© 1995 American Heart Association, Inc.
Articles |
C/T Polymorphism in the 5' Untranslated Region of the Apolipoprotein(a) Gene Introduces an Upstream ATG and Reduces In Vitro Translation
From the Falk Cardiovascular Research Center (B.R.Z., G.E.L., D.P.W., R.M.L.), Stanford University Medical School, Stanford, Calif, and MRC Lipoprotein Team (B.L.K.), Hammersmith Hospital, Ducane Road, London, England.
Correspondence to Richard M. Lawn, Falk Cardiovascular Research Center, Stanford University Medical School, Stanford, CA 94305-5246.
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Abstract Elevated plasma levels of lipoproteinz(a) [Lp(a)] are a significant independent risk factor for arteriosclerosis. Interindividual levels of Lp(a) vary nearly 1000-fold and are mainly due to inheritance that is linked to the locus of the apolipoprotein(a) [apo(a)] gene. A search was made for sequence variants in the 5' flanking region of the apo(a) gene that affect its expression. A C to T transition at position +93 from the transcription start site was found with a frequency of 14% in the study population. In transient transfection assays in HepG2 cells, luciferase reporter gene constructs with a T at this position were associated with a 58% reduction in luciferase activity compared with the more common allele. This single base variant had no significant effect on the binding of nuclear regulatory proteins; however, it introduced an additional upstream ATG initiation codon with its own in-frame stop codon. Furthermore, equivalent levels of mRNA were produced in HepG2 cells transfected with reporter gene constructs containing either a T or a C at position +93. In vitro translation experiments using transcripts derived from either variant apo(a) promoter revealed a 60% reduction in translation associated with the T allele. Hence, the additional ATG created by the T at position +93 in the 5' flanking region of the apo(a) gene impairs the efficiency of translation from the bona fide ATG initiation codon.
Key Words: apolipoprotein • ATG initiation codon • lipoprotein(a) • polymorphism • atherosclerosis • translation
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Introduction |
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Lipoprotein(a) [Lp(a)] consists of an LDL-like particle containing a single copy of apolipoprotein B-100 (apoB-100) covalently attached to apolipoprotein(a) [apo(a)].1 2 The apo(a) and plasminogen genes display striking homology in their cDNA sequence.3 While plasminogen contains five tandemly repeated kringle domains and a protease region, apo(a) has multiple tandem repeats of a kringle-4–like domain, followed by a single copy of kringle 5 and a protease domain that share 75% to 94% DNA sequence identity with plasminogen.3 There is great size heterogeneity of the apo(a) protein4 ; numerous isoforms exist that are encoded for by alleles that contain approximately 15 to 40 copies of the kringle-4–like domain.5
During the 3 decades since its discovery,6 most epidemiological studies have determined that high levels of Lp(a) are a significant independent risk factor for premature coronary artery disease,7 8 9 cerebrovascular disease,10 and restenosis of coronary lesions.11 12 The association of Lp(a) with atherosclerosis is further strengthened by pathological evidence that apo(a) is concentrated in fatty streak lesions13 14 15 and by the development of an apo(a) transgenic mouse model that develops atherosclerosis while on a high-fat diet.16
Interindividual plasma levels of Lp(a) vary nearly 1000-fold.17 Levels in an individual remain quite constant throughout life and are relatively resistant to alterations in diet and treatment with most lipid-lowering medications.18 Metabolic studies performed in human subjects indicate that levels of Lp(a) are governed chiefly by biosynthetic rate.19 20 21 Family studies indicate that plasma levels of Lp(a) are genetically determined and that isoforms are inherited in an autosomal codominant fashion.4 6 22 In addition, plasma levels of Lp(a) appear to be linked almost solely to the apo(a) gene locus.23 24
Plasma levels of Lp(a) are, in general, inversely correlated with
apo(a) protein size.4 These size differences have been
estimated to account for 41% to 69% of the variability of plasma
Lp(a) concentration.21 24 25 The more than 30
different-size alleles of the apo(a) gene that have been
identified5 26 can be accounted for by different numbers
of the kringle-4–like encoding sequence, producing apo(a) species with
apparent molecular mass ranging from 
300 to 800 kD. A recent study
with primary baboon hepatocytes showed that size variants substantially
affect residence time of apo(a) in the endoplasmic reticulum, thus
suggesting that regulation of movement of apo(a) between intracellular
compartments might influence plasma levels of Lp(a).27
Despite the overall inverse trend between apo(a) protein size and plasma levels, it appears that other sequence variants unrelated to the number of kringle-4–like repeats may exist that affect Lp(a) levels.26 In one study, alleles from two different kindreds with apparently identical numbers of kringle-4–like repeats had a 10-fold variation in expression.26 Another recent report shows up to a 200-fold difference in the Lp(a) concentrations associated with isoforms of the same size.17
We have recently cloned and sequenced 1.4 kb of the 5' flanking region
of the apo(a) gene and have demonstrated that it possesses promoter
activity in vitro.28 Deletion analysis revealed that
sequences from 98 nucleotides preceding to 130 nucleotides following
the transcription start site are sufficient to direct transcription in
hepatocytes. DNase I protection, mutagenesis, and nuclear protein
mobility shift analyses demonstrated that the most significant
transcription control element in this region is a binding site for
hepatocyte nuclear factor 1
, spanning nucleotides +26 to
+47.29
We hypothesized that sequence differences in this 5' flanking region might account for differential expression of apo(a) and contribute to the overall variation in plasma concentrations of Lp(a). To test this hypothesis, we studied the promoter activity in a transient transfection assay of the 1.4-kb 5' flanking region of alleles, which were matched for encoded protein size yet expressed widely varying amounts of apo(a). This initial study revealed a C/T transition in the 5' untranslated region that introduces an additional upstream ATG initiation codon and decreases luciferase expression from reporter gene constructs in vitro.
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Subjects
The subjects selected for this study were obtained from kindreds studied at the Hammersmith Hospital. These kindreds were related either to hyperlipidemic patients attending the Hammersmith or Charing Cross Hospital Lipid Clinics in London or to normolipidemic laboratory staff. All individuals were characterized in terms of their Lp(a) level and apo(a) isoform. In addition, immunoblots were scanned to assign the apo(a) concentration associated with each apo(a) isoform after electrophoresis in 3% polyacrylamide gels containing 0.5% agarose.17 Twenty-two alleles were then selected for study. Frequencies of the polymorphisms described were determined in individuals from the Lipid Clinic at the University of California, San Francisco. Dr J.P. Kane kindly provided access to DNA samples from the University of California, San Francisco, Lipid Clinic.
Polymerase Chain Reaction (PCR) of the 1.4-kb 5' Flanking Region
The 1.4-kb 5' flanking region of the apo(a) gene was amplified
in a 100-µL reaction with 1 µg of genomic DNA; 6 µL of 25 mmol/L
MgCl2; 1 µL each of 10 mmol/L dGTP, dATP, dTTP, and dCTP;
10 µL of 10x PCR buffer (Promega); 3 µL (75 picomoles) each of 5'
oligonucleotide MB22
(5'-GATCACGCGTGCGGAAAGATTGATACTATGC-3', Mlu I
site underlined) and 3' oligonucleotide PCR78
(5'-TCAGAGATCTCTTCCTTATGTTCCCTTTTGGGACTGG-3',
Bgl II site underlined); and 1.5 U of Taq
polymerase (Cetus). PCR conditions were 30 cycles of 30 seconds at
95°C, 30 seconds at 48°C, and 60 seconds at 72°C. PCR78 contains
a single base mismatch with apo(a) (underlined) to eliminate the normal
ATG.
Assignment of Alleles Used in Luciferase Reporter Gene Constructs
The G to A polymorphism at position -772 (numbered from
the transcription start site)28 was screened for by
restriction endonuclease digestion of the 1.468-kb PCR product of the
5' flanking region with Taq I and analysis on a 1%
agarose gel stained with ethidium bromide. Alleles possessing an A at
this position lose one of two Taq I sites in this region.
The number of TTTTA repeats at the 5' end of the 1.4-kb flanking
region, ending at nucleotide position -1231, was determined by
amplifying 1 µg of genomic DNA in a 25-µL reaction with 2 µL of
25 mmol/L MgCl2; 0.5 µL of 35S dATP (NEN);
0.5 µL of each of 10 mmol/L dGTP, dTTP, and dCTP; 0.25 µL of 10
mmol/L dATP; 0.5 µL (12.5 picomoles) each of oligonucleotides PCR 65
(5'-GCGGAAAAGATTGATA-3') and PCR 66 (5'-ACGTCAGTGCACTTCAA-3'); and 1 U
of Taq polymerase (Cetus). PCR conditions were 30 cycles of
30 seconds at 95°C, 15 seconds at 45°C, and 90 seconds at 72°C.
PCR products were precipitated with 2.5 vol absolute ethanol and 0.1
vol 3 mol/L NaAc, pH 5.5. The pellet was resuspended in Sequenase stop
solution (USB), electrophoresed in an 8% denaturing acrylamide gel
alongside a control sequencing ladder, and autoradiographed. Fragments
of 96 to 116 bp (5-bp increments) containing 7 to 11 TTTTA repeats,
respectively, were detected.
Plasmid Constructions
The PCR products containing sequences from position -1292 to
+157 of the apo(a) gene flanked by synthetic Mlu I and
Bgl II sites were digested with Mlu I and
Bgl II and cloned into Mlu I/Bgl
II–digested pGL-2 Basic (Promega), a promoterless luciferase reporter
gene vector. Subcloning of Bpu1102 I–Bgl II
fragments [+4 to +157] excised from these reporter gene constructs
(see text) was accomplished by the usual molecular biological
techniques30 (Fig 2A
).
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Cell Culture, DNA Transfection, and Assays of Luciferase and
ß-Galactosidase Activity
HepG2 cells were maintained in culture, plated into 60-mm
dishes, and transiently transfected with 10 µg luciferase expression
plasmid and 2 or 5 µg pSV ß-galactosidase control plasmid (Promega)
per dish using Lipofectin (GIBCO-BRL) as described
previously.28 Luciferase and ß-galactosidase activity in
cell lysates was assayed by measurement of luminescence as
described.28
Gel Mobility Shift Assays
Double-stranded oligonucleotides containing either a C or a T at
position +93, MS+93C/MS+93T
(5'-ATGTAAGTCAACAACAAC/TGTCCTGGGATTG-3'), were synthesized
with a 4-bp 5' overhang and labeled with suitable
[-32P]dNTP by the Klenow fill-in reaction.
Approximately 20 000 cpm (0.1 ng) of labeled oligonucleotides were
used in mobility shift assays with 1 to 4 µg of nuclear extracts from
HepG2 cells. The binding reactions were performed as previously
described.29
Screening for the +93 C/T Polymorphism by Allele-Specific
Oligonucleotide Hybridization
The 1.4-kb 5' flanking region of the apo(a) gene was amplified
from 1 µg of genomic DNA as described above, electrophoresed in a 1%
agarose gel, and transferred to a 0.45-µm nylon membrane (MSI). The
DNA was fixed to the membrane by UV irradiation. Hybridization was
carried out with [
32P]dATP end-labeled ASO+93C
(5'-CAACAACGTCCTGG-3') and ASO+93T
(5'-CCAGGACATTGTTGA-3') in 5x SSPE, 5x Denhardt's
solution,31 and 0.5% SDS for 3 hours at 42°C. The
membrane was washed with 5x SSPE and 0.1% SDS at 50°C and subjected
to autoradiography at -70°C.
Ribonuclease Protection Assay
For analysis of mRNA, total RNA was prepared with a
commercial kit (RNazol) from HepG2 cells transfected with the
pGL2-basic vector into which either the +93 C or +93 T containing
1.4-kb 5' flanking region of apo(a) had been cloned. An anti-sense
riboprobe that protected a 196-bp fragment of luciferase message was
generated by in vitro transcription using T7 polymerase and 50 µCi of
[-32P]dUTP. The template used for this riboprobe was
an EcoO109 I/Cla I–digested fragment of the
pGL2-basic vector (Promega). The template construct for an anti-sense
riboprobe for ß-actin was kindly donated by G. Crabtree. A 300-bp
riboprobe was generated by in vitro transcription using SP6 polymerase
and 1 µCi of [-32P]UTP. The protection assay was
performed as described30 with 65 µg total RNA,
5x105 cpm luciferase probe, and 104 cpm
ß-actin probe. A phosphorimager was used to quantify the actin and
luciferase bands in the dried gel. Values for luciferase mRNA counts
were divided by the values for actin mRNA in each lane as an internal
control. To correct for transfection efficiency, that ratio was further
adjusted for ß-galactosidase activity measured in lysates of
transfected cells. Statistical comparison between the values for the
+93 C and the +93 T promoter region was made using the unpaired
t test.
In Vitro Transcription and Translation
Fragments comprising sequences from +4 to +157 (with either C or
T at +93) of the apo(a) flanking region fused to the luciferase gene
were excised from the pGL2-basic vector (Promega) with
Bpu1102 1 and BamH I 1 and subcloned into
Bluescript SK+. This plasmid was linearized with BamH I 1
and transcribed with T7 polymerase as previously
described.32 In vitro translation was performed with 1
µg of the resulting mRNAs in rabbit reticulocyte lysates as described
by the supplier (Promega). Luciferase activity of 5 µL of each lysate
was assayed by measurement of luminescence as previously
described.29
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To test whether there was allelic variation in the 1.4-kb 5' flanking region of the apo(a) gene that affected transcription, we studied 22 alleles characterized for protein isoform size and contribution of each protein isoform to total plasma Lp(a) concentration. In the majority of cases, alleles were assigned by following the inheritance of two polymorphisms previously noted to exist in this region.28 A G to A transition at position -772 (numbered from the transcription start site) was identified by the use of restriction endonuclease digestion with Taq I, as the A polymorphism eliminates a Taq I site. Alleles could also be distinguished by their number of TTTTA repeats ending at position -1231, which was determined by gel electrophoresis of amplified genomic DNA (not shown).
We determined the frequency of these polymorphisms in individuals of diverse ethnic background obtained from the Lipid Clinic at the University of California, San Francisco. Fifty-four alleles were analyzed for the presence of a G or A at position -772. The frequency of alleles with a G is 0.39; with an A, 0.61. The number of TTTTA repeats, studied in 102 individuals, varied from 7 to 11. The frequencies were 0.01, 0.64, 0.19, 0.14, and 0.02 for repeats of 7, 8, 9, 10, and 11, respectively.
The 1.4-kb 5' flanking region from 14 alleles of interest, all of which
were distinguishable on the basis of number of TTTTA repeats or the G/A
polymorphism, were subcloned into promoterless luciferase reporter gene
vectors and transfected into HepG2 cells; cell extracts were then
assayed for luciferase activity. (Eight alleles that were
indistinguishable by polymorphisms were sequenced to detect possible
differences.) All luciferase activities were compared with a single
control allele, containing 8 TTTTA repeats and a G at position -772,
which was obtained from a healthy laboratory worker with an Lp(a) level
of 41 mg/dL. Most alleles had luciferase activities that were within
the range of usual assay variation (±20%). We found a single variant
allele that gave consistently diminished luciferase activity of
42.2±0.5% (mean±SEM; n=17) compared with the control allele (Fig 1B). This variant allele was present in two unrelated individuals
in our study population who had plasma Lp(a) concentrations of 35.5 and
26 mg/dL, respectively. This allele from both individuals was sequenced
to completion and found to differ in sequence from the control allele
at three sites, having 10 rather than 8 TTTTA repeats at -1231, an A
rather than a G at -772, and a T rather than a C at +93. The frequency
of this +93 T variant was determined by hybridization with
allele-specific oligonucleotides and found to be 14% of the 54 alleles
tested.
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Several of the cases were also of note. Two pairs of individuals had more than 10-fold differences in plasma Lp(a) concentration with roughly similar isoform sizes yet had no significant difference in in vitro luciferase expression. This observation suggests that elements outside the region studied can also influence apo(a) expression (see "Discussion").
To determine whether the C to T transition at +93 alone was responsible
for the reduction in luciferase expression from this allele, a 153-bp
Bpu1102 I-Bgl II fragment containing sequences +4
to +157 of the apo(a) gene with a C at position +93 was excised from
the control allele–luciferase vector construct and replaced by the
corresponding fragment containing T at position +93 (Fig 1A
).
Luciferase activity in HepG2 cells transfected with the control vector
containing the subcloned 153-bp fragment with T at position +93 was
nearly identical (38.0±4.0%) to the luciferase activity found in
lysates from cells transfected with the plasmid containing the entire
variant 1.4-kb 5' flanking region (42.2± 0.5%) (Fig 1).
Position +93 is within a region of the apo(a) gene that we had
previously shown to be essential for transcription and is located
within a DNase I protected footprint29 (Fig 2). Therefore, to determine whether a T at position +93
altered the binding affinity of nuclear proteins, gel mobility shift
assays were performed by using end-labeled synthetic oligonucleotides
spanning positions +79 to +105, containing either C or T at position
+93. Nuclear extracts prepared from HepG2 cells formed two specific
complexes with these oligonucleotides. No significant differences were
noted in the gel-shift pattern associated with the C and T
oligonucleotides (Fig 3). Gel-shift assays performed in
the presence of different amounts of unlabeled oligonucleotides as
competitors showed no difference in the ability of C or T
oligonucleotides to compete for specific binding of nuclear proteins
(Fig 3). This suggests that the presence of a T at position +93 does
not alter the affinity of binding of nuclear proteins to this
oligonucleotide.
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The T in the variant allele introduces an additional ATG 50-bp upstream
from the ATG that initiates translation (+142). The additional upstream
ATG site is in a different reading frame from that of the apo(a) coding
sequence and is followed by an in-frame stop codon (Fig 2). It is
therefore possible that this sequence difference might affect the
efficiency of translation rather than transcription. To estimate
transcription activity, we used a ribonuclease (RNase) protection assay
to quantify the amount of luciferase message expressed by the +93 C and
+93 T alleles. There was no significant difference in luciferase
message expressed by the two alleles (63.3±1.1 and 73.3±3.5 arbitrary
density units [mean±SEM, n=3] for +93 C and +93 T, respectively)
(Fig 4). This strongly suggests that the reduction in
luciferase expression from the variant apo(a) promoter observed in
transient transfection assays results from impaired translation rather
than transcription.
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To test this hypothesis, equal amounts of mRNA derived from apo(a) promoter–luciferase gene constructs, containing either a C or a T at position +93, were translated in a rabbit reticulocyte extract system. Luciferase activity of the lysate of the in vitro translation of the +93 T–derived mRNA was 40.1±0.2% (mean±SEM, n=4) that of the +93 C–derived mRNA. These results provide evidence that the additional upstream ATG site in the variant apo(a) gene impairs translation.
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In great contrast to other lipoproteins, plasma concentration of Lp(a) has an extremely wide variation, nearly 1000-fold among individuals.17 Levels of Lp(a) are relatively unaffected by environmental factors and are almost entirely inherited. Virtually all of the individual variation in plasma levels of apo(a) is closely linked to the apo(a) locus.23 24 In addition, metabolic studies indicate that catabolic rates among individuals are very similar.19 20 21 Hence, it appears that transcription, translation, and posttranslational processing of apo(a) are the major determinants of plasma levels of this atherogenic lipoprotein. It has been estimated that 41% to 69% of the marked variability in apo(a) levels is linked with isoform size variation,21 24 25 which partially results from extended transit time through the endoplasmic reticulum by the larger species.27 A number of studies in both humans and cynomolgus monkeys have shown a relation between Lp(a) concentrations and hepatic apo(a) mRNA levels.33 34 35 We proposed that some of the variation in apo(a) levels would reside in differences in transcription due to sequence differences in the 1.4-kb 5' flanking region of the apo(a) gene, which has previously been demonstrated to possess promoter activity.28 We therefore compared the transcription activity of the 5' flanking region of apo(a) alleles that encode for identically sized proteins yet were associated with very different levels of apo(a). In cases where isoforms matched exactly for mobility were not available, isoforms were generally selected for study because the apo(a) level associated with them was either unusually high or low, given the general inverse correlation between isoform size and level of Lp(a). This strategy was intended to maximize the likelihood of finding such differences.
We now describe a polymorphism in the 5' flanking region of the apo(a) gene, which consists of a single base transition from C to T at position +93. The T variant was found in 14% of the alleles screened. Luciferase activity expressed by reporter gene constructs containing a T at this position was reduced by 58%, initially suggesting that this variant affects transcription. Even though it occurs near the end of a DNase I footprint,29 this sequence variant has no apparent effect on the binding of nuclear factors to this region of the apo(a) promoter. (In contrast, changing six bases within this footprint region was sufficient to abolish nuclear protein binding and result in increased transcription activity [mut b29 ].) Rather, this single base C to T transition introduces an additional upstream ATG initiation codon that is followed by an in-frame stop codon. In our reporter gene constructs, the ATG introduced by this transition is also followed by an in-frame stop codon that is situated immediately upstream of the luciferase coding sequence. Since virtually identical amounts of luciferase mRNA were detected after transfection of HepG2 cells with reporter gene constructs containing either variant, the presence of a T at position +93 appears to have no significant effect on efficiency of transcription. In vitro translation with mRNA from both the +93 C and +93 T alleles showed that the creation of an additional upstream ATG impairs translation.
Previous studies with site-directed mutagenesis have demonstrated that
the presence of an additional ATG upstream of the translation start
site decreases the efficiency of translation.36 37 38 39 The
degree to which translation is affected is dependent on numerous
factors. Sequences surrounding an ATG determine the favorableness of an
upstream ATG as an initiation codon.39 A high level of
translation from the bona fide ATG is maintained in the case when
translation from the upstream ATG is terminated upstream of or within
the downstream initiator codon. The upstream ATG site found in the 5'
untranslated region of the apo(a) gene appears within a moderately
favorable sequence context and would encode a predicted polypeptide of
30 residues. Translation from the bona fide ATG appears to occur with
approximately 50% efficiency, which is consistent with previous in
vitro studies of the luciferase gene in which removal of an upstream
ATG codon resulted in a twofold increase in the level of luciferase
expression.38 The apo(a) gene also contains two other
upstream ATG codons in the 5' untranslated region, both of which are
closely followed by in-frame termination codons (Fig 3). Neither one of
these ATGs is situated within a favorable context for the initiation of
translation, and for these two reasons neither is predicted to have
functional significance.
Several mutations have been described that alter one of the three nucleotides of the ATG initiation codon of a number of genes. Such mutations appear to underlie some recognized disease states in humans, including ß-thalassemia,40 41 42 phenylketonuria,43 leukocyte adhesion deficiency, and hereditary elliptocytosis.44 At least one other disease state, ß-thalassemia, has been reported to be due to the introduction of an additional upstream ATG codon.45
We have also further studied two previously described polymorphisms of the 5' untranslated region of apo(a).28 A G to A variation located at position -772 was screened for by restriction endonuclease digestion with Taq I. In our study population, the frequency of alleles with G was 0.39. In contrast to preliminary analysis,28 we have now found this polymorphism to have no effect on transcription activity.29 We also studied the number of TTTTA repeats ending at position -1231. In 102 individuals, we observed a range of between 7 and 11 such repeats, with 8 repeats having the highest frequency. The number of such repeats did not affect the expression of luciferase in transient transfection assays.
Although the G to A polymorphism and the number of TTTTA repeats did not alter transcription activity of the apo(a) promoter in our studies, it is possible that one or both of these polymorphisms may be found in linkage disequilibrium with other variations of the apo(a) gene that do affect its regulation. In contrast, the C to T transition at +93 reduces the efficiency of translation in our in vitro system and may well do so in vivo. However, demonstrating that this polymorphism reduces levels of Lp(a) in vivo is not straightforward. Most significantly, as variation in apo(a) levels associated with similar isoform size has been reported to be as high as 200-fold,17 the in vivo effects of this mutation, which causes a twofold variation, might not be readily observed. Indeed, the extremely wide range of heritable levels of apo(a) plasma concentration could be due to a number of common polymorphisms at different sites in the gene. Thus, polymorphisms at a single site may significantly alter gene expression in vitro yet be difficult to follow by measurements of plasma concentration in population or family studies. In addition, the methodologies used to determine Lp(a) concentrations and to account for the amount of apo(a) expressed by each allele may be too imprecise at present to monitor in vivo effects of this magnitude. The numbers of subjects and advances in methodologies that would be required to demonstrate association of the +93T polymorphism with decreased levels of Lp(a) are beyond the scope and intent of this study.
The major finding of this report is the discovery of the first alteration in the flanking region of the apo(a) gene that affects its expression. In addition, we predict that significant regulatory elements exist outside the 1.4-kb region currently sampled. We noted several instances of subjects with roughly similar isoform size who had more than 10-fold differences in plasma concentration yet had identical sequence or in vitro activity in this region. Investigations are currently under way to define regulatory regions further upstream and within introns of the apo(a) gene that would contribute to variations in human blood levels of this atherogenic protein.
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Acknowledgments |
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This research was supported by National Institutes of Health (NIH) program project grant HL-48638-01 and the University of California Tobacco Related Disease Research program grant 2RT-0339 to Dr Lawn. Dr Zysow is supported by NIH clinical investigator development award HL-02701-02, Dr Lindahl by a fellowship from the NIH Fogarty International Center, and Dr Wade by a fellowship from the American Heart Association (California Affiliate).
Received July 25, 1993; accepted October 10, 1994.
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