Characterization of Cq3, a Quantitative Trait Locus That Controls Plasma Cholesterol and Phospholipid Levels in Mice

FULL PAPER Physiology Characterization of Cq3, a Quantitative Trait Locus that Controls Plasma Cholesterol and Phospholipid Levels in Mice Jun-ichi SUTO1) 1)Department of Molecular Biology and Immunology, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305–8634, Japan (Received 16 May 2005/Accepted 1 December 2005) y ABSTRACT. Cq3 was identified in C57BL/6J (B6) × KK-A F2 mice as a quantitative trait locus (QTL) that controls plasma cholesterol and phospholipid levels, and normolipidemic B6 allele was associated with increased lipids. Cq3 was statistically significant in F2-a/a, y y but not in F2-A /a; probably because the Cq3 effect was obscured by introduction of the A allele, which in itself has a strong hyperlip- idemic effect. Because the peak LOD score for Cq3 was identified near D3Mit102 (49.7 cM) on chromosome 3, linkage analyses with microsatellite markers located at 49.7 cM were performed in KK × RR F2, B6 × RR F2, and KK × CF1 F2. However, even a suggestive QTL was not identified in any of the three F2. By testing all pairs of marker loci, I found a significant interaction between Cq3 and the KK KK B6 B6 Apoa2 locus, and F2 mice with the Apoa2 /Apoa2 ; D3Mit102 /D3Mit102 genotype had significantly higher cholesterol levels than did F2 mice with other genotypes. The results showed that the ‘round-robin’ strategy was not always applicable to the search for QTL genes; probably because specific gene-to-gene interaction limited the validity of the strategy to the utmost extent. KEY WORDS: Apoa1bp, Apoa2, cholesterol, cholesterol QTL (Cq), phospholipid. J. Vet. Med. Sci. 68(4): 303–309, 2006 Plasma lipid levels are quantitative traits that are con- between KK and RR strains [17]. In addition, Cq3 had an trolled by multiple genes under the influence of environ- effect on plasma T-CHO and phospholipid (PL) levels in y y mental stimuli. To dissect genetic basis, quantitative trait C57BL/6J × KK-A F2-a/a (n=93), but not in F2-A /a (n=97) locus (QTL) analyses have been carried out extensively dur- [19]. The aims in study are to explain the reasons why Cq3 ing the last decade, particularly on mice [19, 23]. Col- was identified only in F2-a/a, and why the ‘round-robin’ leagues and I previously carried out QTL analyses on strategy was not applicable to the case of Cq3. plasma cholesterol levels in three sets of F2 mice that were formed in a ‘round-robin’ manner from C57BL/6J, KK (- MATERIALS AND METHODS Ay), and RR strains [19–21]. We identified six QTLs on chromosomes 1 (Cq1, Cq2, and Cq6), 3 (Cq3), and 9 (Cq4 Mice and genetic cross: In the present study, results from and Cq5). These QTLs have plausible candidate genes, that studies on the following four sets of F2 mice were analyzed. y y is, sterol O-acyltransferase 1 (Soat1) for Cq1, apolipopro- [B6 × KK-A F2] Inbred mouse strains KK-A (yellow hair, tein A-II (Apoa2) for Cq2 and Cq6, apolipoprotein A-I bind- AyaBBCc) and B6 (black hair, aaBBCC) were purchased ing protein (Apoa1bp) for Cq3, and apolipoprotein A-I from Clea Japan (Tokyo), and F2 mice (see below) were pro- (Apoa1) and apolipoprotein A-IV (Apoa4) for Cq4 and Cq5. duced as previously described [19]. Briefly, 190 F2 mice y Of these QTLs, Cq2 (identified in C57BL/6J × KK-A F2) were chosen from >200 F2 to minimize age differences colocalized with Cq6 (identified in C57BL/6J × RR F2), and between individual mice in the experiments. The 190 F2 y Cq4 (identified in C57BL/6J × KK-A F2) colocalized with comprised 93 animals with the a/a genotype at the agouti Cq5 (identified in KK × RR F2). I concluded that Apoa2 is locus (black hair, hereafter called F2-a/a, 49 males and 44 the gene causative of Cq2 and Cq6, because the KK and the females) and 97 animals with the Ay/a genotype (yellow b y RR have same Apoa2 allele that is considered to increase hair, hereafter F2-A /a, 49 males and 48 females). F2 mice plasma cholesterol level [22]. In the similar way, I con- were sacrificed at the ages of 26 to 29 weeks. cluded that Apoa4 is the gene causative of Cq4 and Cq5, [KK × RR F2] The KK strain was purchased from Clea because the only KK has unique nucleotide substitution that Japan, and the RR strain has been bred and maintained at the is considered to increase plasma cholesterol level [22]. National Institute of Agrobiological Sciences (NIAS, Thus, the ‘round-robin’ strategy is a valid method for Tsukuba, Japan). One hundred forty-five F2 females were searching QTL genes. produced and analyzed as previously described [20]. F2 In contrast to Cq2/Cq6 and Cq4/Cq5, Cq3 was identified mice were sacrificed at the age of 170 days. y only in C57BL/6J × KK-A F2 (more correctly in F2-a/a, not [B6 × RR F2] One hundred eighty-seven F2 females were y in F2-A /a, see below), and was not in KK × RR F2 and produced and analyzed as previously described [21]. F2 C57BL/6J × RR F2 [19–21]. This was at variance with the mice were sacrificed at the age of 130 ± 5 days. expectation that the QTL identified in the cross between [KK × CF1 F2] The inbred CF1 strain has been bred and y C57BL/6J and KK-A strains will be identified either in the maintained at the NIAS. Eighty-nine F2 females were pro- cross between C57BL/6J and RR strains or in the cross duced and analyzed in this study. F2 mice were sacrificed at 304 J-I. SUTO the age of 20 weeks. Stat123/Win software (Shinko Trading Co. Ltd. Publication Hereafter, I define the B6 strain as having B alleles, the Department, Tokyo) and SPSS software (SPSS for Win- CF1 strain as having C alleles, the KK strain as having K dows Release 7.5.1 J, SPSS Inc., Chicago, IL). alleles, and the RR strain as having R alleles, throughout the genome. RESULTS All mice were maintained in our laboratory throughout the experimental period under specific pathogen-free condi- For several phenotypic traits, scatter plots of the T-CHO tions, with a regular light cycle of 12 hr light/12 hr dark, and level and body weight (at the time of blood collection) in y with the temperature controlled at 22 ± 3°C. They had free parental KK-A males, KK males, B6 females, F1, and F2 are access to the diet [rodent pellet chow, CE-2 (342.2 kcal/100 shown in Figs. 1A and 1B. In general, T-CHO levels ran g, containing 4.4% crude fat), Clea Japan] and tap water. parallel with those of body weight; that is, heavier mouse y Plasma lipid measurement: In B6 × KK-A F2, plasma strains tended to have a higher T-CHO. T-CHO and body concentrations of triglyceride (TG), T-CHO, HDL-CHO, weight in KK-Ay males were higher than those in KK males. LDL+VLDL-cholesterol (LDL-CHO), free-cholesterol (F- Thus, introduction of the Ay allele simply increased the CHO), non-esterified fatty acid (NEFA), and PL were deter- amount of lipids in circulation. Similar trends were also y mined. In KK × RR F2, B6 × RR F2, and KK × CF1 F2, TG found in the comparison between F1-a/a and F1-A /a, and y and T-CHO were determined. At autopsy, after 24 hr fast- between F2-a/a and F2-A /a. Essentially, all the other lipid ing (KK × CF1 F2 was fasted for 4 hr), blood was drawn classes showed distribution patterns similar to that of T- from the heart into microtubes with heparin or EDTA as CHO [19]. anticoagulant. To separate the plasma from whole blood, Cq3 was further mapped in 93 F2-a/a mice with addi- the tubes were centrifuged at 7,000 rpm for 5 min at 4°C tional microsatellite markers to refine the 95% CI. Signifi- immediately after drawing. All samples were placed at cant QTL was obtained only for F-CHO (LOD score 4.5), –80°C until use. Plasma lipid was determined enzymati- and suggestive QTLs were identified for T-CHO (LOD cally with clinical chemical kits (Test Wako, Wako Pure score 4.2), HDL-CHO (3.8), LDL-CHO (3.0), and PL (3.6). Chemical Industries, Osaka, Japan). A peak LOD score of Cq3 for F-CHO was located at 49.7 QTL analysis: QTL analysis was carried out with the cM on chromosome 3, placing the 95% CI between 47 and Mapmaker/EXP version 3.0b and the Mapmaker/QTL ver- 54 cM (Figs. 2A). The LOD score rose abruptly from below sion 1.1b computer programs [7]. The chromosomal region a suggestive level to a significant level between D3Mit29 with a logarithm of odds (LOD) score of more than 4.3 (45.2 cM) and D3Mit102 (49.7 cM). The B allele was asso- (threshold of statistical significance at α=0.05) was recog- ciated with increased trait values in T-CHO (Table 1), and nized as indicating significant linkage, and the region with a also in F-CHO, HDL-CHO, LDL-CHO, and PL (data not LOD score between 2.8 and 4.3 was recognized as indicat- shown). Thus, Cq3 had an effect on HDL-CHO, LDL- ing suggestive linkage [8]. The α level for suggestive link- CHO, and PL, but not on TG (Table 1) and NEFA. In con- age implies the expectation that there will be one false trast, although no significant Cq3 effects were observed in y positive in a genome-wide search.

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