Familial combined hyperlipidemia (FCHL), originally described

Haplotypes of the ApoA-I/C-III/A-IV Gene Cluster and Familial Combined Hyperlipidemia Esa Tahvanainen, Päivi Pajukanta, Kimmo Porkka, Sari Nieminen, Liisa Ikävalko, Ilpo Nuotio, Marja-Riitta Taskinen, Leena Peltonen, Christian Ehnholm Abstract Familial combined hyperlipidemia (FCHL) is the most frequent familial lipoprotein disorder associated with premature coronary heart disease. However, no genetic defect(s) underlying FCHL has been identified. A linkage between FCHL and the apoa-i/c-iii/a-iv gene cluster has been reported but not verified in other populations. A recent study identified FCHL susceptibility haplotypes at this gene cluster. To study whether such haplotypes are also associated with FCHL susceptibility in Finns, we studied 600 well-defined Finnish FCHL patients and their relatives belonging to 28 extended FCHL families by using haplotype, linkage, sib-pair, and linkage disequilibrium analyses. The genotypes of the MspI polymorphisms were associated with total serum cholesterol (P 0.01) and apob (P 0.05) levels in spouses, which represent the general Finnish population. However, no evidence of direct involvement of any of these loci or their specific haplotypes in the expression of FCHL in the Finnish FCHL families was found. (Arterioscler Thromb Vasc Biol. 1998;18:1810-1817.) Key Words: apoa-i apoc-iii apoa-iv familial combined hyperlipidemia coronary heart disease Familial combined hyperlipidemia (FCHL), originally described by 3 independent groups, 1 3 is characterized by an elevation of cholesterol and/or triglyceride levels. In Western societies, the condition is found in 1% to 2% of the population and in 10% of survivors of myocardial infarction, designating FCHL as one of the most frequent familial dyslipidemias associated with premature coronary heart disease (CHD). In affected subjects, a common characteristic is an increase in small, dense LDL particles. 4 6 A consistent metabolic finding in FCHL is an increased apob plasma level, 7,8 which is due to either increased production or lowered clearance of apob-containing lipoproteins. 9 11 Association between the apoa-i/c-iii/a-iv gene cluster on chromosome 11 and FCHL has been the subject of several studies. An association between FCHL and an XmnI restriction fragment polymorphism in the 5 -flanking region of the apoa-i gene was reported, 12 and the finding was later confirmed by linkage (logarithm of the odds [lod] score, 6.86) without recombinants in 7 families. 13 Further evidence for an association between this gene cluster and FCHL has been provided by other groups, 14 16 but the findings have also been challenged, 17 20 and the positive linkage result has not been confirmed. An explanation for this controversy may be provided by a recent study, 21 in which the contribution of the apoa-i/c-iii/a-iv gene cluster is presented as an epistatic interaction between different haplotypes, a finding that may also partially explain the paradigm of the complex and varying phenotypes of FCHL. A specific combination of haplotypes (1-1-2/2-2-1, the high-risk haplotype combination ) derived from polymorphic XmnI and MspI sites in the 5 -flanking region of the apoa-i gene and the SstI site in the 3 -untranslated region of the apoc-iii gene was reported to be more frequent in hyperlipidemic patients (frequency, 0.06) than in normolipidemic relatives (frequency, 0.03, P 0.05) or spouses (frequency, 0.005, P 0.01). The aim of the current study was to test whether these specific haplotypes are also associated with FCHL in Finnish FCHL families. Methods Study Subjects The collection of carefully documented pedigrees with several affected individuals has been reported. 22 The study design was approved by the ethics committees of the participating centers, and each subject gave informed consent before participation in the study. A total of 28 pedigrees with 600 subjects were included in the study in 3 phases. In the first phase, probands fulfilling the following criteria were selected from hospital records: age 30 to 55 years for men and 30 to 65 years for women; at least a 50% stenosis in 1 or more coronary arteries confirmed by coronary angiography; and serum total cholesterol and/or serum triglycerides higher or equal to that of the age- and sex-specific 90th percentile. Exclusion criteria for the proband were type I diabetes, hypothyreosis, and hepatic or renal disease. In the second phase, the proband and all first-degree relatives were examined. Families with several affected individuals and at least 2 different lipid phenotypes, of which 1 was the Received January 19, 1998; revision accepted June 25, 1998. From the Department of Biochemistry, National Public Health Institute, Helsinki (E.T., S.N., L.I., C.E.); the Department of Human Molecular Genetics, National Public Health Institute, Helsinki (P.P., L.P.); the Department of Medicine, Helsinki University Central Hospital, Helsinki (K.P., M.-R.T.); and the Department of Medicine, University of Turku, Turku (I.N.), Finland. Correspondence to Christian Ehnholm, Department of Biochemistry, National Public Health Institute, Mannerheimintie 166, 00300 Helsinki, Finland. E-mail Christian.Ehnholm@ktl.fi 1998 American Heart Association, Inc. Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org 1810

Tahvanainen et al November 1998 1811 TABLE 1. Characteristics of the Study Population Variable Affected Family Members: Hyperlipidemics (n 146) Nonaffected Family Members: Normolipidemics (n 314) Spouses (n 145) Age, y* 39 15 26 18 43 13 Sex, M/F 76/70 172/142 69/77 BMI, kg/m 2 * 26.7 4.3 22.6 4.7 25.9 4.8 WHR* 0.92 0.10 0.83 0.09 0.89 0.11 Cholesterol, mmol/l* 6.58 1.25 4.91 0.98 5.46 1.02 LDL-C, mmol/l* 4.11 1.02 3.17 0.81 3.46 0.83 HDL-C, mmol/l* 1.26 0.31 1.38 0.34 1.39 0.34 Triglycerides, mmol/l* 2.54 1.81 1.11 0.50 1.34 0.65 ApoB, mg/100 ml* 127 32 82 24 95 26 ApoA-I, mg/100 ml 135 42 137 28 144 28 ApoC-III, mg/100 ml* 15.1 6.4 8.61 2.7 9.18 3.3 C indicates cholesterol. Probands (n 28) were included in affected family members, ie, the hyperlipidemics group. *The means of the groups differ significantly (ANOVA P 0.05); sex tested by 2. combined lipid phenotype IIB in the proband or his/her first-degree relative, were included in the third phase, for which all accessible relatives and their spouses were examined. The characteristics of the study population are shown in Table 1. Biochemical Analyses The biochemical analyses and baseline data have been described in detail. 22 In short, for each family member over the age of 5, blood was drawn after an overnight fast for the measurement of serum lipids and DNA isolation. Subjects using lipid-lowering agents were asked to interrupt their medication for 4 weeks before blood sampling. Serum samples were stored frozen at 70 C until analyzed. Serum total cholesterol and triglycerides were determined with automated enzymatic methods. 23 HDL cholesterol was determined as described. 24 Familial hypercholesterolemia was excluded from each pedigree by determining the LDL receptor status of the proband by using the lymphocyte culture method. 25 None of the study subjects had tendon xanthomas. Genotyping DNA was isolated by a phenol-chloroform extraction method. 26 Three polymorphisms, XmnI, 27 MspI, 28 and SstI, 29 at nucleotide positions 2500C/T and 78G/A of the apoa-i gene and at 3175G/C of the apoc-iii gene, were determined. In the solid-phase minisequencing method used, 30 variable nucleotides are identified by a single nucleotide primer extension reaction catalyzed by DNA polymerase from a polymerase chain reaction (PCR) product on a solid support. Three different primers were used to study each polymorphism: each DNA fragment, containing a nucleotide to be tested, was first amplified by PCR by using a pair of primers, and then the product was analyzed by a detection primer required for minisequencing. The primers that were used to amplify the target sequence were the same as those used previously 21 for MspI and SstI polymorphisms, but for polymorphism XmnI, we used primers 5 -AGCCACCAAACAGTCCAACC-3 and 5 -TCAGGGACTTC- TCAGTTTCC-3, which are located closer to the XmnI site. The detection primers were 5 -ATCAGCTCTGTCCAGAAAGAC-3, 5 -AGTGAGCAGCAACAGGGCC-3, and 5 -ATTGCAGGACCC- AAGGAGCT-3 for XmnI, MspI, and SstI polymorphisms, respectively. The alleles were coded so that allele 1 refers to an absence of restriction site with the XmnI and SstI polymorphisms but the presence of the restriction site with the MspI polymorphism. 21 With this coding scheme, allele 1 was found to be the most common allele for all polymorphisms. Linkage and Sib-Pair Analyses The linkage analyses were computed by using LINKAGE 31,32 and FASTLINK 31,33 35 program versions 5.1 and 2.3, respectively. The possible heterogeneity of FCHL was tested, and an affected sib-pair study on nuclear families was done by the programs HOMOG and SIBPAIR. 32,36 The linkage analyses were calculated by using both a dominant and a recessive mode of inheritance. Based on the estimated FCHL prevalence, population gene frequencies of 0.004 and 0.089 for the dominant and recessive models, respectively, were used. Frequency of phenocopies was estimated to be 0.005, and penetrance, 0.90 in the calculations. To determine individuals status, the age- and sex-specific 90th percentiles of lipids derived from FINMONICA, a large population-based survey done in 1992, 37 were used for both probands and family members. The fractile cut points for subjects under the age of 25 were derived from the Cardiovascular Risk Factors in Young Finns study. 38 To reduce problems caused by unknown penetrance, subjects were coded as affected in linkage, sib-pair, haplotype relative risk (HRR), and tests of transmission disequilibrium (TDT) analyses if they had cholesterol and/or triglycerides 90th age- and sex-specific percentiles; healthy, if they had both cholesterol and triglycerides 30th age- and sex-specific lipid percentiles; and unknown, if they had lipid values between the 30th and 90th age- and sex-specific percentiles. If a spouse of a family member in the second generation in any of the 28 families was also affected according to the lipid criteria, the offspring of these couples were not included in the calculations to avoid bilineal introduction of the trait. Allele frequencies of 0.214, 0.198, and 0.224, which were estimated by random sampling from families for the less common alleles of Sst I, MspI, and XmnI polymorphisms, respectively, were used in the linkage analyses. Power calculations for linkage analysis were done by using the SLINK program. 39,40 With the use of a dominant model with linkage parameters given above and a marker with 4 alleles with equal frequencies, the average simulated lod score was 20.30 ( 0.00); for a recessive model, the lod was 11.33 ( 0.00). TDT, HRR, and Linkage Disequilibrium (LD) TDTs and HRR analyses were done through ANALYZE, an accessory program to the LINKAGE package, which simplifies the use of programs TDTLIKE and HRRLAMB for this purpose. 41,42 The HRRLAMB program selects the first affected individual from each pedigree for which both parents are typed and then tests the nonrandom segregation 41 of the transmitted and untransmitted alleles by a likelihood ratio test. 42 The likelihood ratio based TDT 42 considers all the alleles jointly and tests whether 1 of them is transmitted from heterozygous parents preferentially to affected offspring. LD analyses between

1812 The ApoA-I/C-III/A-IV Gene Cluster and FCHL polymorphisms were estimated by a permutation procedure, 43 and Hardy-Weinberg equilibrium of genotypes and haplotypes was tested by an exact test 44 ; both methods are part of the program package ARLEQUIN. 45 Haplotypes were determined by combining the use of haplotype estimation algorithms of the GENEHUNTER program package, 46 version 1.0, and inspection of the pedigrees. The nonrandom distribution of alleles between probands and spouses was tested by Fisher s exact test; the distribution of genotypes and haplotypes by 2 test; and haplotype combinations by an exact test analogous to Fisher s exact test extended to a 2 k contingency table, where k is the number of haplotype combinations. 47 Logistic Regression Analyses and ANOVAs To study the effects of specific haplotype combinations in the pedigrees, we used logistic regression modeling and variance analysis. Subjects were coded as hyperlipidemic if they had cholesterol and/or triglycerides 90th age- and sex-specific percentiles. Triglyceride values were logarithmically transformed (base e) in all analyses. The regression analyses and ANOVAs were done with the Statistical Package for the Social Sciences (SPSS Inc), version 6.1.3. In the logistic regression analysis, the association of the haplotype combinations with FCHL phenotype was tested after adjusting for sibship. Two alternative models were tested for each haplotype combination and for all haplotype combinations together: a model in which only the sibship was known for each individual and a second model in which haplotype combinations were added in the model. The statistical significance of the comparison between these 2 models was tested on the basis of the change in log likelihood. The effects of the haplotype combinations on triglyceride, cholesterol, and apob levels were studied by variance analysis adjusted for sibship, age, sex, and body mass index. In addition, the contribution of genotypes of each polymorphism and of haplotype combinations on serum total cholesterol, triglyceride, and apob levels was studied in spouses by ANOVA. Results Linkage and Sib-Pair Analyses The results of 2-point linkage analyses between FCHL phenotype and the polymorphisms remained nonsignificant with the use of both a dominant and a recessive model. The maximum lod scores of SstI, MspI, and XmnI polymorphisms with the dominant mode of inheritance were 0.00 (0.50), 0.00 (0.50), and 0.29 (0.22), respectively. (The recombination fractions are given in parentheses.) With the recessive model, the maximum lod scores were 0.08 (0.32), 0.00 (0.50), and 0.04 (0.36), with the same order of markers. In multipoint analyses with MLINK, the maximum lod scores were 0.27 (0.30) with a dominant mode of inheritance and 0.29 (0.28) with a recessive model. In the nonparametric affected sib-pair analyses, there was no excess allele sharing detectable. There was no significant heterogeneity detectable by the HOMOG program. TDT, HRR, and LD Analyses No significant differences in the frequencies of alleles, genotypes, haplotypes, or haplotype combinations were observed between probands and spouses. The frequencies are also given for family members divided according to hyperlipidemic or normolipidemic status, although the statistical comparisons between these latter groups cannot be done by direct comparisons between frequencies because they do not represent independent study subjects (Tables 2 and 3). The distribution of single-locus genotypes, haplotypes, and haplotype combinations did not differ significantly from Hardy- TABLE 2. The Genotype and Allele Frequencies of the XmnI, MspI, and SstI Polymorphisms in Probands and Hyperlipidemic and Normolipidemic Relatives and Spouses Frequency in Polymorphisms Probands Hyperlipidemics Normolipidemics Spouses Xmn I Genotyes 1/1 0.54 0.53 0.63 0.60 1/2 0.43 0.46 0.35 0.38 2/2 0.04 0.01 0.02 0.01 n 28 109 291 134 Alleles 1 0.75 0.76 0.80 0.79 2 0.25 0.24 0.20 0.21 Msp I Genotypes 1/1 0.61 0.59 0.63 0.69 1/2 0.32 0.37 0.34 0.29 2/2 0.07 0.03 0.03 0.01 n 28 115 309 137 Alleles 1 0.77 0.78 0.80 0.84 2 0.23 0.22 0.20 0.16 Sst I Genotypes 1/1 0.50 0.52 0.67 0.55 1/2 0.50 0.44 0.32 0.44 2/2 0.00 0.04 0.01 0.01 n 28 116 308 134 Alleles 1 0.75 0.74 0.83 0.77 2 0.25 0.26 0.17 0.23 Weinberg equilibrium determined from total allele and haplotype frequencies, with 1 exception: in spouses, the frequencies of haplotypes deviated from those estimated from allele frequencies (P 0.05). That finding was due to the fact that 2 polymorphisms, XmnI and MspI, were in LD (D 0.52 for rare alleles, P 0.05). No significant LD between FCHL TABLE 3. The Haplotype Frequencies in Probands and Hyperlipidemic and Normolipidemic Relatives and Spouses Frequency in Haplotype Probands Hyperlipidemics Normolipidemics Spouses 111 0.50 0.53 0.64 0.61 112 0.19 0.21 0.13 0.17 121 0.04 0.07 0.06 0.04 122 0.02 0.01 0.00 0.00 211 0.07 0.04 0.04 0.09 212 0.02 0.02 0.00 0.01 221 0.15 0.13 0.11 0.07 222 0.02 0.01 0.02 0.01

Tahvanainen et al November 1998 1813 TABLE 4. The Mean Values of Serum Total Cholesterol, Triglyceride, and ApoB Levels in Spouses According to XmnI, MspI, and SstI Genotypes Genotype Cholesterol, mmol/l Mean SD (n) TG, mmol/l Mean SD (n) ApoB, mg/100 ml Mean SD (n) Xmn I 1/1 5.43 1.00 (79) 1.31 0.62 (79) 96.0 23 (79) 1/2 5.56 1.13 (47) 1.38 0.69 (47) 96.0 31 (48) 2/2 5.55 0.78 (2) 1.75 1.48 (2) 68 0 (1) Msp I 1/1 5.29 0.91 (91)* 1.28 0.67 (91) 91.7 24 (91) 1/2 5.90 1.21 (38)* 1.44 0.55 (38) 104.8 29 (39) 2/2 5.55 0.78 (2)* 1.75 1.48 (2) 68.0 0 (1) Sst I 1/1 5.43 0.96 (69) 1.27 0.59 (69) 93.1 23 (69) 1/2 5.42 1.09 (58) 1.33 0.67 (58) 96.0 29 (59) 2/2 7.05 0.00 (1) 2.20 0.00 (1) 151.5 0 (1) TG indicates triglyceride. *P 0.01. Covariates were age, sex, and BMI. ANOVA P 0.05. and any of the polymorphisms was detected by the HRRLAMB program. The segregation of these loci did not differ significantly from random in TDTs. Logistic Regression Analyses and ANOVAs In spouses, the genotypes of the MspI polymorphism were associated with total cholesterol (P 0.008) and apob (P 0.036) levels (Table 4). The total serum cholesterol level was 5.29 mmol/l in 1/1 homozygotes (n 91), 5.90 mmol/l in heterozygotes (n 38), and 5.55 in 2/2 homozygotes (n 2). The total serum apob level was 91.7 mg/100 ml in 1/1 homozygotes (n 91), 104.8 mg/100 ml in heterozygotes (n 39), and 68.0 mg/100 ml in 2/2 homozygotes (n 1). With the XmnI and SstI polymorphisms, no statistically significant differences in mean lipid values were detected between genotypes. To analyze whether any haplotype combination could have an effect on the expression of the FCHL phenotype or lipid values, all informative sibships (n 199) were tested by logistic regression and variance analysis. Analyses revealed no dependence between FCHL, cholesterol, triglyceride, or apob levels and the presence of any of the haplotype combinations. When the sibship of each individual was known, the FCHL status was correctly predicted, on average, in 61.6% of subjects. When the information for any single haplotype combination was added, the accuracy of prediction increased at best by 4%, to a total of 65.6% with the haplotype combination 1-1-1/1-1-1 (P not significant) (Table 5). When all the haplotypes were included in the model in a single step, the accuracy of prediction increased to 67.2%, but the effect was still nonsignificant (P 0.21). No significant differences in the frequencies of haplotypes or haplotype combinations were observed between probands and spouses. The high-risk haplotype combination 1-1-2/2-2-1 was observed in both affected and healthy family members as well as in spouses. It was found in 3/27 probands and in 2/114 spouses (P not significant). In those 2 spouses carrying that haplotype combination, their apob levels were 74.0 and 79.3 mg/100 ml, serum total cholesterol 4.67 and 5.10 mmol/l, and serum total triglycerides 0.60 and 1.27 mmol/l; all values were below the mean of those not having that haplotype combination. The effects of the haplotype combinations on serum total cholesterol, triglycerides, and apob were analyzed in spouses by ANOVA. The mean value of apob level in the 2 individuals carrying haplotype combination 1-1-1/2-1-2 was 48.5, which was lower than the mean of 156 other spouses (P 0.014), but after correction for multiple testing, that finding cannot be regarded as statistically significant. Discussion Originally based on family inspection, analysis of the number of affected sibs, and the distribution of adjusted lipid values, it was suggested that FCHL constitutes an autosomal dominant trait. 1 3 Segregation analyses have supported major gene effects on triglyceride levels, apob levels, and LDL particle size distribution in affected families. 48,49 The dominant model of inheritance is supported by a recent finding of linkage 50 to chromosome 1q21-q23 in Finnish pedigrees. The linkage of a syntenic locus with FCHL has also been shown in a mutant mouse strain. 51 Although this finding of linkage seems now very important, it has been noted several times in the course of research on FCHL that it is heterogeneous and also that in individual families, several genes can be involved in the expression of the FCHL phenotype. It is thus plausible that there are still other gene defects associated with FCHL in Finnish pedigrees. In fact, due to the broad definition of FCHL, defects causing FCHL may be found at any step along the metabolic pathways of apob-containing lipoproteins; from the synthesis of VLDL and chylomicrons to the clearance of their end products, chylomicron remnants, and LDL from plasma. Widely studied candidate genes of FCHL include the major genes affecting the metabolism of apob-containing lipoproteins. The apob gene locus does not contribute significantly to the development of FCHL, but whether the genes regulating its expression might contribute to the FCHL phenotype is not known. 52 Mutations in the lipoprotein lipase gene and reduced postheparin plasma lipoprotein lipase activity have been found in FCHL patients in some studies 53 55 but not in all. 17 The LDL receptor would be an obvious candidate gene for FCHL, but its defects cause a different phenotype and disease. 56,57 Despite this, another still-unknown gene in close proximity to the LDL receptor gene may be associated with the small, dense LDL phenotype. 58 Likewise, lipoprotein modulators like cholesterol ester transfer protein, 59,60 lipoprotein(a), 61 hormone-sensitive lipase, 62 and hepatic lipase 63 are potential candidates. In Finnish FCHL families, there was no evidence of linkage between FCHL and lipolytic enzymes. 64 Defects in the insulin signaling pathway 65 or in an even more potent adipsin acylation stimulating protein signaling pathway are intensively studied candidate genes for FCHL. Adipsin acylation stimulating protein, also called basic protein I, is a fragment of the third component of plasma complement (C3a-des-Arg) and a strong modulator of triglyc-

1814 The ApoA-I/C-III/A-IV Gene Cluster and FCHL TABLE 5. The Frequencies of Haplotype Combinations in FCHL Family Members Halotype Combination Hyperlipidemics (n 76) Normolipidemics (n 287) eride synthesis in adipocytes. 66 Other cellular candidate genes include basic protein II, which affects cholesterol ester formation in the liver, 67 and microsomal triglyceride transfer protein, which catalyzes the transport of triglyceride, cholesteryl ester, and phospholipid between phospholipid surfaces. 68 One of the most important candidate loci of FCHL, and also the subject of the current study, is the apoa-i/c-iii/a-iv gene cluster on chromosome 11. Taking into consideration previous partially contradictory results and the recent findings of complex genetic contribution of specific haplotypes, we considered it important to test the involvement of the apoa-i/c-iii/a-iv gene cluster in the expression of the FCHL phenotype in Finns. We found that in spouses, representing the control group, the MspI polymorphism in the promoter region of the apoa-i gene was associated with total cholesterol and apob levels. The MspI polymorphism had earlier been associated with elevated HDL cholesterol, 28 apoa-i, 69 triglyceride, 16 and apob levels. 16 We could also confirm the positive LD between MspI and SstI polymorphisms. 70 The G3A nucleotide change that creates the MspI polymorphism has been reported to increase the transcription efficiency of apoa-i, probably by reducing the affinity of a regulatory factor. 71 Spouses (n 114) Probands* (n 27) Predicted, % Significance, P 111 111 0.283 0.396 0.333 0.296 65.6 0.19 111 112 0.212 0.189 0.246 0.222 60.8 0.97 111 121 0.040 0.063 0.079 0.037 61.6 0.59 111 122 0.000 0.004 0.000 0.000 61.6 1 111 211 0.040 0.060 0.123 0.000 60.8 0.90 111 212 0.040 0.004 0.018 0.037 61.6 0.44 111 221 0.140 0.140 0.079 0.111 61.6 0.24 111 222 0.010 0.028 0.000 0.000 61.6 0.44 112 112 0.040 0.011 0.000 0.000 62.4 0.10 112 121 0.051 0.021 0.009 0.000 61.6 0.41 112 211 0.010 0.004 0.053 0.037 61.6 1 112 212 0.000 0.004 0.009 0.000 61.6 1 112 221 0.071 0.021 0.018 0.111 62.4 0.29 112 222 0.000 0.000 0.009 0.000 61.6 1 121 211 0.010 0.014 0.000 0.000 61.6 0.31 121 221 0.030 0.018 0.000 0.037 61.6 1 121 222 0.000 0.004 0.000 0.000 61.6 1 122 211 0.010 0.000 0.000 0.037 61.6 1 122 222 0.000 0.000 0.009 0.000 61.6 1 211 211 0.000 0.000 0.000 0.037 61.6 1 211 222 0.000 0.004 0.000 0.000 61.6 1 221 221 0.000 0.014 0.018 0.000 62.4 0.10 221 222 0.010 0.004 0.000 0.037 62.4 0.10 *Probands were included in the hyperlipidemics group in logistic regression analysis. The percentage of individuals who were correctly assigned to hyperlipidemic or normolipidemic groups after including each haplotype combination in logistic regression analysis. (61.6% of the individuals were correctly assigned if their membership in a specific sibship was known.) Significance of the likelihood-ratio test between the logistic regression models with and without the haplotype. The nonrandom distribution of frequencies of haplotype combinations between probands and spouses was tested by 2, and no significant deviations were noted. We found the specific combination of haplotypes, which has previously been reported to be associated with FCHL status and plasma cholesterol and triglyceride values, in 11% of the probands and in 2% of their spouses, but the result was not statistically significant. None of the further analyses supported the association of any haplotype combination with the FCHL phenotype or lipid levels. There was no linkage between the studied markers and the FCHL phenotype. Because linkage analysis is dependent on the inheritance model used, several model-free statistical tests were performed, including nonparametric affected sib-pair, TDT, HRR, LD, logistic regression, and ANOVA. There was no excess allele sharing, allelic association, or LD between the studied polymorphisms and the FCHL phenotype. There was no statistically significant TD or increased HRR detectable. The fact that the studied polymorphisms showed no statistically significant association with the FCHL phenotype in Finnish FCHL families does not rule out that there could be other important polymorphisms in these genes that may be in LD with these polymorphisms in other populations. Other possible explanations for the difference between the results of the current study and earlier positive findings can probably be

Tahvanainen et al November 1998 1815 found in different family selection, different criteria used for the determination of the FCHL affected status, different population gene frequencies, and partially the use of different statistical approaches. In the study of Dallinga-Thie and coworkers, 21 wherein the complex genetic contribution of the apoa-i/c-iii/a-iv gene cluster to FCHL was presented, association studies were done with all normolipidemic and hyperlipidemic individuals in the FCHL families without adjusting for sibships. In that study, the individuals in the hyperlipidemic group were related and did not represent independent individuals, as is usually required in association analyses. 72 In the hyperlipidemic relatives of that study, the rare alleles of XmnI and MspI polymorphisms were more frequent than in our study (a proper statistical comparison would require independent sampling). Also, the frequencies of haplotype combinations were clearly different between these 2 studies. The frequency of the most common haplotype combination, 1-1-1/1-1-1, was much lower in the Finnish FCHL family members than in the Dutch, and concurrently some haplotype combinations, ie, 1-1-1/1-2-2, 1-1-1/2-1-1, 1-1-1/2-1-2, 1-1-1/2-2-2, 1-1-2/1-2-1, 1-1-2/2-1-2, 1-2-1/2-1-1, 1-2-1/2-2-2, 2-1-1/2-2-2, and 2-2-1/2-2-2, were not encountered in the Dutch FCHL families. Two haplotype combinations, 2-1-1/2-2-1 and 1-2-1/1-2-1, were not seen in Finnish FCHL families. Those haplotypes in which the S2 allele combined with the X2 and M2 alleles were rare in Finnish and absent in Dutch FCHL families. The populations show minor differences in haplotype frequencies, but the frequencies of the haplotypes 2-2-2, 1-2-2, and 2-1-2 were also so low in the Finnish population that the different outcome with these rare haplotypes may have occurred by chance. We also used different patient selection (based on age- and sex-specific cutoff values) and different exclusion methods of familial hypercholesterolemia than in the previous study. 21 Although we cannot exclude the possibility that the genes of the apoa-i/c-iii/a-iv gene cluster act as a minor modifying factor in the pathogenesis of FCHL, we conclude that the current study does not support the direct involvement of these genes in FCHL in the Finnish FCHL families. The current study does not rule out the possibility that there could be functional mutations in other populations that are in LD or show other types of association with the studied polymorphisms. Acknowledgments This work was supported by grants from the Academy of Finland (C.E., L.P., M.-R.T.), the Helsinki University Central Hospital Research Foundation (C.E., L.P., M.-R.T.), the Finnish Heart Foundation (C.E., L.P., M.-R.T.), and the Emil Aaltonen s Foundation (I.N., P.P.). We thank the members of Finnish FCHL families participating in the European Multicenter Study on Familial Dyslipidemias With Premature Coronary Heart Disease (EUFAM Study). Sirpa Koskela s, Leena Pahlama s, Aira Heine s, and Terho Lehtimäki s work with these families and practical arrangements in family data collection are gratefully acknowledged. We wish to thank Elli Kempas, Sirkka-Liisa Runeberg, Leena Lehikoinen, Ritva Marjanen, Ulla Minkkinen, and Anne Jokiaho for excellent laboratory technical assistance and Aki Suomalainen for maintenance of databases and drawings for this article. We thank Petri Kovanen, Wihuri Research Institute, for LDL receptor status determinations. Appendix This investigation is a part of the larger EUFAM Study, supported by the European Commission, contract number BMH4-CT96-1678. EUFAM Study Group Prof Christian Ehnholm, coordinator, Dr Esa Tahvanainen: National Public Health Institute, Department of Biochemistry, Helsinki, Finland; Prof Leena Peltonen, Dr Päivi Pajukanta: National Public Health Institute, Department of Human Genetics, Helsinki, Finland; Prof Marja-Riitta Taskinen, Dr Kimmo Porkka, Dr Kati Ylitalo: Department of Medicine, University of Helsinki, Helsinki, Finland; Assoc Prof Jorma Viikari, Dr Ilpo Nuotio: Department of Medicine, University of Turku, Turku, Finland; Prof Markku Laakso, Dr Jussi Pihlajamäki: Department of Medicine, University of Kuopio, Kuopio, Finland; Prof Anders G. Olsson: Department of Internal Medicine, Linköping University, Linköping, Sweden; Prof Ole Færgeman, Dr Jesper Jensen: Department of Medicine and Cardiology A, Aarhus Amtssygehus, University Hospital, Aarhus, Denmark; Prof Paolo Rubba, Dr Paolo Pauciullo: Universitá degli studi di Napoli Federico II, Il Facoltá di Medicina Interna e Malattie Dismetaboliche, Naples, Italy; Prof Louis Havekes, Prof Rune Frants: TNO-PG, Gaubius Laboratory and the Department of Human Genetics, University of Leiden, Leiden, Netherlands; Prof Gerd Utermann: Institute of Medical Biology and Human Genetics, Innsbrück, Austria. References 1. Goldstein JL, Schrott HG, Hazzard WR, Bierman EL, Motulsky AG. Hyperlipidemia in coronary heart disease, II: genetic analysis of lipid levels in 176 families and delineation of a new inherited disorder, combined hyperlipidemia. J Clin Invest. 1973;52:1544 1568. 2. Nikkilä EA, Aro A. Family study of serum lipids and lipoproteins in coronary heart disease. Lancet. 1973;1:954 959. 3. Rose HG, Kranz P, Weinstock M, Juliano J, Haft JL. Inheritance of combined hyperlipoproteinemia: evidence for a new lipoprotein phenotype. Am J Med. 1973;54:148 160. 4. Bredie SJ, Kiemeney LA, de Haan AF, Demacker PN, Stalenhoef AF. Inherited susceptibility determines the distribution of dense low-density lipoprotein subfraction profiles in familial combined hyperlipidemia. Am J Hum Genet. 1996;58:812 822. 5. Hokanson JE, Austin MA, Zambon A, Brunzell JD. Plasma triglyceride and LDL heterogeneity in familial combined hyperlipidemia. Arterioscler Thromb. 1993;13:427 434. 6. Hokanson JE, Krauss RM, Albers JJ, Austin MA, Brunzell JD. LDL physical and chemical properties in familial combined hyperlipidemia. Arterioscler Thromb Vasc Biol. 1995;15:452 459. 7. Dejager S, Bruckert E, Chapman MJ. Dense low density lipoprotein subspecies with diminished oxidative resistance predominate in combined hyperlipidemia. J Lipid Res. 1993;34:295 308. 8. Grundy SM, Chait A, Brunzell J. Meeting summary: familial combined hyperlipidemia workshop. Arteriosclerosis. 1987;7:203 207. 9. Cabezas MC, de Bruin TW, de Valk HW, Shoulders CC, Jansen H, Erkelens DW. Impaired fatty acid metabolism in familial combined hyperlipidemia: a mechanism associating hepatic apolipoprotein B overproduction and insulin resistance. J Clin Invest. 1993;92:160 168. 10. Cabezas MC, de Bruin TW, Jansen H, Kock LA, Kortlandt W, Erkelens DW. Impaired chylomicron remnant clearance in familial combined hyperlipidemia. Arterioscler Thromb. 1993;13:804 814. 11. Arner P. Is familial combined hyperlipidaemia a genetic disorder of adipose tissue? Curr Opin Lipidol. 1997;8:89 94. 12. Hayden MR, Kirk H, Clark C, Frohlich J, Rabkin S, McLeod R, Hewitt J. DNA polymorphisms in and around the Apo-A1-CIII genes and genetic hyperlipidemias. Am J Hum Genet. 1987;40:421 430. 13. Wojciechowski AP, Farrall M, Cullen P, Wilson TM, Bayliss JD, Farren B, Griffin BA, Caslake MJ, Packard J, Shepherd J, Thakker R, Scott J. Familial combined hyperlipidaemia linked to the apolipoprotein AI-CIII-AIV gene cluster on chromosome 11q23 q24. Nature. 1991;349: 161 164. 14. Tybjaerg-Hansen A, Nordestgaard BG, Gerdes LU, Faergeman O, Humphries SE. Genetic markers in the apo AI-CIII-AIV gene cluster for combined hyperlipidemia, hypertriglyceridemia, and predisposition to atherosclerosis. Atherosclerosis. 1993;100:157 169.

1816 The ApoA-I/C-III/A-IV Gene Cluster and FCHL 15. Xu CF, Talmud P, Schuster H, Houlston R, Miller G, Humphries S. Association between genetic variation at the APO AI-CIII-AIV gene cluster and familial combined hyperlipidaemia. Clin Genet. 1994;46: 385 397. 16. Dallinga-Thie GM, Bu XD, van Linde-Sibenius Trip M, Rotter JI, Lusis AJ, de Bruin TW. Apolipoprotein A-I/C-III/A-IV gene cluster in familial combined hyperlipidemia: effects on LDL-cholesterol and apolipoproteins B and C-III. J Lipid Res. 1996;37:136 147. 17. Marcil M, Boucher B, Gagne E, Davignon J, Hayden M, Genest J Jr. Lack of association of the apolipoprotein A-I-C-III-A-IV gene XmnI and SstI polymorphisms and of the lipoprotein lipase gene mutations in familial combined hyperlipoproteinemia in French Canadian subjects. J Lipid Res. 1996;37:309 319. 18. Wijsman EM, Motulsky AG, Guo SW, Yang M, Austin MA, Brunzell JD, Deep S. Evidence against linkage of familial combined hyperlipidemia to the apo AI-CIII-AIV gene complex. Circulation. 1992;86(suppl I):I-420. Abstract. 19. Patsch W, Sharrett AR, Chen IY, Lin-Lee YC, Brown SA, Gotto AM, Boerwinkle E Jr. Associations of allelic differences at the A-I/C-III/A-IV gene cluster with carotid artery intima-media thickness and plasma lipid transport in hypercholesterolemic-hypertriglyceridemic humans. Arterioscler Thromb. 1994;14:874 883. 20. Wijsman EM, Brunzell JD, Jarvik GP, Austin MA, Motulsky AG, Deep SS. Evidence against linkage of familial combined hyperlipidemia to the apolipoprotein AI-CIII-AIV gene complex. Arterioscler Thromb Vasc Biol. 1998;18:215 226. 21. Dallinga-Thie GM, van Linde-Sibenius Trip M, Rotter JI, Cantor RM, Bu X, Lusis AJ, de Bruin TW. Complex genetic contribution of the apoai- CIII-AIV gene cluster to familial combined hyperlipidemia: identification of different susceptibility haplotypes. J Clin Invest. 1997;99:953 961. 22. Porkka KVK, Nuotio I, Pajukanta P, Ehnholm C, Suurinkeroinen L, Syvänne M, Lehtimäki T, Lahdenkari A-T, Lahdenperä S, Ylitalo K, Antikainen M, Perola M, Raitakari OT, Kovanen P, Viikari JSA, Peltonen L, Taskinen M-R. Phenotype expression in familial combined hyperlipidemia. Atherosclerosis. 1997;133:245 253. 23. Syvänne M, Vuorinen-Markkola H, Hilden H, Taskinen M-R. Gemfibrozil reduces postprandial lipemia in non-insulin-dependent diabetes mellitus. Arterioscler Thromb. 1993;13:286 295. 24. Taskinen M-R, Kuusi T, Helve E, Nikkilä E, Yki-Järvinen H. Insulin therapy induces antiatherogenic changes of serum lipoproteins in noninsulin dependent diabetes. Arteriosclerosis. 1988;18:168 177. 25. Cuthbert JA, East CA, Bilheimer DW. Detection of familial hypercholesterolemia by assaying functional low-density-lipoprotein receptors on lymphocytes. N Engl J Med. 1986;314:879 883. 26. Vandenplas S, Wud I, Grobler-Rabie A, Brebner K, Ricketts M, Wallis G, Besler A, Boyd C, Mathew C. Blot hybridization analysis of genomic DNA. J Med Genet. 1984;21:164 172. 27. Shoulders CC, Narcisi ME, Jarmuz JJ, Bayliss JD, Scott J. Characterization of genetic markers in the 5 flanking region of the apoa-i gene. Hum Genet. 1993;91:197 198. 28. Jeenah M, Kessling A, Miller N, Humphries S. G to A substitution in the promoter region of the apolipoprotein A-I gene is associated with elevated serum apolipoprotein A-I and high density lipoprotein cholesterol concentration. Mol Biol Med. 1990;7:233 241. 29. Karathanasis SK, Zannis VI, Breslow JL. Isolation and characterization of cdna clones corresponding to two different human apoc-iii alleles. J Lipid Res. 1985;26:451 456. 30. Syvänen AC, Sajantila A, Lukka M. Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing. Am J Hum Genet. 1993;52:46 59. 31. Lathrop GM, Lalouel J-M, Julier CA, Ott J. Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci U S A. 1984;81: 3443 3446. 32. Ott J. Analysis of Human Genetic Linkage. 2nd ed. Baltimore, Md: Johns Hopkins University Press; 1991. 33. Lathrop GM, Lalouel J-M, White RL. Construction of human genetic linkage maps: likelihood calculations for multilocus analysis. Genet Epidemiol. 1986;3:39 52. 34. Cottingham RW Jr, Idury RM, Schaffer AA. Faster sequential genetic linkage computations. Am J Hum Genet. 1993;53:252 263. 35. Schaffer AA, Gupta SK, Shriram K, Cottingham RW Jr. Avoiding recomputation in linkage analysis. Hum Hered. 1994;44:225 237. 36. Terwillinger JD. Program SIBPAIR: sibpair analysis on nuclear families (used with the program package ANALYZE). Available at http:// linkage.rockefeller.edu. Assessed September 14, 1998. 37. Vartiainen E, Puska P, Jousilahti P, Korhonen HJ, Tuomilehto J, Nissinen A. Twenty-year trends in coronary risk factors in North Karelia and in other areas of Finland. Int J Epidemiol. 1994;23:495 504. 38. Porkka KVK, Viikari J, Rönnemaa T, Marniemi J, Åkerblom HK. Age and gender specific serum lipid percentiles of Finnish children and young adults: the Cardiovascular Risk in Young Finns study. Acta Paediatr. 1994;83:838 848. 39. Ott J. Computer-simulation methods in human linkage analysis. Proc Natl Acad Sci U S A. 1989;86:4175 4178. 40. Weeks DE, Ott J, Lathrop GM. SLINK: a general simulation program for linkage analysis. Am J Hum Genet. 1990;47(suppl):A204. 41. Terwilliger JD, Ott J. A haplotype-based haplotype relative risk approach to detecting allelic associations. Hum Hered. 1992;42:337 346. 42. Terwilliger JD. A powerful likelihood method for the analysis of linkage disequilibrium between trait loci and one or more polymorphic marker loci. Am J Hum Genet. 1995;56:777 787. 43. Slatking M, Excoffier L. Testing for linkage disequilibrium in genotypic data using the EM algorithm. Heredity. 1996;76:377 383. 44. Guo S, Thompson E. Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrix. 1992;48:361 372. 45. Schneider S, Kueffer J-M, Roessli D, Excoffier L. ARLEQUIN: a Software Package for Population Genetics. Geneva, Switzerland: Genetics and Biometry Laboratory, Department of Anthropology, University of Geneva; 1996. 46. Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES. Parametric and nonparametric linkage analysis: a unified multipoint approach. Am J Hum Genet. 1996;58:1347 1363. 47. Raymond M, Rousset F. An exact test for population differentiation. Evolution. 1995;49:1280 1283. 48. Cullen P, Farren B, Scott J, Farrall M. Complex segregation analysis provides evidence for a major gene acting on serum triglyceride levels in 55 British families with familial combined hyperlipidemia. Arterioscler Thromb. 1994;14:1233 1249. 49. Jarvik GP, Brunzell JD, Austin MA, Krauss RM, Motulsky AG, Wijsman E. Genetic predictors of FCHL in four large pedigrees: influence of apob level major locus predicted genotype and LDL subclass phenotype. Arterioscler Thromb. 1994;14:1687 1694. 50. Pajukanta P, Nuotio I, Terwilliger JD, Porkka KVK, Ylitalo K, Pihlajamäki J, Suomalainen AJ, Syvänen A-C, Lehtimäki T, Viikari J, Laakso M, Taskinen M-R, Ehnholm C, Peltonen L. Linkage of familial combined hyperlipidaemia to chromosome 1q21 q23. Nat Genet. 1998; 18:369 373. 51. Castellani LW, Weinreb A, Bodnar J, Goto AM, Doolittle M, Mehrabian M, Demant, Lusis AJ. Mapping a gene for combined hyperlipidemia in a mutant mouse strain. Nat Genet. 1998;18:374 377. 52. Coresh J, Beaty TH, Kwiterovich PO Jr, Antonarakis SE. Pedigree and sib-pair linkage analysis suggest the apolipoprotein B gene is not the major gene influencing plasma apolipoprotein B levels. Am J Hum Genet. 1992;50:1038 1045. 53. Hoffer MJ, Bredie SJ, Boomsma DI, Reymer PW, Kastelein JJ, de Knijff P. Demacker PN, Stalenhoef AF, Havekes LM, Frants RR. The lipoprotein lipase (Asn2913Ser) mutation is associated with elevated lipid levels in families with familial combined hyperlipidaemia. Atherosclerosis. 1996;119:159 167. 54. Gagne E, Genest J Jr, Zhang H, Clarke LA, Hayden MR. Analysis of DNA changes in the LPL gene in patients with familial combined hyperlipidemia. Arterioscler Thromb. 1994;14:1250 1257. 55. Babirak SP, Iverius P-H, Fujimoto WY, Brunzell JD. Detection and characterization of the heterozygote state for lipoprotein lipase deficiency. Arteriosclerosis. 1989;9:326 334. 56. Teng B, Thompson GR, Sniderman AD, Forte TM, Krauss RM, Kwiterovich PO Jr. Composition and distribution of low density lipoprotein fractions in hyperapobetalipoproteinemia, normolipidemia and familial hypercholesterolemia. Proc Natl Acad Sci U S A. 1986;80: 6662 6666. 57. Francke U, Brown MS, Goldstein JL. Assignment of the human gene for the low density lipoprotein receptor to chromosome 19: synteny of a receptor, a ligand, and a genetic disease. Proc Natl Acad Sci U S A. 1984;81:2826 2830. 58. Nishina PM, Johnson JP, Naggert JK, Krauss RM. Linkage of atherogenic lipoprotein phenotype to the low density lipoprotein receptor locus on the short arm of chromosome 19. Proc Natl Acad Sci U S A. 1992;89: 708 712. 59. Guerin M, Bruckert E, Dolphin PJ, Chapman MJ. Absence of cholesteryl ester transfer protein-mediated cholesteryl ester mass transfer from high-

Tahvanainen et al November 1998 1817 density lipoprotein to low-density lipoprotein particles is a major feature of combined hyperlipidaemia. Eur J Clin Invest. 1996;26:485 494. 60. Tato F, Vega GL, Tall AR, Grundy SM. Relation between cholesterol ester transfer protein activities and lipoprotein cholesterol in patients with hypercholesterolemia and combined hyperlipidemia. Arterioscler Thromb Vasc Biol. 1995;15:112 120. 61. Cabezas CM, de Bruin TW, Van Linde-Sibenius Trip M, Kock LA, Jansen H, Erkelens DW. Lipoprotein(a) plasma concentrations associated with lipolytic activities in eight kindreds with familial combined hyperlipidemia and normolipidemic subjects. Metab Clin Exp. 1993;42:756 761. 62. Reynisdottir S, Eriksson M, Angelin B, Arner P. Impaired activation of adipocyte lipolysis in familial combined hyperlipidemia. J Clin Invest. 1995;95:2161 2169. 63. Gehrish S, Tesche R, Kostka H, Julius U, Jarose W. Point mutations in the promoter of hepatic lipase (HTGL) in familial combined hyperlipidemia (FCHLL). Circulation. 1995;92(suppl I):I-493. Abstract. 64. Pajukanta P, Porkka KV, Antikainen M, Taskinen MR, Perola M, Murtomäki-Repo S, Ehnholm S, Nuotio I, Suurinkeroinen L, Lahdenkari AT, Syvänen AC, Viikari JS, Ehnholm C, Peltonen L. No evidence of linkage between familial combined hyperlipidemia and genes encoding lipolytic enzymes in Finnish families. Arterioscler Thromb Vasc Biol. 1997;17:841 850. 65. Aitman TJ, Godsland IF, Farren B, Crook D, Wong HJ, Scott J. Defects of insulin action on fatty acid and carbohydrate metabolism in familial combined hyperlipidemia. Arterioscler Thromb Vasc Biol. 1997;17: 748 754. 66. Baldo A, Sniderman AD, St-Luce S, Avramoglu RK, Maslowska M, Hoang B, Monge JC, Bell A, Mulay S, Cianflone K. The adipsinacylation stimulating protein system and regulation of intracellular triglyceride synthesis. J Clin Invest. 1993;92:1543 1547. 67. Kwiterovich P Jr, Motevalli M, Miller M. Acylation-stimulatory activity in hyperapobetalipoproteinemic fibroblast: enhanced cholesterol esterification with another serum basic protein, BP II. Proc Natl Acad Sci U S A. 1990;87:8980 8984. 68. Sharp D, Blinderman L, Combs KA, Kienzle B, Ricci B, Wager-Smith K, Gil CM, Turck CW, Bouma M-E, Rader DJ, Aggerbeck LP, Gregg RE, Gordon DA, Wetterau JR. Cloning and gene defects in microsomal triglyceride transfer protein associated with abetalipoproteinaemia. Nature. 1993;365:65 69. 69. Paul-Hayase H, Rosseneu M, Robinson D, Van Bervliet JP, Deslypere JP, Humphries SE. Polymorphisms in the apolipoprotein (apo) AI-CIII-AIV gene cluster: detection of genetic variation determining plasma apo AI, apo CIII and apo AIV concentrations. Hum Genet. 1992;88:439 446. 70. Marasco O, Melina F, Mele E, Quaresima B, Zingone A, Focarelli E, Picciotti E, Martelli ML, Fotino L, Vigna MF, Baudi F, Dominijanni A, Angotti E, Pujia A, Perrotti N, Colonna A, Mattioli PL, Porcellini A, Costanzo F, Avvedimento VE. Linkage disequilibrium of three polymorphic RFLP markers in the apolipoprotein AI-CIII gene cluster on chromosome 11. Hum Genet. 1993;91:169 174. 71. Angotti E, Mele E, Costanzo F, Avvedimento EV. A polymorphism (G3A transition) in the 78 position of the apolipoprotein A-I promoter increases transcription efficiency. J Biol Chem. 1994;269:17371 17374. 72. Lander ES, Schork NJ. Genetic dissection of complex traits. Science. 1994;265:2037 2048.