Hum Genet (1994) 93:639-648 human .. genet, cs

�9 Springer-Verlag 1994

Linkage analysis of the genetic determinants of high density lipoprotein concentrations and composition: evidence for involvement of the apolipoprotein A-II and cholesteryl ester transfer protein loci

Xiangdong Bu a, Craig H. Warden 2, Yu-Rong Xia 2, Cynthia De Meester 2, Donald L. Puppione 3, Scott Teruya 2, Beth Lokensgard 2, Siamac Daneshmand 2, Jane Brown 1, Richard J. Gray t, Jerome I. Rotter 1, Aldons J. Lusis 2

1 Divisions of Medical Genetics and Cardiology, Departments of Medicine and Pediatrics, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA 2Departments of Medicine and Microbiology and Molecular Genetics, and Molecular Biology Institute, University of California, Los Angeles, CA 90024, USA 3 Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90024, USA

Received: 3 September 1993 / Revised: 29 December 1993

Abstract. We have tested for evidence of linkage between the genetic loci determining concentrations and composi- tion of plasma high density lipoproteins (HDL) with the genes for the major apolipoproteins and enzymes partici- pating in lipoprotein metabolism. These genes include those encoding various apolipoproteins (apo), including apoA-I, apoA-II, apoA-IV, apoB, apoC-I, apoC-II, apoC- III, apoE, and apo(a), cholesteryl ester transfer protein (CETP), HDL-binding protein, lipoprotein lipase, and the low density lipoprotein (LDL) receptor. Polymorphisms of these genes, and nearby highly polymorphic simple se- quence repeat markers, were examined by quantitative sib-pair linkage analysis in 30 coronary artery disease families consisting of a total of 366 individuals. Evidence for linkage was observed between a marker locus D16S313 linked to the CETP locus and a locus determin- ing plasma HDL-cholesterol concentration (P = 0.002), and the genetic locus for apoA-II and a locus determining the levels of the major apolipoproteins of HDL, apoA-I and apoA-II (P = 0.009 and 0.02, respectively). HDL level was also influenced by the variation at the apo(a) lo- cus on chromosome 6 (P = 0.02). Thus, these data indi- cate the simultaneous involvement of at least two differ- ent genetic loci in the determination of the levels of HDL and its associated lipoproteins.

Introduction

The plasma concentrations of high density lipoprotein (HDL)-cholesterol and the most abundant HDL protein,

Correspondence to: J. I. Rotter, Division of Medical Genetics, SSB-3, Departments of Medicine and Pediatrics, 8700 W. Beverly Blvd., Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA

apolipoprotein A-I (apoA-I), are inversely correlated with the risk for coronary artery disease (Gordon et al. 1972; Schaefer 1984; Miller 1987; Tall 1990; Karathanasis 1992). The variations in levels of human HDL are due largely to genetic factors, although environmental influ- ences are also of considerable importance (Rao et al. 1983; Hamsten et al. 1986; Lusis 1988; Breslow 1989, 1991; Hunt et al. 1989; Vogler et al. 1989; Berg 1989; Tall 1992). Studies of rare human mutations have revealed that apoA-I is required for normal HDL assembly, as individ- uals who fail to express apoA-I have greatly reduced HDL-cholesterol levels (reviewed in Karathanasis 1992). In contrast, null mutations of the apoA-II gene, encoding the second most abundant protein of human HDL, do not exhibit dramatic effects on HDL-cholesterol levels (Deeb et al. 1990). Null mutations of cholesteryl ester transfer protein (CETP), on the other hand, are associated with markedly increased levels of HDL-cholesterol (Brown et al. 1989; Inazu et al. 1990). CETP mediates the transfer of cholesteryl esters from HDL to intermediate density lipoproteins (IDL) and low density lipoproteins (LDL). Deficiency states of lipoprotein lipase activity (LPL), in- volving mutations either of the LPL or apoC-II genes, are associated with decreased HDL levels (Doolittle et al. 1992). In one study, variation in HDL levels was shown to be associated with genetic variation at the apolipopro- tein(a) structural gene locus on chromosome 6 (Hegele et al. 1991). A variety of structural gene mutations that abol- ish the activity of lecithin-cholesterol ester transferase (LCAT), the enzyme responsible for esterification of HDL-cholesterol, also greatly decrease levels of HDL- cholesterol (Taramelli et al. 1990). Another potentially very interesting gene as regards its possible role in HDL metabolism is the HDL binding protein (HDLBP), whose structural locus has been mapped to chromosome 2 (Xia et al. 1993). Although this is a cellular HDL-binding pro- tein that recognizes apoA-I, its importance in the metabo-

640

l i sm or overal l regula t ion o f H D L levels is still poor ly un- ders tood (Graham and Oram 1987). Popula t ion associa- t ion and l inkage studies with po lymorph i sms o f the genes for the major apol ipopro te ins and enzymes involved in l ipoprote in me tabo l i sm have sugges ted that variat ions in the genes for apoA- I I (Scot t et al. 1985; Thorn et al. 1993; Warden et al. 1993c), C E T P (Kondo et al. 1989; Kess l ing et al. 1991; He iba et al. 1993), LPL (Heinzmann et al. 1991), and the a p o A I - C I I I - A I V gene complex (Wile et al. 1989; Kess l ing et al. 1991; Xu et al. 1993) inf luence ei- ther HDL-cho les t e ro l levels or H D L composi t ion .

The in v ivo regula t ion o f H D L and its major associ- ated apol ipopro te ins in man is complex . Many known or yet to be known genet ic loci are involved. Mouse models or t ransgenic mouse mode l s can, at least partly, decom- pose this complexi ty . Recent studies of natural ly occur- r ing var ia t ions in mice have revealed that the apoA-I I gene (Mehrabian et al. 1993) and ch romosomal loci not cor responding to any known candidate genes (Warden et al. 1993a) de te rmine HDL-cho les t e ro l levels. Our recent data f rom an apoA- I I t ransgenic mouse indica ted that mice overexpress ing the apoA- I I gene had e levated HDL- choles terol concentra t ions but, nevertheless , exhibi ted in- c reased a therosclerot ic lesion deve lopmen t as compared to normal mice (Warden et al. 1993b). Other studies with genet ica l ly mod i f i ed mice (either t ransgenic mice or gene- targe ted mice) have demons t ra ted that express ion o f apoA- I (Walsh et al. 1989; Rubin et al. 1991), CETP (Ag- el lon et al. 1991), and apoE (Plump et al. 1992; Zhang et al. 1992) each inf luence H D L levels.

We have recent ly repor ted observat ions , based on mouse /human synteny, that the apoA- I I locus is l inked to a locus de te rmin ing apoA- I I levels in man (Warden et al. 1993c). We now repor t a sys temat ic survey of candidate genes ( those genes shown to be b iochemica l ly involved in H D L me tabo l i sm or to encode for HDL-assoc ia t ed apol ipopro te ins ) by l inkage analysis in an enlarged panel o f 30 famil ies enr iched for coronary artery disease. Those genes examined inc lude those encoding the major apo- l ipoprote ins [apo A-I , A-II , A-IV, B, C-I, C-II , C-III , E, and apo(a)] , and prote ins involved in l ipoprote in metabo- l i sm (CETP, LPL, L D L receptor, HDL-b ind ing protein). Ana lys i s pe r fo rmed by the quanti ta t ive s ib-pai r l inkage method (Haseman and Els ton 1972; A m o s et al. 1989) y ie lded two signif icant f indings: a marker locus D 16S313 l inked to the CETP gene is l inked to a locus de termining HDL-cho les t e ro l levels, and the apoA- I I gene is l inked to a locus de te rmin ing the levels o f the major H D L apol ipo- prote ins (apoA-1 and apoA-I I ) .

Materia ls and methods

Pedigrees

At the time of analysis, the study sample consisted of 30 multiplex multigenerational Caucasian pedigrees ascertained through a proband with documented (surgically or angiographically) coro- nary artery disease (CAD) identified at Cedars-Sinai Medical Cen- ter in Los Angeles. Family inclusion criteria required that at least one other blood relative be affected with CAD. A total of 366 in- dividuals from these 30 families agreed to participate in this study

(6 to 33 members per family). The investigation was approved by the institutional Human Subjects Protection Committee.

Plasma biochemical analysis

Overnight fasting blood from participating family members was drawn into EDTA-containing tubes. Plasma concentrations of cho- lesterol and triglyceride (TG) were measured in duplicate using en- zymes and reagents described previously (Moll et al. 1986; War- den et al. 1993c). Surveillance samples measured quarterly using these protocols met the criteria of standardization required by the Centers for Disease Control, Atlanta, GA. Plasma apoA-II concen- trations were determined in triplicate using a turbidimetric assay with standards and antisera obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN.).

Genetic markers

DNA was isolated from peripheral blood cells or transformed lym- phocytes and typed for polymorphisms at or near the following candidate genes: the apoA I-CII1-A IV gene cluster (chromosome 11), apoA-II (chromosome 1), apoB (chromosome 2), the apoE- CII-CI gene cluster (chromosome 19), LPL (chromosome 8), LDL receptor (chromosome 19), CETP (chromosome 16), HDL-binding protein (chromosome 2), and apo(a) (chromosome 6). These poly- morphisms are described in Table 1.

As regards genotyping at the apoC-III tetranucleotide repeat (Zuliani and Hobbs 1990a), novel primers were designed by the Oligo program to amplify the apoC-III tetranucleotide repeat in a single step. Sequences were hC3F (AGG CAG GAG AAT GGG TTG AA) and hC3R (CGG GAG AGA TGA CAG AGT TG). Polymerase chain reaction (PCR) was carried out in 96 well plates in an MJ research thermocycler. Reaction conditions were 95~ l min; 59~ 30 s; and 72~ 1 min; with 25 cycles.

Statistical analysis

The values of HDL and its two major apolipoprotein components, apoA-I and apoA-II, were adjusted by multiple regression analysis for age, sex, and body mass index (BMI). The methodology of ro- bust sib-pair test of linkage was used to test the hypotheses as to whether there was evidence for linkage between a genetic locus controlling a quantitative trait of interest and a specific polymor- phic marker locus (Haseman and Elston 1972; Amos et al. 1989). The underlying basis for this approach is to compare the quantita- tive variation in a trait between siblings as a function of the num- ber of marker alleles they share identical-by-descent (IBD). The actual sib-pair linkage analyses and ordering of marker loci are performed by utilizing the SIBPAL subroutine program of SAGE (1992), version 2.1. This program also calculates the relative prob- ability of the IBD status of each sib-pair. Additional linkage analy- ses were performed on those sib-pairs in which an exact determi- nation of the number of alleles IBD was possible from the pedigree structure. The intraclass correlations between those sib-pairs shar- ing exactly two and zero marker alleles IBD were also calculated. The ILINK subroutine program of LINKAGE 5.2 (Lathrop et al. 1984) was used to estimate the genetic distance between the re- striction fragment length polymorphism (RFLP) markers, which are usually at or only a few kilobases from the actual candidate gene locus, and additional more informative marker(s), such as (CA)n microsatellite repeats.

In addition to utilizing a robust linkage technology that mini- mizes assumptions regarding mode of inheritance, we have tried to avoid false positive results by the following analytic measures: (1) We have excluded from our analyses those sibs with extreme trait values, i.e. greater than the mean _+3 S.D. (2) We excluded those sib-pairs with very large squared trait differences, i.e., greater than the mean +3 S.D. (3) We used the more conservative unweighted least-squares option during sib-pair analysis. (4) Finally, to be

641

Table 1. Candidate gene polymorphisms typed in coronary artery disease (CAD) families

Gene Marker Heterozygosity Chromo- Reference index some

ApoA I-C III-A IV RFLPs detected by XbalA, XbalB, and XmnI XbalA = 0.354 11 Reviewed in Mehrabian et al. 1992 gene cluster (A) XbalB = 0.412

Xmnl = 0.50 0.70-0.95 11

0.74 1

0.73 2 0.80 19

0.94 6 0.39 6 O.46 19 0.11 16

0.47 16

0.57 16

0.27 2

0.81 8 0.44 8 0.39 8

ApoA I-C III-A IV gene cluster (B)

ApoA II

ApoB ApoE-C II-C I

gene cluster Apo(a) Manganese SOD 2 LDL receptor CETP (A)

CETP (B)

D16S313

HDL binding protein (HDLBP)

LPL3GT LPL5GT LPL

Simple sequence tetranucleotide repeat within the apoC-III gene Simple sequence repeat (CA) within the second intron VNTR at the 3" end of the gene Simple sequence repeat (CA) within the apoCII gene Variable number of kringle repeats of gene

RFLP detected by TaqI

Simple sequence repeat (CA) within gene A TaqI RFLP identified by Southern analysis with cDNA probe A TaqI RFLP identified by Southern analysis with cDNA probe Simple sequence repeat (CA) mapping 6 cM from the CETP gene An RFLP detected by cDNA hybridization

Microsatellite 3" to the LPL gene Microsatellite 5" to the LPL gene A HindIII RFLP identified by Southern analysis with LPL cDNA

Zuliani and Hobbs, 1990a; this report Weber and May, 1989

Boerwinkle et al. 1989 Weber and May, 1989

Lackner et al. 1991 Xiang et al. 1987 Zuliani and Hobbs, 1990b Drayna et al. 1987

Drayna et al. 1987

Hudson et al., 1992

Xia et al. 1993; and this report

Wood et al. 1993 Wood et al. 1993 Heinzmann et al. 1988

RFLP, Restriction fragment length polymorphism; apo, apolipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; CETP, cholesteryl ester transfer protein; LPL, lipoprotein lipase

maximally conservative, we performed all analyses on both the raw and log transformed data, with and without regression adjust- ments of age, sex, and body mass index (BMI, kg/m2). A true pos- itive result will usually be robust to different transformations for a relatively normally distributed trait. In addition, in results not shown, we varied the estimated gene frequencies of the marker al- leles, and determined that the results reported herein for this data set were robust to varying estimates of allele frequencies.

Resu l t s

These families are enriched for coronary artery disease, as they were ascertained on the basis of at least two members with coronary artery disease (as defined by angiography, myocardial infarction, or coronary artery bypass surgery). The quantitative lipid phenotypes of these family mem- bers are shown in Table 2. The distr ibutions of HDL- cholesterol and plasma apoA-I and apoA-II levels in all members of these pedigrees are shown in Fig. 1. A mong the unrelated family members , including probands, their spouses, and other marrying- in but genetically unrelat- ed family members , the means (_+ S.D.) of HDL, apoA-I, and apoA-II levels (mg/dl) were 55 + 18, 186 _+ 40, and 36 _+ 7, respectively. Table 3 summarizes the correlations between the various phenotypes measured and the levels of the plasma apoA-I, apoA-II, and HDL-cholesterol . As expected, the levels of apoA-I and apoA-II were signifi- cantly correlated with each other (r = 0.343, P<0.001),

Table 2. Mean (+SD) phenotype values of the members of the CAD families

Trait n Mean + SD

Age (years) 366 52 + 18 Body-mass-index (kg/m 2) 366 25 + 4 LDL particle size (~) 279 267 + 8 Total cholesterol (mg/dl) 365 201 + 41 LDL cholesterol (mg/dl) 364 123 + 36 HDL cholesterol (mg/dl) 364 55 + 17 Triglyceride (mg/dl) 365 104 + 70 Apo B (mg/dl) 360 128 + 34 ApoA-I (mg/dl) 361 179 + 39 ApoA-II (mg/dl) 333 35 + 6 Lipoprotein(a) (mg/dl) 360 18 + 23

with HDL-cholesterol (r = 0.713, P<0.001; r = 0.343, P<0.001), as well as with total cholesterol (r = 0.141, P = 0.015; r = 0.274, P<0.001). Lesser magni tude but still sig- nif icant correlations were also observed between Lp(a) levels and HDL, as well as between Lp(a) and apoA-I lev- els (r = -0 .123, -0 .131 ; P<0.05).

A n informative microsatell i te po lymorphism within the apoA-II gene was found to be significantly l inked to genes determining the serum levels of both apoA-I and apoA-II by sib-pair analysis. We have previously reported

642

100 90 80 7o

b-, Z 60

50 O O 40

30 20 10 0

20 40 60 80 100 120 14050 100 150 200 250 300 350 15 20 25 30 35 40 45 50

HDL (mg/dl) ApoAI (mg/dl) ApoAlI (mg/dl)

Fig. 1. Distributions of HDL, apoA-I, and apoA-II levels (mg/dl) in the coronary artery disease families

Table 3. Correlations between plasma apo-A-I, apo-AII, and HDL-cholesterol concentration and other lipid variables a

Variable (mg/dl) ApoA I ApoA II HDL Cholesterol

n Correlation P value n Correlation P value n Correlation P value

Total cholesterol b 294 0.141 0.015" 274 0.274 0.001" HDL-cholesterol b 294 0.713 0.001" 274 0.343 0.001" LDL-cholesterol b 294 -0.042 0.470 274 0.042 0.493 Triglyceride b 294 -0.127 0.030* 274 0.170 0.005* ApoB b 294 -0.122 0.037* 269 0.092 0.131 ApoAI b - - - 269 0.343 0.001" ApoAII b 269 0.343 0.001" - - -

Lp(a) b 292 -0.131 0.025* 267 -0.036 0.557

300 0.117 0.043*

300 -0.136 0.018" 300 -0.374 0.001" 294 --0.240 0.001" 294 0.713 0.001" 274 0.343 0.001" 292 -0.123 0.036*

* P<0.05 a All variables adjusted for age, sex, and body mass index b Units: mg/dl

Table 4. P-values of sib-pair linkage analyses for ApoA-I levels (mg/dl) with apoA-II locus

Genetic Loci No. of P values sib- pairs Log ApoA-I

(ApoA-I)

ApoA-1 linkage (SIBPAL program)

(a) Without age, sex, bmi adjustment:

apoA-II 99 0.06 0.140

(b) With age, sex, bmi adjustment:

apoA-II 91 0.009 0.030

ApoA-1 linkage on those sib-pairs sharing exactly two, one, and zero apoAH alleles

(a) Without age, sex, bmi adjustment:

apoA-II 59 0.068 0.133

(b) With age, sex, bmi adjustment:

apoA-II 59 0.015 0.038

bmi, Body mass index

the l inkage of the apoA-II gene with a locus determining apoA-II levels after adjustment for sex, age, and body- mass index (Warden et al. 1993c). An additional 12 sib- pairs are included in the present study, yielding a similar

P-value of 0.02 for the total combined data. Fol lowing ad- jus tment for age, sex and body mass index, a P-value of 0.009 was obtained for the l inkage of log(apoA-I) levels with the apoA-II gene in 91 sib-pairs (Table 4). A P-value of 0.03 was obtained for the untransformed apoA-I levels. Similar significant P-values (P = 0.015 and 0.038 for log(apoA-I) and apoA-I levels, respectively) were ob- tained by limiting the analysis to the 59 sib-pairs sharing exactly two, one, and zero apoA-II alleles IBD (Fig. 2). Figure 3 shows the correlations of apoA-I levels among sib-pairs sharing two apoA-II alleles IBD (r = 0.71) and zero apoA-II alleles IBD (r = 0.27). The levels of HDL- cholesterol did not exhibi t s ignif icant ev idence for l ink- age with the apoA-II locus at the current sample size (P = 0.106, 99 sib-pairs), though this result is in the cor- rect direct ion for l inkage. We also examined the evi- dence for l inkage of a locus de te rmin ing apoA-I levels with two highly po lymorphic markers located 6 cM (D1S74) and 12 cM (D1S75) from the apoA-I I gene on h u m a n chromosome 1. Non-s ign i f ican t P-va lues (P = 0.19 and 0.67, respect ively) were obta ined with these two markers, indicating no evidence for l inkage at these distances.

Evidence for l inkage was also observed between a mi- crosatellite polymorphism (D 16S313) l inked to the gene for CETE and a locus determining HDL-cholesterol level and apoA-I level by the sib-pair l inkage method (Table 5). Analysis of a subset of the pedigrees (9 pedigrees) with a

S i b - p a i r s q u a r e d a p o A - I ( m g / d l ) d i f f e r e n c e

643

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14,ooo

12,ooo

lO,OOO

8,ooo

6,ooo

4,000

2,000

-A

r = - 0 . 2 3 N=59

P = 0 . 0 3 8

A

-A

/x

&

/,,

1----- 1

N u m b e r o f a p o A - I I a l l e l e s s h a r e d I B D

A

2x

2

Fig. 2. Significant regression of squared trait differ- ences for unadjusted plasma apoA-I levels versus the number of apoA-II alleles that a sib-pair shares identi- cal-by-descent (IBD). This figure includes only those sib-pairs whose apoA-II marker IBD genotypes could be definitely assigned to sharing exactly two, one, and zero alleles at the apoA-II locus

250

200"

a. 150

..c, 100"

A0,71 "/f" g �9 �9

r=0,27 �9 �9 ~

e~

~5

Sib 2, ApoA-I (mg/dl)

Fig. 3 A, B. Intraclass correlations of apoA-I levels between sib- pairs. A Sib-pairs sharing exactly two (11) apoA-II marker alleles IBD; B Sib-pairs sharing zero (O) apoA-II marker alleles IBD

TaqI RFLP of the CETP gene [referred to in Table 1 as CETP(B)] yielded possible evidence for linkage (P = 0.07 and P = 0.02) of the CETP gene with log-transformed plasma HDL-cholesterol and apoA-I levels, following ad- justment for age, sex, and body mass index. To increase informativeness, a microsatellite polymorphism in the vicinity of the CETP gene (D 16S313) was typed among a larger set of pedigrees (104 sib-pairs). The estimated dis- tance between the microsatellite and the CETP gene was estimated at 6.2 cM by the ILINK program of LINKAGE

5.1 (Lathrop et al. 1984). Following adjustment for sex, age, and BMI, significant evidence for linkage was ob- served between D16S313 locus and log-transformed HDL-cholesterol levels by sib-pair analysis (P = 0.002; Table 5). The levels of significance for the apoA-I levels versus the CETP locus were more variable, and achieved statistical significance only if more extreme values (i.e. possible outliers) were included in the analysis (Table 5). No evidence for linkage was observed between D16S313 locus and the locus controlling apoA-II levels (P = 0.41). Figure 4 shows the intraclass correlations of HDL and apoA-I levels between sib-pairs sharing two or zero D16S313 alleles identical-by-descent. Consistent with the above sib-pair linkage analyses, sib-pairs sharing both D 16S313 alleles IBD have a much higher intraclass corre- lations for these quantitative traits than those of sib-pairs sharing no D16S313 alleles IBD.

Tentative evidence for linkage was also observed be- tween polymorphisms of the structural gene for apt(a), encoding a polypeptide of the Lp(a) particle, and a locus determining unadjusted HDL levels (P = 0.02, 104 sib- pairs; Table 6). However, after adjusting the HDL levels for age, sex, and BMI, the evidence of linkage did not quite reach statistical significance (P = 0.08). The results were not significant with log transformation. In contrast, a less informative RFLP marker for the manganese super- oxide dismutase 2 gene, which is about 10 cM from the

Table 5. P-values of sib-pair linkage analyses for HDL and apoA-I levels with the D16S313 locus, which is linked to CETP

Adjustment No. of P-values No. of P-values No. of P-values sib-pairs sib-pairs sib-pairs

Log HDL Log apoA-I (HDL) (apoA-I)

Log apoA-II (apoA-II)

Without age, sex, 104 0.030 0.174 99 0.054 0.080 97 0.40 0.40 bmi adjustment (0.015)*

With age, sex, 99 0.002 0.026 95 0.140 0.074 93 0.41 0.31 bmi adjustment (0.048)* (0.015)*

* P values in parentheses are obtained from the analyses using all sib-pairs (including possible outliers)

644

~ 100

o -~ 50-

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O3

B �9 r=-0.21

0o o ~ o ~ Sib 2, HDL cholesterol (mg/dl)

�9 �9 �9

300 C

200 �9

<

' - - 1 0 0

6'3

o o o o ~ ~ ~

Sib 2, ApoA-I (mg/dl)

Fig. 4 A-D. Intraclass correlations of HDL and apoA-I levels. A HDL levels and sib-pairs sharing exactly two (m) CETP marker al- leles IBD; B HDL levels and sib-pairs sharing zero (O) CETP marker alleles IBD; C apoA-I levels and sib-pairs sharing exactly two (1) CETP marker alleles IBD; D apoA-I levels and sib-pairs sharing zero (O) CETP marker alleles IBD

D r=-O.01

J

Q

apo(a) locus, did give a significant P value for linkage af- ter the covariate adjustment (P = 0.04, Table 6). This level of significance was robust to the log transformation (P = 0.01), and appeared to show tentative evidence for linkage with a locus determining apoA-I levels as well (P = 0.05).

Similar sib-pair linkage analysis was performed and no significant evidence for linkage was observed with nine polymorphisms at or near six additional candidate gene loci examined (Table 6). These included a polymor- phism of an HDL-binding protein gene located in the dis- tal region of the long arm of chromosome 2.

Discussion

We have utilized a linkage approach using quantitative sib-pair analysis to test for evidence for linkage between loci determining plasma HDL levels (both cholesterol and apolipoproteins) and polymorphic markers at loci for most of the candidate genes thought to be involved in HDL metabolism. Two significant findings were observed (Table 7). First, markers linked to the CETP locus on chromosome 16 demonstrated significant evidence for linkage to a locus controlling HDL-cholesterol levels (P = 0.002) and tentative evidence for linkage to a locus con- trolling apoA-I levels. Second, a microsallelite marker within the apoA-II gene on chromosome 1 was linked to a locus determining apoA-I (P = 0.009) and apoA-II (P = 0.02), but at the current sample size not to a locus con- trolling HDL-cholesterol (P = 0.11). In addition, an infor-

mative size polymorphism of the apo(a) gene on chromo- some 6 was linked to a locus controlling HDL levels (P = 0.02). Although significant linkage was not observed with polymorphic markers at the other candidate gene loci studied, the involvement of these genes in genetic control of HDL-cholesterol cannot be formally excluded at the current sample size. If any of these additional loci con- tribute, they must do so in a quantitatively smaller fash- ion.

A large number of population association studies with candidate genes for lipoprotein metabolism have been re- ported (Lusis 1988; Humphries 1988; Boerwinkle and Hixson 1990; Kessling et al. 1991; Mehrabian and Lusis 1992). Clearly, such studies are useful in the preliminary identification of genes contributing to variations in lipoprotein metabolism. However, population association studies are subject to certain important potential prob- lems. The associations observed in different populations can be highly variable, not only because of differences in allele frequencies, but also because of differences in link- age disequilibrium (Mehrabian and Lusis 1992). In con- trast to association studies, few linkage studies involving candidate genes for quantitative lipoprotein variations have been reported. Linkage studies are subject to fewer poten- tial problems, particularly the problem of mixed ethnic populations.

The underlying basis for the quantitative sib-pair link- age method is to compare the quantitative variation in a trait between siblings as a function of the alleles they share identical-by-descent (IBD) at the test locus (Hase- man and Elston 1972; Amos et al. 1989). When compared with parametric maximum likelihood methods, this ap- proach has a major advantage of not requiring specifica- tion of a genetic model in order to detect linkage. This is particularly important for studying a complex trait such as HDL metabolism. However, the consequence of utilizing this nonparametric linkage method is that relatively larger sample sizes are required. Nevertheless, in this era of vari- able number tandem repeats (VNTR) and (CA)n repeats, very informative markers will make quantitative trait loci (QTL) mapping feasible in terms of attainable sample size. Carey and Williamson(1991) demonstrated that the power of sib-pair linkage analysis of quantitative traits can be substantially increased by using selected samples, i.e., ascertaining a proband with an extreme score on the quantitative trait. As was discussed in the Methods and materials section, these 30 pedigrees were ascertained via a proband with documented (surgically or angiographi- cally) coronary artery disease (CAD) and at least one other blood relative available for study was required to be affected with CAD. The family members of these pedi- grees will thus have a higher probability of abnormal lipoproteins(i.e., extreme trait values) associated with dis- ease. Thus, sib-pair linkage analysis with such a data set will increase the power of the analysis.

The observation that genetic variations of the CETP gene locus contribute to HDL-cholesterol levels is consis- tent with biochemical understanding of the CETP protein. CETP mediates the transport of cholesteryl esters from HDL particles to IDL and LDL, and rare CETP deficien- cies are associated with significant increases in plasma

Table 6. P-values of sib-pair linkage analy- ses for other candidate loci

645

Candidate loci a No. of P-values No. of P-values sib-pairs sib-pairs

Log HDL Log apoA-I (HDL) (apoA-I)

Without age, sex, bmi adjustment

ApoAI-CIII-AIV(A) b 67 0.10 0.19 65 0.19 0.21 ApoAI-CIII-AIV(B) 95 0.26 0.47 88 0.10 0.09 apoB 101 0.88 0.86 96 0.39 0.31 apoE-CII-CI 35 0.99 1.00 30 0.90 0.96 apolipoprotein(a) 104 0.10 0.02 99 0.08 0.13 Manganese SOD 2 58 0.08 0.13 58 0.11 0.12 LDL-receptor 103 0.30 0.16 98 0.93 0.91 HDL binding protein 115 0.75 0.54 110 0.80 0.69 LPL3GT 86 0.66 0.41 84 0.70 0.74 LPL5GT 102 0.99 0.97 97 0.96 0.93 Lipoprotein lipase 82 0.92 0.85 81 0.96 0.97

With age, sex, bmi adjustment

ApoA I-C III-A IV(A) b 66 0.04 0.12 65 0.13 0.15 ApoA I-C III-A IV(B) 92 0.51 0.68 87 0.21 0.15 apoB 100 0.63 0.73 95 0.25 0.20 poCI-CII-E 35 0.95 0.98 30 0.81 0.95 apolipoprotein(a) 102 0.26 0.08 97 0.24 0.23 Manganese SOD 2 57 0.01 0.04 57 0.05 0.06 LDL-receptor 102 0.11 0.11 97 0.93 0.90 HDL binding protein 110 0.78 0.41 105 0.90 0.65 LPL3GT 81 0.64 0.51 79 0.67 0.73 LPL5GT 97 0.89 0.88 92 0.79 0.58 Lipoprotein lipase 77 0.98 0.97 76 0.95 0.95

a see Table 1 for definitions of candidate loci. b Based on the haplotypes generated by three RFLPs

Table 7. Summary of sib-pair linkage analyses for HDL and its as- sociated apolipoprotein levels with D16S313 (CETP linked) and with apoA-II

Quantitative trait Candidate gene locus

D16S313 (CETP) apoA-II

Without age, sex, bmi adjustment

HDL cholesterol Linked Unlinked a apoA-I levels Borderline Linked apoA-II levels Unlinked Linked

With age, sex, bmi adjustment

HDL cholesterol Linked Unlinked a apoA-I levels Borderline Linked apoA-II levels Unlinked Linked

a Unlinked in current sample, although the P-value is in the correct direction for indicating linkage

HDL-cho les t e ro l levels (Inazu et al. 1990). Moreover , previous associat ion (Kondo et al. 1989) and l inkage (Heiba et al. 1993) studies have sugges ted that the CETP locus contr ibutes to HDL-cho le s t e ro l levels. One cons idera t ion in the interpretat ion o f this f inding is that the gene for LCAT, invo lved in the es ter i f icat ion o f HDL-choles te ro l ,

res ides near the CETP gene on c h romosome 16. Unfor tu- nately, po lymorph i sms at the LCAT gene have not been ident i f ied in humans. Never theless , associa t ion studies showing that po lymorph i sms o f the CETP gene are asso- c ia ted with al terat ions in HDL-cho le s t e ro l levels suppor t the concept that the CETP locus d i rec t ly contr ibutes , at least in part, to the obse rved var ia t ions in HDL-cho le s - terol. This is so since l inkage d i sequi l ib r ium is un l ike ly to extend more than a few cent imorgans , and in fact usual ly only a few hundred k i lobases (a f ract ion o f a cen t imorgan in humans) , as has been demons t ra ted in the studies o f cyst ic f ibrosis and o f Hunt ington d isease (Kerem et al. 1989; Andrew et al. 1992). However , in our data set, the D16S313 locus was est imated to be 6.2 cM from the CETP locus and it may still be poss ib le that the var ia t ions at the D16S313 locus affecting H D L ref lect the var ia t ions at the LCAT locus rather than at the CETP locus, or even that both loci contribute.

The f inding that the apoA- I I gene locus is l inked to genes control l ing the levels of both a p o A - I and apoA- I I but not to HDL-cho les t e ro l is unexpected . We recent ly observed s ignif icant l inkage be tween the apoA- I I gene lo- cus and a locus control l ing the levels of p l a sma apoA- I I and p la sma free fatty acids in both mice and humans (Warden et al. 1993c). Genet ic studies and studies with t ransgenic animals have revea led that the level of apoA- I I

646

production has a profound effect on plasma HDL-choles- terol levels in mice, whereas apoA-I levels are only mini- mally influenced (Doolittle et al. 1990; Warden et al. 1993b). In contrast, the present findings indicate that apoA-II gene variations do not significantly affect levels of HDL-cholesterol but do significantly affect levels of apoA-I. (Alternatively, the effect on apoA-I is relatively greater than that on HDL). The explanation is unclear, but the difference in the findings in mice and humans may be related to certain important differences in HDL metabo- lism. For example, mice have little or no plasma CETP, and mouse apoA-II is a monomer rather than a dimer (dis- cussed in Hedrick et al. 1993). It should be noted that all our linkage results should still be considered somewhat tentative due to our studies involving several statistical comparisons. While the apoA-II locus appears to be linked to a locus (or loci) determining both apoA-I and apoA-II levels, this linkage methodology cannot distinguish whether the effects on apoA-I and apoA-II are independent effects of the locus, or indirectly linked, i.e., one quantitative trait mediating the other, and that the linkage of both is due to their consequent biochemical correlations.

The observation of tentative linkage between a locus controlling HDL levels and the apt(a) locus should be considered preliminary. As discussed above, this could simply be a type-I error. Further studies will be required to confirm this result. The biochemical basis of the ob- served linkage between the apt(a) gene and HDL levels is totally unknown. Interestingly, in one previous associa- tion study, HDL levels were shown to vary with genetic variation at the apt(a) locus (Hegele et al. 1991). As shown in Table 3, borderline significant correlations(neg- ative) between the Lp(a) level and the levels of apoA-I, apoA-II, or HDL-cholesterol were observed in our data. Nevertheless, the metabolism of the various lipoprotein particles is highly interrelated, and undoubtedly many in- teractions have remained undetected.

No evidence of linkage between HDL levels and the remaining candidate genes surveyed was observed. Some of these tested loci have revealed significant relationships with HDL levels or its associated apolipoprotein levels in previous association studies, including the apoAI-CIII- AIV gene cluster (reviewed in Humphries 1988; Lusis 1988) and the LPL gene (Heinzmann et al. 1991). There are several possible explanations: (1) the current sample size may have been inadequate to detect such a linkage if it exists; (2) the informativeness of the polymorphisms and closeness of the marker to the candidate genes may have been inadequate; (3) the associations previously ob- served may have been artifactual; (4) the current popula- tion studies may not exhibit variations that occur in other populations; and (5) the previously reported associations may indicate only quantitatively minor polygenic effects rather than major gene effects tested by the linkage meth- ods utilized herein. If the prior association results have been consistent over a sufficient number of studies and populations, the latter hypothesis is the most intriguing, and emphasizes the importance of a linkage approach to complement population association studies.

In conclusion, our results indicate that: (1) the CETP gene contributes to interindividual variation in HDL-cho-

lesterol levels; (2) variation of the apoA-II gene deter- mines, in part, HDL apolipoprotein composition, i.e., apoA-I and apoA-II levels; and (3) the apt(a) gene may also be involved in determining HDL levels. In consider- ing the limitations of robust sib-pair linkage analysis methodology and multiple candidate gene loci tested (i.e., relatively large number of statistical tests) in this paper, we feel that all these findings require further confirma- tion. Although we have examined many of the biochemi- cally defined genes likely to Contribute to HDL levels and the remaining few are to be examined in ongoing studies, this does not exhaust the potential of other, as yet unknown, genes. Indeed, it seems likely that many loci presently un- known (for example, those encoding regulators of gene transcription) are likely to participate in the control of HDL levels (Warden et al. 1993a).

Acknowledgements. The authors thank Judy Tyler (Cedars-Sinai Medical Center) for assistance with aspects of this work. This work was supported by NIH grants HL42481 (A.J.L. and J.I.R.) and HL28481 (A.J.L.). J.I.R. was also supported by a grant from the Stuart Foundations and the Cedars-Sinai Board of Governors' Chair in Medical Genetics. C.W. was supported, in part, by a grant-in aid from the AHA/GLAA. X.B. was supported, in part, by NIH endocrinology training grant DK07426. The results of sib- pair analysis were obtained by using the program package SAGE, which is supported by U.S. Public Health Service Resource Grant 1 P41 RR03655 from the Division of Research Resources.

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