Experiment
The hypothesis that genetic factors affecting vascular geometry also affect the location and extent of atherosclerotic plaque development was the objective of this study.
Apolipoprotein E-null mice with a 129S6/SvEvTac strain background, 129S6/SvEvTac-Apoetm4Mae (129-apoE), develop atherosclerotic plaques faster in the aortic arch but slower in the aortic root than mice with a C57BL/6J background, B6.129P2-Apoetm1Unc (B6-apoE) . The aortic root lesion sizes are also significantly smaller in the 129-apoE mice than those in the B6-apoE mice in both sexes. The aortic arch lesion sizes in 129-apoE mice, in contrast, are significantly bigger than those in B6-apoE mice. The shape of the aortic arch also differs between these two strains. The angle of the aortic arch in 129-apoE mice is significantly greater than in B6-apoE mice, reflecting a broader curvature.
Male 129-apoE mice were crossed with female B6-apoE mice,to generate F1 mice which were intercrossed to generate F2 progeny, (B6.129P2-Apoetm1Unc/J x 129S6/SvEvTac-Apoetm4Mae)F2; 138 males and 128 females. (Reciprocal crosses between female B6-apoE and male 129-apoE mice were not productive for unknown reasons.)
The aortic arches of 24 wk old F2 mice were dissected and their images captured. Plaque areas in the aortic arch and the diameters of the ascending aorta (DA1), transverse aorta (DA2), and descending aorta (DA3) were measured using Image J 1.40. The angle B (DA1) A (DA2) C (DA3) was defined as the angle of the aortic arch. The plaque area in the aortic arch was measured using the captured image and was used as a representation of atherosclerosis in the aortic arch. DNA was isolated from the livers and SNP analysis was performed using Illumina SNP panels. 235 SNP markers were used. QTL analyses were performed using R/qtl software. Peak LOD scores were determined by 1000 permutations, p>0.05 being significant, p>0.63 suggestive. QTL analysis was performed using the F2 mice.
QTL analysis of the males only data identified QTL Arlsq1 (aortic root lesion size QTL 1), mapping to Chr 9 at 59 Mb, nearest SNP rs13480208, LOD=5.63. The B6 allele at rs13480208 was associated with an increased root lesion size, and mice homozygous for the B6 allele had twice larger plaque sizes than mice homozygous for the 129 allele at this locus. The QTL accounted for 16% of trait variance.
In females, 4 suggestive QTL on Chrs 1, 6, 9 and 14 contributing to root lesion size were identified.
Using combined data from both F2 males and females with sex treated as an interactive covariate, Table 2, the following significant and suggestive QTL were identified :
A highly significant QTL, Arlsq2 ( aortic root lesion size QTL 2 ), contributing to root lesion size was detected on Chromosome 9 peaking at 61 Mb, nearest SNP rs13480208, LOD=6.9. The confidence interval spanned from 47 - 71 Mb, with the locus contributing to 8.9% of the trait variance. The B6 allele was associated with an increased root lesion size.
Two suggestive QTL for root lesion size mapped to Chr 2, peaking at 154 Mb, nearest SNP rs6376291, LOD=3.8, accounting for 4.3% of trait variance, with B6 the high allele; and Chr 15, peaking at 68 Mb near SNP rs13482628, LOD=4.5, accounting for 6.9% of trait variance, with 129 the high allele.
Two significant QTL were detected in males only data that affected arch lesion size.
QTL Aath1, aortic arch atherosclerosis 1, mapped to Chromosome 1 peaking at 163 Mb nearest SNP rs13476024, LOD=5.3, accounting for 16% of the trait variance in arch lesion size in males.
QTL Aath2, aortic arch atherosclerosis 2, mapped to Chromosome 1 at 105 Mb nearest SNP rs3685643, LOD=3.9.
The 129 allele of both Aath1 and Aath2 contributed to an increased arch lesion size with the alleles having an additive effect on the trait. Neither QTL was identified in female mice.
QTL, Aath3, aortic arch atherosclerosis 3, affecting arch lesion in female mice only mapped to Chromosome 15 peaking at 92 Mb, LOD=4.0, accounting for 14% of trait variance in arch lesion size in females. The 129 allele at this locus contributed to an increased arch lesion size additively.
When the data from both sexes was combined, F2 males and females with sex treated as an interactive covariate, the following significant QTL were identified [Table 2]:
QTL Aath6, aortic arch atherosclerosis 6, mapped to Chromosome 1 peaking at 163 Mb nearest SNP rs3707322, LOD=6.8. The confidence interval spanned between 151 -173 Mb, with the locus accounting for 16% of the trait variance in arch lesion size.
QTL Aath7, aortic arch atherosclerosis 7, mapped to Chromosome 1 at 105 Mb nearest SNP rs13476012, LOD=5.0. The confidence interval spanned between 75-135 Mb.
QTL Aath8, aortic arch atherosclerosis 8, mapped to Chromosome 15 peaking at 96 Mb nearest SNP rs6326790, LOD score 4.7. The confidence interval spanned from 72-102 Mb, with the locus accounting for 7.4% of trait variance. The 129 allele was the high allele for all 3 QTL based on the combined data.
A significant QTL Aaa1, aortic arch angle 1, was identified on Chromosome 1 peaking at 121 Mb nearest SNP rs13476098, LOD=5.6. The confidence interval spanned between 81 -139 Mb, with the locus accounting for 5.5% of trait variance. The 129 allele was the high allele.
Two suggestive QTL for aortic arch angle mapped to Chr 4 (peaking at 145 Mb, nearest SNP rs3023025, LOD=3.5, accounting for 6.2% of trait variance, with B6 the high allele); and Chr 15 (peaking at 76 Mb nearest SNP rs3701224, LOD=3.5, accounting for 2.2% of trait variance, with 129 the high allele).
The mean DA1-3 was predominantly governed by a QTL, Adq1 (aortic diameter QTL 1) on Chromosome 9 at 33 Mb, nearest SNP rs3665206, LOD=8.5, identified in male mice. The QTL accounted for 25% of trait variance. The B6 allele at rs3665206 contributed to a larger diameter of the aorta with an additive effect.
A peak was not detected in female mice, however when the data from both sexes was combined, QTL Adq2 (aortic diameter QTL 2) was mapped to Chr 9, peaking at 33 Mb, nearest SNP rs3665206, LOD=9.9. The confidence interval spanned between 31-37 Mb with the locus accounting for 4.7% of trait variance. The B6 allele contributed to a larger diameter of the aorta with an additive effect. [Table 2].
In combined data a significant body weight QTL, Bwq12 (body weight QTL 12) mapped to Chromosome 2 peaking at 166 Mb near SNP rs13476892, LOD=5.1. The confidence interval spanned between 158 and 166 Mb with the locus contributing to 3.2% of the trait variance. The B6 allele was the high allele. Mapping results for the geometric traits were not altered when body weight was used as a covariate.
In combined data three significant cholesterol QTL were identified on Chrs 1, 2 and 9.
[Table 2].
QTL Pcho7, plasma cholesterol 7, mapped to Chr 1, peaking at 169 Mb, near SNPrs3707322, LOD=6.6. The confidence interval spanned between 155-181 Mb, with the locus accounting for 9.8% of the trait variance. The 129 allele was the high allele conferring increased levels of plasma lipids.
QTL Pcho8, plasma cholesterol 8, mapped to Chr 2, peaking at 4 Mb, near genome marker gnf02.001.197, LOD=4.8. The confidence interval spanned between 4 and 22 Mb, accounting for 3.2% of trait variance with the 129 allele as the high allele.
QTL Pcho9, plasma cholesterol 9, mapped to Chr 9, peaking at 55 Mb, near SNP rs13480208, LOD=6.2. The confidence interval spanned between 43-71 Mb, accounting for 11.0% of the trait variance with the 129 allele the high allele.
In addition 2 female specific QTL contributing to plasma cholesterol levels were identified in the analysis of female only mice: QTL Pchol10 (plasma cholesterol 10) mapped to Chr 2, peaking at 8 Mb with a LOD score of 5; QTL Pchol11 (plasma cholesterol 11) mapped to Chr 7, peaking at 123 Mb with a LOD score of 3.7
In the combined data data three significant triglyceride QTL were identified on Chrs 1, 7, and 9. [Table 2].
QTL Tgl5, triclyceride 5, mapped to Chr 1, peaking at 177 Mb near SNP rs13476259, LOD=7.3. The confidence interval spanned between 167-183 Mb, accounting for 12.8% of trait variance, with the 129 allele the high allele.
QTL Tgl6, triglyceride QTL 6, mapped to Chr 7, peaking at 37 Mb near SNP rs6228386, LOD=6.1. The confidence interval spanned between 25-57 Mb, accounting for 7.1% of trait variance, with the B6 allele the high allele.
QTL Tgl7, triglyceride QTL 7, mapped to Chr 9, peaking at 67 Mb near SNP rs3693209, LOD=6.0. The confidence interval spanned 31-81 Mb, accounting for 10.0% of trait variance, with the 129 allele the high allele.
In addition a male specific plasma triglyceride QTL Tgl8 (triglyceride QTL 8) mapped to Chr 7, peaking at 49 MB with a LOD score of 4.6 in the analysis of male only mice.
In the combined data QTL Phdlc5, plasma HDL cholesterol 5, mapped to Chr 1, peaking at 179 Mb, near SNP rs13476259, LOD=7.8. The confidence interval spanned between 167-185 Mb accounting for 9.6% of trait variance, with the 129 allele the high allele. [Table 2]
The overlapping QTL for curvature of the aortic arch and atherosclerosis support the ontogeny of the aortic arch formation as a potential risk factor for atherosclerosis.
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