Experiment
In the current study quantitative trait locus (QTL) mapping was performed for plasma total-cholesterol (CHO) and triglyceride (TG) concentrations in reciprocal F2 male intercross populations between DDD/SgnRbrc (DDD) and C57BL/6J (B6) mice. DDD mice have significantly higher plasma lipid concentrations than B6 mice. Male B6 mice have higher triglyceride concentrations than female B6 mice.
For QTL analysis 150 (DDD x B6)F2 and 150 (B6 x DDD)F2 male mice were generated. Mice were weaned at 4 weeks of age. Plasma lipid concentrations were determined at the age of 11-14 weeks in DDD, B6, and F1 mice and at the age of 11-12 weeks in F2 mice. Mice were euthanized and blood serum was collected from the heart. Plasma CHO and TG concentrations were determined buy enzymatic methods using clinical kits. A total of 117 microsatellite loci were genotyped; their respective chromosomal positions were retrieved from MGI. Genomic DNA was extracted from the tail of DDD mice for exome capture and sequencing.
QTL mapping was performed using R/qtl version 1.384. Single QTL scans were performed by computing at 1 cM intervals across the entire genome using the cross direction as a covariate. Threshold LOD scores for significant (p<0.05) and suggestive (p<0.63) linkages were determined by performing 1000 permutations. After single QTL scans were performed, two QTL scans were performed. Lastly, the combined effects of covariates and all QTL including those that were significant and suggestive were assessed using multiple QTL models.
Table 2: Significant and suggestive QTL identified in genome-wide scans of F2 males:
QTL Cholq1 (cholesterol QTL 1) mapped to Chromosome 1, peaking at 80.5 cM nearest marker D1Mit356 with a LOD score of 32.7. The 95% confidence interval spanned from 77.5 to 85.5 cM. The DDD allele was associated with higher plasmid lipids and was inherited in an additive mode of inheritance.
QTL Cholq17 (cholesterol QTL 17) (syn Choldq6) mapped to Chromosome 17, peaking at 35.1 cM nearest marker D17Mit152 with a LOD score of 3.4. The 95% confidence interval spanned from 17.1 to 51.1 cM. The B6 allele was associated with higher plasmid lipids and was inherited in an additive mode of inheritance.
Curator Note: Choldq6 was oiginally mapped in J:253639 using crosses between congenic mice DDD.Cg-Ay and B6.Cg-Ay. Since the original mapping population differs from the mapping poulation used here we have asigned the QTL mapped to Chr 17 in the cuurent study with unique nomenclature.
QTL Cholq7 (cholesterol QTL 7) mapped to Chromosome 19, peaking at 8.0 cM nearest marker D19Mit68 with a LOD score of 5.0. The 95% confidence interval spanned from 3.0 to 19.0 cM. The DDD allele was associated with higher plasmid lipids and was inherited in a recessive mode of inheritance.
Three suggestive QTL for plasma CHO were identified mapping to Chromosome 3 (23.8 cM, LOD=2.3), Chromosome 5 (59.8 cM, LOD=2.9, and Chromosome 9 (37.0 cM, LOD=2.2).
For triglyceride concentrations (TG) one significant QTL, Trigq6 (triglyceride QTL 6) mapped to Chromosome 1, peaking at 84.5 cM nearest marker D1Mit356 with a LOD score of 12.5. The 95% confidence interval spanned from 77.5 to 93.5 cM. The DDD allele was associated with higher plasmid lipids and was inherited in a dominant mode of inheritance.
Curator Note: We have assigned QTL nomenclature to the triglyceride QTL mapped in the current study that is in keeping with the nomenclature sequence of existing trigylceride QTL.
Four suggestive QTL for TG were identified mapping to Chromosome 5 (50.8 cM, LOD=2.6), Chromosome 12 (47.0 cM, LOD=2.9), Chromosome 14 (60.3 cM, LOD=2.3) and Chromosome 15 (53.9 cM, LOD=2.4 cM).
Pairwise analysis failed to identify any significant interactions between both traits. Multiple regression analysis indicated that the detected QTL explain 56.7 and 27.5% of the variation in plasma CHO and TG concentrations, respectively.
Next, single QTL scans were performed that included body weight and cross direction as added covariates. Significant QTL were detected on Chomosomes 5 and 12.
QTL Cholq4 (cholesterol QTL 4) mapped to Chromosome 5 peaking at 59.8 cM nearest marker D5Mit239 with a LOD score of 3.7. The 95% confidence interval spanned from 17.8 to 75.8 cM. The DDD allele was associated with higher plasma lipids.
QTL Trigq7 (triglyceride QTL 7) mapped to Chromosome 12 peaking at 47.0 cM nearest marker D12Mit259 with a LOD score of 3.7. The 95% confidence interval spanned from 13.0 to 62.0 cM. The B6 allele was associated with higher triglyceride levels.
In two-QTL scans there were no significant pairwise interactions for both traits.
Next, cross direction was included as an interactive covariate and a significant QTL was identified on Chromosome 17 in linkage with plasma CHO.
QTL Cholq8 (cholesterol QTL 8) mapped to Chromosome 17 peaking at 60.7 cM with a LOD score of 2.6. (the threshold LOD score for QTL x covariate interaction was 2.4). The 95 % confidence interval spanned from 50.1 to 60.7 cM and slightly overlapped that of Cholq17.
Because of the prominent phenotypic effect exerted by the Chromosome 1 QTL on both traits, composite interval mapping was performed including the nearest marker, D1Mit356, for Cholq1/Trigq6, and cross direction as added covariates.
Six significant QTL were identified for CHO:
QTL Cholq9 (cholesterol QTL 9) was mapped to Chromosome 3 peaking at 19.8 cM nearest marker D3Mit25 with a LOD score of 4.2. The 95% confidence interval spanned from 10.8 to 35.8 cM. The B6 allele was associated with higher plasma lipids and was inherited in an additive mode of inheritance.
QTL Cholq10 (cholesterol QTL 10) was mapped to Chromosome 4 peaking at 23.1 cM nearest marker D4Mit286 with a LOD score of 3.6. The 95% confidence interval spanned from 9.1 to 37.1 cM. The B6 allele was associated with higher plasma lipids and was inherited in an additive mode of inheritance.
QTL Cholq11 (cholesterol QTL 11) was mapped to Chromosome 9 peaking at 37.0 cM nearest marker D9Mit207 with a LOD score of 4.7. The 95% confidence interval spanned from 12.0 to 59.0 cM. The DDD allele was associated with higher plasma lipids and was inherited in an additive mode of inheritance.
QTL Cholq4 (Chr 5: 59.8 cM, LOD=3.7), Cholq17 (Chr 17: 37.1 cM, LOD=5.0) and Choldq7 (Chr 19:5.0 cM, LOD=3.7) were also identified.
Next data was combined from the current study on males with previously analyzed data on (B6 x DDD.Cg-Ay F2) females (n=598) [J:253639] and QTL mapping analysis was performed. When sex was added as a covariate an additional significant QTL was identified, Cholq12 (cholesterol QTL 12) mapped to Chromosome 11 peaking at 61.4 cM nearest marker D11Mit124 with a a LOD score of 3.5.
When including sex as an interactive covariate, searching for QTL x sex interactions for CHO, one additional significant QTL, Cholq13 (cholesterol QTL 13) mapped to Chromosome 19 peaking at 10.0 cM with a LOD score of 3.6. The 95% confidence interval spanned from 3.0 to 26.0 cM. The DDD allele tended to be associated with decreased CHO concentrations in females, whereas the DDD allele significantly associated with increased CHO concentrations in males.
Curator Note: Text notes that Cholq13 overlapped with the previously identified QTL Cholq7, however the combined mapping population data differs from the mapping population used to map Cholq7 so we have assigned unique nomenclature to the QTL identified in the meta data.
Five potential candidate genes map within the 95% confidence interval of Cholq7 and Cholq13: Lrp5, Pitpnm1, Naa40, Mark2 and Bscl2.
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