Experiment
In the current study, plasma lipid phenotypes were characterized in crosses between DDD.Cg-Ay (DDD-Ay) and B6.Cg-Ay (B6-Ay) female mice. Strains DDD-Ay and B6-Ay were referred together as A-y mice; their control littermates, DDD/SgnRbrc (DDD) and C57BL/6J (B6) were referred together as non-Ay mice. Plasma triglyceride (TG) levels were significantly higher in the DDD-Ay strain than in the B6-Ay strain. In contrast, plasma total cholesterol (CHO) levels did not differ substantially between the two strains.
F2 females were weaned at 4 weeks of age. At 16 weeks of age the body weights were determined for mice that had fasted for 4 hours. Mice were then euthanized and whole blood was drawn from the heart. Plasma and triglyceride levels were determined enzymatically. Genomic DNA was isolated from the tails of mice. Microsatellite sequence length polymorphisms were identified after PCR amplification of genomic DNA.
Initially, the F2 Ay and F2 non-Ay mice were independently analyzed for a single QTL using R/qtl. Threshold LOD scores for suggestive (p<0.63) and significant (p<0.05) linkages were determined by performing 1000 permutations for each trait. After single QTL scans, pairwise evaluations of potential interactions between loci were performed. Next data on F2 non-Ay and F2 Ay were combined and analyzed. The presence or absence of possible statistical interactions between genotypes at the QTL and agouti locus (non-Ay and Ay) was evaluated by two-way ANOVA.
Table 1:
Results of the genome wide scan for single QTL in the F2, non-Ay mice revealed:
Three suggestive QTL for triglyceride (TG) levels mapped to Chromosome 1 (86 cM, LOD=2.00), Chromosome 16 (37 cM, LOD=2.47) and Chromosome 18 (59 cM, LOD=2.31) respectively.
A significant QTL linked to total plasma cholesterol, Cholq1 (cholesterol QTL 1) mapped to Chromosome 1 peaking at 79.0 cM nearest marker Apoa2 with a LOD score of 23.10. The 95% confidence interval spanned from 76.0 to 85.0 cM. The DDD allele was associated with higher cholesterol levels.
A second significant QTL, Cholq2 (cholesterol QTL 2) mapped to Chromosome 8 peaking at 50.0 cM nearest marker D8Mit183 with a LOD score of 3.58. The 95% confidence interval spanned from 21.0 to 50.0 cM. The B6 allele was associated with higher cholesterol levels. Potential candidate gene Lcat mapped within the 95% CI of QTL Choldq2.
And a suggestive QTL linked to total cholesterol mapping to Chromosome 16 (22.0 cM, LOD=2.19).
Results of the genome wide scan for single QTL in the F2< Ay mice revealed:
A significant QTL, Cholq14 (cholesterol QTL 14), mapped to Chromosome 1, also peaking at 79.0 cM nearest marker Apoa2 with a LOD score of 13.79. The 95% confidence interval spanned from 75.0 to 85.0 cM. The DDD allele was associated with higher cholesterol levels.
A suggestive QTL linked to cholesterol mapped to Chromosome 11 (56.0 cM, LOD=2.46). Three suggestive QTL linked to triglyceride levels were mapped to Chromosome 1 (70.0 cM, LOD=2.52), Chromosome 3 (31.0 cM, LOD=2.39) and Chromosome 11 (47.0 cM, LOD=2.43) respectively.
And three suggestive QTL linked to triglyceride levels mapping to Chromosome 1 (70.0 cM, LOD=2.52), Chromosome 3 (31.0 cM, LOD=2.39) and Chromosome 11 (47.0 cM, LOD=2.43).
In the QTL analysis of combined mice (F2 non-Ay plus F2 Ay) in single QTL scans:
A significant QTL, Trigq5 (triglyceride QTL 5), linked to triglyceride levels mapped to Chromosome 1 peaking at 81 cM nearest marker Apoa2 with a LOD score of 4.11. The 95% confidence interval spanned from 66.0 to 89.0 cM. The DDD allele was associated with increased triglyceride levels. Gene Apoa2 is presented as a plausible candidate gene for both Choldq1 and Tridg1.
A suggestive locus linked to TG levels mapped to Chromosome 11 (37.0 cM, LOD=3.02).
A significant QTL, Cholq15 (cholesterol QTL 15) linked to total plasma cholesterol levels mapped to Chr 1 overlapping the previously identified QTL Choldq1 with a LOD score of 35.73 peaking nearest marker Apoa2. Another, Cholq16 (cholesterol QTL 16) mapped to Chr 8 overlapping the previously identified QTL Choldq2 with a LOD score of 4.63 peaking nearest marker D8Mit183.
A suggestive locus identified in the combined data, also linked to CHO mapped to Chromosome 11 ( 60.0 cM, LOD=2.90).
CHO levels at Chromosome 1 were further analyzed using the marker nearest QTL Choldq1 as covariates. When the nearest marker (D1Mit291 or D1Mit16) was included as an additive covariate LOD scores shrank to near zero and further significant QTL were not identified in either F2 mapping population.
However, when the nearest marker was used as an interactive covariate:
A significant QTL, Cholq3 (cholesterol QTL 3) was identified mapping to Chr 17 peaking at 15.0 cM nearest marker D17Mit176 with a LOD score of 5.67 in the F2 non-Ay population.
Another significant QTL, Cholq4 (cholesterol QTL 4) was also identified on Chr 5 in the F2, non-Ay on Chr 5 peaking at 77.0 cM nearest marker D5Mit221 with a LOD score of 3.94.
Two significant QTL were identified in the F2 Ay mapping population:
QTL, Cholq5 (cholesterol QTL 5) mapped to Chr 6 peaking at 75.0 cM nearest marker D6Mit259 with a LOD score of 4.22; and QTL Cholq6 (cholesterol QTL 6) mapped to Chr 17 peaking at 46.0 cM nearest marker D17Mit93 with a LOD score of 3.31.
When the agouti genotype was used as an additive or an interactive covariate in the combined F2 data set, no significant QTL were newly identified for both TG and CHO phenotypes. No significant interactions were identified for either phenotype in pairwise scans.
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