Complex inheritance of the 5-lipoxygenase locus influencing atherosclerosis in mice

Abstract

We previously mapped a locus on chromosome 6 with a large effect (LOD > 6) on aortic lesion size in a (C57BL/6J x CAST/Ei) F(2) cross and identified arachidonate 5-lipoxygenase (5LO) as a candidate gene in this region. Subsequent studies with the 5LO knockout model showed effects on atherosclerosis and aortic aneurysms. We now report detailed genetic analysis of the chromosome 6 locus. We created a panel of overlapping and reciprocal subcongenic lines from the B6.CAST Ldlr(-/-) chromosome 6 congenic strain (CON6.Ldlr(-/-)) and analyzed aortic lesion size in different subcongenic lines. Our results revealed that there are at least two subregions, designated as Ath37 and Ath38 that affect the size of aortic lesions independently of 5LO. We also showed that homozygote 5LO null mice develop smaller atherosclerotic lesions. We conclude that the relation between the mouse chromosome 6 locus and atherosclerosis is complex and is due to at least two genes with large effects within this region. This complexity should be considered when interpreting results of knockout studies.

Figure 1.

The genotype and phenotype of the subcongenic lines. (A) The genotype of the 16 CON6 subcongenic lines for 10 microsatellite markers originally used to derive the congenic strain. The solid and open boxes represent the B6 and the CAST genotypes, respectively. The short name of the markers is given at the top, where 152 indicates D6Mit152; 123, D6Mit123; 102, D6Mit102; 104, D6Mit104; 44, D6Mit44; 193, D6Mit193; 61, D6Mit61; 111, D6Mit111; 198, D6Mit198; and 14, D6Mit14. B6 refers to C57BL/6J Ldlr^−/−, and CON6 refers to B6.CAST chromosome 6 congenic on an Ldlr^−/− background. The numbers at the bottom are the centimorgan positions of each marker obtained from the Mouse Genome Informatics database. (B) An overview of the aortic lesion size in each subcongenic line. (Top) The scatter plot of lesion size measured for each subcongenic mouse. The shaded line corresponds to the mean of each group. The x-axis is the line number for each subcongenic. (Bottom) The schematic of each subcongenic. Hatched boxes depict the CAST congenic region. The location of the 5LO and PPARγ genes are shown with broken arrows across the subcongenic lines. Mice used for these analyses were all female and were fed a Western diet beginning at 3 months of age for 8 weeks. All mice are on an Ldlr^−/− background.

Figure 1.

The genotype and phenotype of the subcongenic lines. (A) The genotype of the 16 CON6 subcongenic lines for 10 microsatellite markers originally used to derive the congenic strain. The solid and open boxes represent the B6 and the CAST genotypes, respectively. The short name of the markers is given at the top, where 152 indicates D6Mit152; 123, D6Mit123; 102, D6Mit102; 104, D6Mit104; 44, D6Mit44; 193, D6Mit193; 61, D6Mit61; 111, D6Mit111; 198, D6Mit198; and 14, D6Mit14. B6 refers to C57BL/6J Ldlr^−/−, and CON6 refers to B6.CAST chromosome 6 congenic on an Ldlr^−/− background. The numbers at the bottom are the centimorgan positions of each marker obtained from the Mouse Genome Informatics database. (B) An overview of the aortic lesion size in each subcongenic line. (Top) The scatter plot of lesion size measured for each subcongenic mouse. The shaded line corresponds to the mean of each group. The x-axis is the line number for each subcongenic. (Bottom) The schematic of each subcongenic. Hatched boxes depict the CAST congenic region. The location of the 5LO and PPARγ genes are shown with broken arrows across the subcongenic lines. Mice used for these analyses were all female and were fed a Western diet beginning at 3 months of age for 8 weeks. All mice are on an Ldlr^−/− background.

Figure 2.

Fine mapping of the CON6 distal locus. (A) The phenotype classification and genotype of the subcongenic lines 12 and 14 used to narrow the boundaries of the fine-map distal locus. Shown are the subcongenic line number, the phenotype of each line, and the genotype information for each line for seven microsatellite markers located in the fine-mapped region. The number 61 indicates D6Mit61; 111, D6Mit111; 220, D6Mit220; 301, D6Mit301; 258, D6Mit258; 291, D6Mit291; and 198, D6Mit198. The solid and open boxes represent the B6 and the CAST genotypes, respectively. (B) The CON6 locus has at least two genes affecting atherosclerosis. The map of the CON6 locus along with the location of the two major loci affecting atherosclerosis are shown. The size of the fine-mapped proximal and distal novel loci for atherosclerosis is indicated below their respective intervals. The dashed line indicates the interval where genes with small effect are located. As mentioned in the text there may be at least two genes with opposing effects in this interval. The relative locations of the six candidate genes (PPARγ, 5LO, Adipor2, A2m, CD163, and Olr1) are shown with solid squares on the map.

Figure 2.

Fine mapping of the CON6 distal locus. (A) The phenotype classification and genotype of the subcongenic lines 12 and 14 used to narrow the boundaries of the fine-map distal locus. Shown are the subcongenic line number, the phenotype of each line, and the genotype information for each line for seven microsatellite markers located in the fine-mapped region. The number 61 indicates D6Mit61; 111, D6Mit111; 220, D6Mit220; 301, D6Mit301; 258, D6Mit258; 291, D6Mit291; and 198, D6Mit198. The solid and open boxes represent the B6 and the CAST genotypes, respectively. (B) The CON6 locus has at least two genes affecting atherosclerosis. The map of the CON6 locus along with the location of the two major loci affecting atherosclerosis are shown. The size of the fine-mapped proximal and distal novel loci for atherosclerosis is indicated below their respective intervals. The dashed line indicates the interval where genes with small effect are located. As mentioned in the text there may be at least two genes with opposing effects in this interval. The relative locations of the six candidate genes (PPARγ, 5LO, Adipor2, A2m, CD163, and Olr1) are shown with solid squares on the map.

Figure 3.

5LO null mice have reduced atherosclerosis. Scattergram of aortic lesions in 5LO^−/− (n = 6) and C57BL/6J (n = 9) mice. Two-month-old, chow-fed female mice of each group were fed with the high-fat, atherogenic diet for 18 weeks prior to lesion assessment. Numbers within each plot indicate the mean leasion size for each group. B6 5LO^−/− mice developed ∼10-fold smaller aortic lesions as compared to B6 wild-type mice.

Figure 4.

Aneurysms in subcongenic mice. The frequency plot for the incidence and severity of aneurysms in female subcongenic Ldlr^−/− mice homozygous for the CAST 5LO allele (left) and the B6 5LO allele (right). The number of mice analyzed for the CAST and the B6 group are 76 and 116, respectively. The severity of aneurysms for each mouse was estimated using a semiquantitative method (see materials and methods) and represented as a “relative score” on the plot.