© 1996 American Heart Association, Inc.
Articles |
Polymorphic Markers in Apolipoprotein C-III Gene Flanking Regions and Hypertriglyceridemia
the Department of Medicine, Baylor College of Medicine, Houston, Tex (A.P.S., L.S.); the Human Genetics Center, University of Texas Health Science Center, Houston (G.P.P., E.B.); and the Department of Laboratory Medicine, Landeskrankenanstalten Salzburg, Austria (W.P.).
Correspondence to Eric Boerwinkle, PhD, Human Genetics Center, University of Texas Health Science Center, Houston, TX 77225. E-mail eboerwin@gsbs.gs.uth.tmc.edu.
Abstract
Hypertriglyceridemia and hyperlipidemia are common disorders associated with coronary artery disease and premature death. The proteins encoded by the apolipoprotein (apo) A-I/C-III/A-IV gene cluster are involved in the metabolism of both triglycerides and cholesterol. In a large sample of individuals from the ARIC study, six polymorphic markers were typed and plasma lipid values were measured to determine whether the well-established association between the Sst I S2 allele in the 3'-untranslated region of the apo C-III gene and hypertriglyceridemia was due to disequilibrium with variation in the 5' regulatory region of the apo C-III gene. The Sst I polymorphism was significantly associated with hypertriglyceridemia (P=.006) but not with carotid artery wall thickness, plasma apo C-III levels, or elevated cholesterol. The frequency of the S2 allele was 0.14 in those with high triglyceride levels and 0.05 in those with low triglyceride levels. None of the 5' flanking polymorphisms were significantly associated with any of the plasma lipids studied. There was substantial linkage disequilibrium between the Sst I polymorphism and each of the 5' apo C-III polymorphisms; however, the significant association between the apo C-III haplotypes and hypertriglyceridemia (odds ratio, 4.0; P<.0001) was solely attributable to the effects of the Sst I polymorphism (odds ratio, 3.96). As a part of these analyses, we also defined a unique haplotype that is inversely associated with the occurrence of hypertriglyceridemia, suggesting further molecular analyses of this important gene region.
Key Words: apo C-III • triglycerides • cholesterol • haplotypes • NIDDM
Hypercholesterolemia and hypertriglyceridemia have been associated with atherosclerosis,1 myocardial infarction,2 3 4 and premature death.5 Although hyperlipidemia is common, the causes are not well understood, but both genetic and environmental factors have been implicated.
Variation in the apo A-I/C-III/A-IV gene cluster on chromosome 11q23.36 is a candidate for contributing to the occurrence of both hypertriglyceridemia and hypercholesterolemia. Apos A-I and A-IV are associated with HDL particles, whereas apo C-III is found in HDL, LDL, and VLDL particles. The in vivo function of apo C-III is poorly understood, but levels of hepatic apo C-III synthesis are elevated in individuals with hypertriglyceridemia. In transgenic mice, elevated apo C-III levels cause apo E to be displaced from VLDL particles.7 In vitro, apo C-III has been shown to inhibit lipoprotein lipase activity.8 Additional copies of the human apo C-III gene in transgenic mice are associated with hypertriglyceridemia,9 whereas the absence of apo C-III in knockout mice leads to reduced triglycerides.10 Variation within and around the apo C-III gene has been associated with elevated lipid levels and cardiovascular disease.11 In particular, the S2 allele of an Sst I polymorphism in the 3'-untranslated region of the apo C-III gene has been consistently associated with hypertriglyceridemia.12 13 14 Since the Sst I polymorphism is in the 3'-untranslated region of the apo C-III gene, it does not become part of the functional apo C-III protein. Rather, it is more likely to be in linkage disequilibrium with an as yet unknown mutation(s). The observed relationship between plasma apo C-III levels and triglyceride levels15 suggests that the promoter region, a primary site of transcriptional control, is an excellent candidate for the unknown function mutation. In this article, we make use of the large Atherosclerosis Risk in Communities (ARIC) study to investigate the association between apo C-III gene variation and hypertriglyceridemia with and without accompanying hypercholesterolemia.
Methods
Sample Definition
Subjects were selected from the ARIC Study, which includes 
16 000 adults ranging in age from 44 to 65 years and residing in four US cities.16 To avoid race-specific differences, only white men and women were included in the present study. Individuals taking primary lipid-lowering drugs were excluded because of lack of information about the values of triglycerides and cholesterol before medication.
Individuals for this analysis were selected according to their plasma triglyceride and cholesterol levels. The LL group consisted of 50 normolipidic individuals (LDL cholesterol <160 mg/dL and plasma triglyceride <250 mg/dL). The LH group consisted of 48 individuals with normal triglycerides and elevated plasma cholesterol levels (LDL cholesterol 
160 mg/dL and triglycerides <250 mg/dL). The HL group consisted of 122 individuals with elevated triglycerides and normal cholesterol (250 mg/dL
plasma triglycerides <400 mg/dL and LDL cholesterol <160 mg/dL or plasma triglycerides >400 mg/dL and total cholesterol <250 mg/dL). The HH group contained 123 individuals with both elevated plasma triglyceride and cholesterol levels (250 mg/dL plasma triglycerides <400 mg/dL and LDL cholesterol 160 mg/dL or plasma triglycerides 400 mg/dL and total cholesterol 250 mg/dL). For the hypercholesterolemic-hypertriglyceridemic group, all ARIC participants who qualified were included in this study, whereas an equal number of male and female participants were randomly selected for each of the three remaining lipid phenotypes. These values of triglycerides, LDL, and total cholesterol correspond to approximately the 90th percentile for men and 95th percentile for women of this age group.17
Descriptive characteristics of the study subjects are presented in Table 1
. As expected, individuals with elevated triglycerides, with or without accompanying elevated cholesterol, have significantly higher body mass index, lower plasma HDL cholesterol, and lower plasma apo A-I levels. In addition, the frequency of non–insulin-dependent diabetes mellitus (NIDDM) was significantly elevated in those individuals with hypertriglyceridemia. Plasma apo B levels were elevated in both the high-cholesterol groups and the high-triglyceride groups, so that the highest apo B levels occurred in the HH group. Carotid artery wall thickness (CAWT) was measured by B-mode ultrasonography.18 The combined intimal plus media thickness at six sites was used in this analysis. Average CAWT determined by this method was not significantly different among the four lipid phenotype groups (P=.16).
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Laboratory Methods
Blood was drawn and lifestyle variables were recorded during the baseline visit of the ARIC study.16 The blood was separated at the field centers by centrifugation and frozen for later analysis. Genomic DNA was extracted from frozen buffy coats by a salting-out procedure.19 Both allele-specific amplification and allele-specific oligonucleotide hybridization procedures were used to genotype the polymorphic sites. The oligonucleotide sequences, amplification conditions, and allele-specific oligonucleotide hybridization procedures were carried out as previously described.20 21 Analysis of the Sst I polymorphism was carried out by Southern blotting using previously described probes and methods.22
Plasma cholesterol and triglycerides were measured by enzymatic procedures23 24 with a Cobas-Bio or Cobas-Fara centrifugal analyzer (Roche Diagnostics) and corresponding kit (Catalogue Nos. 236691 and 701912, Boehringer Mannheim Diagnostic). Cholesterol was determined in both total HDL and HDL3 subfractions after separation by a polyanion precipitation procedure.25 LDL cholesterol was calculated by Friedewald's equation.26 Plasma apo A-I and B levels were determined by radioimmunoassay.27 28 Lipoprotein(a) levels were determined by an enzyme-linked immunosorbent assay.29 Apo C-II and C-III concentrations were measured by immunodiffusion with antiserum-containing plates and standards obtained by polyanion precipitation (Daiichi Pure Chemical, Tokyo). These measures were determined only in those individuals with hypertriglyceridemia (the HL and HH groups).
Statistical Methods
The association between apo C-III alleles and genotypes with plasma lipid phenotypes was assessed with a contingency 
2 or a Monte Carlo estimation of Fisher's exact probability.30 Fit of the observed genotype frequencies to Hardy-Weinberg expectations was determined with a 2 goodness-of-fit test. Haplotypes were constructed with maximum likelihood methods.31 The standardized pairwise linkage disequilibrium statistic (D')32 was calculated to summarize the degree of association between the apo C-III markers. The hypothesis that mean plasma lipid, lipoprotein, or apolipoprotein levels differed among apo C-III genotypes was tested with a two-way ANOVA accounting for unbalanced data.33 Odds ratios were calculated by use of established methods.34
Results
Comparison of Apo C-III Allele Frequencies Among Lipoprotein Phenotype Groups
Individuals were typed for six polymorphic sites of interest flanking the apo C-III gene: -641, -630, -625, -482, -455, and Sst I. All sites were diallelic, with the more common allele called "1" and the rarer allele called "2." The observed genotype and allele frequencies for each site in all four lipid phenotype groups are shown in Table 2. The genotypic frequencies at polymorphic sites -641, -630, -625, -482, and -455 were not significantly different among the four lipoprotein phenotype groups. The frequencies of the -641 alleles were f1=60.2% and f2=39.8%; the -630 alleles, f1=60.0% and f2=40.0%; the -625 alleles, f1=60.0% and f2=40.0%; the -482 alleles, f1=67.8% and f2=32.2%; and the -455 alleles, f1=65.2% and f2=34.8%.
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The Sst I allele frequencies, on the other hand, were significantly different among the four groups (2=12.53, df=3, P=.006). The greatest difference was observed between the high-triglyceride groups (HH and HL) versus the low-triglyceride groups (LH and LL) (2=19.31, df=1, P<.001). The frequency of the rarer S2 allele was 14.3% in those with high triglyceride levels and 5.2% in those with low triglyceride levels. Of the 68 people carrying the Sst I S2 allele, 58 had hypertriglyceridemia, and all individuals homozygous for the S2 (5/5) allele had hypertriglyceridemia. No differences were observed between the HH group and the HL group (2=0.98, df=1, P=.32), the LH group and the LL group (2=0.56, df=1, P=.45), or the high cholesterol groups (HH and LH) and the low cholesterol groups (HL and LL) (2=1.29, df=1, P=.26) at the Sst I polymorphism.
The present sample contained 39 individuals with diagnosed non–insulin-dependent diabetes mellitus (NIDDM), and 38 of the 39 individuals had hypertriglyceridemia. The frequency of the Sst I S2 allele in the subjects with NIDDM was not significantly different from that in the nondiabetics without hypertriglyceridemia (5.3% versus 5.2%), as had been reported previously.35 When the individuals with NIDDM were excluded from the analysis, the frequency of the Sst I S2 allele rose in the individuals with hypertriglyceridemia, making the difference in S2 frequency between the low- and high-triglyceride groups more pronounced (5.2% versus 16.1%) (2=25.76, df=1, P<.001).
Effects of the Apo C-III Polymorphisms on Plasma Lipid Levels
Analyses were performed within each lipid group to determine whether any of the apo C-III flanking markers affected plasma lipid, apolipoprotein, or CAWT values. None of the 5' flanking markers exhibited significant effects. Average total cholesterol, LDL cholesterol, HDL cholesterol, apo A-I, apo B, apo C-II, and apo C-III levels and CAWT were not significantly different among 5' apo C-III genotypes (data not shown). As expected, average plasma triglyceride levels were significantly different among Sst I genotypes (P=.0033;S1/S1=244.7±119.6 mg/dL, S1/S2=289.6±95.2mg/dL, and S2/S2=365.8±141.8 mg/dL). None of the other lipid or lipoprotein levels were significantly different among Sst I genotypes. In particular, average apo C-III levels were not significantly different among Sst I genotypes. In addition, the apo C-III Sst I polymorphism and the promoter polymorphisms were not associated with plasma apo C-II/C-III, apo C-II/A-IV, apo E/C-III, or apo E/IV ratios (data not shown).22 Therefore, the observed association between the apo C-III Sst I polymorphisms and hypertriglyceridemia is not mediated through the effect of the gene on plasma apo C-III levels or lipoprotein particle modeling. Likewise, average CAWT was not significantly different among Sst I genotypes.
Haplotype Effects
Haplotypes were constructed by use of a maximum likelihood method.31 Extensive linkage disequilibrium was observed across the entire region; the values of the standardized pairwise linkage disequilibrium coefficients were always >.92. The pairwise disequilibrium statistics are given in Table 3. Disequilibrium between loci in the apo A-I/C-III/A-IV gene cluster has been reported by others.36 37 Of the 626 chromosomes completely typed for the -641, -630, and -625 polymorphisms, 624 of the chromosomes were in complete association; the three locus haplotypes were either 111 (frequency, 60.1%) or 222 (frequency, 39.9%). Therefore, these three systems were combined, arbitrarily labeled -630, and considered simultaneously in further analyses. Of the 534 chromosomes in nondiabetic subjects completely typed for the -630, -482, -455, and Sst I polymorphisms, 9 of 16 possible haplotypes were present in this sample. The frequencies of the apo C-III haplotypes within each of the four lipid phenotype groups are presented in Table 4. The most frequent haplotype within each of the lipoprotein phenotype groups was 1111. The average frequency of the 1111 haplotypes in the entire sample was 59.7%.
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Analyses relating apo C-III haplotype frequencies to hypertriglyceridemia were limited to nondiabetics, because the association with the Sst I polymorphism was limited to individuals without NIDDM. Apo C-III haplotype frequencies were significantly different among triglyceride groups (P<.001). The haplotype 2222 was more frequent in those individuals with elevated triglyceride levels, while the haplotype 2121 was less frequent in the high-triglyceride groups. The association between apo C-III gene variation and hypertriglyceridemia was attributable predominantly to the effects of the Sst I polymorphism. When the Sst I polymorphism was excluded from the analysis, there was no significant association between haplotypes on the basis of the other three apo C-III polymorphisms and triglyceride groupings. The odds ratio for the presence of the Sst I S2 allele alone in predicting hypertriglyceridemia was 3.96 (95% confidence interval, 3.27 to 4.65), whereas the odds ratio for haplotype 2222 was 4.0 (95% confidence interval, 3.39 to 4.81). Therefore, addition of other apo C-III polymorphisms did not add significantly to the prediction of hypertriglyceridemia.
Discussion
We have characterized the association between apo C-III gene variation and hypertriglyceridemia in a sample of 343 individuals from the ARIC study. The primary objective of this study was to determine whether the well-known association between the 3' Sst I polymorphism could be accounted for by its association with biologically significant mutations in the 5' regulatory region of the apo C-III gene. In our sample, the Sst I polymorphism was significantly associated with hypertriglyceridemia, and this association was particularly marked when individuals with clinically recognized NIDDM were excluded. The frequency of the rare Sst I allele in those with hypertriglyceridemia was 14.3% compared with 5.2% in those with normal levels of plasma triglycerides. Five sites in the 5' region of the apo C-III gene were typed and analyzed. Even though there was significant linkage disequilibrium between these 5' sites and the 3' Sst I polymorphism, the 5' loci were not significantly associated with plasma triglyceride phenotype, taken either individually or as haplotypes. Addition of the 5' sites to a model predicting triglyceride phenotype status was not significant after the Sst I polymorphism was considered. Therefore, the observed association between the apo C-III Sst I polymorphism and hypertriglyceridemia observed in these data was not attributable to functional variation in the 5' regulatory region of the apo C-III gene.
Apo A-I/C-III/A-IV genes are candidates for hypertriglyceridemia and hyperlipidemia because of the central role of the products of the gene in the structure and function of several lipoprotein particles. Several studies have found associations between a broad spectrum of apo A-I/C-III/A-IV gene variation and hypertriglyceridemia,1 38 39 but the results have been inconsistent. However, the association between the Sst I polymorphism in the 3' untranslated region of the apo C-III gene and hypertriglyceridemia is relatively strong and has been generally consistent among various studies. A summary of the results from other studies considering the Sst I polymorphism is presented in Table 5. The S2 allele of the Sst I polymorphism has been associated with hypertriglyceridemia in most studies of Caucasians,1 2 5 11 12 20 43 American Indians,48 Arabs,13 and Japanese.14 Consistent with the observed association between the S2 allele and hypertriglyceridemia is a report that the S2 allele is also associated with lower levels of HDL in young adults.49 However, other studies of Japanese47 and Caucasians40 have found conflicting results. In addition, the S2 allele has also been associated with clinically recognized coronary heart disease4 11 but not with NIDDM.35
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In the present study, the frequency of the S2 allele in individuals with hypertriglyceridemia was 14.2% compared with 5.2% in individuals with normal triglyceride levels. These frequencies are similar to those observed in other samples of Caucasians (Table 5
). Normal Danish men1 had an S2 allele frequency of 9%, whereas in individuals with elevated triglycerides, the frequency was 16%. In the Framingham study,11 the reported S2 frequency was 8% in control subjects and 12% in individuals with CHD. A normal English population2 had a reported S2 frequency of 6.7%. The S2 frequency in lipid clinic patients20 was 25.9%, compared with 8.3% in their first-degree relatives.
Because the Sst I polymorphic site is located in the 3' untranslated region of the apo C-III gene, it is generally considered not to alter the function or regulation of the apo C-III gene but rather is in linkage disequilibrium with a second, as yet unknown, biologically functional mutation related to hypertriglyceridemia. The established relationship between plasma apo C-III levels and triglyceride metabolism9 10 15 50 51 suggests that the promoter region of the apo C-III gene is a likely candidate region for the functional mutation. Within the apo C-III promoter region, there are several sequences that are homologous to known positive and negative enhancers of protein expression. Mutational analysis of the promoter region suggested that nucleotides -821 and -685 are needed for maximum expression of apo C-III in the liver.52 A phosphoenolpyruvate carboxykinase insulin/phorbol ester response element has been shown to be active in regulating apo C-III expression.53 The transcription factor NF-
B has been shown to bind to several sites in the promoter region.54 Footprint analysis has shown that there are several active regions: nucleotides -890 to -686 bind both intestinal and hepatic transcription factors, whereas nucleotides -686 to -553 bind only hepatic transcription factors.55 In this study, polymorphic sites at nucleotides -641, -630, -625, -482, and -455 in the apo C-III promoter region were typed in four groups of subjects differing according to plasma triglyceride and cholesterol levels. None of these sites were significantly associated with plasma triglyceride levels taken individually or as haplotypes, indicating that they are not the functional changes responsible for the observed association between the 3' Sst I polymorphism and triglyceride levels. At this juncture, it is perhaps worth pointing out that there is ample precedent for regulatory elements that reside 3' to the structural gene,56 57 and perhaps we have been too eager to implicate the 5' region of genes for the observed associations between plasma lipid levels and nonfunctional DNA variation.
Haplotypes20 based on the -625, -482, and Sst I polymorphisms in the apo C-III gene were reportedly strongly associated with hypertriglyceridemia. The 222 haplotype was reported to have a relative risk for being hypertriglyceridemic of 3.14. However, it is interesting to note that the Sst I S2 allele by itself had a relative risk of 3.85. Therefore, it appears that the data20 are very similar to those reported here, in that the Sst I polymorphism was significantly associated with hypertriglyceridemic status and that addition of the 5' polymorphisms did not strengthen the observed association. However, the interpretations of these data are different. It has been suggested20 that the causative mutation responsible for the observed Sst I association is in the 5' regulatory domain of the apo C-III gene, and more recently these authors have suggested that -455 is the site of the causative mutation because it alters a putative insulin/phorbol ester response element. In the present study, we conclude that the causative mutation is not one of the variable sites typed in the 5' regulatory domain of the apo C-III gene. In particular, the -455 polymorphism, which resides within the insulin/phorbol ester response element, was not significantly associated with the occurrence of hypertriglyceridemia in this sample and therefore cannot be the causative variation responsible for the Sst I association.
In our study, the observed association between the Sst I polymorphism and hypertriglyceridemia was strengthened when individuals with NIDDM were excluded from the analyses. There were 39 individuals with NIDDM, of whom 38 had hypertriglyceridemia. The allele frequencies for the Sst I polymorphism were not significantly different between those with NIDDM and individuals with normal triglyceride levels (5.3% versus 5.2%). A second study35 also reported that the frequency of the Sst I S2 allele in individuals with NIDDM is similar to that in individuals from the general population,35 even though diabetics usually have elevated triglyceride levels. These data suggest that there is heterogeneity in the genetic factors contributing to hypertriglyceridemia in the general population; one set of factors is clearly linked to the apo C-III gene, and another set is contributing to the onset of NIDDM.
The 211 haplotype based on the -625, -482, and Sst I polymorphisms has been reported to be protective20 with respect to the development of hypertriglyceridemia and had a relative risk of 0.28. In our study, this haplotype corresponds to haplotypes 2111 and 2121, because of the additional data from the -455 polymorphism (underlined). Confirming the previous report, these two haplotypes combined are less frequent in the sample of hypertriglyceridemic compared with normotriglyceridemic individuals (P=.03). However, when the haplotypes are analyzed separately, the 2121 haplotype appears to be protective (odds ratio, 0.30; P=.005), whereas 2111 does not (odds ratio, 1.01; P=.79). Therefore, these data suggest that there is a protective mutation in linkage disequilibrium with the 2121 haplotype.
Acknowledgments
This work was supported by grants HL-40613 and HL-27341 from the National Institutes of Health and contracts N01-HC-55015, N01-HC-55016, N01-HC-55018, N01-HC-55019, N01-HC-55020, N01-HC-55021, and N01-HC-55022 from the National Heart, Lung, and Blood Institute. Dr Boerwinkle is an Established Investigator of the American Heart Association and the recipient of a Research Career Development Award from the National Institutes of Health (HL-02453). We gratefully acknowledge the statistical contributions of Kim Lawson. We would also like to acknowledge the assistance of the ARIC investigators: Carol Summers, Catherine Burke, Deanna Horwitz, and Carmen Woody from the Forsyth County, North Carolina, field center; Agnes L. Hayes, Roberta J. Howell, Jane G. Johnson, and Patricia F. Martin from the Jackson, Miss, field center; Barbara Kuehl, Anne Murrell, Bryna Lester, and Jennifer Hill from the Minneapolis, Minn, field center; Joel G. Hill, Patricia M. Crowley, Joyce B. Chabot, and Patricia Hawbaker from the Washington County, Maryland, field center; Valerie Stinson, Pam Pfile, Hoang Pham, and Teri Trevino from the Central Hemostasis Laboratory, Houston, Tex; Karima Ghazzaly, Dandra Sanders, Charles Etta Rhodes, and Doris Epps from the Central Lipid Laboratory, Houston, Tex; Regina DeLacy, Delilah Cook, Carolyn Bell, Teresa Crotts, and Suzanne Pillsbury from the Bowman-Gray School of Medicine; and Larry Crum, Peter DeSaix, Mal Foley, and Tom Goodwin from the Coordinating Center, Chapel Hill, NC.
Received December 11, 1995; revision received March 1, 1996; References
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