Loci controlling plasma non-hdl and HDL cholesterol levels in a C57BL/6J CASA/Rk intercross

Transcription

1 Loci controlling plsm non-hdl nd HDL cholesterol levels in C57BL/6J CASA/Rk intercross Ephrim Sehyek, 1 Elizbeth M. Duncn, Hnnh J. Yu, Lynn Petukhov, nd Jn L. Breslow Lbortory of Biochemicl Genetics nd Metbolism, The Rockefeller University, 1230 York Avenue, New York, NY Abstrct Plsm non-hdl nd HDL cholesterol levels re predictors of crdiovsculr diseses. We crried out genetic cross between two lbortory inbred mouse strins, C57BL/6J nd CASA/Rk, to detect loci tht control the plsm levels of non-hdl nd HDL cholesterol. With regrd to non-hdl cholesterol, chow-fed CASA/Rk mles nd femles hd 87% nd 25% higher levels, respectively, thn did C57BL/6Js. The levels of non-hdl cholesterol in F1s were similr to C57BL/ 6J. There ws no strin difference in HDL cholesterol levels. An intercross between F1s ws performed, nd plsm non-hdl nd HDL cholesterol ws mesured in 185 mle nd 184 femle mice. In both mle nd femle F2 mice, plsm non-hdl nd HDL cholesterol levels were unimodlly distributed; however, in both cses the vlues for femles were significntly lower thn for mles. Therefore, linkge nlysis ws performed with sex s covrite. Significnt linkge for non-hdl cholesterol ws found on chromosome 6 t 49 cm (LOD 5.17), chromosome 4 t 55 cm (LOD 4.22), nd chromosome 8 t 7 cm (LOD 3.68). Significnt linkge for HDL cholesterol ws found on chromosome 9 t 14 cm (LOD 7.52) nd chromosome 8 t 76 cm (LOD 4.69). A significnt episttic interction involving loci on chromosomes 2 nd 5 ws lso observed for non- HDL cholesterol. In summry, linkge nlysis in these cross-identified novel loci confirmed previously identified loci in control of plsm non-hdl nd HDL cholesterol nd disclosed novel interction in controlling non-hdl cholesterol levels in the mouse. Sehyek, E., E. M. Duncn, H. J. Yu, L. Petukhov, nd J. L. Breslow. Loci controlling plsm non-hdl nd HDL cholesterol levels in C57BL/6J CASA/Rk intercross. J. Lipid Res : chromosome logrithm of odds crdio- Supplementry key words vsculr disese Plsm levels of non-hdl nd HDL cholesterol hve been shown to modulte the risk for crdiovsculr diseses. Incresed plsm levels of non-hdl cholesterol, especilly in the form of LDL cholesterol, nd decresed levels of HDL cholesterol re ssocited with incresed risk for these diseses. Multiple studies hve shown lrge individul-toindividul vrition in plsm non-hdl nd HDL cholesterol levels (1). The cuses of this vrition nd the regultion of non-hdl nd HDL cholesterol levels re only prtilly understood. Current understnding supports complex interction between environmentl nd genetic determinnts. Yet, wheres much is known bout the nture nd effect of environmentl fctors including cigrette smoking, totl dietry ft intke, types of dietry ftty cids, physicl ctivity, nd lcohol consumption, reltively little is known bout the genetic bsis of this vrition. Dt from twin nd fmily studies hve shown tht 50% of the interindividul vribility in LDL cholesterol nd HDL cholesterol cn be scribed to genetic determinnts; however, only some of the genes involved hve so fr been identified (2 6). Studies in pedigrees tht segregte muttions in criticl genes lrgely expnd the understnding of the physiologicl nd metbolic spects of lipoprotein crriers of non-hdl nd HDL cholesterol. Yet, lthough in some popultions vrints of these genes re sufficiently common to hve n impct on non-hdl nd HDL cholesterol levels, it is likely tht other genes re involved. Previous mpping studies in the mouse hve identified loci in linkge with plsm non-hdl nd HDL cholesterol levels. For exmple, Ko et l. used humn polipoprotein B (pob) trnsgenics in C57BL/6 129 cross to identify loci on chromosome 6 nd chromosome 4 in linkge with plsm pob levels, signture polipoprotein for plsm non-hdl cholesterol lipoproteins (7). Mehrbin et l. used C57BL/6J CAST/Ei cross to mp loci on chromosomes 2, 3, 5, 8, 9, 14, 16, 17, nd 18 in linkge with plsm HDL cholesterol (8). Here we describe two mouse strins, C57BL/ 6J nd CASA/Rk, tht, when fed chow diet, disply different plsm levels of non-hdl cholesterol. To exmine the genetic bsis of this difference, n intercross ws performed Mnuscript received 2 Mrch 2003 nd in revised form 27 My Published, JLR Ppers in Press, June 16, DOI /jlr.M JLR200 Abbrevition: LOD, logrithm of odds. 1 To whom correspondence should be ddressed. e-mil: sehyee@rockefeller.edu Copyright 2003 by the Americn Society for Biochemistry nd Moleculr Biology, Inc Journl of Lipid Reserch Volume 44, 2003 This rticle is vilble online t

2 nd linkge for non-hdl cholesterol s well s HDL cholesterol ssessed in the F2 progeny. We identified three loci, on chromosomes 6, 4, nd 8, in linkge with plsm non- HDL cholesterol, nd two loci on chromosomes 9 nd 8 in linkge with HDL cholesterol. Moreover, nlysis of loci interctions identified significnt episttic interction tht ffects plsm non-hdl cholesterol levels. MATERIALS AND METHODS Animls, diet, nd lipoprotein seprtion The inbred mouse strins C57BL/6J nd CASA/Rk were purchsed from The Jckson Lbortories (Br Hrbor, ME). CASA/ Rk mles were mted with C57BL/6J femles to generte F1 nimls. F1 mles were intercrossed with F1 femles to generte 369 F2 nimls (185 mles nd 184 femles). All nimls were bred nd housed in single humidity- nd temperture-controlled room with 12 h drk-light cycle (6 AM 6 PM light-drk cycle) t the Lbortory Animl Reserch Center t Rockefeller University nd fed with single lot of Picolb Rodent Chow 20 (ctlog #5053) pellet contining 0.02% w/w cholesterol. At the ge of 11 weeks, food ws removed from the cges t 10 AM nd the nimls were llowed ccess to wter. At 3 PM, the mice were nesthetized with ketmine/xylzine, til tipped for DNA extrction, nd blood smples collected by hert puncture into EDTA-contining tubes. Plsm ws immeditely seprted, plsm density ws djusted to d gm/ml, nd non-hdl nd HDL frctions (d g/ml nd d g/ml, respectively) were isolted fter overnight spinning t 40,000 rpm (Beckmn L8-55M ultrcentrifuge, rotor type 42.2 Ti) t 4 C. The non-hdl nd HDL frctions were seprted nd kept t 80 C for nlysis of cholesterol concentrtion s described below. All experiments were pproved by the Institutionl Animl Cre nd Reserch Advisory Committee. Plsm totl, non-hdl, nd HDL cholesterol mesurements Totl plsm cholesterol, non-hdl, nd HDL cholesterol levels were determined enzymticlly using Sigm kit. It is of note tht plsm totl cholesterol, non-hdl, nd HDL cholesterol levels in C57BL/6J, CASA/Rk, nd F1 mles nd femles were mesured in one ssy, wheres the mesurements in F2 mles nd F2 femles were determined in nother ssy. The profile of plsm lipoprotein cholesterol ws determined by on-line postcolumn nlysis of Superose 6 gel-filtrtion s described previously (9). Genotyping Til tips from prentls, F1, nd F2 mice were digested with proteinse K, nd DNA ws precipitted with ethnol. Fluorescently lbeled primers corresponding to 350 mrkers shown to be polymorphic between C57BL/6Jnd CAST/Ei by Ikoubov et l. (10) were tested to see if they were lso polymorphic between C57BL/6J nd CASA/Rk. This resulted in the identifiction of 255 mrkers tht were used for genotyping in the current cross. The verge spcing between these mrkers ws 5.9 cm. Mrkers were subjected to PCR mplifiction using fluorescently lbeled primers, nd PCR products were nlyzed by cpillry electrophoresis using the ABI 3700 DNA sequencer. All PCR rections nd electrophoresis were utomted using the Tecn, Genesis RST 100, nd Robbins Scientific Hydr 384 robots nd crried out by the Strr Center Genotyping Core Fcility t the Rockefeller University. Allele scores were nlyzed using the ABI Genotyper 3.6 NT softwre. The mrker positions in cm correspond to mpping dt found in the Mouse Genome Informtics Dtbse t Sttisticl nlyses Differences in plsm non-hdl, HDL, nd totl cholesterol levels between prentls nd F1s nd comprisons of plsm non-hdl nd HDL cholesterol levels for F2s with the vrious combintions of genotypes t D6Mit63, D4Mit46, D8Mit171, D9Mit325, nd D8Mit91 were nlyzed using one-wy ANOVA with Tukey s posttest. Differences in plsm non-hdl nd HDL cholesterol between F2 mles nd femles were nlyzed using the unpired Student t-test. Linkge, intervl mpping (using the mximum likelihood lgorithm), nd loci interctions (using the Hley-Knott regression) were nlyzed with the R/qtl softwre pckge, Version , with sex s covrite. R/qtl ws lso used to permute the ctul dt sets for F2s to determine the significnt LODs t the 95% genome-wide threshold level. In ddition, R/qtl ws used to identify locus-locus interctions by computing joint LOD score. This joint LOD score represents composite of the proportion of the trit vrince explined by ech locus, which together correspond to the dditive component of the interction nd the proportion explined through epistsis. Finlly, R/qtl ws used to permute the dt sets for F2s to determine the threshold LOD score for the joint nd epistsis LODs t which 95% significnce is chieved. This softwre pckge, developed by Krl Bromn nd Gry Churchill, is publicly vilble t The C57BL/6J nd CASA/Rk lleles t chromosome 6, 4, nd 8 for non-hdl cholesterol nd chromosomes 9 nd 8 for HDLcholesterol were ssessed seprtely in F2 mles nd F2 femles for dditivity nd dominnce using the Mp Mnger QTXb10 version 0.19 softwre. RESULTS To determine the plsm levels of non-hdl nd HDL cholesterol, we subjected the plsm of chow-fed mle nd femle C57BL/6J, CASA/Rk, nd F1s to density ultrcentrifugtion, nd mesured the concentrtions of non- HDL cholesterol (d g/ml) nd HDL cholesterol (d g/ml) in ech group. As shown in Tble 1, CASA/Rk mles nd femles displyed plsm non-hdl cholesterol levels tht were 87% nd 25% higher thn the corresponding vlues in C57BL/6J mles nd femles, respectively. Furthermore, the levels of non-hdl cholesterol in F1 mles nd femles were similr to those in C57BL/6J nimls, ruling out codominnt effect. As for HDL cholesterol, lthough femles tended to disply levels tht were lower thn the corresponding vlues in mles, no significnt differences were found between C57BL/6J, CASA/Rk, nd F1. For further chrcteriztion of cholesterol distribution mong different clsses of lipoproteins, we subjected the plsm of C57BL/6J, CASA/Rk, nd F1 mles nd femles to superose gel chromtogrphy. As shown in Fig. 1, when compred with C57BL/6J nd F1, CASA/Rk mles nd femles displyed incresed VLDL cholesterol, with no mjor differences in either LDL or HDL cholesterol levels. To exmine the inheritnce of plsm non-hdl nd HDL cholesterol levels, F1 nimls were intercrossed, nd the concentrtions of non-hdl nd HDL cholesterol were determined in 369 F2 mice (185 mles nd 184 femles). Sehyek et l. Genetics of lipoprotein cholesterol levels 1745

3 TABLE 1. Plsm non-hdl cholesterol, HDL cholesterol, nd totl plsm cholesterol levels in prentl nd F1 nimls Non-HDL Cholesterol HDL Cholesterol b Totl Cholesterol c mg/dl Mles C57BL/6J (n 5) CASA/Rk (n 4) F1 (n 5) Femles C57BL/6J (n 10) CASA/Rk (n 5) F1 (n 5) One-wy ANOVA: Overll mles: P ; C57BL/6J versus CASA/Rk, P 0.01; CASA/Rk versus F1, P Overll femles: P ; C57BL/6J versus CASA/Rk, P 0.05; CASA/Rk versus F1, P b Overll mles nd overll femles: NS. c Overll mles: NS; CASA/Rk versus F1, P Overll femles: NS. To control for possible gender effects, we exmined the concentrtions of non-hdl nd HDL cholesterol in F2 mles nd femles nd found tht, compred with F2 femles, F2 mles disply significntly higher non-hdl nd HDL-cholesterol levels (non-hdl cholesterol of mg/dl vs mg/dl; P , nd HDL cholesterol of mg/dl vs mg/dl; P , in mles nd femles, respectively). Figure 2 shows unimodl distribution of plsm non-hdl nd HDL cholesterol in F2 mles nd femles, with lower levels in femles. These findings suggest tht in F2s, plsm non-hdl nd HDL cholesterol levels re probbly controlled by more thn one gene. Moreover, the dt clerly show gender effect on plsm non-hdl nd HDL cholesterol levels, nd justify linkge nlysis with sex s covrite. To identify loci tht control plsm levels of non-hdl nd HDL cholesterol, whole genome scn ws done on the F2 mice. For non-hdl cholesterol, quntittive trit locus mpping using the R/qtl progrm with sex s covrite reveled significnt linkge on chromosomes 6, 4, nd 8. The intervl mps for these chromosomes re shown in Fig. 3. With regrd to chromosome 6, the nlysis reveled pek of linkge t 49 cm, with mximum LOD score of 5.17 tht fell between mrkers D6Mit37 t 46 cm nd D6Mit63 t 50 cm. With regrd to chromosome 4, the nlysis showed pek of linkge t 55 cm with mximum Fig. 1. Lipoproteins cholesterol profile in C57BL/6J, CASA/Rk, nd F1 mles nd femles. Animls fed with chow diet were fsted, plsm smples isolted, the plsm of nimls in ech group were pooled, nd plsm lipoprotein cholesterol profiles were nlyzed s described in Mteril nd Methods (n 10 nimls per group). Fig. 2. Distribution of plsm non-hdl cholesterol nd HDL cholesterol in F2 mles nd femles. F2 nimls (185 mles nd 184 femles) were fsted, plsm smples collected, nd non-hdl cholesterol, nd HDL cholesterol mesured s described in Mterils nd Methods Journl of Lipid Reserch Volume 44, 2003

4 LOD score of 4.22 tht fell between mrkers D4Mit46 t 51 cm nd D4Mit204 t 62 cm. As for the locus on chromosome 8, the nlysis found pek of linkge t 7 cm with mximum LOD score of 3.68 tht fell between mrkers D8Mit58 t 1 cm nd D8Mit171 t 8 cm. Linkge nlysis of HDL cholesterol with sex s covrite reveled significnt linkge on chromosomes 9 nd 8. The intervl mps for these chromosomes re shown in Fig. 4. With regrd to chromosome 9, the nlysis reveled pek of linkge t 19 cm with mximum LOD score of 7.52 tht fell between mrkers D9Mit325 t 14 cm nd D9Mit140 t 25 cm. Finlly, for chromosome 8, the nlysis disclosed pek of linkge t 69 cm with mximum LOD score of 4.69 tht fell between mrkers D8Mit91 t 67 cm nd D8Mit56 t 73 cm. At the peks of linkge for plsm non-hdl cholesterol, the genotypic mens in F2s, represented by the closest mrkers, were clculted for F2 mles nd F2 femles nd re shown in Tble 2. For the loci on chromosome 6 nd chromosome 4, homozygotes for the C57BL/6J llele hd higher plsm non-hdl cholesterol levels thn did heterozygotes nd homozygotes for the CASA/Rk llele. At the chromosome 6 locus, the phenotypic effect of the CASA/Rk llele best fits dominnt mode of inheritnce, wheres t the chromosome 4 locus, the CASA/Rk llele best fits dominnt nd codominnt mode of inheritnce in mles nd femles, respectively. At the chromosome 8 locus, homozygotes for the CASA/Rk llele hd higher Fig. 3. Chromosomes 6, 4, nd 8 linkge mps of plsm non- HDL cholesterol in F2s. The mrker positions correspond to mpping dt found in the Mouse Genome Informtics Dt-bse. Fig. 4. Chromosomes 9 nd 8 linkge mps of plsm HDL cholesterol in F2s. The mrker positions correspond to mpping dt found in the Mouse Genome Informtics Dt-bse. Sehyek et l. Genetics of lipoprotein cholesterol levels 1747

5 TABLE 2. Genotypic effect on non-hdl cholesterol level in F2 mles nd femles Locus Locus Closest Mrker Plsm Non-HDL Cholesterol in Mles Plsm Non-HDL Cholesterol in Femles Chr Pek (cm) Nme cm BB BC CC BB BC CC 6 49 D6Mit b 55 D4Mit c 7 D8Mit One-wy ANOVA: Mles: overll NS; femles: overll P ; BB versus BC, P 0.001; BB versus CC, P b Mles: overll P ; BB versus BC, P 0.05; BB versus CC, P 0.05; femles: overll P ; BB versus CC, P c Mles: overll P ; BB versus CC, P 0.05; femles: overll NS. plsm non-hdl cholesterol levels thn did heterozygotes nd homozygotes for the C57BL/6J llele. At this locus, the phenotypic effect of the CASA/Rk llele best fit codominnt mode of inheritnce. In mles, the loci on chromosomes 6, 4, nd 8 pper to explin 5%, 4%, nd 5% of the vrince in non-hdl cholesterol levels, while in femles the sme loci pper to explin 12%, 10%, nd 3% of the vrince, respectively. At the peks of linkge for HDL cholesterol, the genotypic mens in F2s, represented by the closest mrkers, were clculted for F2 mles nd F2 femles nd shown in Tble 3. For the locus on chromosome 9, homozygotes for the CASA/Rk llele hd higher plsm HDL cholesterol levels thn did heterozygotes nd homozygotes for the C57BL/ 6J llele. At this locus, the phenotypic effect of the CASA/ Rk llele best fit codominnt mode of inheritnce. For the chromosome 8 locus, homozygotes for the C57BL/6J llele hd higher plsm HDL cholesterol levels thn did heterozygotes nd homozygotes for the CASA/Rk llele. At this locus, the phenotypic effect of the CASA/Rk llele best fit dominnt nd codominnt mode of inheritnce in mles nd femles, respectively. Finlly, in F2 mles, it ppers tht the locus on chromosomes 9 nd the locus on chromosome 8 ech explin 8% of the vrince in HDL cholesterol levels, while in F2 femles the sme loci pper to explin 10% nd 4% of the vrince, respectively. Locus interctions in determining plsm non-hdl nd HDL cholesterol levels were next determined. The R/qtl softwre progrm output includes the positions of the intercting loci, the joint LOD score of the interction, the new LOD score of locus 1 in the presence of locus 2, the new LOD score of locus 2 in the presence of locus 1, nd n LOD score of epistsis. For non-hdl cholesterol, significnce for joint LOD score ws chieved for the interctions of four pirs of loci. These were primrily of n dditive nture (dt not shown). In contrst, s shown in Tble 4, fifth interction ws observed between loci on chromosomes 2 nd 5, for which the joint LOD ws not significnt but the epistsis LOD of 7.47 ws significnt. For HDL cholesterol, significnce for joint LOD ws chieved for the interctions of 19 pirs of loci, which were primrily of n dditive nture (dt not shown). DISCUSSION In the present study, we describe two mouse inbred strins, C57BL/6J nd CASA/Rk, which on chow diet disply different plsm levels of non-hdl cholesterol. Utilizing these strins in genetic cross, we hve mpped loci in linkge with plsm non-hdl nd HDL cholesterol levels. This nlysis reveled three loci on chromosomes 6, 4, nd 8 in linkge with plsm levels of non-hdl cholesterol, nd two loci on chromosomes 9 nd 8 in linkge with plsm levels of HDL cholesterol. In ddition, for plsm non-hdl cholesterol levels, significnt episttic interction ws detected. Previous studies in the mouse hve identified loci in linkge with non-hdl cholesterol levels. Ko et l. performed n intercross between C57BL/6J 129 mouse strins with the F2s hemizygous for humn pob trnsgene (7). TABLE 3. Genotypic effect on HDL cholesterol level in F2 mles nd femles Locus Locus Closest Mrker Plsm HDL Cholesterol in Mles Plsm HDL Cholesterol in Femles Chr Pek (cm) Nme cm BB BC CC BB BC CC 9 19 D9Mit b 69 D8Mit One-wy ANOVA: Mles: overll P ; BB versus BC, P 0.05; BB versus CC, P 0.001; femles: overll P ; BB versus BC, P 0.01; BB versus CC, P b Mles: Overll P ; BB versus BC, P 0.01; BB versus CC, P 0.01; femles: overll P ; BB versus CC, P Journl of Lipid Reserch Volume 44, 2003

6 Locus 1 Chr: cm TABLE 4. Loci interction in determining plsm non-hdl cholesterol levels Locus 2 Chr:cM Joint Locus 1 LOD LOD Locus 2 LOD Epistsis LOD 2:26 5: Genome-wide 95% significnce for joint-lod nd epistsis-lod thresholds of 9.51 nd 7.45, respectively. Utilizing humn pob s surrogte mrker for plsm non-hdl lipoproteins, they found linkge on chromosome 6 tht peked t D6Mit55 (49.7 cm) nd on chromosome 4 tht peked t D4Mit27 (42.5 cm). Their pek on chromosome 6 coincides with our non-hdl cholesterol pek on chromosome 6 t 49 cm, while their pek on chromosome 4 is bout 13 cm proximl to our pek on chromosome 4 t 55 cm. It is of note tht in both studies, the 129 nd CASA/Rk lleles decrese the plsm levels of pob nd non-hdl cholesterol, respectively. In the Ko study, the genotypic effects of both the chromosome 6 nd chromosome lleles pper to ct in codominnt fshion. In contrst, in our study the chromosome 6 nd chromosome 4 CASA/Rk lleles pper to ct minly in dominnt mode (Tble 2). Possible interprettions of this difference re: i) the 129 nd CASA/Rk segregte different lleles t the loci on chromosome 6 nd chromosome 4, ii) the 129 nd CASA/Rk segregte the sme llele tht cts differently under the two genetic bckgrounds, nd iii) the linkge in the two crosses is to different genes in ech intervl. The first possibility is conceivble given tht 129 nd CASA/Rk represent different subspecies of Mus musculus, seprted by pproximtely one million yers of evolution (11). The non-hdl cholesterol locus we mpped to chromosome 8 hs not been reported in previous studies. Previous studies in the mouse hve identified loci in linkge with HDL cholesterol levels. Mehrbin et l. fed C57BL/6J CAST/Ei F2s with chow diet for 13 weeks nd mesured HDL cholesterol, then switched the nimls to n therogenic diet for n dditionl 5 weeks nd remesured HDL cholesterol (8). After chow feeding, they found linkge on chromosome 9 tht peked t D9Mit2 (17 cm), nd on chromosome 8 tht peked t D8Mit14 (67 cm). Their peks on chromosome 9 nd chromosome 8 coincide with our HDL cholesterol peks on chromosome 9 t 19 cm nd on chromosome 8 t 69 cm. In their cross, the chromosome 9 locus CAST/Ei llele incresed nd the chromosome 8 locus CAST/Ei llele decresed HDL cholesterol levels. Both loci ppered to ct in codominnt fshion. This is similr to wht we observed for the CASA/Rk lleles in our cross. This is quite plusible since the inbred strins CAST/Ei nd CASA/Rk were both derived from the sme pool of cstneus wild-type mice t the Jckson lbortories ( notes/456e.html) nd my hve fixed the sme lleles t the chromosome 9 nd chromosome 8 loci. It is noteworthy in our study tht prentl CASA/Rks hve higher non-hdl cholesterol levels thn prentl C57BL/6Js, yet QTL nlysis in the F2s reveled dominnt CASA/Rk lleles t both chromosome 6 nd chromosome 4 tht decrese non-hdl cholesterol levels (Tbles 1, 2). This type of finding hs been observed in other QTL nlyses (12), nd presumbly mens tht the CASA/Rk lleles t these loci re phenotypiclly silent in the context of the CASA/Rk genome, but decrese non-hdl cholesterol levels in the presence of one or more C57BL/6J lleles. This implies the presence of significnt gene interctions. The Ensembl mouse genome dtbse ws serched for known genes in the intervls of loci for non-hdl cholesterol on chromosome 6 (38 68 cm), chromosome 4 (31 81 cm), nd chromosome 8 (1 18 cm). The chromosome 6 intervl contins the peroxisome prolifertor ctivted receptor (ppr- ) gene t 50.5 cm, which encodes n importnt trnscriptionl fctor tht regultes glucose nd ftty cids metbolism. This intervl lso contins the pob mrna-editing protein (pobec1) gene t 59 cm, which encodes n importnt constituent of the complex tht posttrnscriptionlly edits pob-100 mrna into pob-48 mrna. The ppr- gene is very close to the chromosome 6 pek t 49 cm, wheres the pobec1 gene is 10 cm distl. The chromosome 4 intervl contins the leptin receptor (lep-r) gene t 47 cm, which encodes the receptor for leptin, hormone produced by dipose tissue tht regultes stiety nd energy metbolism; the crnitine o-plmitoyltrnsferse II (cptii) gene t 51 cm, which encodes mitochondril ftty cid trnsporter; the sterol crrier protein 2 (scp2) gene t 51 cm, which encodes protein tht trnsfers brnched chin ftty cids into the peroxisomes for -oxidtion; nd the cytidine deminse (cd) gene t 67 cm, which encodes n enzyme tht ctlyzes the posttrnscriptionl editing of pob-100 mrna into pob-48 mrna. The cptii nd scp2 genes re very close to the chromosome 4 pek t 55 cm, wheres the lep-r nd cd genes re 8 cm proximl nd 12 cm distl to the pek, respectively. The chromosome 8 intervl contins the insulin receptor substrte-2 (irs-2) gene t 6 cm, which encodes n importnt protein in the insulin signling pthwy. This gene is very close to the chromosome 8 pek t 7 cm. The Ensembl dtbse ws lso serched for cndidte genes in the intervls of loci for HDL cholesterol on chromosome 9 (4 29 cm) nd chromosome 8 (52 73 cm). The chromosome 9 intervl contins the poa-i, poc-iii, poa-iv, nd poa-v locus t 27 cm. The genes encoded t this locus re known to ply importnt physiologicl roles in plsm HDL cholesterol metbolism. ApoA-I is the mjor structurl protein of HDL, nd poc-iii nd poa-v hve been shown in both mouse nd humn studies to regulte triglyceride levels, which re often inversely correlted with HDL cholesterol levels. This locus is 8 cm distl to the chromosome 9 pek t 19 cm. The chromosome 8 intervl did not contin ny genes known to ply prominent role in lipoprotein or lipid metbolism. In summry, cross between two strins tht differ in plsm non-hdl cholesterol reveled loci on chromosomes 6, 4, nd 8 tht control the plsm levels of non-hdl cholesterol, nd loci on chromosomes 9 nd 8 tht control the plsm levels of HDL cholesterol. Moreover, we found significnt episttic interction tht controls the levels of plsm non-hdl cholesterol. It is hoped tht the discov- Sehyek et l. Genetics of lipoprotein cholesterol levels 1749

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