We have outlined a novel strategy for fine mapping atherosclerosis loci using association on a sensitized genetic background. In this proof of concept paper, we have provided strong evidence that the strategy works, with the potential of greatly narrowing the regions of the mouse genome that contribute to differences in atherosclerosis susceptibility. We have previously reported the successful application of the EMMA algorithm for correction of the significant population structure existing among inbred strains of mice, allowing association analysis using common inbred strains of mice5
. As compared to linkage, association analysis has much improved mapping resolution because it utilizes the many historical recombinations that have occurred during the generation of inbred strains rather than the much smaller number of recombinations that occur in a genetic cross.
In order to induce significant atherosclerotic lesions, we utilized a sensitized genetic background (the human apolipoprotein B transgene). Indeed, the lesions we observed were about 10-fold larger than those obtained using a high fat, cholic acid diet alone. It is noteworthy that our results among the BXH subset of RI strains surveyed with the human apoB transgene were highly concordant with previous studies from the laboratory of Paigen and coworkers and our laboratory. This suggests that the genetic factors contributing to fatty streaks and more advanced lesions show considerable overlap. The concordance of our data with previous genetic studies of atherosclerosis in mice also validates the F1 hybrid strategy for introducing a sensitizing mutation onto multiple genetic backgrounds. The strategy has the drawback that it will miss recessive mutations contributing to the trait that are carried by the partner strain (that is, the strain to which the sensitizing mutation carrying strain is crossed).
We have used our association strategy to fine map loci that have previously been identified using linkage, focusing on Ath30
. Our original study in BXH.Apoe−/−
mice identified the Ath30
locus on Chr 1 with a 95% confidence interval of 8Mb, from 72 to 80 Mb, containing over 300 genes25
. Using association we are now able to reduce the interval of the Ath30
QTL to a 2 Mb region, between 74.8 to 76.8 Mb on Chr 1, containing only 31 genes. This allows us to eliminate several positional candidates from our initial cross, such as 2310007B03Rik
. Analysis of SNPs with coding changes identified 13 of the 31 genes in the interval, with missense variants.
Since the locus does not map with plasma lipids, we hypothesized that the gene(s) underlying the Ath30
locus may be vessel wall specific and thus performed eQTL analysis of aorta. Two genes had highly significant local eQTL, Des
encodes a proteoglycan that has been previously implicated as a candidate gene for atherosclerosis in human lesions33
and galactosidase, beta 1-like is an uncharacterized gene.
To further examine the possible role of these candidates in atherogenesis we compared their expression in cells derived from aortas from wild-type and Apoe−/− mice. These results indicate that Des mRNA levels are induced prior to initiation of extensive atherosclerosis at 4 weeks of age and elevated expression continues into more advanced lesion development at 24 weeks of age. There were no effects on Glb1l.
The original studies identifying Ath30
indicated that this locus was sex-biased, with females, but not males, exhibiting a significant QTL.25
. The explanation for the failure to observe sex-bias in the present association study is unclear. However, it is known that the nature of the sensitizing mutation can affect sex differences (for example,34, 35
and, whereas, the previous study used an Apoe−/−
sensitizing mutation, this study employed a human APOB transgene. The fact that the sex bias was not observed in our present study could well be due to differences in sensitizing mutation.
In conclusion, our results suggest that the mouse may provide another unbiased approach to identify the genes and pathways contributing to this common forms of atherosclerosis (and other complex traits), complementing human studies. Analyses in mice may also help overcome some of the limitations of human studies, particularly the analysis of gene-by-gene and gene-by-environment interactions.