Am. J. Hum. Genet. 50:1038-1045, 1992

Pedigree and Sib-Pair Linkage Analysis Suggest the Apolipoprotein B Gene Is Not the Major Gene Influencing Plasma Apolipoprotein B Levels Josef Coresh, * T Terri H. Beatyt Peter 0. Kwiterovich, Jr., * and Stylianos E. Antonarakis* i$ *Departments of Pediatrics and Medicine, School of Medicine, TDepartment of Epidemiology, School of Hygiene and Public Health, and $Center for Human Genetics, Johns Hopkins University, Baltimore

Summary Previous studies suggest that plasma apolipoprotein B-100 (apoB) level is strongly influenced by genetic factors. Characterizing alleles that influence plasma apoB level would help define genetic risk factors for coronary artery disease. This study examined the role of variability in the apolipoprotein B gene (APOB) in determining plasma apoB level. Twenty-three informative families from the Johns Hopkins Coronary Artery Disease Family Study were studied. Linkage analysis between three polymorphisms in the APOB gene (XbaI at codon 2488, MspI at codon 3611, and EcoRI at codon 4154) and a putative major gene with a codominant allele for elevated apoB levels gave evidence against linkage (LOD score of 7.9 at a recombination fraction of .001). None of the families had a LOD score greater than 0.5, while five families had a LOD score less than 0.5. Sib-pair analysis also showed no relationship between the proportion of genes identical by descent at the APOB locus and either crude or adjusted plasma apoB levels. Thus, in 23 informative families, there was no evidence for the presence, in APOB, of common alleles that influence plasma apoB levels. These results suggest that APOB is not the major locus influencing plasma apoB levels. -

-

Introduction

Plasma apolipoprotein B-100 (apoB) level is emerging as a strong predictor of coronary artery disease (Kladetzky et al. 1980; Sniderman et al. 1980; Campeau et al. 1984; Ford et al. 1990; Hearn et al. 1990) and of myocardial infarction (Avogaro et al. 1978; Sniderman et al. 1982; Al-Muhtaseb et al. 1989; Sandkamp et al. 1990). Several studies have demonstrated a significant genetic component in the familial aggregation of high apoB levels (Berg 1984; Beaty et al. 1986; Hamsten et al. 1986; Amos et al. 1987; Hasstedt et al. 1987; Pairitz et al. 1988; Tiret et al. 1990). A major gene influencing apoB levels in all or a subset of families with high apoB levels has been suggested by Received September 19, 1991; revision received December 20, 1991.

Address for correspondence and reprints: Josef Coresh, Department of Epidemiology, Room 6509, Johns Hopkins University, School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, MD 21205-2179. i 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297 /92/ 5005-0020$02.00

1038

some of these studies (Amos et al. 1987; Hasstedt et al. 1987; Pairitz et al. 1988; Tiret et al. 1990). The apolipoprotein B gene (APOB), which codes for apoB, is a logical candidate locus that may control the variability of plasma apoB levels in the population. Using linkage and sib-pair analysis methods, the present study examined the hypothesis that the APOB locus influences apoB levels. APOB is the first logical candidate for being the major gene influencing apoB levels. This gene codes for both apoB-48 and apoB-100 (Powell et al. 1987; Chen et al. 1988; Higuchi et al. 1988). Mutations in either the promoter or the coding regions of the gene could influence apoB levels. Rare mutations in the APOB gene have been identified that result in markedly abnormal plasma apoB levels. Mutations that lead to the production of truncated forms of apoB cause hypobetalipoproteinemia, a codominant disorder characterized by markedly decreased levels of plasma apoB (Scott 1990). Another mutation, an

arginine-to-glutamine substitution at residue 3500, leads to familial defective apoB-100, a phenotype

1039

APOB Gene and Plasma apo B Level characterized by hypercholesterolemia and markedly elevated apoB levels because of defective binding of apoB to LDL receptors (Soria et al. 1989). Finally, APOB is large and is under complex regulation (Scott 1990); thus it is a likely candidate for the accumulation of mutations that would influence apoB level. Population studies have attempted to establish the contribution of APOB to the variation in lipid levels and to the risk of coronary disease. The results of these studies have been mixed. Several studies have found restriction sites within APOB to be associated with variation in apoB levels (Berg 1986; Law et al. 1986; Tikkanen et al. 1988; Friedl et al. 1990), while others did not (Hegele et al. 1986; Aburatani et al. 1988; Darnfors et al. 1989; Myant et al. 1989; Genest et al. 1990; Tikkanen et al. 1990). The disagreement between these studies is not surprising given the limitations of population-based studies, the small size of many of the samples, and the ethnic diversity of the subjects. The present study examined selected families in an attempt to definitively establish the role of APOB in influencing apoB levels. Families were ascertained through probands who underwent coronary arteriography at an early age in a study of genetic factors influencing the risk of coronary heart disease (Kwiterovich et al. 1992). Segregation analysis was used to select those families that were most likely to carry a major gene influencing plasma apoB levels. Several markers in the APOB locus were used to increase the available information on genetic recombination. Both pedigree linkage analysis, which is more powerful, and sib-pair linkage analysis, which is less powerful but more robust, were used to analyze these family data. Material and Methods Study Population

Segregation analysis of plasma apoB levels adjusted for age, sex, body-mass index, smoking, and alcohol intake showed that 57 of 116 families in the Johns Hopkins Coronary Artery Disease Family Study gave support for a major-gene model with modest polygenic component over a polygenic model with a nontransmitted factor; the other 59 families supported the latter model over the former (Coresh 1992). Segregation analysis of adjusted apoB level in these 57 families clearly supported a codominant-majorgene model which explained 55% of the variation in apoB levels (Coresh 1992). The segregation analysis

model assumed the existence of two alleles, termed "L" and "H." at a single locus. The prevalence of the L allele was 67%, and the mean adjusted apoB levels (adjusted to the values for a 45-year-old male with a body-mass index of 26 kg/iM2 who does not smoke cigarettes or drink alcohol) for individuals of types LL, HL, and HH were 124.3, 164.1, and 208.3 mg/ dl, respectively. The common SD was 25.8 mg/dl. There was no evidence for a significant residual polygenic heritability; however, a significant spouse correlation was detected. This residual spouse correlation could not be incorporated into PAP (Hasstedt and Cartwright 1979), the computer package used for genotype prediction, or into LINKAGE (Lathrop and Lalouel 1988),the computer package used for linkage aialysis. Omitting the spouse correlation had no impact on the estimators for other parameters in the model (data not shown). Under this best-fitting model, the probability of each individual having an LL, HL, or HH phenotype was calculated using PAP (Hasstedt and Cartwright 1979; Hasstedt 1982). The most probable genotype of each individual was selected, and pedigrees were drawn for all families. Families were eligible for this linkage study if they contained at least one individual whose most probable genotype was HL (heterozygous) and if this person had either two children, or one child and one parent, with data. Thirty-four (60%) of the 57 families satisfied this criterion. Families not satisfying this criterion were not studied, since they would contain little or no linkage information. The XbaI, MspI, and EcoRI polymorphisms in APOB (table 1) were determined for all members of the 34 families selected under the phenotype criteria. Twenty-three of those families were informative, i.e., contained at least one individual who satisfied the following: (1) most probable genotype of HL, (2) heterozygous for at least one of the APOB markers, and (3) at least two children or one child from a phase-known mating. ApoB Measurement

Subjects enrolled in the study were instructed to fast for at least 12 h before blood samples were drawn. Blood was collected into tubes containing EDTA (final concentration 1.5 mg/ml blood). Tubes were placed on wet ice and brought to the laboratory where they were centrifuged. Plasma was removed and used for lipoprotein and apolipoprotein measurements. The content of LDL apoB in unfractionated plasma was measured by radioimmunodiffusion using an apoB kit

1040

Coresh et al.

Table I PCR Amplification of APOB Polymorphisms

FRAGMENT SIZE

REGION RFLP

Xbal

....

Mspl ....

(bp)

CODON SITE (polymorphism)

AMPLIFIED (codon-codon)

(-) Allele

(+) Allele

2488 (ACC-ACT: Thr- Thr) 3611 (CGG-CAG:

2424-2527

309

3565-3645

240

4071-4209

414

192 117 138 102 249 165

Arg-*Glu) EcoRI ...

4154 (GAA-AAA:

Glu-Lys)

(M-Partigen; Calbiochem-Behring Corp, La Jolla, CA) as described elsewhere (Kwiterovich et al. 1987). This assay permits a better estimate of LDL apolipoprotein B than do other methods, which provide a better assessment of total plasma apolipoprotein B (VLDL, IDL [intermediate-density lipoprotein], and LDL) (Bachorik and Kwiterovich 1988). The coefficient of variation for the plasma apoB measurement was 4.0%-5.9%. DNA Analysis

DNA was isolated using sucrose-triton lysis followed by phenol extraction (Maniatis et al. 1982). Polymorphisms were initially determined by Southern blotting (Southern 1975). Later, for convenience, the DNA region surrounding each polymorphism was amplified using PCR (Saiki et al. 1985). Amplification was performed with 200-600 ng of genomic DNA template, 200 nM of each primer, 200 pM each dNTP, and 0.625 units of Taq polymerase/25-gl reaction mixture for 40 cycles (at 941C for 30 s, 540C for 30 s, and 720C for 30 s) following a 6-min denaturation at 940C. The oligonucleotides used and the amplification products obtained are shown in table 1. Ten microliters of the reaction products were digested overnight with 5 units of the appropriate restriction endonuclease, at the manufacturer's recommended conditions. The reaction products were separated using a 4% agarose gel (3% NuSieve and 1% agarose) at 80 mV for 3 h and were visualized with ethidium bromide. Any ambiguous or inconsistent results were repeated. Statistical Analysis

Linkage analysis was conducted using the computer package LINKAGE (provided courtesy of Jurg Ott). The recombination fraction between the three mark-

OLIGONUCLEOTIDE 5' TGGTGAAATTCAGGCTCTGG 5' CCACCAATCAGAAATGTAGG 3' TCCATGGCAAATGTCAGCTC 5' AGGAACCTTAGGTGTCCTTC

3' 3' 3' 3'

5' GAGAAGTGTCTTCAAAGCTG 3' 5' GGACTTTCGAATATACCTGG 3'

ers XbaI, MspI, and EcoRI was assumed to be zero, since no recombination events were observed and since the markers are all within APOB. LOD scores for each family were calculated for recombination fractions between 0 and .5. Segregation parameters used were those derived from the best-fitting model identified by a previous segregation analysis (Coresh 1992), i.e., prevalence of the low allele, L, of .67; genotypic means of 124.3, 164.1, and 203.8 mg/dl for LL, HL, and HH, respectively; and a common variance of 612.7. The cleavage sites for XbaI, MspI, and EcoRI were present in 52.9%, 89.8%, and 83.7%, respectively, of the alleles (prevalence of the plus allele). These allele frequencies were calculated using data from 610 family members in the Johns Hopkins Coronary Artery Disease Family Study. Sib-pair linkage analysis was conducted using the SIBPAL routine in S.A.G.E. v2.0 (Elston et al. 1986). For each sib pair, a maximum-likelihood algorithm was used to determine the proportion of genes identical by descent at the APOB locus; the algorithm incorporated marker data on the whole sibship and parents. To test for linkage, regression of the squared sib-pair apoB differences on the estimated proportion of genes identical by descent at the APOB locus was used. If there was linkage, sibs with more APOBs identical by descent would have more similar apoB levels (smaller squared apoB difference) than would sibs without linkage, and the regression coefficient would be negative. Lack of linkage would yield a regression coefficient of zero. A one-sided test that the regression coefficient is less than zero was performed. In addition to the analysis of the XbaI, MspI, and EcoRI polymorphisms individually, the combined marker phenotype was analyzed. Each polymorphic site had two alleles ( + and - ), which gave rise to three possible marker phenotypes ( + +, + -, and

APOB Gene and Plasma apo B Level

1041

Table 2 LOD Scores for Linkage of APOB, with Plasma ApoB Level for Each Family and at Different Recombination Fractions

LOD SCORE

AT

RECOMBINATION FRACTION OF

FAMILY ID (size)

.000

.001

.05

.10

.20

.30

.40

17 (12) ............. 20 (8) ............. 35 (10)............. 38 (11)............. 69 (9) ............. 72 (7) ............. 77 (7) ............. 98 (5) ............. 111 (7)............. 118 (11) ............. 147 (7)............. 148 (6)............. 158 (10) ............. 184 (11) ............. 186 (12) ............. 200 (8) ............. 210 (5) ............. 220 (8)............. 237 (12) ............. 243 (5)............. 245 (8)............. 268 (7) ............. 279 (12) ............. Total (198) ........

.06 .03 -1.41 -.49 .26 .30 -.55 -.03 -.30 -.06 -.89 -.16 .39 - 1.69 -2.23 -.03 .08 -.30 -.37 -.49 -.06 .28 .23 - 7.43

.06 .03 -1.40 -.48 .26 .30 -.54 -.03 -.29 -.06 -.88 -.16 .39 - 1.67 -2.22 -.03 .08 -.29 -.37 -.49 -.06 .28 .23 - 7.34

.08 .02 -.92 -.20 .24 .24 -.36 -.02 -.21 -.05

.09 .02 -.63 -.08 .22 .19 -.25 -.02 -.15 -.05 -.37 -.10 .32 -.69 - 1.14 .01 .04 -.08 -.15 -.26 -.02

.09 .02 -.29 .00 .18 .10 -.12 -.01 -.07 -.04 -.18 -.05

.06 .01 -.12 .01 .12 .04

.03 .01 -.03 .00 .06 .01 -.01 .00 .00 -.01 -.02 -.01 .09 -.03 -.11 .00 .00 .00 -.01 -.01 .00 .08 .06 .11

-.53 -.13 .36 - 1.03 - 1.6 -.01 .06

-.15 -.24 -.36 -.04 .27 .21 - 4.34

.25 .19

-2.65 NOTE. -Totals differ from the sum of some columns because of rounding error.

- - ). Three marker phenotypes at three sites gave rise to 27 APOB marker phenotypes. These marker phenotypes result from pairs of the eight possible haplotypes. The prevalence of each of the eight haplotypes was estimated using the 191 individuals who, among the 610 individuals in the Johns Hopkins Coronary Artery Disease Family Study for whom the XbaI, MspI, and EcoRI polymorphisms were typed, were homozygous for all three markers (data not shown). The square root of the prevalence of the homozygote genotype in the population was used to estimate the haplotype frequency. Haplotypes which were not observed among the 610 individuals were assigned a prevalence of .001. The frequencies of the eight haplotypes were .407, .30, .09, .04, .16, .001, .001, and .001 for + ++,

- ++,

+ -+,

+ +-,

- +-,

+--,

- - +, and - - -, respectively. The eight haplotypes result in (8 x 9)/2 = 36 genotypes, and the matrix showing which haplotype combinations give rise to which marker phenotypes was entered into SIBPAL.

.25 -.31 -.60 .02 .02 -.02 -.06 -.13 .00 .21 .15 - .88

-.05 .00 -.02 -.03 -.08 -.02 .17 -.12 -.30 .01 .01 .00 -.02 -.06 .00 .15 .11 - .12

Results

LOD scores for each ofthe 23 families at recombination fractions of 0 to .5 are shown in table 2. The total LOD score of - 7.3 at a recombination fraction of .001 indicates that, in the total study population, linkage of the APOB and the major gene for elevated apoB levels can be ruled out. In fact, the total LOD score was negative for all recombination fractions of .3 or less. Eight families had a positive LOD score, while 15 had a negative LOD score at a recombination fraction of .001. Furthermore, none of the families gave strong support for linkage; the highest LOD score for a single family was .4. On the other hand, three families had a lod score lower than - 1. Thus, there is little evidence for linkage heterogeneity in these families. Sib-pair analysis allows for an assumption-free test for linkage between the APOB locus and the gene for elevated apoB levels. Regression of the squared apoBlevel difference between sibs on the proportion of genes identical by descent at the APOB locus gave a

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Coresh et al.

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U) u a)

+

a) 9-0--

4

4

4.

10000 - X

4

0.

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+I

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.875 .75 .625 .375 .5 Proportion APOB Genes Identical by Descent .25

.125

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Squared apoB difference vs. proportion of APOBs identical by descent for 230 sib pairs. Plasma apoB difference was adjusted Figure I for age, gender, body-mass index, smoking, and alcohol consumption. The proportion of genes identical by descent was calculated based on the combined marker phenotype of the XbaI, MspI, and EcoRI polymorphisms in the APOB. Straight line is from simple linear regression.

positive rather than a negative regression coefficient. The P value for linkage with MspI was 81, with XbaI was .84, with EcoRI was .78, and with the combined haplotype was .88. Figure 1 shows the regression of the squared adjusted apoB differences on the proportion of genes identical by descent that were estimated using the combined marker information. Using the unadjusted apoB level or the square root of the adjusted apoB level gave similar results (data not shown). .

Discussion

Both full-pedigree and sib-pair linkage analysis indithat APOB is not linked to plasma apoB levels in this study population. The LOD score for linkage of APOB with the major locus influencing apoB levels was 7.3 at a recombination fraction of .001. None of the 23 families studied gave substantial evidence for linkage (LOD score greater than .5), while three families had a LOD score less than 1.0. Sib-pair analysis showed that the proportion of APOB alleles identical by descent was not associated with the similarity of apoB levels within a sib-pair. cate

-

-

Pedigree linkage analysis must rely on the results of the segregation analysis. The segregation analysis that led to the model used in the present paper assumed that variation in plasma apoB level was the result of a major-gene effect, familial correlations, and a residual individual variation, all of which were assumed to be additive. The major locus was assumed to have two alleles, and no interaction with other genes or environmental factors was considered. Residual individual variation is assumed to be normally distributed. Despite the common use of these assumptions in segregation analysis, it is very difficult to rigorously test these assumptions.

It is not known how sensitive the results of linkage analysis are to the validity of the underlying model. Under the null hypothesis of no linkage, the maximum-likelihood estimator of the recombination fraction converges to 1/2 even when the trait-related parameter values are misspecified (Williamson and Amos 1990). Thus, misspecification of the trait-related parameters should not lead to spurious inferences of linkage. The effect of model misspecification for qualitative traits has been discussed by several authors

1043

APOB Gene and Plasma apo B Level

(Greenberg 1990; Neuman and Rice 1990). Further study is required to establish the sensitivity of linkage analysis of quantitative traits to the validity of the underlying model. Thus, the results of the pedigree linkage analysis must be interpreted in the context of the model underlying the analysis. The model used for linkage analysis in the present study was chosen on the basis of a detailed segregation analysis of a larger study population of 116 families. The families eligible for this study were those supporting a Mendelian major-gene model with a modest polygenic component rather than a polygenic model with a nontransmitted factor. Thus, plasma apoB level in these families would be more likely to be under control of a major gene of the type modeled. The linkage analysis was repeated using the parameters estimated from all 116 families (Coresh 1992), to test the sensitivity of the results to using parameters fitted to the 57 Mendelian families. The analysis yielded a negative LOD score as well (LOD = - 3.1 at a recombination fraction of .001). The smaller LOD score was mainly due to the larger residual SD (28.5 compared with 24.8), which resulted in poorer discrimination between genotypes. The sib-pair analysis described here provides further evidence that the lack of linkage between the APOB locus and plasma apoB levels is not merely due to the misspecification of the model of inheritance. The families used in the present study were of moderate size (5-12 members). As a result, the ability to test for linkage heterogeneity was limited. However, of the 23 families studied, none exhibited a LOD score above 1, but three had a lod below - 1. Rare APOB alleles, which lead to abnormal plasma apoB levels and segregate in some families, are known (e.g., hypobetalipoproteinemia and familial defective apoB100). None of 20 of the probands of these families that were screened by allele-specific oligonucleotide hybridization carried the familial defective apoB mutation (data not shown). The proportion of families in the general population in which such alleles are segregating is unknown. Errors in the marker typing and in determining paternity can lead to apparent recombination events. However, the expected magnitude of both is much smaller than the observed recombination rate; the maximum LOD score occurred at a recombination fraction of .4. The LOD score was - 2.6 at a recombination fraction of .1, which is much higher than the expected error rates. We have found PCR amplifica-

tion followed by digestion to be a rapid and reliable method for the determination of RFLPs. The present study indicates that, despite the importance of APOB lipoprotein assembly and metabolism, it is not likely to be the major gene causing variation in apoB levels. The approach described in the present paper- segregation analysis followed by pedigree and sib-pair linkage analysis -should allow for examination of the role that other candidate genes play in influencing apoB levels. Natural candidates include the regulatory proteins that have been shown to bind to the APOB promoter, as well as proteins involved in lipoprotein secretion and metabolism.

Acknowledgments This research was in partial fulfillment of the requirements for the doctoral degree (to J.C.) at the Johns Hopkins University. We thank Hazel Smith for the innumerable hours spent recruiting patients and collecting data for the study, Jeff Jenkins for the meticulous data management, and Dr. Szklo for carefully reviewing the manuscript. The results reported in this paper were obtained by using the program package S.A.G.E., which is supported by U.S. Public Health Service resource grant RR03655 from the Division of Research Resources. The research was also supported by NIH grant HL31497. J.C. was supported by Medical Scientist Training Program grant GM07309.

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Pedigree and sib-pair linkage analysis suggest the apolipoprotein B gene is not the major gene influencing plasma apolipoprotein B levels.

Previous studies suggest that plasma apolipoprotein B-100 (apoB) level is strongly influenced by genetic factors. Characterizing alleles that influenc...
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