Ath-2, a Second Gene Determining Atherosclerosis Susceptibility and High Density Lipoprotein Levels in Mice

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1 Copyright 1989 by th Gntics Socity of rica th2, a Scon Gn Dtrining throsclrosis Suscptibility an High Dnsity Lipoprotin Lvls in Mic vrly Paign, Muril N. Nsbitt; Dian Mitchll, Dborah lb an Rn C. LoufS Chilrn s Hospital Oaklan Rsarch Institut, Chilrn s Hospital Mical Cntr, Oaklan, California 9469, Dpartnt of iology, Univrsity of California at San Digo, La Jolla, California 9293, an $Vtran s inistration Hospital, Los ngls, California 924, an Dpartnt of Micin R26, Univrsity of Washington, Sattl, Washington Manuscript rciv Jun 24, 1988 ccpt for publication January 1 1, 1989 I STRCT Strain C57L/6J an /J iffr at two gns trining athrosclrosis suscptibility. Th first gn, th1, was scrib arlir an this rport charactrizs th2. Th allls at th2 ar r for rsistanc an s for suscptibility to athrosclrosis. Th rsistant phnotyp in fal icis charactriz by high plasa high nsity lipoprotincholstrol lvls (74 g/l & SEM 2) an vry fw lsions/ous aftr 14 wks of consuption of an athrognic it (.1 k SEM.1 in a prtrin rgion of th aorta). Th suscptibl phnotyp in fal ic is charactriz by low lvls of high nsity lipoprotincholstrol(35 g/l k SEM 1) an 1.2 lsions/ous k SEM.2 in th sa rgion of th aorta. In th2 htrozygots, rsistanc is oinant to suscptibility. Rcobinant inbr strains riv fro C57L/6 an wr charactriz for poal, poa2 an suscptibility to athrosclrosis. th1 an th2 intract with ach othr so that rsistant allls at ithr locus confr a rsistant phnotyp to th anial. Th ap position of th2 is not known, but th2 os not ap nar gns trining th apolipoprotins for I, 11, or E. N th ous, svral gns controlling th structur an quantity of high nsity lipoprotin (HDL) hav bn scrib (LEOEUF t al. 1983; LUSIS t al. 1983; PIGEN t al. 1987). Th structural gn for apolipoprotin 1, na poal, aps on chrooso 9, an th structural gn for apolipoprotin 11, na poa2, aps on chrooso I (LUSIS t al. 1983). utation in or nxt to th poa2 gn controls th rat of synthsis of apo 11, an strains with highr plasa lvls of apo I1 hav largr HDL particls (DOOLITTLE t al. 1987). thir gn, na thi for athrosclrosis suscptibility, trins plasa HDLcholstrol lvls an athrosclrotic lsion foration in ic f an athrognic it (PIGEN t al. 1987). For thos inbr strains carrying th s or suscptibl alll of thi, lvls of HDLcholstrol in th rang of 335 g/l wr obsrv in fal ic f an athrognic it, an athrosclrotic lsions wr prsnt in th aorta an coronary artris. In contrast, thos strains carrying th r or rsistant alll of thi ha HDLcholstrol lvls of 658 g/l whn f an athrognic it an fw, if any, lsions in th aorta an coronary artris. thi aps at a istanc of 4.9 f SE 1.8 cntiorgans fro poa2 on chrooso 1 (PIGEN t al. 1987a, c). In this rport, w onstrat that strains C57L/ 6 an /J iffr not only at thi but also at a scon gn. This novl gn, which is signat th2, is unlink to th1 or to loci ncoing apolipoprotins 1, 11, or E. Gntics 122: (May, 1989) MTERILS ND METHODS Matrials: Th athrognic it contain 15% fat, 1.25% cholstrol, an.5% cholic aci as scrib prviously (PIGEN t al. 1985, 1987b). Noral chow was Purina chow containing 4% fat. Th sourcs for chicals hav bn scrib prviously (PIGEN t al. 1985, , ). nials: Strains C57L/6J an /J wr obtain fro Th Jackson Laboratory, ar Harbor, Main. Crosss to obtain th1 an th2 htrozygots wr carri out in our laboratory using C57L/6 as fals. Th RI strains wr riv fro C57L/6J an /J by MNN. Originally 25 RI strains wr a using /J as th othr ( X strains 125) an 25 RI strains wr a using C57L/6 as th othr ( X strains 125). So RI strains hav fail to br so only 45 ar currntly availabl. ll anials wr hous in a tpraturcontroll facility with a 12 hr light an ark cycl an wr sacrific by crvical islocation. Exprintal sign: Mic that wr 24 onths of ag wr aintain on th athrognic it for 14 wks, sacrific aftr an ovrnight fast, an harts an th bginning portion of th aorta wr rov for valuation of athrosclrotic lsions. Fasting bloo sapls wr obtain aftr 4 wks of athrognic it consuption to valuat HDL lvls. Fal ic wr us unlss al sx is spcifically stat. Histology: Th foralinfix harts an aortas wr b in glatin an lor sctions prpar on a cryostat as scrib (PIGEN t al. 1987). Sctions wr stain with oil r an countrstain with hatoxylin an light grn. throsclrotic valuation was prfor by counting th nubr of lsions in th aortic wall in a stanariz rgion of th aorta (PIGEN t al. 1987). Each lsion was count as a singl plaqu vn if it was obsrv in svral conscutiv sctions. Th tissu was co so that

2 164. Paign t al. TLE 1 HDLcholstrol lvls an lsion foration in strains C57L/6, an F, progny Man SEM Mic HDLcholstrol g/l Sx Chow ous High fat it No. of lsions/ C57L/6 Fal 71 f 2 (11) 35 f 1 (16) 1.2 f.2 (2) Mal 63 f 2 (1) 63 f 4 (IO).2 f.1 (1) /J Fal (14) 69 f 2.1 (16) 74f 2 f.1 (12) Mal 84 f 2 (8) 75 f 4 (8) (8) FI (C57L/6 X ) Fal (1) 81 f 3 (1) 71 f 4 (1) FI (C57L/6 X X5) Fal 71 f 4 (5) (5) Rsults ar ans f SEM. Th nubr of anials asur is inicat in parnthss. th iniviual counting th lsionswas not awar of th intity of th hart. nalytical thos: Th trination of apolipoprotin I an I1 isofors by isolctric focusing was carri out as scrib prviously (LUSIS t al. 1983). HDLcholstrol was trin as scrib prviously (PIGEN t al. 1987b) by agaros gl lctrophorsis of plasa aftr slctiv prcipitation of vry low an low nsity lipoprotins with polythyln glycol (IZZO, GRILLO an MURW 1981). Us of agaros gls to asur HDLcholstrol pritt th us of th sa ous for HDLcholstrol trination at four wks an valuation of lsions in th aorta at 14 wks.chicalassayofhdlcholstrolusually rquirs sacrific of th ous to obtain sufficint plasa. Th HDLcholstrol valus obtain by nsitotry of agaros gls wr convrt to g/l HDLcholstrol by coparison to a stanar of pool plasa fro C57L/6 fal ic f chow. Th HDLcholstrol valu of th stanar was trin bychicalassay (RUDEL an MORRIS 1973). Phnotypic scoring: For lsion foration in th aorta, th phnotyp of rsistanc to athrosclrosis was fin as.4 lsions pr ous aftr 14 wks of th athrognic it; th phnotyp of suscptibility to athrosclrosis was fin as or than.8 lsion pr ous. Sinc this work bgan, w vlop a bttr thoofvaluating th athrosclrosis phnotyp by taking into account th siz of lsions as wll as th nubr of lsions (PIGEN t al. 1987). Whnsizoflsion is valuat, th iffrnc btwn suscptibl an rsistant phnotyps is uch gratr than is apparnt fro lsion nubr alon. For HDLcholstrol, th phnotyp of rsistanc to athrosclrosis was fin as gratr than 55 g/l HDLcholstrol aftr 4 wks on th athrognic it; th phnotyp of athrosclrosis suscptibility was fin as lss than 5 g HDLcholstrol. RI strainsinotxhibitintriatphnotyps,an thr was no abiguity in scoring th phnotyp of ach strain. RESULTS throsclrosis suscptibility an HDL lvls in strains C57L/6,, an F1 progny: Parntal strains C57L/6 an /J an thir F1 progny wr valuat for th phnotyps of athrosclrosis suscptibility an HDLcholstrol lvls (Tabl 1). oth parntal strains an F1 progny hav approxiatly qual quantitis of HDLcholstrol on noral chow. How vr, fal C57L/6 show a cras in HDL lvls to approxiatly half whn f an athrognic it. This lowr lvl of HDL is associat with a suscptibility to lsion foration aftr 14 wks on th athrognic it. Thus, fal C57L/6 ic xhibit th phnotyp of suscptibility to athrosclrosis, as rport arlir, whil strain /J is rsistant. In fal F1 (C57L/6 X ) progny, rsistanc is oinant sinc HDLcholstrol lvls ar high an lsion foration is rar (Tabl 1). s scrib blow in gratr tail, rsistanc is also oinant in an F1 btwn C57L/6 an strain X5, which carris suscptibl allls at thl but rsistant allls at th2. Mal ic of both parntal strainshow no cras in HDL lvls in rspons to th athrognic it. This ffct of al sx on HDL lvls in C57L/6 ic was rport arlir (PIGEN al. t 19876). Furthr stuis wr liit to fal ic. Cosgrgation of HDLcholstrol lvls an suscptibility to lsion foration: In orr to trin whthr th two phnotyps of HDLcholstrol lvls an athrosclrosis suscptibility wr trin by th sa gntic factor, an analysis of th X an X rcobinant inbr (RI) st of strains was conuct. RI strains ar a uniqu tool in ous gntics which can b us to tst whthr two phnotyps ar trin by th sa gntic factor, whthr a phnotyp is trin by a singl or ultipl gns, an what gns ar link to ach othr (ILEY 1981 ; TYLOR 1978). RI strains ar construct by crossing two prognitor strains an stablishing a sris of nw inbr strains fro thir progny. Each nw RI strain consists of a uniqu ixtur of gns in a hoozygous stat riv fro th two parntal strains. If th phnotyps of athrosclrosis suscptibility an HDLcholstrol lvls ar trin by th sa gntic factor, thn ths phnotyps shoul cosgrgat. Evn on iscorant RI strain (.g., on that was suscptibl to athrosclrosis but ha high lvls of HDLcholstrol) woul suffic to show that th phnotyps wr trin by sparat gns.

3 throsclrosis an HDL in Mic 165 TLE 2 Lsions an HDLcholstrol lvls in RI strains Phnotyp Strain Lsions & SE (n) HDL C SE (n) Lsions HDL X1 (5) (5) 79 f 7 R R X3.4 f.2 (5) 85 f 8 R (3) R X9.2 f.2 85 (6) f 8 (3) R R X7.1 f.1 (9) 67 f 4 R (5) R X8 (4) 76 f 5 (4) R R X12 (4) 79 f 4 R (3) R X15 65 f 5 (4) R R X18.2 f.2 (5) 65 f 5 R (5) R X19.4 f.2 (11) 63 f 5 (5) R R X2 (5) 62 f 5 R (4) R X22 97 f 6 (3) R R X25 (5) 75 f 6 R (4) R X9.4 f.2 (5) 63 f 1 R (3) R X12.3 f.3 (4) 75 f 6 R (4) R X18.2 f 68.2 (5) f 6 (5) R R X19 (4) 73 f 6 R R X22 (5) 79 f 5 (5) R R X23 (4) 89 (4) f 6 R R X25.1 f.1 (1) 89 f 8 (3) R R X4 1.1 f.3 (8) 4 f 3 (8) S S X6.9 If 36 (7).3 f 2 (7) S S X9 1.2 f.3 (1) 42 f 4 (5) S S XIO 2.6 f.2 (IO) 39 f 4 (5) S S X13.9 f.2 (9) 48 f 3 (5) S S X f.2 (7) 34 f 2 (7) S S X17.9f.2 (12) 37 f 3 (5) S S X21.9 f.4 (6) 46 f 2 (6) S S X2 1.8 f.4 (9) 36 f 3 (5) S S X4 2.2 f.5 (9) 48 f 2 (3) S S X7 1.7 f.4 (1) 38 f 2 (5) S S X8 1.3 f.3 (4) 41 f 3 (4) S S X f.5 (4) 4 f 5 (4) S S X16.8 f.3 (5) 44 f 3 (5) S S Rsults ar ans f SEM. Th nubr of anials asur is inicat in parnthss. Prviously, w onstrat that th two phnotyps cosgrgat for 17 RI strains xain (PIGEN t al. 1987). In this rport, w xain 3 aitional RI strains. Fal ic of RI strains riv fro C57L/6 an wr tst for HDLcholstrol lvlsinic f th athrognic it for 4 wks an for athrosclrotic lsion foration in th aorta aftr 14 wks of it consuption. Th phnotyps of lsion foration an HDLcholstrol lvls ar copltly concorant in ths RI strains also (Tabl 2) inicating that ths phnotyps ar trin by th sa gn or by closly link gns. If th two phnotyps ar trin by iffrnt gns, th absnc of rcobinants aong 47 RI strains can b us to calculat th axiu istanc btwn th of 1.7 cm at 95% confinc accoring to th forula givn in TYLOR (1 978). Sinc HDLcholstrol lvls an suscptibility to athrosclrosis cosgrgat, thr aitional strains (X15, X22, X19) wr charactriz by HDLcholstrol lvls only (Tabl 2). TLE 3 Rsistant an suscptibl phnotyps in RI strains sort by gnotyp at poa2 No. of RI strains with phnotyp Gnotyp at poa2 Total Rsistant Suscptibl poa2"' (fro rsistant parnt, ) poa2"'" (fro suscptibl parnt, C57L/6) TLE 4 Intraction of thi an ths to prouc athrosclrosis suscptibility phnotyps thi allls th2 allls Strains throsclrosis phnotyp rr rr Rsistant rr ss C3H, L/c Rsistant ss rr 5 Rsistant ss ss C57L/6, 14 X strains" Suscptibl Thos with suscptibl phnotyp in Tabl 2. Strain istribution pattrn of gns for apolipoprotin I1 an athrosclrosis suscptibility: Strains C57L/6 an iffr in th lctrophortic allls at poa2 (LUSIS t al. 1983). Sinc arlir work ha shown that C57L/6 carris suscptibl allls at thi, w thought that ight carry rsistant allls at this locus. If this wr tru, thn all strains carrying th poa261b fro th /J parnt shoul hav carri th thl'l' allls an bn rsistant to athrosclrosis xcpt for fw a strains with a crossovr vnt btwn thi an poa2; likwis strains carrying th poapl" fro th C57L/6 parnt shoul hav carri th thl'l' allls an bn suscptibl to athrosclrosis (xcpt for crossovrs). Th po2 allls wr trin for th RI strains, an Tabl 3 suarizs th athrosclrosis suscptibility phnotyp sort by th poa2 allls. Nin of 1 strains carrying poa2'lb wr rsistant as xpct; th on xcption coul carry a crossovr vnt btwn th1 an poa2. Howvr, for th 23 strains carrying th pa2~" allls, 13 wr suscptibl to athrosclrosis an 1 wr rsistant. Such a istribution of rsistant an suscptibl strains is not consistnt with a singl gn iffrnc in athrosclrosis suscptibility apping at th1. Th bst xplanation for such a strain istribution pattrn is th prsnc of a scon gn for athrosclrosis suscptibility sgrgating in th RI sts riv fro C57L/6 an /J. This gn, which w hav na th2, ust intract with thi so that rsistant allls at ithr locus giv a rsistant phnotyp (Tabl 4). Suscptibility can occur only whn both thi an th2 hav suscptibl allls. Th

4 166. Paign t al. r I I I I I I 1 IJ n=16 X 5 n=8 I I I I I I I I I I I I I 1 I I I I I I I OYI I I I I I I I F1 (6 X x5) n=6 C CI U I I I a I I I I I I I I I I I I I I I R1 Strains F n =33 I I HDLCholstrol (g/l) FIGURE 1.HDLcholstrol lvls in C57L/G,, X 5, F1, backcross of FI (G X X 5 to G, an RI strains. Each circl rprsnts a singl ous (E) t r th avrag HDLcholstrol in an R1 strain (F). N rfrs to th nubr of anials (E) or th nubr of strains (F). biological concpt ay b that having high lvls of HDLcholstrol whn consuing an athrognic it is critical to prvnting lsions an that whthr thos high lvls ar a rsult of th rsistant alll at th1 or th rsistant alll at th2 os not attr as long as th high HDLcholstrol lvls ar aintain. Tsting for th xistnc of th2: If th2 xists an if rsistant allls at th2 rsult in a phnotyp of athrosclrosis rsistanc rgarlss of th allls at th1, that pricts that a backcross of an RI strain that is th1"l" th2'l' to C57L/6 will prouc offspring that ar th2"l" an th2"" in qual nubrs. Strain X5 was thought to b such a strain sinc it carri th poa2, an thus prsuably th thl"", fro th C57L/6 parnt. Mals of strain X5, wr at to C57L/6. Fal F1 ic wr tst for HDLcholstrol at four wks an lsion foration in th aorta at 14 wks. Th F1 ic wr inistinguishabl phnotypically fro th X5 parnt or fro strain. (Tabl 1, Figur i). s shown by ths ata, th th2'l" htrozygots rsbl th rsistant parnt so that th rsistant alll of th2 is oinant to suscptibl. Sinc th th2 htrozygot was istinguishabl fro an th2 suscptibl ous, it was possibl to us a backcross to C57L/6 to onstrat th xistnc. a 2 s a w $.CI 2 & 4 h 8 a VI Wcl 9i b 4 h: b h 8 VI E i% 8 a.i a. E x. t In v 4 In 4 2 xx r 5 ux x 4 xcc x x a x c c r 3 rd 2 v a432 r " 2 z lri < x E I x < q x I < ~ x x x~ < x x ~ x cc x ax< ax< < c xb : L y Mb2 o a% c, 5.8 CY 2.: Y 82s.g.CQa. +Sf.= z 'J c7cgx o &Q$.s O.4, 4 bt.5 a cr ~ x E.5% b o a z su 6%; ; 5%;;: 3 ~ 4 a + ; ; w L1 9 $5 Z Z. C Q 3 T C I? 5 % a $ U X S g p 5 C$'.Fa? $ E3.8 >. 3ctr a o w g a 2% c 22 cf" b3 aj j 55 x bc 5 Ycu a% gw 'c1 E O h 'E 2 :g g n o O'z H Z c.2 32 a <.E 5 7 E E c 5 E 4 Zb 5% U W ha % 5 5 b a E+ E E '$2u L 222U g 7 22% pul TSU? u.22 ;i c x.y E 7zw 2 9 a= &a 25 Tk '2 iq$ q. w ,; o f 2 & E 4 ps'z i? Zc $35 z ;ge.' : $5 4: &c 2.2 b clic 32 o" 4.g ri ZQI g 32

5 throsclrosis an HDL in Mic 167 TLE 6 Lack of linkag of th2 to poal or to GpiI, a gn nar po StI'ill th2 poa I Gpi1 X4 X6 X9 X1 X13 X14 X 17 X21 X2 X4 X7 X8 X 14 X16 X5 Misnlatchs /15 inicats C57L/6lik; inicats /Jlik. po is approxiatly 4 cm cntroric to Cpi1 on chrooso 7. of th2. Mal F1 (C57L/6 X X5) wr backcross to C57L/6 fals. Fal offspring wr tst for HDLcholstrol lvls at 4 wks. ong th 39 backcross progny, 17 ha HDL lvls charactristic of th suscptibl phnotyp an 22 ha HDL lvls charactristic of th rsistant phnotyp (Figur 1). Th sparation of backcross progny into approxiatly qual nubrs of suscptibl an rsistant ic ata confirs th xistnc of a scon gn trining athrosclrosi suscptibility. Figur 1 suarizs th HDLcholstrol ata fro all xprints. Th istribution of HDLcholstrol lvls is pict for parntal strains C57L/ 6 (1) an /J (1 ) an for RI strain X5 (1C). Th F1 progny fro a cross btwn C57L/6 an X5 rsbl th X5 parnt (ID). Th backcross progny sparat into two istinct groups rsbling th parntal strains (IE) as o th Rl strains (IF). It shoul b ntion that th istribution of RI strains into 14 suscptibl an 19 rsistant rsult fro a slctiv rathr than rano tsting of RI strains. W tst all strains which carri th poa2 fro th C57L/6 parnt, bcaus ths wr inforativ for th ap position of th2, but tst only half of thos strains carrying th poa2 fro th parnt. Th 45 strains woul b xpct to sgrgat 3 rsistant to 1 suscptibl for athrosclrosis phnotyp bas on th intraction of thi an th2. Mapping th2: caus rsistant allls at thi ask th gnotyp at th2, th th2 gnotyp is known for only 1 5 Rl strains; thos 14 strains Tabl 2 that ar suscptibl to athrosclrosis an thus carry suscptibl allls at both thi an th2 an X5 which carris th2'as onstrat by th backcross. Th strain istribution pattrn of ths 15 strains was copar to th strain istribution pattrn of othr gntic arkrs. s a first stp, th RI strains wr typ for th lctrophortic allls at poal an poa2 (Tabl 5). llls for two prviously app gntic arkrs on ithr si of poa2 ar also shown. thi is thought to ap cntroric to poa2, btwn poa2 an Xv74 (. PIGEN, unpublish ata). Th istanc btwn poa2 an Xv74 is 6 cm (95% uppr confinc liit, 21 cm). Sinc th2 was not link to th poa2 gn (Tabl 3), th first qustion was whthr it was link to th poal gn, which cos for th othr ajor protin coponnt of HDL. Coparison of athrosclrosis suscptibility phnotyp with poal allls rval 6 isatchs in 15 strains inicating no linkag (Tabl 6). Th nxt qustion was whthr suscptibility was link to th gn for po on chrooso 7 (LUSIS t al. 1987). No polyorphiss btwn C57L/6 an ar known for po, so th narby arkr Gpi I was us to inicat linkag. Gpi1 aps 12 cm fro th cntror an po aps btwn th cntror an Gpi approxiatly 4 cm fro GpiI. Coparison of athrosclrosis suscptibility phnotyp with Gpi1 allls rval 7 isatchs in 15 strains inicating no linkag (Tabl 6). In fact, th athrosclrosis suscptibility phnotyp in ths 15 strains show no clos linkag to any of th or than 15 arkrs typ in th X an X sts. DISCUSSION Ths ata suggst th xistnc of a scon gn trining athrosclrosis suscptibility in th ous. Th bst proof for th xistnc of th2 woul hav bn a ap position; howvr, th fact that backcross progny show rsistant an suscptibl phnotyps in approxiatly qual nubrs is sufficint vinc that th2 xists. It was quit isappointing that no linkag to known gns was foun. Th X RI strains ar typ for ovr 15 arkrs which swp approxiatly 7% of th gno, so that uch of th gno was liinat fro consiration as a ap position for th2. In th futur th typing of aitional gntic arkrs in ths RI strains ay provi a linkag. thi an th2 intract so that rsistant allls at ithr locus confr a rsistant phnotyp to th ous. This intraction of two gns is intrsting bcaus such intractions of gns affcting athrosclrosis suscptibility ay b coon in th huan population. If on xain th X RI strains for th ffct of thi allls on athrosclrosis suscptibility without any knowlg of th2, on woul conclu that thi suscptibl allls wr or prvalnt aong suscptibl strains, but that suscptibl allls wr also obsrv in rsistant strains. Two siilar

6 168. Paign t al. associations of huan gntic arkrs with an athrognic lipi profil isas hav bn rport. Th first is a RFLP polyorphis in th po 1 po C I11 gn coplx. particular alll at th pol sit is or prvalnt in prsons who hav low lvls of HDLcholstrol an incras risk of hart isas (ORDOVS t al. 1986). Howvr th sa allls ar also foun in iniviuals with noral lvls of HDL. This puzzling obsrvation coul b xplain if low lvls of HDL rsult fro a two gn syst such as thi an th2. Low lvls of HDL woul rsult fro th corrct allls at both gns; howvr only th RFLP at pol is known. ong prsons carrying th corrct alll, th phnotyp of low HDL pns upon th allls at th scon locus. Likwis, 9% of prsons with typ 111 lipoprotinia carry th E2/ E2 lctrophortic variant of apo E (DVIGNON, GREGG an SING 1988). Howvr, ost E2/E2 iniviuals hav noral lipoprotin profils suggsting that th typ I11 lipoprotinia phnotyp rquirs E2/E2 as wll as th corrct alll at a scon, as yt unknown, gn. oth thl an th2 trin lvls of HDLcholstrol on th athrognic it. Th xistnc of ths two gntic variants ay prit stuis of cholstrol transport to tst th hypothsis of rvrs cholstrol transport in vivo. It woul b ial to conuct ths stuis in strains congnic xcpt for th thl or th2 locus. Th construction of such congnics has bgun in our laboratory. This work was support by grant 1858 fro th Council for Tobacco Rsarch, by grants HL3287 an HL28481 fro th Hart, Lung, an loo Institut, National Instituts of Halth, ioical Rsarch Support Grant RR5467 fro th National Instituts of Halth, by rit rviw grant 1 fro th Vtrans inistration, Los ngls, by grant fro th rican Hart ssociation with funs contribut in part by th laa, Orang, an Santa arbara County Chaptrs, an grants 85 N132, 85N136, an Snior Invstigator 816F13 fro th California ffiliat of th rican Hart ssociation. LITERTURE CITED ILEY, D., 1981 Rcobinant inbr strains an bilinal congnic strains, pp in Th Mous in ioical Rsarch: History, Gntics an Wil Mic, it by H. L. FOSTER, J. D. SMLL an J. G. Fox. caic Prss, Nw York. LTT, C., K. MILEHM, M. HS, M. N. NESITT, M. E. HRPER an M. I. SIMON, 1983 Chrooso apping of th ink cll focusinucing an xnotropic nv gn faily in th ous. Proc. Natl. ca. Sci. US SO: DVIGNON, J., R. E. GREGC ND C. F. SING, 1988 polipoprotin E polyorphis an athrosclrosis. rtriosclrosis 8: DOOI.ITTI.F., M. H.,. J. Lusrs, J. LUCERO an R. C. LEOEUF, 1987 Th apolipoprotin I1 structural gn controls I1 synthtic rat an siz of high nsity lipoprotins. Circulation 76: IV222. IZZO, C., F. GRILLO an E. MURDO, 1981 Iprov tho for trination of high nsity lipoprotins by using polythyln glycol 6. Clin. Ch. 27: LEOEUF, R. C., D. L. PUPPIONE, V. N. SCHUMKER an.j. LUSIS, 1983 Gntic control of lipi transport in ic. I. Structural proprtis an polyorphiss of plasa lipoprotins. J. iol. Ch. 258: LUSIS,. J.,.. TYLOR, R. W. WNGENSTEIN an R. C. LEOEUF, 1983 Gntic control of lipi transport in ic. 11. Gns controlling structur of high nsity lipoprotins. J. iol. Ch. 258: Lusrs,. J.,.. TYLOR, D. QUON, S. ZOLLMN an R. C. LEOEUF, 1987 Gntic factors controlling structur an xprssion of apolipoprotins an E in ic. J. iol. Ch. 262: ORDOVS, J. M., E. J. SCHEFER, D. SLEM, R. H. WRD, C. J. GLUECK, C. VORGNI, P. W. E. WILSON an S. K. KRTHNIS, 1986 polipoprotin I gn polyorphis associat with pratur coronary artry isas an failial hypoalphalipoprotinia. N. Engl. J. M. 314: PIGEN,., D. LEE, P.. HOLMES an D. MITCHELL 1987a Gntic analysis of urin strains C57L/6J an C3H/HJ to confir th ap position of thi, a gn trining athrosclrosis suscptibility. ioch. Gnt. 25: PIGEN,., P.. HOLMES, D. MITCHELL an D. LEE, 1987b Coparison of athrosclrotic lsions an HDLlipi lvls in al, fal, an tstostrontrat fal ic fro strains C57L/6, L/c, an C3H. throsclrosis PIGEN,., D. MITCHELL, P.. HOLMES an D. LREE, 1987c Gntic analysis of strains C57L/6 an L/cJ for th1, a gn triningathrosclrosis suscptibility in ic. ioch. Gnt. 25: PIGEN,., D. MITCHELL, K. REUE,. MORROW,. J. Lusts an R. C. LEOEUF, 1987 th1, a gn trining athrosclrosis suscptibility an high nsity lipoprotin lvls in ic. Proc. Natl. ca. Sci. US 84: PIGEN,.,. MORROW, C. RNDON, D. MITCHELL an P.. HOLMES, 1985 Variation in suscptibility to athrosclrosis aong inbr strains of ic. throsclrosis 57: PIGEN,.,. MORROW, P.. HOLMES, D. MITCHELL an R.. WILLIMS, 1987 Quantitation assssnt of athrosclrotic lsions in ic. throsclrosis 68: RUDEL, L. L., an M. D. MORRIS, 1973 Dtrination of cholstrol using ophthalalhy. J. Lipi Rs SELDIN, M. F., H. C. MORSE 111, L.. D'HOOSTELERE, J. L. RITTEN an. D. STEINERG, 1987 Mapping of alphaspctru on istal ous chrooso 1. Cytognt. Cll Gnt. 45: SELDIN, M. F., H. C. MORSE 111, R. C. LEOEUF an. D. STEIN ERG, 1988 Establishnt of a olcular gntic ap of istal ous chrooso 1: furthr finition ofa consrv linkag group syntnic with huan chrooso lq. Gnoics 2: TYLOR,.,, 1978 Rcobinant inbr strains: us in gn apping, pp in Origzns of Inbr Mic, it by H. C. MORSE 111. caic Prss, Nw York. Counicating itor: D. ENNETT