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1 From the Lipid Research-Atherosclerosis Division, Departments of Pediatrics and Medicine, Johns Hopkins University School of Medicine, Baltimore.
2 Presented at the symposium Fat Intake During Childhood, held in Houston, June 8–9, 1998. 3 Supported in part by grant HD32193 from the National Institutes of Health and by a grant from the Dorothy Wagner Wallis Charitable Trust. 4 Address reprint requests to SQ Ye, Core Molecular Biology Lab, Lipid Research-Atherosclerosis Division, Department of Pediatrics, CMSC 604, Johns Hopkins University, 600 North Wolfe Street, Baltimore, MD 21287. E-mail:syq{at}mail.jhmi.edu.
ABSTRACT
Genes influence quantitative variations in plasma lipoprotein concentrations. For example, intake of dietary saturated fat and cholesterol raises the average serum cholesterol concentration, leading to a higher risk of coronary artery disease in populations. However, not all individuals within the population are susceptible: genetic factors appear to render individuals either "dietary responsive" or "dietary nonresponsive." In this review, we focus on current knowledge about the influence of genetic polymorphisms in certain genes on the lipoprotein response to dietary fat and cholesterol. Our preliminary studies in the Dietary Intervention Study in Children suggest a significant dose-response relation between the decrease in LDL cholesterol from baseline to 36 mo of follow-up in both the intervention group (who consumed a low-fat, low-cholesterol diet) and the usual care group (who consumed a regular diet) and the presence of the APOA1*A allele at the M1 site and the + site at the M2 site of the gene encoding apolipoprotein (apo) A-I. The DNA polymorphisms on the genes encoding apo A-IV, apo B, apo C-III, apo E, lipoprotein lipase, cholesteryl ester transfer protein, lecithin:cholesterol acyltransferase (phosphatidylcholine–sterol O-acyltransferase), and LDL receptor were found by others to be associated with the plasma lipoprotein response to dietary intervention. Possible mechanisms involved in these effects are discussed and certain discrepancies in the literature about some genetic effects on responsiveness are analyzed. An improved understanding of the influence of specific genes on lipoprotein responsiveness to dietary fat and cholesterol may allow us to identify and counsel certain individuals to avoid high-fat diets so that they may reduce their risk of developing hyperlipidemia and coronary artery disease.
Key Words: DNA polymorphisms • apolipoprotein A-I • apolipoprotein A-IV • apolipoprotein B • apolipoprotein C-III • apolipoprotein E • lipoprotein lipase • cholesteryl ester transfer protein • lecithin:cholesterol acyl transferase • LDL receptor • dietary cholesterol • dietary fat • dietary saturated fat • children • adolescents • triacylglycerol • very-low-density lipoprotein • low-density lipoprotein • high-density lipoprotein
INTRODUCTION
Elevated serum LDL-cholesterol concentrations in the general population result, in part, from a high intake of saturated fats and cholesterol (1). However, individuals vary widely in the response of their LDL-cholesterol concentrations to dietary fat and cholesterol (2). These variations are not explained solely by differences in compliance of patients following dietary regimens, suggesting strongly that genetic, hormonal, and environmental factors are also involved (3). Genetically determined variability of individual response to dietary fat and cholesterol intake has been documented (4). For example, genetic polymorphisms, which are usually derived from one or more mutations in DNA at a particular locus, can influence response to a dietary challenge. Such mutations either create or abolish sites for a specific restriction enzyme. Although intake of dietary saturated fat and cholesterol raises the average serum cholesterol concentration of a population, leading to a higher risk of coronary artery disease (CAD), not all individuals within the population are susceptible because genetic factors appear to render individuals either "dietary responsive" or "dietary nonresponsive." This review updates current evidence in humans for the influence of both structural and nonstructural DNA polymorphisms in genes relevant to lipid metabolism on the responsiveness, or lack of responsiveness, to dietary fat and cholesterol.
LIPOPROTEIN METABOLISM
Human plasma lipoproteins are particles consisting of hydrophobic lipids in their cores, surrounded by a shell of polar lipids and apolipoproteins. Core lipids include triacylglycerol and cholesteryl esters. Shell lipids include unesterified cholesterol and phospholipids. The protein moieties of lipoproteins are called apolipoproteins. To date, 16 apolipoproteins have been isolated and characterized: apolipoprotein (apo) A-I, apo A-II, apo A-IV, apo(a), apo B, apo C-I, apo C-II, apo C-III, apo C-IV, apo D, apo E, apo F, apo G, apo H, apo I, and apo J (5–7). The major lipoproteins are classified according to either their electrophoretic migration or their separation after ultracentrifugation as follows: chylomicrons, VLDL (preß mobility), LDL (ß mobility), and HDL ( mobility). Lipoprotien(a) or Lp(a) (preß mobility) results from the connection of apo B on LDL to apo(a) by a disulfide band. A general schema of human lipoprotein metabolism is presented in Figure 1.
FIGURE 1. . Human lipoprotein metabolism. On the left is depicted the pathway of exogenous (dietary) lipoprotein metabolism. On the right lower portion is depicted the pathway of endogenous (hepatic) lipoprotein metabolism. On the right top portion is depicted the reverse cholesterol transport process or HDL metabolism. Each pathway or process is elaborated in the text. SR-B1, scavenger receptor, class B, type I, a putative HDL receptor; A-I, A-II, B, C-II, and E denote apolipoproteins of the same name.
Plasma lipoproteins are synthesized and secreted by intestine and liver. The apolipoproteins solubilize highly hydrophobic lipids, permitting their transport in blood. Certain apolipoproteins function as ligands for specific receptors that facilitate the uptake and removal of lipoprotein particles or regulate the movement of particular lipids into and out of specific target cells and tissues. Other apolipoproteins serve as cofactors for enzymes important in lipoprotein metabolism.
Dietary triacylglycerol, cholesteryl ester, and phospholipids are emulsified in the intestine by bile salts, their fatty acids are hydrolyzed by pancreatic lipases, and the resultant molecules (fatty acids, 2 monoacylglycerols, and cholesterol) are taken up by intestinal cells. Triacylglycerol and cholesteryl ester are reformed in the intestinal cells and packaged into chylomicrons for excretion into lymph and then blood. Apo B-48, A-1, and A-IV are on the surface of chylomicrons. Apo B-48 is essential for secretion of chylomicrons from the intestine. Once in the circulation, apo C-II, a cofactor for lipoprotein lipase (LPL) is transferred from HDLs to chylomicrons, a process facilitated by apo A-IV. Triacylglycerols in chylomicrons are hydrolyzed within minutes by LPL, which is located on the surface of endothelial cells lining the capillaries of adipose and other peripheral tissues, such as muscle. As the triacylglycerols are hydrolyzed, chylomicron remnants are produced, which are rapidly taken up by the liver through the interaction of apo E on the surface of the chylomicron remnant with the chylomicron receptor. The released fatty acids are either taken up and stored in adipose tissue for future use or utilized by muscle for energy.
Once inside the liver, the cholesteryl esters in the chylomicron remnants are hydrolyzed and the liberated unesterified cholesterol down-regulates the genes for both the LDL receptor and the rate-limiting enzyme of cholesterol biosynthesis, hydroxymethylglutaryl-CoA (HMG-CoA) reductase. Thus, the potential effect of dietary cholesterol to increase LDL concentrations is offset by the inhibition of hepatic cholesterol synthesis. However, in 1 in 4 individuals, HMG-CoA reductase is not down-regulated as efficiently, leading to higher concentrations of LDL cholesterol (2). The saturated fatty acids from the chylomicron remnants, or those that are eventually mobilized from adipose tissue, appear to down-regulate the LDL receptor, leading to an increase in LDL concentrations.
In liver, the triacylglycerol-rich VLDLs are assembled and secreted as particles containing triacylglycerol and cholesteryl ester in the core, surrounded by apo B-100, E, C-I, C-II, and C-III. The fatty acids attached to the glycerol moiety of the triacylglycerol may be derived from either the endogenous synthesis of fatty acids from acetyl CoA or the mobilization of fatty acid from adipose tissue back to the liver. Acetyl CoA derived from amino acids, sugars, and fatty acid oxidation can be used to synthesize fatty acids. Apo B-100 is essential for the secretion of VLDL. Once in the circulation, the triacylglycerol in VLDL is hydrolyzed by LPL and apo C-II, producing fatty acids and the VLDL remnant. The triacylglycerol in the VLDL remnant can be further hydrolyzed to smaller particles called intermediate density lipoproteins (IDLs), which are either taken up by the interaction of apo E with the LDL (B,E) receptor on the surface of the liver or converted by the action of hepatic lipase (hepatic triacylglycerol lipase) into the cholesteryl ester–rich LDL.
LDLs are then bound and internalized as the result of the interaction of apo B-100 with the LDL (B,E) receptor. The cholesteryl esters in the core of LDL are hydrolyzed in lysosomes, producing unesterified cholesterol, which can down-regulate the LDL receptor and HMG-CoA reductase genes through the sterol regulatory element (8). About two-thirds of LDL is removed by the liver and the rest by peripheral tissues. LDL also serves as the major carrier of vitamin E. Elevated concentrations of LDL, and its oxidized derivative, promote atherogenesis by causing endothelial dysfunction, proliferation of arterial smooth muscle cells, and conversion of monocytes into macrophages in the arterial wall, inducing foam cell formation (9).
HDL is secreted from both intestine and liver as nascent particles. These nascent particles contain phospholipids and some unesterified cholesterol in the core, surrounded by a coat of apo A-I. Nascent HDL particles appear to have preß mobility on electrophoresis, and 9 subclasses of nascent HDL have been described (10). Once in the bloodstream, nascent HDLs are converted to a mature form of HDL through the interaction of the enzyme phosphatidylcholine–sterol O-acyltransferase (also called lecithin-cholesterol acyltransferase, or LCAT) and its cofactor, apo A-I. Unesterified cholesterol is removed from peripheral cells and esterified through the action of LCAT and apo A-I, producing cholesteryl ester in the core of more mature HDL particles. During the process of lipolysis, apo C-I, C-II, and C-III can be transferred, along with phospholipids, from VLDL to the mature HDL particle. As more cholesteryl ester is formed, the subfraction of HDL called HDL3 is converted to HDL2. The structure and chemical composition of HDL can also be modified by cholesteryl ester transfer protein (CETP), in which a molecule of cholesteryl ester from HDL is exchanged for a molecule of triacylglycerol on VLDL or a VLDL remnant. The triacylglycerol content of the HDL2 particles is often higher than that in HDL3. HDL2 can be converted back to HDL3 through hydrolysis of triacylglycerol by hepatic lipase.
The cholesteryl ester in the core of HDL can be delivered to sterol-producing cells, such as liver, adrenal glands, ovary, and testis, through the interaction of apo A-I with a putative HDL receptor known as SRB-I (scavenger receptor, class B, type I) (7). This process, often referred to as reverse cholesterol transport, promotes the delivery of cholesteryl ester to the cell. HDL is subsequently released from cells back into the bloodstream to gather more cholesteryl ester. At some point, the entire HDL particle is internalized for catabolism.
The function of Lp(a) is not well understood. The apo(a) in Lp(a) is homologous to plasminogen and can interfere with the thrombolytic effects of the plasmin system. The size of apo(a) varies considerably (from a molecular weight of 200000 to 800000) due to the genetically determined variability in kringle IV repeats. Lp(a) is not modulated by diet.
APOLIPOPROTEIN GENES
Most of the 16 apolipoproteins reported to date (5–7) have been well characterized with respect to structure and functions. Either the complete genes or the complementary DNA for these apolipoproteins, except for apo G and apo I, has been cloned. Based on Online Mendelian Inheritance in Man (OMIM), 20 allelic variants have been found for APOA1, 1 for APOA2, 4 for APOA4, 2 for the apo(a) gene, 17 for APOB, 12 for APOC2, 2 for APOC3, 29 for APOE, and 1 for APOH (5–7). This section will focus on the effect of known common DNA polymorphisms in the apolipoprotein genes, not uncommon single-gene defects, on the responsiveness of apolipoproteins to dietary fat and cholesterol.
APOA1/APOC3/APOA4 gene complex
The APOA1/APOC3/APOA4 gene complex is located on chromosome 11q. This gene complex modulates apolipoprotein and lipid metabolism (11–13) and there appears to be genetic regulation of the expression of the APOA1 and APOA4 genes by the APOC3 gene at the transcriptional level (12, 13).
APOA1
APOA1 encodes apo A-I, an amphipathic protein involved in cholesterol removal from cells and transport back to liver (reverse cholesterol transport) (14). Low apo A-I or HDL-cholesterol concentrations are inversely associated with CAD (15). Although 20 allelic variants of apo A-I have been identified (7), the effect of mutations in the APOA1 promoter [in the -76 base pair (bp) and 83 bp region] on the responsiveness to dietary fat and cholesterol has received the most attention. Both the G-to-A substitution at -75 bp (M1 site) and the C-to-T and G-to-A substitutions at 83 bp (M2 site) can be detected simultaneously in a single restriction fragment length polymorphism (RFLP) analysis with MspI (16–18). The 3 genotypes present at the M1 site have been designated G/G, G/A, and A/A and those at the M2 site as +/+, +/-, and -/- (12). In a normolipidemic population, the frequencies of these alleles are as follows: G/G +/+, 0.556; G/A +/+, 0.313; A/A +/+, 0.049; G/G +/-, 0.058; G/A +/-, 0.025; and A/A +/-, 0.000 (17). These alleles influence the variability in plasma HDL-cholesterol and apo A-1 concentrations and appear to be correlated with CAD (16–18).
Lopez-Miranda et al (19) studied 50 healthy, adult males without a family history of CAD who were switched from a National Cholesterol Education Program Step I diet (containing 12% monounsaturated fat) to a diet containing 22% monounsaturated fat. These investigators showed that, after changing diets, those with the APOA1*A allele had significantly higher LDL-cholesterol concentrations than did those who were homozygous for the APOA1*G allele.
In a pilot study of 303 subjects from the Dietary Intervention Study in Children, we examined the effect of the APOA1 promoter DNA polymorphisms on lipoprotein responsiveness to dietary fat and cholesterol. We found a significant dose-response relation between the decrease in LDL cholesterol from baseline to 36 mo of follow-up in both the intervention group (who consumed a National Cholesterol Education Program Step II diet) and the usual care group (who consumed a regular diet) and the presence of the APOA1*A allele at the M1 site and the + site at the M2 site. Mean reductions in LDL cholesterol were most marked in A/A homozygotes (-17.3%), least marked in G/G homozygotes (-11.0%), and intermediate in A/G heterozygotes (-15.5%). The mean reductions of LDL cholesterol were -14.8% for +/+ homozygotes, -9.8% for +/- heterozygotes, and -9.1% for -/- homozygotes (20). There was also an interaction of the effect of these 2 DNA polymorphisms. These effects were present in both the intervention and the usual care groups, compatible with the interindividual variability in the relation between dietary intake of saturated fat and cholesterol present in both groups (PO Kwiterovich Jr, SQ Ye, et al, unpublished observations, 1998). These pilot data are congruent with previous studies indicating that these gene loci account for a significant portion of the variability in plasma lipid response to dietary alteration (21).
The mechanisms responsible for the observed effect are currently unknown. These mutations may have a direct effect on liver or intestinal APOA1 expression. There were reports that the APOA1*A allele is associated with a 4–7-fold increase in the transcription rate of APOA1 in vitro (22, 23). Although increased apo A-I synthesis may promote reverse cholesterol transport, how this might result in decreased LDL-cholesterol concentrations in those subjects with the APOA1*A allele is not clear. One possibility is that this mutation is in linkage disequilibrium with a functional mutation in a neighboring gene. As well, the APOA1 promoter may interact with a transcription factor that also affects another gene important in LDL metabolism.
APOA4
Apo A-IV is synthesized in the intestine as a 46-kDa apolipoprotein during fat absorption. It is believed to affect reverse cholesterol transport, the assembly and metabolism of HDL, and the transfer of apo C-II, the cofactor for LPL, from HDL to chylomicrons (24, 25). A substitution of guanine (allele 1) with a thymidine (allele 2) in the third base at codon 360 results in a substitution of histidine for glutamine. This results in the loss of the Fnu4HI restriction site and is responsible for the electrophoretically distinct isoform apo A-IV-2 (26). There are 3 genotypes: 1/1, 1/2, and 2/2.
The influence of these alleles on baseline concentrations of plasma lipids, apolipoproteins, and lipoproteins is presently unclear. Those with the 1/2 or 2/2 genotype have significantly smaller increases in plasma LDL-cholesterol concentrations when placed on a high-cholesterol diet than do persons with the 1/1 (normal) genotype (27), an observation that was confirmed in 2 other studies (28). Thus, subjects with the 1/2 or 2/2 genotype are less responsive to dietary intervention in terms of LDL-cholesterol lowering than are their normal counterparts. The APOA4*2 allele accounts for 6–7% of the variance in dietary response (28). The frequency of the APOA4*2 allele is 0.08. Mata et al (29) found a sex difference in the effect of apo A-IV polymorphism on dietary responsiveness. In males (n = 83), there was no association between APOA4 genotype and baseline lipid concentrations, whereas in women, 1/2 heterozygous subjects (n = 12) had higher HDL-cholesterol and lower triacylglycerol concentrations at baseline than did 1/1 homozygous subjects (n = 48). In men, reductions in LDL cholesterol with a low-fat diet were less for those with the 1/2 genotype (-7%; n = 17) than for those with the 1/1 genotype (-16%; n = 76), but no such association between APOA4 genotype and diet-induced lipid changes was found in women.
CAD is increasing in developing countries, particularly in urban areas. The impact of urbanization and the APOA4 polymorphism on plasma lipoprotein concentrations was studied in 222 men and 236 women from rural and urban Costa Rica (30). The APOA4 allele frequencies were 0.937 for APOA4*1 and 0.062 for APOA4*2. Significant interactions between the APOA4 polymorphism and area of residence (rural or urban) were detected for HDL cholesterol (P = 0.003), apo A-I (P = 0.05), LDL particle size (P = 0.01), and ratio of LDL to HDL cholesterol (LDL:HDL) (P = 0.005). Urban compared with rural carriers of the APOA4*2 allele had significantly lower plasma HDL-cholesterol (0.95 compared with 1.17 mmol/L) and apo A-I (980 compared with 1140 mg/L) concentrations, significantly higher LDL:HDL (3.35 compared with 2.39), and significantly smaller LDL particles (258 compared with 263
After taking these lifestyle characteristics into account, the area of residence x genotype interactions for plasma lipoprotein concentrations were no longer statistically significant. Lifestyles associated with an urban environment, such as increased smoking and saturated fat intake, elicit a more adverse plasma lipoprotein profile among Costa Rican carriers of the APOA4*2 allele than among 1/1 homozygotes. Therefore, under such conditions, those with the APOA4*2 allele may be more susceptible to CAD.
The mechanism of the APOA4*2 mutation on these observed effects is not known. The apo A-IV-2 variant binds to lipoproteins with higher affinity than does apo A-IV-1, which may result in delayed hepatic clearance of chylomicron remnants (31). Different apo A-IV isoforms may also differentially affect either lipid absorption in intestine or the metabolism of chylomicrons and chylomicron remnants.
APOC3
Apo C-III is a major apolipoprotein in triacylglycerol-rich lipoproteins, ie, VLDL and chylomicrons (22). Apo C-III appears to inhibit the hydrolysis of triacylglycerol by LPL. Recent studies in human apo C-III transgenic mice indicate that apo C-III promotes hypertriglyceridemia (32), supporting an important role for apo C-III in lipid metabolism in vivo. In addition to inhibition of LPL by apo C-III, increased concentrations of apo C-III may displace apo E from triacylglycerol-rich remnants, thus decreasing their uptake and removal from the circulation.
The C-to-T polymorphism at nucleotide 1100 in the APOC3 gene (C/C, C/T, and T/T genotypes) was reported to affect the consistency of changes in plasma cholesterol in response to dietary fat challenge (33). This mutation does not alter the amino acid sequence but is associated with significantly higher triacylglycerol concentrations. Humphries et al (33) examined the effect of this mutation in APOC3 on both the consistency and magnitude of change in plasma cholesterol concentrations in response to 2 separate changes from a high-saturated-fat diet to a low-saturated, high-polyunsaturated-fat diet in 55 free-living, healthy white men and women. Those with the C/C genotype (and thus the lowest baseline triacylglycerol concentrations) showed a more consistent response to dietary change, as estimated by a higher correlation with change in plasma cholesterol, than did those with the C/T or T/T genotypes. However, the C/C genotype was not associated with the magnitude of the change in cholesterol concentrations. The molecular mechanism or mechanisms of these effects on consistency of response is not known.
The SstI polymorphism, arising from a C-to-G substitution in the 3' untranslated region of the APOC3 gene, distinguishes 2 alleles: APOC3*S1 and APOC3*S2. The *S2 allele has been associated with elevated plasma triacylglycerol, cholesterol, and apo C-III concentrations. In 90 young men, Lopez-Miranda et al (34) examined the effect of the S2 mutation on response of LDL cholesterol to dietary monounsaturated fat. The frequency of the *S2 allele was 0.14. Subjects were fed a low-fat diet for 25 d, followed by a diet rich in monounsaturated fatty acid (22% monounsaturated fatty acid, 38% total fat) for 28 d. There were no significant differences in baseline LDL-cholesterol concentrations between subjects with the S1/S1 and S1/S2 genotypes. After consumption of the diet high in monounsaturated fatty acids, significant increases in LDL cholesterol (0.13 mmol/L, P < 0.027) were noted in the S1/S1 subjects, whereas a significant decrease was observed in the S1/S2 subjects (-0.18 mmol/L, P < 0.046). Significant genotypic effects were seen for diet-induced changes in LDL cholesterol (P < 0.00034), total cholesterol (P < 0.009), and apo B (P < 0.0014).
A study of the effect of the interaction between this mutation and that present in position -76 of the APOA1 gene promoter region (G-to-A) revealed that both mutations have an additive effect on diet-induced changes in total cholesterol, LDL cholesterol, and apo B. Thus, plasma LDL-cholesterol responsiveness to the diet may be explained, at least in part, by variation at the APOC3 gene locus.
APOB
Apo B exists in 2 major forms: apo B-100 synthesized in liver and apo B-48 from intestine (35). Apo B is essential for the synthesis and secretion of both chylomicrons and VLDL, in addition to serving as the principal ligand for the interaction of LDL with LDL receptor (36). Plasma lipoprotein change induced by dietary fat and cholesterol has been related to certain apo B genetic polymorphisms.
Boerwinkle and Chan (37) described a 3-codon insertion-deletion DNA polymorphism in the 27 amino acid signal peptide region of the human APOB gene. Two polymorphic alleles differ by the insertion or deletion of 9 bp that encodes amino acids -16 to -14 (Leu-Ala-Leu) in the apo B signal peptide. The 3 APOB signal peptide genotypes were designated as Ins/Ins, Ins/Del, and Del/Del.
Xu et al (38) investigated the insertion-deletion polymorphism in 106 normal Finnish individuals who had originally participated in the North Karelia Dietary Intervention Studies (39, 40), in which a switch to a diet low in saturated fat and cholesterol from a regular diet produced a significant lowering of plasma LDL-cholesterol, HDL-cholesterol, apo B, and apo A-I concentrations. The relative frequency of the APOB*Ins allele in this Finnish sample was 0.73. Ins/Ins Finns had higher baseline triacylglycerol concentrations (1.3 ± 0.07 mmol/L) than did those with the Ins/Del (1.0 ± 0.4 mmol/l) or Del/Del (0.8 ± 0.4 mmol/L) genotype. After the low-fat diet, a significant genetic dietary difference was found for plasma triacylglycerol; namely, individuals with the Ins/Ins genotype had a mean reduction in plasma triacylglycerol (0.11 ± 0.46 mmol/L), whereas those with one or more APOB*Del alleles showed a mean increase in plasma triacylglycerol (0.7 ± 0.53 mmol/L). It will be of interest to determine whether this difference is due to the effect of APOB signal peptide mutations on apo B secretion.
The MspI polymorphism (CGGG CAGC) in exon 26 of apo B results in an amino acid change (Arg3611Gln). This mutation was found to explain 6.3% of the phenotypic variance in change of apo A-I in response to a low-fat diet (24% fat) in a study of healthy men and women from eastern Finland (41). The common allele (APOB*M+) was associated with a reduction in apo A-I concentrations, whereas the less common allele (APOB*M-) was associated with an increase in apo A-I concentrations, in response to a 15% reduction in dietary fat.
The effects of the XbaI and EcoRI APOB gene polymorphisms on the lipoprotein responsiveness to dietary fat and cholesterol have also been studied (28, 42). The XbaI polymorphism is due to a single base variation in exon 26 of the APOB gene that does not lead to changes in the amino acid sequence. The EcoRI polymorphism is due to a single base variation in the coding region of APOB, presumably near the LDL receptor recognition site, that leads to the substitution of lysine for glutamic acid. The APOB*X+ allele (XbaI restriction site present) was found in some studies to be associated with higher serum cholesterol or triacylglycerol concentrations and with a greater dietary response.
Recently, Lopez-Miranda et al (43) studied the effect of the APOB XbaI polymorphism on interindividual variability during postprandial lipemia. They subjected 51 healthy young male volunteers (20 with the X-/X- genotype and 31 with either the X+/X- or X+/X+ genotype) to a vitamin A fat-loading test. All of the subjects had the apo E E3-E3 phenotype. The investigators found that subjects with the homozygous X- genotype had significantly greater retinyl palmitate and apo B-48 postprandial responses for both the large and the small triacylglycerol-rich lipoprotein fractions than did homozygous X+ subjects. They concluded that subjects with the X-/X- genotype at the APOB locus had a greater postprandial response than did subjects with the APOB*X+ allele. These differences observed in postprandial lipoprotein metabolism might explain some of the reported associations of this APOB polymorphism with CAD risk.
In an analysis of candidate genes' roles in the lipid and lipoprotein response to dietary challenge, Friedlander et al (44) found that overall genetic variation in the APOB gene region (as defined by haplotypes derived from signal peptide insertion-deletion polymorphisms and MspI, XbaI, and EcoRI restriction site polymorphisms) accounted for 24% of the phenotypic variance in LDL-cholesterol response to diet.
APOE
Apo E is present in both apo B–containing lipoproteins (VLDL, IDL, and LDL) and in apo A-1–containing lipoproteins (HDL) (45, 46). The smallest VLDL remnant, IDL, can be taken up by the liver through the interaction of apo E with the LDL (B,E) receptor. During fat absorption, apo E is transferred from HDL to chylomicrons, and as the triacylglycerols in chylomicrons are hydrolyzed by LPL, the resulting chylomicron remnant particle is removed through the interaction of apo E with the remnant receptor.
Three common polymorphisms (alleles) of apo E arise through variation in the amino acids at residues 112 and 158 (45–47). This results in 3 homozygous genotypes, E4/E4, E3/E3, and E2/E2, with frequencies of 2%, 56%, and 1%, respectively, and 3 heterozygous genotypes, E4/E3, E3/E2, and E4/E2, with frequencies of 23%, 15%, and 3%, respectively. The frequency can vary between different cultural groups. Variation at the APOE locus has been estimated to account for 8–10% of the total variation in plasma cholesterol concentrations in various populations. These variations are detectable by HhaI RFLP analysis (48).
The apo E phenotype influences the uptake of lipoproteins via interactions with apo B and E (LDL) receptors and separate hepatic chylomicron remnant receptors. For example, the APOE*E2 gene product binds poorly to chylomicron remnant receptors and has been shown to delay fat clearance in vivo (46). Conversely, the APOE*E4 gene product increases fat clearance in vivo (46). Several studies have shown correlations between apo E phenotype and total and LDL-cholesterol concentrations in subjects consuming their usual diet (45, 47, 49). Other studies observed correlations between apo E phenotypes and lipid responses to high-fat, high-cholesterol dietary interventions (46, 50, 51). These studies suggest that patients possessing the APOE*E4 allele have a greater response to dietary restriction of cholesterol and fat.
In a prospective study of 103 healthy men designed to evaluate the influence of apo E phenotype on lipoprotein response to a low-fat (24% fat) compared with a high-fat (46% fat) diet, Dreon et al (52) found that the diet-induced reduction in LDL cholesterol did not reduce LDL particle number, but rather resulted in a shift from large, buoyant, cholesteryl ester–rich LDL particles (Sf: 7–12) to smaller, denser LDL particles (Sf: 0–7). The magnitude of this effect was significantly related to apo E phenotype, with progressively greater reductions in concentrations of large LDL in persons with different apo E phenotypes as follows: E3-E2 < E3-E3 < E4-E3 and E4-E4. These results suggest that the relative magnitude of LDL-cholesterol reductions induced by a low-fat diet in subjects with differing apo E phenotypes may depend on whether cholesterol is transported predominantly in larger or smaller LDL particles.
Martin et al (53) reported a relation between APOE genotype and HDL-cholesterol response to dietary cholesterol feeding. They found that the effect of cholesterol feeding on total HDL cholesterol produced no change in E3/E2 subjects, a small increase in E3/E3 subjects, and the largest increase in E4/E3 subjects.
In a special Turku CAD risk factor intervention project for babies, Routi et al (54) examined the effect of prospective, randomized cholesterol-lowering dietary intervention and apo E phenotype on serum Lp(a) concentrations in infants aged 7–24 mo. They found that serum Lp(a) values differed significantly according to apo E phenotype; the median Lp(a) values increased in the following phenotype order: E2-E2, E3-E2, E4-E2, E4-E3, and E4-E4 genotype. Their results suggested that apo E phenotypes influence serum Lp(a) concentrations, but that the effect of the cholesterol-lowering dietary intervention was not significant in subjects aged 24 mo.
Lapinleimu et al (55) also studied the effects of apo E phenotype on changes in serum lipid concentrations in a 6-mo, prospective, randomized trial of a low-saturated-fat, low-cholesterol diet in 846 infants aged 7 mo at the start of the study. They found that the apo E phenotype influenced serum cholesterol concentration markedly by 7 mo of age, when serum cholesterol concentrations were higher in apo E4–positive infants (E2-E4, E3-E4, and E4-E4) than in apo E4–negative infants [159 ± 31 mg/dL (4.10 ± 0.81 mmol/L) compared with 150 ± 29 mg/dL (3.89 ± 0.74 mmol/L)]. The HDL-cholesterol concentration was slightly lower in apo E4–positive infants than in apo E4–negative infants [34 ± 8 mg/dL (0.88 ± 0.20 mmol/L) compared with 35 ± 7 mg/dL (0.91 ± 0.19 mmol/L)]. Between 7 and 13 mo of age, the mean serum cholesterol concentration of infants in the intervention group was unchanged, ie, the mean apo B concentration increased slightly and the mean apo A-I concentration decreased. In the control infants, the mean serum cholesterol concentration increased by 9 ± 25 mg/dL (0.24 ± 0.65 mmol/L), the mean apo B concentration increased markedly, and the mean apo A-I concentration was stable. Although between the ages of 7 and 13 mo, a reduced saturated fat and cholesterol diet effectively prevented the age-associated increases in serum cholesterol and non-HDL-cholesterol concentrations that were obvious in the control infants, these dietary effects occurred independently of apo E phenotype.
Type 2 diabetes is associated with postprandial lipoprotein clearance defects that are correlated with the fasting hypertriglyceridemia widely observed in type 2 diabetes patients. Reznik et al (56) studied whether these postprandial disturbances are also found in normotriglyceridemic type 2 diabetes patients in the fasting state and whether the apo E polymorphism influences postprandial metabolism of intestinally derived lipoproteins. The vitamin A fat-loading test was used in 18 normotriglyceridemic type 2 diabetes patients and 7 normotriglyceridemic obese control subjects, and postprandial triacylglycerol and retinyl palmitate concentrations were evaluated in total plasma and in the chylomicron (Sf > 000) and nonchylomicron (Sf < 1000) fractions isolated by ultracentrifugation. Type 2 diabetes patients exhibited an amplified response of both triacylglycerol and retinyl palmitate in the 3 fractions compared with that of the obese control subjects. Incremental triacylglycerol response to the oral fat load was strongly correlated with fasting triacylglycerol level (r = 0.80, P < 0.0001) in the whole study population. Postprandial lipoprotein profiles were distinguished in type 2 diabetes patients according to apo E phenotype: despite normal fasting triacylglycerol concentrations in E3-E3 (n = 6), E2-E3 (n = 6), and E3-E4 (n = 6) patients, postprandial retinyl palmitate response was 2-fold to 3-fold higher in E2-E3 and E3-E4 patients than in those with the common E3-E3 phenotype. In contrast, lower increases in postprandial triacylglycerol and lower fasting and postprandial HDL- and HDL3-cholesterol concentrations were observed in E3-E4 patients than in E3-E3 patients, possibly reflecting modifications in lipid content of the postprandial lipoproteins driven by a differential lipid transfer activity depending on apo E isoform. These data indicate an enhanced postprandial lipemia in normotriglyceridemic type 2 diabetes patients and illustrate the influence of apo E polymorphism on lipoprotein clearance in these patients. Postprandial alterations of lipoprotein remnants may thus accelerate atherogenesis even in normotriglyceridemic type 2 diabetes patients.
LIPID-PROCESSING ENZYME GENES
Four major enzymes involved in lipid and lipoprotein metabolism have been well characterized: LPL, hepatic lipase, LCAT, and CETP. According to OMIM (7), 36 allelic variants were reported for the LPL gene, 19 for the LCAT gene, and 3 for the CETP gene. This section will deal with the effects of common DNA polymorphisms of the LPL, CETP, and LCAT genes on responsiveness to dietary fat and cholesterol. Although 15 polymorphisms of the hepatic lipase gene have been described (7, 57, 58), no data on their effect on responsiveness to dietary fat and cholesterol are available.
LPL gene
LPL is expressed in several tissues, such as adipose, adrenal gland, bone, brain, breast, colon, eye, heart, lung, ovary, parathyroid, placenta, testis, uterus, and whole embryo. LPL is bound to the endothelial surfaces of these tissues after synthesis. In the presence of apo C-II, LPL functions to hydrolyze the triacylglycerol of circulating chylomicrons and VLDL.
The HindIII polymorphism in the LPL gene (H-/H-, H+/H-, H+/H+ genotypes) is associated with plasma triacylglycerol and HDL-cholesterol concentrations and CAD (28). This mutation is located in intron 8 and has no effect on protein structure. In a dietary intervention study, Humphries et al (33) found that the HindIII polymorphism of the LPL gene was consistently associated with differences in the magnitude of the total cholesterol response to the change in the proportion of saturated fatty acids in the diet. Individuals with the H-/H+ or H-/H- genotype had a larger response than did those with the H+/H+ genotype (mean change of 14.2% compared with 9.3%). On changing from a saturated-fat-enriched to a polyunsaturated-fat-enriched diet, individuals with the H-/H+ or H-/H- genotype experienced a significant decrease in triacylglycerol concentrations (12.7%) whereas those with the H+/H+ genotype experienced only a small decrease (4.6%). These results might explain why dietary differences do not consistently explain the association between LPL genotype and lipid traits in studies of different communities and countries.
CETP gene
CETP mediates the transfer and exchange of cholesteryl ester and triacylglycerol between the plasma lipoproteins and plays an important role in HDL cholesteryl ester and apo A-I catabolism and in the determination of HDL size and subclass distribution (59). In some primary and secondary hyperlipidemias [eg, familial hypercholesterolemia (FH), dysbetalipoproteinemia, hypertriglyceridemia, and nephrotic syndrome] and during postprandial lipemia, accelerated CETP-mediated cholesteryl ester transfer results in increased cholesteryl ester net mass transfer from HDL to VLDL or chylomicrons, probably contributing to reduced HDL cholesteryl ester concentrations and cholesteryl ester enrichment of potentially atherogenic chylomicron and VLDL remnants (59). The TaqI polymorphism of the gene encoding CETP (B2 with TaqI cutting site, B1 without TaqI cutting site) was associated with HDL-cholesterol concentrations (28). Subjects with a low to normal HDL-cholesterol concentration have a low frequency of the *B2 allele (24%), whereas subjects with very high HDL-cholesterol concentrations have a high frequency of the *B2 allele (62%). The group with low HDL cholesterol also had higher CETP activity and higher plasma triacylglycerol concentrations (28). In one study, Clifton et al (28) found that changes in HDL cholesterol in response to dietary fat and cholesterol were strongly related to baseline HDL cholesterol and that TaqI polymorphism was unrelated to dietary response.
LCAT gene
LCAT plays an important role in lipoprotein metabolism, especially in reverse cholesterol transport. The enzyme is synthesized in the liver and circulates in blood plasma as a complex with components of HDL. Cholesterol from peripheral cells is transferred to HDL particles, esterified with sn-2 fatty acids of phosphatidylcholine through the action of LCAT, and incorporated into the core of lipoprotein. The cholesteryl ester is thereby transported to the liver. Thus, LCAT facilitates the removal of excess cholesterol from peripheral tissues to the liver. A lack of LCAT activity can be expected to lead to accumulation of free cholesterol in the tissues. Hoeg et al (60) reported that overexpression of LCAT in transgenic rabbits and baboons significantly reduces atherosclerosis induced by dietary fat and cholesterol, indicating that LCAT may be a good target for gene therapy for CAD.
Kammerer et al (61) investigated the effects of a polymorphic PvuII site in the LCAT gene on serum HDL-cholesterol and apo A-I concentrations in a population of 750 pedigreed baboons. They also tested for genotype x diet interactions by using data on HDL-cholesterol and apo A-I concentrations with 2 diets (laboratory feed compared with a diet high in cholesterol and saturated fat). A significant (P < 0.001) association between LCAT genotype and HDL-cholesterol concentrations was observed. With both diets, animals homozygous for the less common allele had HDL-cholesterol concentrations that averaged 18–19% lower than those in animals homozygous for the more common allele. HDL-cholesterol concentrations of the heterozygotes were intermediate. The LCAT RFLP accounted for 5% of the variation in HDL-cholesterol concentrations with the 2 diets. However, these investigators observed no strong evidence of an LCAT genotype x diet interaction effect.
LIPOPROTEIN RECEPTOR GENES
At least 5 lipoprotein receptors have been described: LDL receptor, lipoprotein receptor related protein, VLDL receptor, scavenger receptor, and a putative HDL receptor (SRB-I) (15). All of their complementary DNAs or genes have been cloned. At least 56 allelic mutations in the LDL receptor gene have been described (7), whereas the mutations of other lipoprotein receptors have not been extensively examined yet. Little data on the effects of DNA polymorphisms in other lipoprotein receptors on the responsiveness to dietary fat and cholesterol are available.
The LDL receptor is a transmembrane cellular glycoprotein that is pivotal in cholesterol homeostasis (62). Several different mutations, including deletions, insertions, missense mutations, and nonsense mutations, have been described (63). FH is a Mendelian dominant disorder that results from the expression of a mutant allele or alleles in the LDL receptor gene. Those heterozygous for FH, with a mutation in 1 of their 2 LDL receptor alleles, produce one-half the normal number of LDL receptors, resulting in a 2-fold elevation in plasma LDL cholesterol. FH has a population frequency of 1/5000 and accounts for 5% of patients with premature CAD.
Patients with two FH mutants alleles are referred to as FH homozygotes, although most have 2 different mutations at the LDL receptor locus (genetic compounds). FH homozygotes develop planar xanthomas by the age of 5 y and usually develop coronary atherosclerosis and aortic stenosis in the first decade, often leading to death by 20 y of age. However, little information is available on the effects of these mutations on responsiveness to dietary fat and cholesterol.
Children and adults with heterozygous FH experience decreases in total cholesterol concentrations of 10–15% with adherence to American Heart Association type II diets (64). Clifton et al (28) examined the role of a PvuII polymorphism in the LDL receptor gene in modulating the dietary response of volunteers with mild polygenic hypercholesterolemia. PvuII detects the A-to-G transition in the intron near exon 16 of the LDL receptor. The presence of the PvuII site results in a 14-kb fragment (*A allele) and the absence results in 16.5-kb fragment (*B allele). In contrast, when either a standard high-fat diet or a vegetarian diet was given, the 15 men with 2 alleles were less responsive than the 8 men with a single *B allele, particularly in HDL-cholesterol (A/A compared with A/B: 1.5% compared with -20%; P = 0.03). Similar results were seen when the typical Australian diet (35% fat, ratio of polyunsaturated to saturated fat of 0.2) was contrasted with a low-fat diet; with HDL-cholesterol changes of 1% compared with -26% for the A/A compared with A/B genotype (P < 0.05). However, the results of the second trial conducted by the same investigators were inconsistent. Clearly, more studies are needed.
CONCLUSIONS
The effects of DNA polymorphisms on lipoprotein responsiveness to dietary fat and cholesterol have drawn increasing attention in recent years because an understanding of the molecular mechanisms underlying these effects may be useful in identifying and counseling certain individuals to avoid high-fat diets, which may specifically increase their chance of developing hyperlipidemia and thus increase their risk of CAD. In this article, we reviewed the evidence relating lipoprotein response to dietary fat and cholesterol and DNA polymorphisms in the genes encoding apo A-I, apo A-IV, apo B, apo C-III, apo E, LPL, CETP, LCAT, and LDL receptor. Preliminary studies in the Dietary Intervention Study in Children showed a significant dose-response relation between the decrease in LDL cholesterol from baseline to 36 mo of follow-up in both the intervention group (who consumed a low-fat, low-cholesterol diet) and the usual care group (who consumed a regular diet) and the presence of the APOA1*A allele at the M1 site and the + site at the M2 site of the APOA1 promoter.
Although more and more data are available on the effects of genetic polymorphisms in genes related to lipid metabolism and the responsiveness to dietary fat and cholesterol, no consistent effects of most reported genetic factors have been seen. The major problems related to these discrepancies are sample size, effects of age and sex, different ethnic and cultural (dietary) backgrounds of the participants, different dietary protocols used, and the difficulty of ensuring compliance. More clinical trials in large populations with standardized protocols are needed to study further the effect of these polymorphisms on the responsiveness to dietary fat and cholesterol.
The molecular mechanisms underlying the effects of each DNA polymorphism on the responsiveness to dietary fat and cholesterol are largely unknown. The mechanism of the association of the apo E isoforms with dietary response is presumably due to the well-understood effects of the Cys-Arg changes in these isoforms. The apo E4 isoform has the highest fractional efficiency of cholesterol absorption from the gut and highest binding affinity of lipoproteins to the LDL receptor, leading to a larger down-regulation of the LDL receptor compared with the apo E2 or E3 isoform. However, certain DNA polymorphisms may be a marker of another sequence change elsewhere at the gene locus.
Not every gene relevant to lipid metabolism has been assessed. Other genes involved in determining the strong genetic effects that determine response to dietary change have yet to be identified.
ACKNOWLEDGMENTS
Figure 1 was kindly provided by H Bryan Brewer Jr, Molecular Disease Branch, National Heart, Lung, and Blood Institute, National Institutes of Health.
REFERENCES