private mutation during inbreeding that impacts CD11a production. These defects could be explained by a CC042 private variant in the Itgal gene, which encodes CD11a and is found within the Tip2 interval. This 15-bp deletion leads to aberrant mRNA splicing and is predicted to result in a truncated protein product. The ItgalCC042 genotype was associated with all measured disease traits, indicating that this variant is a major determinant of susceptibility in CC042 mice.
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