Experiment
In the current study loci controlling plasma lipid concentrations were identified by performing a quantitative trait locus analysis on genotypes from 233 mice( 127 males, 106 females) from an F2 cross between KK/HlJ (KK) and I/LnJ (I/L), two strains known to differ in their high-density lipoprotein (HDL) cholesterol levels.
(KKHlJ x I/LnJ) F2 mice were weaned at 21 d of age onto standard mouse chow. Blood from the tail vein was collected from 12 to 14-wk-old mice. Plasma HDL and total cholesterol were measured using a VT350 Automatic Chemical Analyzer. Non-HDL cholesterol was calculated as the difference between the total cholesterol and HDL cholesterol. DNA was extracted from liver using a Maxwell 16 System. A custom single-nucleotide polymorphism (SNP) array was designed with 137 SNPs evenly spaced across the genome.
QTL mapping was performed using R/QTL (version 1.25215; http:// www.rqtl.org). Coordinates for markers were based on the genetic map previously described (Cox et al. 2009). Main effect QTLs were computed over 1-cM increments across the entire genome using composite interval mapping. To aid in identifying QTL that might be sex-specific, additional analyses were performed in each sex separately. All significant and suggestive QTL were combined in a multi-locus model with the percentage of the variation explained by each QTL computed with regression analysis. Significant (P< 0.05) and suggestive (P< 0.63) logarithm of the odds ratio (LOD) threshold levels were chosen calculated using 1,000 permutations for the autosomes and 16,803 permutations for Chr X.
Analysis of means and standard deviations for HDL and non-HDL cholesterol levels showed that there were no statistically significant differences between sexes for HDL cholesterol values (Table 1). There was a statistically significant difference in non-HDL values between males and females.
For HDL levels, two significant and one suggestive QTL were mapped in both male and female mice, Table 2:
QTL Hdlq107, HDL QTL 107, mapped to Chromosome 1, peaking at 74.7 cM nearest marker rs13476229 with a LOD score of 8.9. The 95% confidence interval spanned between 70.6 and 81.6 cM.
[Curator Note: Authors refer to this QTL as previously mapped QTL Cq1, however the cross used here differs from that used (( C57BL/6J x KK-Ay)F2 ) to map Cq1 in J:54322, Suto, 1999. We regard the current study as a separate map study and have assigned the QTL identified here with unique nomenclature.]
QTL Hdlq108, HDL QTL 108, mapped to Chromosome X, peaking at 38.4 cM nearest marker rs13483831 with a LOD score of 9.3. The 95% confidence interval spanned between 33.4 and 40.4 cM.
[Curator Note: Authors refer to this QTL as Hdlq84, however that QTL symbol was previously published as a Chromosome 4 locus. We regard the current study as a separate map study and have assigned the QTL identified here with unique nomenclature.]
A suggestive QTL mapped to Chromosome 6 peaking at 24.8cM nearest marker rs6355719 with a LOD score of 2.9
For all three HDL QTL, mice homozygous for the KK allele showed greater HDL levels, in an additive manner for Hdlq107 (Cq1) and the suggestive Chr 6 QTL and in a dominant manner for Hdlq108 ( Hdlq84), compared with mice homozygous for the I/L allele.
HDL QTL on Chr 1 near 74.7 cM have previously been identified in a number of other mouse crosses, including the one involving KK mice (Suto, 1999) and was determined to be due to a specific polymorphism in the Apoa2 gene (Wang et al. 2004). The result here at Hdlq107 was expected as KK mice harbor the Apoa2b allele whereas I/L mice carry Apoa2a.
For non-HDL levels, one significant and two suggestive QTL were mapped, Table 2:
QTL Nhdlq17, non-HDL QTL 17, mapped to Chromosome 3, peaking at 32.4 cM nearest marker rs6198234 with a LOD score of 4.5. The 95% confidence interval spanned between 29.2 and 35.2 cM. Mice homozygous for the KK and I/L alleles did not differ in non-HDL but underdominance was suggested at this QTL because heterozygous mice had lower non-HDL levels than did KK and I/L homozygous mice.
[Curator Note: Authors refer to this QTL as Nhdlq14, however Nhdlq14 QTL was originally mapped in J:93243 to Chromosome 8 using (C57BL/6J x 129S1/SvImJ)F2 mice. We consider the current study a separate mapping experiment and have assigned the QTL identified here with unique nomenclature.]
Suggestive QTL mapped to Chromosome 1 peaking at 67.7 cM nearest marker rs1685344 with a LOD score of 3.2 and to Chromosome X peaking at 38.3 cM nearest marker rs13483831 with a LOD score of 3.3.
Analyses were performed in male and female populations separately to detect QTL controlling HDL and non-HDL levels that might be sex-specific.
For HDL, one suggestive male-specific QTL on Chr 2, 36.2 cM and two suggestive female-specific QTL on Chr 3, 19.2 cM and Chr 4, 75.1 cM were localized.
For the Chr 2 QTL, I/L homozygous males showed greater HDL levels in an additive manner compared with KK homozygous males; females did not exhibit this pattern. On Chr 3, I/L homozygous females had greater HDL levels, in an additive manner, compared with KK homozygous females, whereas on Chr 4 KK homozygous females displayed greater HDL levels, in an additive manner, compared with I/L homozygous females.
For non-HDL, three suggestive female-specific QTL on Chr 5, 42.8cM, Chr 7 64.0cM, and Chr 14, 41.9cM were detected.
KK homozygous females showed greater non-HDL levels in a dominant manner compared with I/L homozygous females for the Chr 5 QTL and in an additive manner for the Chr 14 QTL. For the Chr 7 QTL, I/L homozygous females showed greater non-HDL compared to KK females in an additive manner. No significant or suggestive male-specific non-HDL QTL were detected.
In the combined analysis evaluation of HDL and non-HDL values based on sex show that the Chr X QTL affects males and females differently (Figure 2, A and B). When HDL and non-HDL values were examined based on the genotype for the nearest marker to Hdlq108 (rs13483831), a significant difference in HDL values was observed based on genotype for males P< 0.0001) with no difference seen for females. Conversely, a significant difference in non-HDL values was observed based on genotype for females (P< 0.0001) with no effect seen for males.
The Chr X QTL also was analyzed for its affect on plasma lipid values in the context of Hdlq107 (nearest marker = rs13476229) by comparing the average lipid values for mice when considering only the genotype at rs13476229 vs. mice when considering both the genotype at Chr 1: rs13476229 and Chr X: rs1348483831. For males, mice hemizygous for the KK allele at Chr X: rs13483831 had greater HDL levels than mice hemizygous for the I/L allele compared with only considering the genotype at Chr 1: rs13476229 (Figure 3A). For non-HDL, there was no statistically significant difference in male mice based on genotypes at Chr 1: rs13476229 and Chr X: rs1348483831.
For females, there was a statistically significant difference in HDL levels for mice that were homozygous for the KK allele at Chr X: rs1348381 vs. those homozygous for the I/L allele, but only in those mice homozygous for I/L at Chr 1: rs13476229 (P< 0.05; Figure 3C). Females possessing the KK allele at Chr X: rs13483831 had higher non-HDL values, whereas those with the I/L allele had lower non- HDL values compared to the values when only considering the genotype at Chr 1: rs13476229 (Figure 3D).
The genomic region (5380 Mbp, 95% confidence interval) containing Hdlq108 was examined for genes involved in cholesterol metabolism. Of the 195 genes located in the interval, there was one gene involved in the cholesterol synthesis pathway, sterol-4-alpha-carboxylate 3- dehydrogenase (Nsdhl).
Because of the known relationship between NSDHL and lipid metabolism, the coding region of Nsdhl was sequenced for both I/L and KK strains. A unique, nonsynonymous coding polymorphism was found in I/L mice that alters a positively charged lysine to a negatively charged glutamic acid. The unique polymorphism is likely responsible for Hdlq108, however further in vivo experiments need to be performed to validate the functionality of the polymorphism.
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