Experiment
A previous study (J:69981) identified the Cia23 and Cia9 loci as the main genetic factors influencing the arthritis development in a F2 intercross between the arthritis susceptible strain C57BL/10.Q (B10.Q) and the resistant NOD.Q strain. In the present study congenic strains of the Cia23 and Cia9 loci were established and a partial advanced cross (PAI) was made between them to pinpoint the interactive effects between the two arthritis susceptibility loci.
The congenic strain for the Cia23, B10.Cg-(D2Mit116-D2Mit91)NOD/ShiLtJH2q/Hlmdl, was constructed by the speed congenic technique and backcrossed to the B10.Q strain for an additional 5 generations. The congenic fragment from NOD.Q was ~54-Mb long, ranging from D2Mit116 to D2Mit91.
The congenic strain for the Cia9 locus, B10.Cg-(D1Mit235-D1Mit17)NOD/ShiLtJH2q/Hlmdl, was constructed using the same technique as the Cia23 congenic. The congenic fragment from NOD.Q was ~150-Mb long, ranging from D1Mit235 to D1Mit17.
Cia23 and Cia9 congenic mice were intercrossed, F2 mice were genotyped, and selected mice were used for breeding to generate the partial advanced intercross (PAI). The aim was to produce all possible combinations of genotypes at the location of the linkage peaks within the Cia23 and Cia9 loci, respectively. Data were collected from 212 PAI mice, 96 Cia23 single and subcongenics, 62 Cia9 single and subcongenics, and 111 wild-type B10.Q mice in five CIA experiments. Single congenics and wild-type mice were used to control for disease variation in the separate experiments.
At the age of 10-12 wks, mice were immunized at the base of the tail with 100 ug of rat collagen type II (CII). Arthritis was evaluated blindly using a scoring system based on the number of inflamed joints in each paw. Briefly, each toe and knuckle was given a score of 1 (total 10/limb), an inflamed wrist or ankle was given a score of 5, giving a maximum score per limb of 15 and a maximal score per mouse of 60. Severity of arthritis was evaluated as maximum disease score and as accumulated score generated by calculating the sum of the disease scores for each mouse, respectively. Mice were bled at day 35 after induction of CIA, and the level of mouse anti-CII IgG Abs were measured in the sera, using ELISA. The levels of complement factor 5 in sera were also detected using the ELISA.
A full genomic scan was performed on 395 backcross animals using 162 microsatellite markers. Genotypes from 502 PAI mice were analyzed from 11 markers spanning 46.0-189.3 Mb on Chromosome 1 and 6 markers from 12.0 to 38.2 Mb on chromosome 2. Genotypes from 162 microsatellite markers were analyzed.
Linkage analysis was performed using R/qtl QTL mapping software in R. Arthritis severity was analyzed as maximum arthritic score and accumulated arthritic score. QTL analysis was calculated under the assumption that gender influenced the arthritis development as an interactive covariate. To investigate gene interactions, two-dimensional genome scan with a two-QTL model was performed.
In the backcross analysis QTL Cia9 (Chr 1, D1Mit349, Max LOD = 6.35 and Cia23 (Chr 2, D2Mit296-D2Mit91, Max LOD=8.82) were confirmed in linkage to arthritis incidence and severity. Cia23 also influenced the frequency of CD11B-positive cells (D2Mit296, LOD=3.25) and the higher expression of CD11B-positive cells correlated with arthritis development.
A suggestive QTL was identified in linkage with arthritis severity mapping to Chromosome 10, peaking with marker D10Mit50, LOD=2.94. No loci linked to immune response against CII, measured as levels of anti-CII Abs, were found.
The dominant protective effect of Cia23 was clearly confirmed in the PAI, seen by almost complete inhibition of arthritis in mice carrying one or two NOD.Q alleles at the Cia23 locus. However, in the mice carrying one or two NOD alleles at Cia9 in combination with the Cia23 alleles, the complete inhibition mediated by Cia23 was broken. Even mice homozygous for NOD at Cia23 could develop disease if combined with the disease-promoting allele at Cia9.
Interestingly, the inheritance was different at the more centromeric part of the congenic region on Chromosome 1 (at marker D1Mit14) where heterozygosity enhanced the disease, whereas homozygosity of the NOD allele in fact protected against arthritis. This argued for the presence of at least two genes in the Cia9 fragment.
Analysis Tools