Hypercholesterolemia in ENU-induced mouse mutants

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1 Hypercholesterolemi in ENU-induced mouse mutnts Mnuel Mohr,* Mrtin Klempt,* Birgit Rthkolb,* Mrtin Hrbé de Angelis, Eckhrd Wolf,* nd Bernhrd Aigner 1, * Institute of Moleculr Animl Breeding nd Biotechnology,* Ludwig-Mximilins-University, Munich, Germny; nd Institute of Experimentl Genetics, GSF Reserch Center for Environment nd Helth, Neuherberg, Germny Abstrct Hypercholesterolemi is cused by multiple environmentl fctors nd genetic predispositions, nd plys n importnt role in the development nd pthogenesis of vrious humn diseses. In this study, we imed to estblish rndomly mutnt mouse lines showing hypercholesterolemi for their further use in the detection of novel custive lleles. In the Munich ENU Mouse Mutgenesis Project, clinicl chemistry blood nlysis ws performed on more thn 15,000 G1 mice nd 230 G3 pedigrees of chemiclly mutgenized mice to detect dominnt nd recessive muttions leding to n incresed plsm totl cholesterol level. Using inbred C3HeB/FeJ mice we identified more thn 100 nimls consistently showing hypercholesterolemi. Trnsmission of the ltered phenotype to the subsequent genertions led to the production of nine hypercholesterolemic lines. A single line showed further obvious devitions in the nlysis of dditionl clinicl chemistry blood prmeters. Thus, the lines produced will contribute to the serch for lleles tht selectively cuse primry hypercholesterolemi. Mohr, M., M. Klempt, B. Rthkolb, M. Hrbé de Angelis, E. Wolf, nd B. Aigner. Hypercholesterolemi in ENU-induced mouse mutnts. J. Lipid Res : Supplementry key words cholesterol ethylnitrosoure high density lipoproteins low density lipoproteins phenotype-driven screen Few pproprite lbortory niml models exist for reserch on multifctoril nd polygenic humn diseses, due to their complex nd/or species-specific phenotypes. Genetic engineering techniques llow the defined ltertion of the mouse genome; however, the resulting phenotype of the mutnt mice cnnot be predicted. A complementry phenotype-driven strtegy for the serch of the genes involved in the ppernce of defined phenotypic ltertion consists of the production of gret pool of rndomly mutnt mice nd the subsequent selection of relevnt lines ccording to the similrities in the pthology of niml model nd humn ptients (1). Mnuscript received 18 June 2004 nd in revised form 17 August Published, JLR Ppers in Press, September 1, DOI /jlr.M JLR200 ENU (N-ethyl-N-nitrosoure) hs been used in vrious mouse mutgenesis progrms to produce rndom muttions. Specific pthologic sttes hve been identified by pproprite routine procedures llowing the screening of lrge numbers of mice for brod spectrum of prmeters (2, 3). Forwrd genetics techniques result in the detection of the chromosoml mpping site nd the subsequent identifiction of the custive muttion in the estblished lines (4). Successful detection of the custive muttions in the phenotypiclly ltered mice hs been demonstrted in lredy exmined ENU-induced mutnt mouse lines (5). In the Munich ENU Mouse Mutgenesis Project, screening profile of clinicl chemistry blood prmeters ws estblished for the nlysis of offspring of chemiclly mutgenized mice in order to detect phenotypic vrints with defects of diverse orgn systems or chnges in metbolic pthwys. Breeding of the ffected mice nd screening of the offspring confirmed the trnsmission of the ltered phenotype to the subsequent genertions, thereby reveling muttion s cuse for the berrnt phenotype (6, 7). Mutnt lines from the Munich ENU project with the custive muttion lredy identified re successfully used in reserch (e.g., Refs. 8, 9). Hypercholesterolemi including incresed vlues of the norml proportion of low density lipoprotein cholesterol (LDL-C) nd high density lipoprotein cholesterol (HDL-C) cts s min fctor in the development of humn diseses such s crdiovsculr disese (10) nd Alzheimer s disese (11). Beneth subset of rre monogenic forms, in most cses hypercholesterolemi occurs s polygenic nd multifctoril disorder (12, 13). Humns show predominntly LDL-C nd re sensitive to diet-induced elevtions of LDL-C. In contrst, mice exhibit high mounts of HDL-C nd low LDL-C level (14). Mny trnsgenic Abbrevitions: C3H, C3HeB/FeJ [inbred mouse strin]; ENU, N-ethyl- N-nitrosoure; Gn, genertion number; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol. 1 To whom correspondence should be ddressed. e-mil: b.igner@gen.vetmed.uni-muenchen.de Copyright 2004 by the Americn Society for Biochemistry nd Moleculr Biology, Inc Journl of Lipid Reserch Volume 45, 2004 This rticle is vilble online t

2 mouse lines showing hypercholesterolemi hve been produced tht mimic the humn sitution (15 17). Here, we present the genertion nd the phenotypic description of nine mutnt lines derived from the phenotype-driven high-throughput hypercholesterolemi screen in the Munich ENU Mouse Mutgenesis Project for their subsequent use in the identifiction of novel lleles leding to incresed plsm totl cholesterol. MATERIALS AND METHODS Mutgenesis nd breeding of mice The experiments were conducted on the inbred C3HeB/FeJ (C3H) genetic bckground. C3H mice re described s highly therosclerosis-resistnt strin (Ref. 18 nd references therein). Ten-week-old mle mice (genertion G0) were injected intrperitonelly with ENU (3 90 mg/kg t weekly intervls). The screen for dominnt muttions ws performed on G1 nimls derived from the mting of the mutgenized G0 mles to wild-type C3H femles. Inheritnce of the observed bnorml phenotype in the inbred C3H genetic bckground ws tested on G2 mice derived from the mting of the ffected G1 mouse exhibiting the ltered phenotype nd wild-type mice. The screen for recessive muttions ws performed on G3 mice produced in two-step breeding scheme from G1 mice. G1 mles known not to hrbor dominnt muttions were mted to wild-type femles for the production of G2 nimls. Subsequently, six to eight G2 femles were bckcrossed to the G1 mle to produce the G3 mice of the pedigree. The nlysis of the inheritnce of n observed bnorml phenotype in G3 mice ws done on G5 mice. Therefore, the ffected G3 mouse presumbly hrboring homozygous recessive muttion ws mted to wildtype mouse for the production of the presumbly heterozygous mutnt G4 mice showing n inconspicuous phenotype. Subsequently, the G5 mice derived from the mting of G4 mice to ech other were tested for the bnorml phenotype. Alterntively, G5 mice derived from the bckcross of G4 mouse to the ffected G3 niml were exmined. Once the custive muttion is identified, the internl nmes of the estblished lines will be replced ccording to the officil nomenclture. Mouse husbndry ws done under continuously controlled specific-pthogen-free (SPF) hygiene stndrd ccording to the Federtion of Europen Lbortory Animl Science Associtions (FELASA) protocols ( recommendtions.htm). Stndrd rodent diet (Altromin, Lge, Germny) nd wter were provided d libitum. All niml experiments were conducted under the pprovl of the responsible niml welfre uthority. Clinicl chemistry nlysis Blood smples from 3-month-old G1 nd G3 mice fsted overnight were obtined by puncture of the retroorbitl sinus under ether nesthesi. Physiologic prmeter vlues were determined in mle nd femle C3H controls. Plsm from Li-heprintreted blood ws nlyzed using n Olympus AU400 utonlyzer (Olympus, Hmburg, Germny) nd the dpted regents (Olympus, Hmburg, Germny). Clibrtion nd qulity control were performed ccording to the mnufcturer s protocols. Totl cholesterol, HDL-C, nd LDL-C were exmined by enzymtic colorimetry tests using the regents OSR6116, OSR6187, nd OSR6183 (Olympus, Hmburg, Germny) with the liner mesurement rnges of mg/dl, mg/dl, nd mg/dl, respectively, for humn smples. In ddition, the clinicl chemistry screen included the following 21 plsm prmeters: ) substrtes: cretinine, ferritin, glucose, totl protein, trnsferrin, triglycerides, ure, nd uric cid; b) electrolytes: clcium, chloride, iron, phosphorus, potssium, nd sodium; c) enzymes: lnine minotrnsferse, lkline phosphtse, mylse, sprtte minotrnsferse, cretine kinse, lctte dehydrogense, nd lipse. Furthermore, the following 13 hemtologic prmeters were mesured in EDTA-treted blood using n ABC blood nlyzer (Scil, Viernheim, Germny): ) red blood cells: hemtocrit, hemoglobin, men corpusculr hemoglobin, men corpusculr hemoglobin concentrtion, men corpusculr volume, red blood cell count, nd red blood cell distribution width; nd b) white blood cells: grnulocytes, lymphocytes, men pltelet volume, monocytes, pltelets, nd white blood cell count. RESULTS ENU-induced phenotypic vrints showing incresed plsm totl cholesterol The serch for mutnt mice showing incresed plsm totl cholesterol concentrtions in the Munich ENU Mouse Mutgenesis Project ws conducted on the inbred C3H genetic bckground 3 months post prtum fter overnight fsting of the nimls. Determintion of the physiologic rnge in mle nd femle controls resulted in men totl cholesterol levels of 132 nd 109 mg/dl, respectively. The 95% rnge of the vlues covered the dt rnge including two stndrd devitions bove nd below the men, indicting the Gussin distribution of the dt. The vlues within this rnge were defined to be physiologic, thereby eliminting outlier dt (15). Thus, hypercholesterolemi ws defined in our test for mle nd femle mice showing vlues bove the cutoff level of 160 mg/dl nd 140 mg/dl, respectively, in two mesurements within 3 week intervl (Tble 1). In the 6.5 yer exmintion period, 15,624 G1 offspring of ENU-treted mice were screened for dominnt muttions. Of the mice screened for dominnt muttions, 60% were mle nimls; 57 mle nd 3 femle mice showed incresed men plsm totl cholesterol levels of the two mesurements between 162 mg/dl nd 358 mg/dl nd between 141 mg/dl nd 180 mg/dl, respectively. For reveling recessive muttions, 6,531 G3 mice were exmined from 233 different pedigrees of ENU-treted nimls. Of these, 35 mle nd 11 femle mice exhibited incresed men plsm totl cholesterol levels of the two mesurements between 160 mg/dl nd 312 mg/dl nd between 175 mg/dl nd 351 mg/dl, respectively. The lower number of femle phenotypic vrints my be cused by sex- TABLE 1. Physiologic rnge of plsm totl cholesterol (mg/dl) in 3-month-old C3H wild-type mice Sex No. Rnge Men SD Men 2 SD 95% Rnge Mle Femle SD, stndrd devition. Cutoff level: mle: 160 mg/dl; femle: 140 mg/dl. Mohr et l. Hypercholesterolemi in ENU mutnts 2133

3 TABLE 2. Screen nd nlysis of the inheritnce for hypercholesterolemi in ENU mutgenized mice Anlysis of Inheritnce b Screen Smple Pedigrees Vrints Vrints Mted Without Offspring Negtive Offspring Mutnts c no. no. (m/f) Dominnt (G1) d 15,624 nd 60 (57/3) 50 (47/3) 22 (19/3) 22 (22/0) 6 (6/0) Recessive (G3) 6, (35/11) 32 (21/11) 17 (7/10) 12 (11/1) 3 (3/0) Totl 22, (92/14) 82 (68/14) 39 (26/13) 34 (33/1) 9 (9/0) m/f, mles/femles; nd, not determined. G1 nd G3 offspring of ENU-treted mice showing hypercholesterolemi (mle: 160 mg/dl; femle: 140 mg/dl) in two mesurements of 3-week intervl. b A muttion s cuse for the ppernce of the bnorml phenotype ws dignosed in phenotypic vrints with offspring showing cholesterol levels bove the cutoff level (mle: 160 mg/dl; femle: 140 mg/dl) in two mesurements of 3 week intervl. Offspring were produced by mting the G1 phenotypic vrints to wild-type mice (screen for dominnt muttions) nd from G4 intercross or G4 G3 bckcross breedings fter mting the G3 phenotypic vrints to wild-type mice (screen for recessive muttions). c Phenotypic vrints inheriting hypercholesterolemi to the subsequent genertions. d Sixty percent of the G1 mice screened for dominnt muttions were mle nimls. specific compenstion mechnisms of muttions ffecting the plsm cholesterol metbolism nd/or by the selective erly loss of femle phenotypic vrints due to the pthologic consequences of hypercholesterolemi (Tble 2). Trnsmission of hypercholesterolemi to subsequent genertions The nlysis of the inheritnce of the hypercholesterolemic phenotype ws performed on G2 nimls from the mting of 50 G1 phenotypic vrints to wild-type mice in the screen for dominnt muttions nd on G4 intercross or G4 G3 bckcross offspring fter breeding 32 G3 phenotypic vrints to wild-type mice in the screen for recessive muttions (see Mterils nd Methods). In the TABLE 3. cse no offspring occurred, the phenotypic vrints were mted to nother wild-type niml. The remining 24 mle phenotypic vrints (10 from the screen for dominnt muttions nd 14 from the screen for recessive muttions) showing plsm cholesterol levels between 163 mg/dl nd 215 mg/dl were not mted, but sperm of these mice ws frozen for long-term storge. From the 82 phenotypic vrints mted, 39 (48%) produced no offspring due to severe illness nd/or sterility in repeted breeding pproches (Tble 2). The lck of offspring occurred in 13 of the 14 femle phenotypic vrints mted irrespective of the extent of the pthologic plsm totl cholesterol vlues ( mg/dl) nd, s tendency, to higher degree in mles showing plsm totl cholesterol levels bove 220 mg/dl (dt not shown). Phenotypic penetrnce nd extent of hypercholesterolemi in the mutnt lines Penetrnce Hypercholesterolemi d Line Men PTC, Founder b Offspring Tested, c m/f m f m f Men mg/dl no. % (no.) mg/dl (SD) CHOHD /65 82 (35) 62 (20) 208 (24) 204 (40) CHOHD /49 29 (6) 12 (3) 182 (15) 157 (16) CHOHD /31 38 (5) 0 (0) 228 (55) CHOHD / (3) 80 (4) 180 (14) 153 (19) CHOHD /33 41 (6) 30 (5) 186 (13) 162 (30) CHOHD /nd 100 (7) nd (3) 219 (6) 202 (63) CHOHR /9 100 (7) 89 (2) 192 (14) 185 ( ) e CHOHR /5 100 (2) 100 (3) 170 (8) f 190 (12) CHOHR / (5) 100 (3) 212 (51) f 202/22 f m/f, mles/femles; nd, not determined; PTC, plsm totl cholesterol; SD, stndrd devition;, no vlues. CHOH, hypercholesterolemic line; D1-D6, dominnt muttion; R1-R3, recessive muttion. b The mutnt lines re listed ccording to the men plsm totl cholesterol level (mg/dl) of both mesurements of the founder G1/G3 mutnt of the line. c Offspring fter mting heterozygous mutnts to wild-type mice (dominnt muttions) nd offspring fter mting heterozygous mutnt mice (recessive muttions). d Hypercholesterolemi in the offspring ws dignosed by the ppernce of totl cholesterol levels bove the cutoff level (mle: 160 mg/dl; femle: 140 mg/dl). 100% phenotypic penetrnce is defined in our breeding scheme by the ppernce of 50% nd 25% hypercholesterolemic offspring in the lines hrboring dominnt nd recessive muttion, respectively. The men plsm totl cholesterol level of the second mesurements of the nlyzed hypercholesterolemic mutnts is shown. e Vlue of only one of the two nimls ws included. f Men plsm totl cholesterol level of the first mesurements of the hypercholesterolemic mutnts Journl of Lipid Reserch Volume 45, 2004

4 This my be due to pleiotropic effects of the muttions nd/or to negtive consequences of the elevted cholesterol levels on the reproductive system of the ffected mice. Of the 82 mted mice showing hypercholesterolemi, 43 (52%) produced offspring. A muttion s cuse for the ppernce of hypercholesterolemi ws dignosed in the phenotypic vrints when plsm totl cholesterol vlues bove the cutoff level (mle: 160 mg/dl; femle: 140 mg/ dl) were detected in the offspring in two mesurements t 3 week intervl. Of the 43 offspring producing phenotypic vrints, 34 (79%) did not trnsmit the bnorml phenotype (Tble 2). In these breeding pproches, offspring showing hypercholesterolemi in the first mesurement (mle: mg/dl; femle: mg/ dl) were observed preferentilly from phenotypic vrints with higher hypercholesterolemi levels, but these results were not reproducible in the second test fter 3 weeks (not shown). Therefore, inheritnce of the bnorml phenotype did not occur in these cses. In summry, nine mle mutnts trnsmitting hypercholesterolemi to the subsequent genertions were reveled (Tble 2). The extent of the pthologic plsm totl cholesterol vlues in the phenotypic vrints ws not indictive of the successful estblishment of mutnt line (dt not shown). In ll, breeding of 10 phenotypic vrints ws necessry to estblish one mutnt line with hypercholesterolemi in our project. The further breeding of the descendnts of the nine confirmed mutnts resulted in the estblishment of the lines CHOHD1 to -D6 nd CHOHR1 to -R3 hrboring dominnt nd recessive muttions, respectively. Mutnt offspring showing hypercholesterolemi bove the cutoff level (mle: 160 mg/dl; femle: 140 mg/dl) were produced by mting heterozygous mutnts to wild-type nimls (lines CHOHD1 to -D6) nd by breeding heterozygous mutnt mice (lines CHOHR1 to -R3). The mle nd femle phenotypic mutnts of the nine lines showed men plsm totl cholesterol levels between 170 mg/dl nd 230 mg/dl nd between 150 mg/dl nd 200 mg/dl, respectively (Tble 3). The phenotypic penetrnce of the incresed totl cholesterol levels in the nine lines ws nlyzed by defining complete phenotypic penetrnce in the cse of the ppernce of 50% nd 25% hypercholesterolemic offspring fter mting heterozygous phenotypic mutnts to wildtype mice nd fter crossing heterozygous mutnt mice in the lines with dominnt nd recessive muttions, respectively. In five of the nine lines (CHOHD4, CHOHD6, CHOHR1, CHOHR2, CHOHR3), high phenotypic penetrnce of hypercholesterolemi ws observed, which fcilittes the effective subsequent phenotypic nd moleculr genetic nlyses. The other four lines (CHOHD1, CHOHD2, CHOHD3, CHOHD5) showed n incomplete penetrnce of vrious degrees (Tble 3). Additionl phenotypic ltertions in the mutnt lines For the further nlysis of the hypercholesterolemic ltertion, in six of the nine lines (CHOHD1, CHOHD5, CHOHD6, nd CHOHR1 to -R3), the LDL-C nd HDL-C levels were mesured in the phenotypic mutnts. The mle nd femle physiologic rnges (men vlue 2 stndrd devitions) were nlyzed in wild-type C3H controls nd resulted in 3 11 mg/dl nd 8 20 mg/dl for LDL-C, nd in mg/dl nd mg/dl for HDL-C, respectively. In the lines nlyzed, most hypercholesterolemic mutnts showed LDL-C nd HDL-C vlues bove the physiologic rnge. Compred with norml levels, the increse of the pthologic LDL-C vlues ws higher thn the increse of the pthologic HDL-C vlues, which indictes the disproportionl increse of LDL-C nd HDL-C (Tble 4). However, the exct nlysis of the proportion of LDL-C nd HDL-C in the prticulr mutnt lines requires the exmintion of dditionl mutnts nd the use of the respective wild-type littermtes s controls. According to published results (19) ( org/phenome), our wild-type HDL-C vlues seemed to be elevted, compred with the physiologic totl cholesterol levels. This my be due to species-specific inccurcies of the detection method, which works with regents for humn smples nd includes immunoinhibition steps (Olympus, Hmburg, Germny). However, overestimtion of HDL-C in the hypercholesterolemic mutnts due to inccurcies of the detection method used even underestimted the rel disproportionl increse of LDL-C nd HDL-C. The overll clinicl exmintion nd the nlysis of dditionl 21 clinicl chemistry plsm prmeters (including the substrtes glucose, totl protein, triglycerides, nd ure nd 13 hemtologic prmeters; see Mterils nd Methods) showed obvious devitions in the hypercholesterolemic mutnts only for line CHOHR3, where the hy- TABLE 4. LDL cholesterol nd HDL cholesterol vlues in the hypercholesterolemic mutnts Men LDL-C c Men HDL-C c Line Mutnts tested, b m/f m f m f no. mg/dl (SD) CHOHD1 13/11 34(7) 31(5) 176(17) 147(13) CHOHD5 2/1 21(3) 22( ) 162(13) 128( ) CHOHD6 3/3 35(6) 42(21) 170(8) 152(71) CHOHR1 4/0 27(10) 145(22) CHOHR2 2 d /3 28(7) 16(1) 141(9) 154(6) CHOHR3 d 5/3 24(8) 35(3) 167(35) 152(18) Wild-type e 20/20 7(2) 14(3) 128(11) 105(10) m/f, mles/femles; SD, stndrd devition;, no vlues. CHOH, hypercholesterolemic line; D1, D5-D6, dominnt muttion; R1-R3, recessive muttion. b The hypercholesterolemic mutnts were produced by mting heterozygous mutnts to wild-type nimls (dominnt muttion) nd by breeding heterozygous mutnt mice (recessive muttion). c Men plsm LDL/HDL cholesterol level of the second mesurements of the hypercholesterolemic mice. d Men plsm LDL/HDL cholesterol level of the first mesurements. e As controls, the physiologic plsm LDL nd HDL cholesterol vlues were nlyzed in 20 mle nd femle wild-type C3H mice ech. The plsm totl cholesterol vlues for these mice were s follows (men/ SD): mle 143/13; femle 118/17. Mohr et l. Hypercholesterolemi in ENU mutnts 2135

5 percholesterolemic mice hd decresed lifetime leding to deth fter bout 5 months. In ddition, they exhibited incresed plsm ure nd cretinine vlues, decresed urine ure nd cretinine concentrtions, severe lbuminuri, nd microcytic nemi. The hypercholesterolemic mutnts of the other eight lines were inconspicuous, indicting tht hypercholesterolemi most likely ws not cused s secondry effect due to preceding ltertions in other metbolic pthwys. Thus, the lines hrbor muttions in genes tht primrily nd selectively influence the plsm cholesterol metbolism. DISCUSSION High-throughput screening of gret number of rndomly mutnt mice for hypercholesterolemi resulted in the estblishment of nine lines hrboring dominnt nd recessive muttions. Eight of the lines showed selectively incresed plsm totl cholesterol levels. Aprt from devitions in the plsm cholesterol level, the clinicl chemistry screen of more thn 15,000 G1 nimls nd of G3 mice from more thn 230 pedigrees in the Munich ENU Mouse Mutgenesis Progrm (6, 7) detected mutnts with devitions of the plsm substrtes ferritin, glucose, totl protein, ure, nd uric cid. More thn hlf of ll mutnts found with plsm substrte bnormlities in our screen showed pthologic totl cholesterol level ( Rthkolb et l., unpublished results). This my indicte the involvement of high number of genes in the homeostsis of the blood totl cholesterol level. Reproduction of the hypercholesterolemic stte succeeded in the rndom renlysis of the ffected nimls. Under our breeding pproches for the estblishment of mutnt lines, nerly 80% of the fertile phenotypic vrints did not trnsmit hypercholesterolemi to the offspring. A high rtio in the filure of the trnsmission of the defect to the offspring ws lso found for other phenotypic devitions in our clinicl chemistry screen (Rthkolb et l., unpublished results). One reson my be the occurrence of non-geneticlly fixed hypercholesterolemi is due to the inherent impossibility of performing complete stndrdiztion of husbndry nd experiment. In ddition, loss of the dditive phenotypic effect by segregtion of multiple muttions in the subsequent genertions lso leds to the filure in the trnsmission of the berrnt phenotype. Nerly hlf of the estblished lines showed n incomplete penetrnce of the mutnt phenotype of vrious degrees. Appernce of hypercholesterolemi due to the interction of multiple unlinked muttions leds to lower numbers of ffected offspring. Future linkge experiments in the lines will revel the number nd the chromosoml positions of the muttions involved in the development of hypercholesterolemi. We did not exmine devitions in the number of offspring per litter nd/or in the sex rtio of the offspring indicting erly loss of mutnts s n lterntive cuse for the decresed frequency of mice showing hypercholesterolemi. The phenotypic nlysis of the muttion my be improved by nlyzing the nimls more frequently nd/or by inducing the ltered phenotype specificlly in the mutnts through pproprite diet chllenges. Extensive vritions in the plsm cholesterol concentrtions hve been found in different genetic bckgrounds (20) ( Therefore, genertion nd subsequent nlysis of hybrids nd/or congenic strins will give rise to vritions in the ppernce of hypercholesterolemi. In ddition to the clinicl chemistry screen in the Munich ENU Mouse Mutgenesis Project, systemtic clinicl chemistry nlysis hs been described in nother ENU mouse mutgenesis progrm (3). The ppernce of dislipidemic lines ws reported on different genetic bckground (BALB/c C3H), in which dditionl muttions my occur to cuse hypercholesterolemi (19). Due to the triggering of rndom muttions by ENU, discovery of genes not yet known to be involved in the plsm cholesterol metbolism my occur in our mouse mutnts. Chromosoml linkge nlysis will give fst overview of the ffected genomic sites. The subsequent identifiction of the custive muttions including the nlysis of their functionl relevnce by reverse genetics methods, s well s the exmintion of the pthologic consequences of the muttions, will contribute to the nlysis of the polygenic cuses of humn hypercholesterolemi. This work ws supported by the Germn Humn Genome Project (DHGP) (B. R.) nd the Ntionl Genome Reserch Network (NGFN) (M. K., M. M.). REFERENCES 1. Brown, S. D., nd R. Blling Systemtic pproches to mouse mutgenesis. Curr. Opin. Genet. Dev. 11: Hrbé de Angelis, M. H., H. Flswinkel, H. Fuchs, B. Rthkolb, D. Soewrto, S. Mrschll, S. Heffner, W. Prgent, K. Wuensch, M. Jung, A. Reis, T. Richter, F. Alessndrini, T. Jkob, E. Fuchs, H. Kolb, E. Kremmer, K. Scheble, B. Rollinski, A. Roscher, C. Peters, T. Meitinger, T. Strom, T. Steckler, F. Holsboer, T. Klopstock, F. Gekeler, C. Schindewolf, T. Jung, K. Avrhm, H. Behrendt, J. Ring, A. Zimmer, K. Schughrt, K. Pfeffer, E. Wolf, nd R. Blling Genome-wide, lrge-scle production of mutnt mice by ENU mutgenesis. Nt. Genet. 25: Noln, P. M., J. Peters, M. Strivens, D. Rogers, J. Hgn, N. Spurr, I. C. Gry, L. Vizor, D. Brooker, E. Whitehill, R. Wshbourne, T. Hough, S. Greenwy, M. Hewitt, X. Liu, S. McCormck, K. Pickford, R. Selley, C. Wells, Z. Tymowsk-Llnne, P. Roby, P. Glenister, C. Thornton, C. Thung, J. A. Stevenson, R. Arkell, P. Mburu, R. Hrdisty, A. Kiernn, A. Erven, K. P. Steel, S. Voegeling, J. L. Guenet, C. Nickols, R. Sdri, M. Nsse, A. Iscs, K. Dvies, M. Browne, E. M. Fisher, J. Mrtin, S. Rstn, S. D. Brown, nd J. Hunter A systemtic, genome-wide, phenotype-driven mutgenesis progrmme for gene function studies in the mouse. Nt. Genet. 25: Silver, L. M Mouse genetics: concepts nd pplictions. Oxford University Press, New York. 5. Russ, A., G. Stumm, M. Augustin, R. Sedlmeier, S. Wttler, nd M. Nehls Rndom mutgenesis in the mouse s tool in drug discovery. Drug Discov. Tody. 7: Rthkolb, B., T. Decker, E. Fuchs, D. Soewrto, C. Fell, S. Heffner, W. Prgent, R. Wnke, R. Blling, M. Hrbé de Angelis, H. J. Kolb, 2136 Journl of Lipid Reserch Volume 45, 2004

6 nd E. Wolf The clinicl-chemicl screen in the Munich ENU Mouse Mutgenesis Project: screening for cliniclly relevnt phenotypes. Mmm. Genome. 11: Rthkolb, B., E. Fuchs, H. J. Kolb, I. Renner-Muller, O. Krebs, R. Blling, M. Hrbé de Angelis, nd E. Wolf Lrge-scle N-ethyl- N-nitrosoure mutgenesis of mice - from phenotypes to genes. Exp. Physiol. 85: Vreugde, S., A. Erven, C. J. Kros, W. Mrcotti, H. Fuchs, K. Kurim, E. R. Wilcox, T. B. Friedmn, A. J. Griffith, R. Blling, M. Hrbé de Angelis, K. B. Avrhm, nd K. P. Steel Beethoven, mouse model for dominnt, progressive hering loss DFNA36. Nt. Genet. 30: Hfezprst, M., R. Klocke, C. Ruhrberg, A. Mrqurdt, A. Ahmd- Annur, S. Bowen, G. Llli, A. S. Witherden, H. Hummerich, S. Nicholson, P. J. Morgn, R. Oozgeer, J. V. Priestley, S. Averill, V. R. King, S. Bll, J. Peters, T. Tod, A. Ymmoto, Y. Hirok, M. Augustin, D. Korthus, S. Wttler, P. Wbnitz, C. Dickneite, S. Lmpel, F. Boehme, G. Perus, A. Popp, M. Rudelius, J. Schlegel, H. Fuchs, M. Hrbé de Angelis, G. Schivo, D. T. Shim, A. P. Russ, G. Stumm, J. E. Mrtin, nd E. M. C. Fisher Muttions in dynein link motor neuron degenertion to defects in retrogrde trnsport. Science. 300: Durrington, P Dyslipidemi. Lncet. 362: Chuhn, N. B Membrne dynmics, cholesterol homeostsis, nd Alzheimer s disese. J. Lipid Res. 44: Genest, J Lipoprotein disorders nd crdiovsculr risk. J. Inherit. Metb. Dis. 26: Rder, D. J., J. Cohen, nd H. H. Hobbs Monogenic hypercholesterolemi: new insights in pthogenesis nd tretment. J. Clin. Invest. 111: Dietschy, J. M., nd S. D. Turley Control of cholesterol turnover in the mouse. J. Biol. Chem. 277: Loeb, W. F., nd F. W. Quimby The clinicl chemistry of lbortory nimls. 2nd edition. Tylor & Frncis, Phildelphi, PA. 16. Fzio, S., nd M. F. Linton Mouse models of hyperlipidemi nd therosclerosis. Front. Biosci. 6: D515 D De Winther, M. P., nd M. H. Hofker New mouse models for lipoprotein metbolism nd therosclerosis. Curr. Opin. Lipidol. 13: Prk, J. Y., J. K. Seong, nd Y. K. Pik Proteomic nlysis of diet-induced hypercholesterolemic mice. Proteomics. 4: Hough, T. A., P. M. Noln, V. Tsipouri, A. A. Toye, I. C. Gry, M. Goldsworthy, L. Moir, R. D. Cox, S. Clements, P. H. Glenister, J. Wood, R. L. Selley, M. A. Strivens, L. Vizor, S. L. McCormck, J. Peters, E. M. Fisher, N. Spurr, S. Rstn, J. E. Mrtin, S. D. Brown, nd A. J. Hunter Novel phenotypes identified by plsm biochemicl screening in the mouse. Mmm. Genome. 13: Pigen, K., nd J. T. Eppig A mouse phenome project. Mmm. Genome. 11: Mohr et l. Hypercholesterolemi in ENU mutnts 2137