Experiment
To gain insight into genetic factors that influence atherosclerosis at different vascular locations, 335 F2 mice from an intercross between 129S6/SvEvTac-Apoetm4Mae (129S6-apoE) and D2.129P2(B6)-Apoetm1Unc/J (DBA/2J- apoE) were analyzed. Average plaque size in the aortic arch was comparable between the two strains in both male and female mice.
The aorta with branch vessels from the F2 mice at 4 months of age were dissected and then imaged; atherosclerotic lesions were measured on the captured images using Image J 1.43 software. Genomic DNA was isolated from livers. Genome wide single scans for QTL that influenced atherosclerotic plaque size were performed in the aortic arch and its branches using 170 SNP genotypes of 191 F2 males and 144 F2 females.
To account for potential gender differences and interactions, the genome scans were performed in both sexes combined using sex as an additive or an interactive covariate, or separately in males and females. The significance thresholds were determined by 10,000 permutations. QTL were considered significant if LOD scores exceed 95% (p<0.05) of the permutation distribution; they were considered suggestive if the scores exceeded 37% (<0.63) distribution. All genetic positions (cM) were converted to physical positions (Mb, NCBI Build 37) using the Mouse Map Converter.
Two significant QTL for aortic arch lesion size were detected in the sex-combined analysis:
QTL Aath4, aortic arch atherosclerosis 4, mapped to Chromosome 2 at 137 Mb, LOD=7.0. The confidence interval spanned between 123-148 Mb. The DBA/2J allele was associated with an increased arch plaque size in comparison with the 129S6 allele. The DBA allele was inherited in an additive mode. Aath4 accounted for 10.7% of the trait variance.
QTL Aath5, aortic arch atherosclerosis 5, mapped to Chromosome 10 at 51 Mb, LOD=5.1. The confidence interval spanned between 30-101 Mb. At this locus the 129S6 allele conferred increased arch plaque size relative to the DBA allele The 129 allele was also inherited in and additive mode. Aath5 accounted for 5.8% of the trait variance
No significant differences were observed between the sex-additive and sex-interactive analyses.
When the 2 sexes were analyzed separately, the male only analysis revealed two significant loci and one suggestive locus:
QTL Aath9, aortic arch atherosclerosis 9, mapped to Chromosome 2 peaking at 153 Mb, LOD=4.0 with a confidence interval between 132-165 Mb. The DBA allele was the high allele, inherited in an additive mode.
QTL Aath10, aortic arch atherosclerosis 10, mapped to Chromosome 10 at 46 Mb, LOD=4.0 with a confidence interval between 26-111 Mb. The 129S6 was allele the high allele, also inherited in an additive mode of inheritance.
A suggestive QTL mapped to Chr 19 at 43 Mb, LOD=3.1.
In the female only analysis, one suggestive QTL was detected on Chr 2 at 132 Mb, LOD=3.3.
Genome scans allowing for pair wise analysis to search for additional QTL and interactions were also carried out. No additional interacting loci were observed.
Aath4 and Aath5 were not present in the previous (B6.129P2-Apoetm1Unc/J x 129S6/SvEvTac-Apoetm4Mae)F2 cross analysis done in J:170863. The simplest inference is that these QTL lie within regions where the sequence of the 129 mice differs from that of the DBA mice, but is shared with that of the B6 mice.
Using the Mouse Phylogeny viewer, regions where DBA differs from 129 and B6 and Aath4 are at 125-129 Mb, 131 Mb, 135, Mb, 139-143 Mb and 148-149 Mb. These regions correspond to Chr 15q21, 2q11-14 and 20p11-13 in humans, each of which contains variations associated with cardiovascular phenotypes.
Regions where DBA/2J sequence differs from both 129S6 and B6 occur in four interval: near 46 Mb, near 55 Mb, between 67- 69 Mb, and 84-93 Mb. These intervals correspond to human Chr 6q21-22, Chr 10q21 and Chr12q23-24; regions associated with stoke, plasma VLDL concentrations, carotid artery disease, heart rate and blood pressure.
To gain insight into candidate genes underlying each QTL SNPs and insertion/deletions that differ between DBA/2J and 129S5, the closest relative to 129S6, potential functional consequences were considered. Results suggested that atherosclerosis susceptibility of Aath4 in DBA may be contributed to by multiple genes, including Mertk and Cd93, that play roles in phagocytosis of apoptotic cells and modulate inflammation. A candidate gene for Aath5 is Stab2, the DBA allele of which is associated with 10 times higher plasma hyaluronan than with the 129 allele.
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