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12, NO. 3, 1992

Structure, Function, Molecular Genetics, and Epidemiology of Apolipoprotein B

Plasma lipids are transported in lipoprotein particles, macromolecular complexes stabilized by specialized proteins known as apolipoproteins. There is a strong association between plasma lipids (cholesterol and triglyceride) and early coronary heart disease (CHD). Apolipoprotein (apo) B-100-containing lipoproteins are important in this respect because they comprise the most atherogenic lipoprotein species; lipoprotein (a), low density lipoproteins (LDL), intermediate density lipoproteins (IDL), and very low density lipoproteins (VLDL). In addition, the plasma level of apoB itself has been shown to be a risk factor for CHD. ApoB-100 is one of the largest proteins known, containing 4536 amino acid residues.'-' It is synthesized in the liver and is secreted in VLDL. When VLDL are converted to IDL and finally to LDL, apoB-100 is the only protein that is not transferred to other lipoprotein particles. ApoB is the sole protein component of LDL, and each LDL particle contains a single molecule of apoB- 100. In postprandial plasma chylomicrons, there is another species of apoB, apoB-48.' ApoB-48 contains 2 152 amino acid residues and is synthesized in the small intestine.'.# ApoB-48 is identical to the N-terminal 48% of apoB-100 in its sequence. It is undetectable in plasma during fasting and is present at a much lower level than apoB-100 in the postprandial state. Therefore all clinical studies correlating apoB levels with specific disease states deal exclusively with plasma apoB-100 levels.

EPIDEMIOLOGY OF APOB-100AND ApoB-100-CONTAINING LIPOPROTEINS Several epidemiologic, clinical, and animal research studies have linked apoB-containing lipoprotein particles with an increased risk of CHD. Well-publicized studies, such as the one in Framingham9and the Multiple Risk Factor Intervention Trial (MRFIT)'" primary prevention study, have clearly shown that individuals with

From rhe Departmenrs qf Cell Biology and Medicine, Buylor College of Medicine. and the Cmrer,for Demographic. cmd Population Genetics. The University of Texas Health Science Center at Houston. Houston Texus. Reprint requests: Dr. Chan, Department of Cell Biology, Baylor College of Medicine. One Baylor Plaza, Houston, TX 77030.

elevated total serum cholesterol levels are at increased risk of early CHD. The primary cholesterol-carrying particle in the blood is LDL, and a similar positive association exists between plasma LDL-cholesterol concentrations and CHD. In both the Framingham and the MRFIT studies, there is approximately a fourfold increased risk of early CHD in the upper tenth percentile of plasma cholesterol compared with the lower tenth percentile. Although more equivocal, these and other studies have shown a corresponding erosion of CHD risk following a reduction in plasma total and LDL-cholesterol levels. Plasma levels of the apolipoproteins have often been shown to be better predictors of CHD than the lipid component of the lipoprotein particles. However, it is important to note that in the case of apoB-100, the protein does not exist in circulation separate from a lipoprotein particle. Cambien et al" examined the relationship between parental history of CHD and plasma lipid, lipoprotein, and apolipoprotein levels in a sample of 4045 middle-aged men. Subjects with a positive family history of CHD had higher total cholesterol, LDL-cholesterol, and apolipoprotein B levels, and lower HDL-cholesterol levels than subjects with a negative family history of CHD. When stepwise discriminate analysis was performed, including the effects of a large number ofdemographic, life-style and biologic variables, only plasma apoB levels were related to family history of CHD after considering age, cigarette smoking, and body mass index. This and similar studies suggest an underlying genetic mechanism influencing plasma apoB levels and the risk of CHD. Biometric genetic studies of CHD rely on the detection of genetic factors contributing to lnterindividual variation of lipid, lipoprotein, and apolipoprotein levels. These analyses use correlations between genetically related and unrelated family members to determine the contribution of genetic and environmental factors to the variation in a CHD risk factor. For apoB levels and two related traits, total serum cholesterol and LDL-cholesterol levels, the relative impact of genetic, shared environmental, and individual specific factors is given in Table 1. For each trait, the contribution of genes is statistically significant and is larger than that of shared environmental factors. For plasma apoB levels, over 50% of the variance is attriutable to genetic factors. In other words, half of the observed individual-to-individual differences in apoB levels is attributable to genetic

Copyright O 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.

31 1

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LAWRENCE CHAN, M.D., and ERIC BOERWINKLE, Ph.D.

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12, NUMBER 3, 1992

TABLE 1. Influence of Genetic, Shared Environmental, and Individual Factors to the Variation in Plasma Cholesterol, LDL-Cholesterol, and ApoB Levels

Total cholesterol 11 LDL-cholesterol1 Apolipoprotein Bn

Genes*

Shured Environments'

Individuul Environmenf

Major GrncY9

53% 52% 52%

7% 16% 14%

40% 32% 35%

Yes# yes:v* Yes"

variation. These analyses also try to find evidence for a single gene with a large effect (called "major genes") on the traits of interest. Several statistical strategies have been proposed for detecting the effects of segregation at a single locus. Nowadays, the most commonly used method is complex segregation analysis.'* A summary of the results of complex segregation analysis of total cholesterol, LDL-cholesterol, and apoB levels is also contained in Table 1. Two studies have reported significant evidence for a single gene with a large effect on plasma apoB level^.'^,'^ In the study by Hasstedt et a l ' h h e unidentified major gene accounted for a full 43% of the variance in plasma apoB levels. However, there is surely more than one gene with a large effect on plasma apoB levels. Clearly, mutations in the LDL-receptor gene leading to familial hypercholesterolemia influence apoB level^.'^ The common apoE polymorphism has an effect on plasma apoB levels in the general population. In one study, Boerwinkle and UtermannI6 estimated that the apoE polymorphism accounted for 12% of the variance in plasma apoB levels; individuals with the ~2 allele had lower apoB levels, and those with the ~4 allele had elevated apoB levels. In addition to mutations in the LDL-receptor and the apoE polymorphism, alterations in the apoB structural gene also have been shown to influence plasma lipid, lipoprotein, and apolipoprotein levels, and the risk of CHD (see later).

PRIMARY STRUCTURE OF APOB-100 The primary amino acid sequence of apoB-100 as deduced from its cDNA sequence was confirmed by extensive primary peptide sequence data.17 Based on its amino acid composition, apoB-100 has an estimated molecular mass of 5 13 kDa. The somewhat higher apparent molecular mass of 550 kDa of the native protein is the result of glycosylation. ApoB-100 contains 8 to 10% carbohydrate in the form of galactose, mannose, N-acetylglucosamine, and sialic acid residues.'' There are 19 potential N-linked glycosylation sites predicted from the primary sequence of the protein. Among these, 16 were found to be actually glycosylated and three unglycosylated.17The carbohydrate moieties are unevenly distrib-

uted; 8 of the 16 sites occur within 700 residues (between Asn27S2 and Asn343X) in the C-terminal half of the molecule. Another strikingly asymmetrically distributed structure in apoB-100 are the disulfide bonds. There are 25 cysteine residues in apoB- 100. Fourteen of these 25 cysteine residues occur within the N-terminal 1000 residues in apoB-100. Sixteen of the cysteine residues have been found to be involved in intramolecular disulfide linkage.'' Using the fluorescent probe, 5-iodoacetamidofluoresceine, to label the free sulfhydryl of LDL, Coleman et a12"found that Cys-22 and Cys-24 are exposed on the surface of the LDL particle and are potentially available for disulfide linkage to apo(a) on lipoprotein(a).

CONFORMATION OF APOB-100 Compared with the soluble apolipoprotein in other particles, apoB- 100 in LDL contains a relatively high Psheet content of 16 to 40% by physical measurements. Use of a predictive algorithm2' suggests that apoB- 100 contains 43% a-helix, 21% P-sheet structure, 20% random coil structure, and 16% p-turns. Two approaches have been taken to analyze the conformation of native apoB-100 on lipoprotein particles: the accessibility of the protein to various proteolytic enzymes and its accessibility to various monoclonal antibodies. When LDL that contained apoB-100 as the sole protein component was subjected to controlled proteolysis by trypsin treatment, some peptides cleaved from the protein were released from the lipoprotein particle, whereas others stayed with the particle. The high proportion of peptides that were recovered reproducibly in the trypsin releasable (34%) or nonreleasable (3 1%) fractions exclusively indicates that apoB- 100 assumes a specific conformation on LDL so that certain regions of the molecule are more accessible to trypsin digestion than others. It also suggests that some parts of the molecule are intrinsically more likely to associate tightly with lipid and LDL than other parts.17 Similar conclusions were also reached by other laboratories using different proteolysis conditions. 22-24 Another method that has been used to probe apoB100 conformation on various lipoprotein particles in-

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*Overall contribution of gene variation to the distribution of risk factor levels. +Contribution of shared household environment to the distribution of risk factor levels. 'Effects of environmental factors specific to the individual. This component also encompasses the influence of measurement error $Is there evidence supporting a single gene with a large effect on risk factor levels'? 11 Iselius." "Hamsten et al.7' #Moll et al.7' **Morton et a1.14 "Hasstedt et al."

APOLIPOPROTEIN B-CHAN,

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volves the use of monoclonal a n t i b ~ d i e s . ~A~ . large ~' number of monoclonal antibodies directed against different parts of apoB-100 have been d e v e l ~ p e d . ~ The ~ - ~ ep' itopes for many of these antibodies have been mapped at the primary sequence leve1.28.29 The degree of accessibility of specific epitopes on different lipoprotein particles provides a map for apoB-100 conformation that partially overlaps but is distinct from that provided by accessibility to proteolytic enzymes.

heparin-binding sequences have been found to be distributed throughout the length of ap0B-100.~'-~~ There is, however, a cluster of these sequences around the putative receptor-binding regions, domains A and B. Heparinbinding sequences in apoB-100 are thought to be involved in receptor binding because of the observations that chemical modification of lysine and arginine residues abolishes both heparin and receptor binding'2," and that heparin releases LDL bound to fibroblast LDL receptors.4h

RECEPTOR-BINDING AND HEPARINBINDING DOMAINS A major function of all apolipoproteins, including apoB-100, is lipid-binding. The mechanism by which the plasma apolipoproteins bind to lipids is still poorly understood. Long stretches of exclusively nonpolar residues present in membrane-spanning proteins are not found in any of the plasma apolipoproteins. Two types of structures within the apolipoprotein primary sequences are thought to be important in lipid binding: amphipathic &-helices and proline-rich hydrophobic sequences with P-sheet potential. Amphipathic a-helices are found in both apoB- 100 and the soluble apolipoproteins (apoA-I, A-11, A-IV, C-I, C-11, C-111, and E),47 whereas the proline-rich hydrophobic sequences are unique to apoB .48 The presence of lipid-associating peptides in apoB100 has been demonstrated by incubating proteolytic fragments of apoB-100 with microemulsions of exogenous lipid^.^'.^' The reassembled lipid-peptide particles can be isolated by isopyknic or density gradient ultracentrifugation. A number of lipid-associating peptides have been identified in apoB- 100 by using this method. These experimentally defined lipophilic peptides are distributed widely throughout the apoB-100 primary sequence. Similar lipophilic sequences have also been found in the soluble apolipoproteins. One experimental approach that specifically examines the functional role of different regions of the apoB100 sequence in vivo is the expression of an apoB minigene construct in transgenic mice. Xiong et a15' microinjected an apoB promoter-driven minigene that contains apoB (1-5612878-3925t4528-4536), designated B4, to generate transgenic mice. The transgene was expressed predominantly in the liver and small intestine, and the B4 miniprotein was secreted into the circulation at high levels. When the lipoprotein fractions were analyzed, B4 was found exclusively in the LDL fraction and none was detected in the HDL or nonlipoprotein fractions. The major apoB sequence region in this construct, apoB2878-3925, constitutes less than 25% of apoB- 100 but it contains about half of the proline-rich hydrophobic sequences in this protein. It does not contain any long amphipathic ~ i - h e l i c e s .Therefore ~~ proline-rich hydrophobic sequences, which are unique to apoB-100, but not amphipathic &-helices, which are also present in other apolipoproteins, may be important in rendering the protein nontransferable and allowing its tight association with LDL.

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LIPID-BINDING SEQUENCES A major function of apoB- 100 is to serve as a ligand for the LDL receptor. Another apolipoprotein that also serves as a ligand for the LDL receptor is apoE. Acidic residues in the LDL receptor at the N-terminal domain are thought to interact with basic residues on the receptor-binding domain of apoE and apoB-100."' In apoE, these basic residues have been mapped to a region near amino acids 140-150." Lysine and arginine residues have also been implicated in apoB-100-LDL receptor interaction; chemical modification of these residues abolishes receptor binding.'2," Monoclonal antibody mapping has localized the receptor-binding domain of apoB-100 to the vicinity of residues 3000-4000.'4~" By sequence alignment, there are two regions in apoB- 100 that are rich in basic amino acids that show similarity to . ~ span amino the apoE receptor-binding d ~ m a i n They acid residues 3 147+3 157 (designated domain A) and 335!9+3367 (designated domain B). A synthetic oligopeptide corresponding to apoB3345+338 1 conferred receptor-binding ability to trypsin-inactivated hypertriglyceridemic VLDL and allowed the latter to be taken up by cultured human fibroblasts and suppress cellular 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, the rate-limiting step in cholesterol biosynthesis.' There is as yet no experimental evidence directly supporting the role of domain A in receptor binding. Sequence comparison among apoB-100 from multiple vertebrate species indicates that while domain B maintains a strong net positive charge and shows high homology across species, domain A is not as well conserved with wide differences in the net positive charge.16 The description of the syndrome of familial defective apoB-1003740has provided interesting information on the receptor-binding function of apoB-100. The LDL isolated from individuals with this familial disorder have impaired receptor-binding ability. There is a strong association of an Arg'SOO-+Glnsubstitution and familial defective apoB-loo,3ysuggesting that this residue is important for receptor binding (see later). ApoB and LDL have been detected in atherosclerotic tissue. The retention of LDL on the endothelial surface is important because it allows modifications such as oxidation and additional lipolysis by lipoprotein lipase to take place, processes that may alter the atherogenicity of LDL. The mechanism for the local accumulation of LDL is unknown but may be related to LDL binding to the extracellular matrix, such as glycosaminoglycans, proteoglycans, or collagen, on the arterial wall. Specific

SEMINARS IN LIVER DISEASE-VOLUME

APOLIPOPROTEIN B GENE STRUCTURE ApoB-100 is one of the largest proteins known. For a gene that encodes a messenger RNA that spans 14 kb, the apoB gene is actually relatively small; being 43 kb pairs long, it is only about three times the length of its exons. The apoB gene contains 29 exons and 28 introns5' and is located on the short arm of chromosome 2." The reason for the relatively small overall size of the apoB gene is the presence of two unusually large exons. Exon 26 spans 7522 base pairs and exon 29, 1906 base pairs. ApoB is a highly polymorphic protein because of its large size. There is considerable heterogeneity in the reported apoB cDNA sequences, much of which appears to be bona fide sequence polymorphisms and not sequencing error^.'^ Such polymorphisms have also been detected in the form of restriction fragment length polymorphism (RFLP)." In addition, there is a length polymorphism involving the signal peptide region of apoB," and an extremely polymorphic variable number of tandem repeat (VNTR) regions approximately 200 basepairs 3' of the apoB genG4 (see later).

BlOGENESlS OF ApoB-48: DISCOVERY OF ApoB mRNA EDITING The mechanism for the biogenesis of apoB-48 was elucidated in 1987. At that time, it was known that apoB-48 shared antigenic determinants with the N-terminal half of apoB-100. The proposed explanations for the biosynthesis of apoB-48 in the small intestine included post-translational cleavage of apoB- 100 and differential splicing of apoB mRNA, which both proved wrong. Detailed analysis of the apoB gene and mRNAs for apoB-100 and apoB-48 led to the discovery of mammalian RNA editing. Surprisingly, apoB-48 mRNA was found to be the product of a single base C+U conversion involving the codon CAA for glutamine-2 153 in apoB100, producing a UAA, an inframe stop codon. Translation of apoB-48 mRNA would produce a protein containing 2152 amino acid residues instead of 4536 residues in apoB- 100.7.8 ApoB mRNA editing is an intranuclear event that occurs post-transcriptionally coincident with splicing and polyadenylation." The editing reaction appears to be a deamination or transamination of a cytidine residue to a uridine residue.s6." It must be very specific because there are 3135 cytidines in mature apoB-100 mRNA, of which only a single cytidine is edited. The edited residue is located in exon 26, the largest exon to date. Apart from its sequence specificity, apoB RNA editing is also t i s s u e - s p e c i f i ~ .In~ ~mammals, ~~~ it occurs very efficiently in the small intestine, which produces almost exclusively apoB-48 mRNA and protein. In nonrodents, editing is essentially nonexistent in the liver, where apoB-100 is the predominant species produced. The tissue specificity is somewhat different in rats and mice. In these animals, in addition to the small intestine, the liver also has good editing activity. About 60 to 70% of the hepatic mRNA is in the edited (apoB-48) form. Interestingly, editing activity is also detected in tissues

12, NUMBER 3, 1992

that produce little or no a p ~ B . ' ~ In , ' ~the chicken, there is apparently no editing of apoB mRNA.60 The discovery of apoB mRNA editing is exciting because it represents a novel mechanism of gene regulation. The portion of apoB- 100 protein missing in apoB48 contains the LDL receptor-binding domains as well as important LDL-binding peptide sequences. ApoB-48 behaves physiologically as a very different protein. It is localized in different lipoprotein particles and it does not interact with the LDL receptor. Therefore the end result of RNA editing is the production of a functionally distinct protein that serves a divergent physiologic role in lipoprotein metabolism. The fact that editing activity is present in tissues that do not produce apoB suggests that RNA editing may well modify other RNAs and serve a function of general biologic importance to the cell.

FAMILIAL DEFECTIVE ApoB-100 One mutation in the apoB gene (discussed earlier in the section on Receptor-Binding and Heparin-Binding Domains) has been shown to be associated with familially transmitted hypercholesterolernia. Vega and G r ~ n d y ~ ~ described a patient with elevated cholesterol whose LDL was cleared from circulation at 50% of the normal rate and whose LDL exhibited reduced affinity for LDL receptors in an in vitro assay. Innerarity et a138showed that the phenotype of hypercholesterolemia and defective apoB binding was inherited as an autosomal codominant trait, and they suggested the name "familial defective apoB-100" (FDB) for this condition. The mutation likely responsible for FDB was identified by directly sequencing genomic clones of the region surrounding the putative receptor binding domain. In the original proband described by Vega and Grundy,17 a unique G to A substitution was found in the large exon, exon 26, at nucleotide position 10708." This substitution changes Arg to Gln at residue 3500 of the apoB-100 polypeptide. In addition, in a large pedigree the apoB100 3500 mutation cosegregated perfectly with overt hypercholesterolemia, and defective apoB binding and clearance. Although not proven, it is very likely that the observed missense mutation at codon 3500 is responsible for the FDB phenotype. The apoB-I00 3500 mutation has been observed in hypercholesterolemic patients from multiple populations. In 1 100 hypercholesterolemic patients from Montreal, Salzburg, San Francisco, and Dallas, 1 1 probands with the alteration were dete~ted.~' Even though the ascertainment criterion used in these studies was complex and different among the four sites, we can infer that roughly 1 in 100 patients in a lipid clinic contains this apoB-100 alteration. There is considerably heterogeneity in the expression of the apoB-100 3500 substitution. Original observations indicated that the hypercholesterolemia associated with FDB was less severe than that attributable to defects in the LDL-receptor gene.'' However, in one study by Tybjaerg-Hansen et aL6' the hypercholesterolemia was more severe. Likewise, some patients with FDB have arcus corneae and tendon xanthomas, a condition usually associated with LDL-re-

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3 14

ceptor defects, but other patients lack such lesions. Insufficient epidemiologic data exist to assess the precise relationship between FDB and CHD. In addition to our lack of knowledge concerning the role of FDB in atherosclerotic CHD, a large number of questions exist concerning the apoB- 100 3500 mutation. For example, the exact mechanism by which this mutation influences LDL-receptor binding is not known but is thought to be due to local conformational changes in and around the receptor binding domain (see discussion in the section on Receptor-Binding and Heparin-Binding Domains). The frequency of FDB and the apoB-100 mutation is also not known. Although there has been considerable speculation and ad hoc estimation, carefully designed epidemiologic studies have not been reported. Published reports have been limited to Caucasians of primarily European descent; to our knowledge, this mutation has not been investigated in other major racial or ethnic groups. Furthermore, the relationship between the apoB-100 3500 mutation and the potential spectrum of physiologic and clinical effects is unknown. Existing studies have ascertained subjects for study in a heterogeneous manner and from high-risk groups. Individuals with the 3500 mutation and a risk profile below the clinical horizon are missed from such studies and potentially bias our knowledge on this subject. Finally, it is likely that other mutations in the apoB gene can lead to FDB, but their identity and impact remain unknown. Nevertheless, studies of the apoB- 100 3500 mutation and other apoB alterations have shed light on the important role of this protein in lipid metabolism and the risk of CHD.

ApoB-100 DNA ASSOCIATION STUDIES Basic molecular methods, such as Southern blot analysis and the polymerase chain reaction, have enabled detection of extensive genetic variability in candidate genes of lipid metabolism, including apoB. Some of this genetic variability has a direct biochemical and physiologic effect on the gene product and on lipid metabolism (such as the codon 3500 mutation described in the last section). However, all variability does not necessarily directly affect the phenotype of interest; it can be "silent" with respect to its phenotypic effects. These "marker" loci with no direct effects may be associated in the population with genetic variability with physiologic effects. This nonrandom association between alleles at two gene loci is known as linkage disequilibrium. Therefore a marker with no direct effect on the phenotype may have a statistical association with lipid levels or a disease endpoint. In contrast, when only the simple association approach is used, a locus with a significant effect may go undetected if it is not in disequilibrium with the marker locus. Alternative approaches that use family data and analyze the cosegregation of a candidate gene with riskfactor levels have been developed (see Amos et alh2). These methods do not rely on linkage disequilibrium. Unfortunately, they have not been applied to plasma apoB levels or have not used the apoB gene, and therefore they will not be discussed further. A summary of 25 different studies examining the

potential association of several common apoB DNA RFLPs with CHD or CHD risk-factor levels is given in Table 2. Although the exact sampling strategy and study population are different for each investigation, one of two study designs is usually employed. In the first design, a sample of unrelated individuals is typed for the polymorphism of interest and the investigators ask whether average cholesterol levels, for example, are significantly different among genotypes. In the second design, individuals are sampled because of a particular disease status, for example, having had a myocardial infarction. These individuals are then matched with a disease-free control group and the investigators ask if apoB allele frequencies are significantly different between cases and controls. The first design attempts to estimate the role of the apoB gene on risk-factor variation in the population at large. The second design examines the potential role of the apoB gene in the CHD disease process itself. Each design has its own strengths and weaknesses. In 14 of the 20 studies a positive association was reported between DNA variation in the apoB gene and CHD or CHD risk-factor levels. These data provide evidence that there is DNA variation in or around the apoB gene that is contributing to CHD risk. However, the identity and characteristics of this variation are unknown. The three most commonly investigated RFLPs in apoB are an XbaI polymorphism at codon 2488, and MspI polymorphism at codon 361 1, and an EcoRI polymorphism at codon 4154. Of these three, the XbaI polymorphism has received the most attention because in a large number of studies there is a consistent and significant association between it and CHD or cholesterol levels. In several studies (see Law et al," Berg,@ Talmud et al ") the X2 or allele, that is, presence of the XbaI restriction site, at this locus is associated with elevated total cholesterol and/or LDL-cholesterol levels and CHD. In an informative set of studies, Series et alhhand Houlston et alh7have shown that the fractional catabolic rate of LDL is different among XbaI genotypes. In addition, it has been shown that the rate of LDL apoB synthesis is different among XbaI genotypes.67 These data suggest that the metabolism of apoB and apoB-containing lipoprotein particles is influenced by common DNA variation in the apoB gene. However, the XbaI polymorphism at codon 2488 of the apoB gene is silent, affecting the third or wobble position of a threonine codon. Therefore it is very unlikely that this polymorphism itself is functionally significant. Rather, it is likely in linkage disequilibrium with some other, unknown, site in or close to this gene. Common DNA variation in the apoB gene is not limited to simple nucleotide substitutions. The second kind of DNA variation consists of the insertion or deletion of nucleotides leading to length variation in a gene region. This type of polymorphism is found at the 3' end of the apoB gene of a VNTR in the form. Boerwinkle el aP4 described a rapid and accurate method that utilizes, the polymerase chain reaction to facilitate typing of the 3' apoB VNTR. This new method can distinguish a large number of alleles in this system, making the apoB 3' VNTR a valuable marker for association and family

+

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APOLIPOPROTEIN B X H A N , BOERWINKLE

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12, NUMBER 3 , 1992

TABLE 2. Summary of 25 Association Studies Employing DNA Variation in the ApoB Gene Year

N,*

Design

BergM Hegele et a175 Law et a14

R CIC R

Talmud et a P

R & CIC

Rajput-Williams et allh

MspI

EcoRI

R

Aburatani et aI7' Leren et aI7'

CIC 0

Houlston et a17'

R

Ferns et alx" Monsalve et alx'

CIC CIC

Aalto-Setala et alx' Series et alhh Wiklund et aI8' Myant et alR4 Xu et alx5

R 0 CIC CIC R

Houlston et alh7

Xbul

R

Genest et alR6 Paulweber et alR7 Myklebost et alXX

CIC CIC 0

Mendis et alRq Renges et a19" Tybjaerg-Hansen et aI9'

CIC CIC CIC

Kessling et a19' Nieminen et alY' Deeb et alq4

R CIC CIC

*Total sample size considered. 'Approximate design of the study: R site present. :LDL cholesterol. +LDL fractional catabolic rate. 11 HDL cholesterol.

=

studies following the segregation of the apoB gene. For example, Krul et a168used the apoB 3' VNTR to show the cosegregation of biochemical and immunologic variants of apoB with the apoB structural gene. However, polymorphisms consisting of length variation are not limited to noncoding regions of the apoB gene. We have described polymorphic length variation in the signal peptide of the human apoB gene.5xh9Eukaryotic signal peptides are required for the co- and posttranslational processing of most secretory proteins, and information specifying cellular or subcellular localization may also reside in the signal peptide sequence. To our knowledge, this polymorphism is the only naturally occurring signal peptide length variation in humans. Following amplification by the polymerase chain reaction and polyacrylamide gel electrophoresis, individuals exhibit amplification products from one or two of three potential alleles. The alleles and their amplification products were named according to the number of amino acid residues in the apoB signal peptide as determined by direct DNA sequencing (Fig. 1). The signal peptide alleles consist of the following: the longest allele (designated 5'PSP-29) encodes 29 amino acids in the signal peptide and contains two copies of the sequence (CTG GCG

random sample; CIC

=

Association

p

Structure, function, molecular genetics, and epidemiology of apolipoprotein B.

SEMINARS IN LIVER DISEASE-VOL. 12, NO. 3, 1992 Structure, Function, Molecular Genetics, and Epidemiology of Apolipoprotein B Plasma lipids are tran...
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