Download Polymorphisms in the apolipoprotein(a) gene and their relationship

 1997 Oxford University Press
Human Molecular Genetics, 1997, Vol. 6, No. 7 1099–1107
Polymorphisms in the apolipoprotein(a) gene and
their relationship to allele size and plasma
lipoprotein(a) concentration
Loretto H. Puckey1, Richard M. Lawn2 and Brian L. Knight1,*
1MRC
Lipoprotein Team, Clinical Sciences Centre, Royal Postgraduate Medical School, Hammersmith Hospital,
London W12 ONN, UK and 2Division of Cardiovascular Medicine, Stanford University School of Medicine,
Stanford, CA 94305, USA
Received February 11, 1997; Revised and Accepted April 14, 1997
Genotypes at five previously described polymorphic
sites at the apolipoprotein(a) gene locus have been
determined for the members of 27 families as well as for
unrelated white Caucasian and Asian-Indian subjects,
and their relationship with isoform size and plasma
lipoprotein(a) concentrations investigated. There was
strong linkage disequilibrium between sites at the
5′-region of the gene and also between this region and
a site in the coding sequence for Kringle 4-37 on the
other side of the polymorphic Kringle 4 repeat region.
There was no evidence that changes at any of the sites
had any direct effect upon lipoprotein(a) concentration.
However, certain haplotypes were present almost
exclusively on apolipoprotein(a) alleles within a restricted range of sizes and associated lipoprotein(a)
concentrations. After correcting for the effect of allele
size, there were clear differences between the lipoprotein(a) concentrations associated with alleles of
different haplotypes, suggesting that there may be
genetically distinct groups of apolipoprotein alleles of
different size and different levels of expression. Factors
that regulate expression apparently exchange at a rate
similar to the rate of change of Kringle 4 repeat number.
INTRODUCTION
Lipoprotein(a) [Lp(a)] is a plasma particle similar in size and
composition to low-density lipoprotein. In addition to
apolipoprotein (apo)B100, Lp(a) contains a second protein,
apo(a), that bears a strong resemblance to plasminogen (1,2).
Apo(a) occurs in >30 differently sized isoforms resulting from
different numbers of a repeated sequence homologous to the
fourth of the triple-looped ‘Kringle’ structures found in
plasminogen (3). There is considerable variation in plasma Lp(a)
concentrations between individuals, >90% of which is
determined by the apo(a) gene locus (4–6). In Caucasian
populations, there is an inverse trend between Lp(a)
concentration and the size of its apo(a) component (7). However,
there is a large variation within this trend, with up to 200-fold
differences in the concentrations associated with the same sized
apo(a) isoforms produced from different independent alleles (8).
In the livers of both monkeys (9) and man (10), there are also
differences in the abundance of apo(a) mRNA transcripts of the
same size, suggesting that at least part of the size-independent
variation in expression is likely to involve regions that regulate
the rate of transcription of the gene.
The immediate 5′-flanking region of the apo(a) gene has been
cloned (11,12), and the first 1.5 kb has been shown to promote
transcription of luciferase reporter gene constructs transfected into
HepG2 cells (11). Three single base substitutions and a variable
region containing between seven and 11 copies of a TTTTA repeat
have been identified in this sequence (11,13,14). One substitution,
a C→T change at position +93 in the 5′-untranslated region of the
first exon, is associated with a 58% decrease in expression resulting
from a reduction in translation due to the creation of a novel
upstream ATG start site (14). Also, an association of 10 or 11
TTTA repeats with small apo(a) isoforms (15) or low plasma Lp(a)
concentrations (15,16) has been observed. However, the other sites
have not been examined in detail.
To assess the physiological significance of these changes, we
have studied the polymorphisms in white Caucasian and
Asian-Indian subjects and have investigated their relationship to
allele size and Lp(a) concentration. We have also examined the
effects of a further polymorphism, in the coding sequence at the
other end of the gene, which changes a Met residue to a Thr close
to the putative lysine-binding site in Kringle 4-37 (17) and has
been reported to be in linkage disequilibrium with the apo(a) size
polymorphism (18). Finally, since there is evidence for the
presence of subgroups of apo(a) alleles (8,13,15), we have
constructed haplotypes from the five polymorphisms to discover
if particular groups, defined in this way, are associated with a
limited range of sizes or concentrations.
RESULTS
Population studies
Five polymorphisms in the human apo(a) gene have been
examined in this study (Fig. 1). One (N–/N+) relates to an NcoI
*To whom correspondence should be addressed. Tel: +44 181 383 3262; Fax: +44 181 383 2077; Email: [email protected]
1100 Human Molecular Genetics, 1997, Vol. 6, No. 7
restriction enzyme site at position 12 605 in the coding sequence.
The others are in the 1400 bp region immediately 5′ to the
translation start site. Three of these are single base differences, at
positions –772 (G/A), +93 (C/T) and +121 (G/A), while the fourth
is a variable region beginning at position –1231, containing
between seven and 11 copies of a TTTTA repeat. Table 1 shows
the allele frequencies and heterozygosity index for each of these
polymorphisms in a population of unrelated white Caucasians
and a population of unrelated Asian-Indians. The only major
difference between the populations was at position –772, where
the G variant was more frequent in the Caucasians and the A
variant was more frequent in the Indians. At the other positions,
the rarer variant was slightly more frequent in the Indian
population. Eight TTTTA repeats were most common in both
populations, with nine and 10 repeats at a lower frequency, similar
to each other. The frequencies in the Caucasian sample were
similar to those described previously for the TTTTA repeats
(15,16) the G/A (+121) polymorphism (13) and the N–/N+
polymorphism (18).
In both populations reported in Table 1, the frequency
distributions of genotypes were in Hardy–Weinberg equilibrium.
Linkage disequilibrium was examined for each pair of polymorphisms by calculating the disequilibrium statistic (∆) and the
relative linkage disequilibrium (rel. D), values of which are given
in Figure 2. As would be expected, strong linkage disequilibrium
was detected between the four polymorphic loci in the immediate
5′-flanking region of the apo(a) gene. There was only one
instance where the ∆ value was not significant, between the sites
at +93 and +121. Linkage disequilibrium was also detected
between the 5′ sites and the NcoI polymorphism in Kringle 4-37.
The only exceptions were the almost complete equilibrium
observed between the NcoI polymorphism and the G/A site at
position –772 in the Indian population and between the NcoI
polymorphism and the G/A site at position +121 in the Caucasian
population.
The apo(a) phenotype was determined for 170 unrelated white
Caucasians. Two bands were detected on phenotyping gels for all
but 17 subjects. The others showed single band phenotypes. The
distribution of the different sized isoforms in the two populations
is shown in Figure 3, and provides evidence for at least two and
probably three major subgroups.
Family studies
To examine the relationship between the different polymorphic
variants in greater detail, genotypes were determined at all five
sites for 269 members of 27 unrelated white Caucasian families.
Haplotypes were constructed by analysis of the segregation of the
variants, with the assumption that there had been no recombination within the apo(a) locus. For each pedigree, only the
independent haplotypes were included in the subsequent analysis.
Unambiguous haplotypes were obtained for 197 independent
alleles. A total of 26 different haplotypes from a possible 40
(65%) were observed, the frequencies of which are given in Table
2. The polymorphic sites were clearly not in equilibrium. The T
variant at position +93 was present exclusively on alleles that
contained the A variant at position –772. Almost all of the A
(+121) variants were also on alleles with the A variant at –772.
In contrast, the N+ variant was more frequent on alleles
containing the G variant at position –772. Most of the alleles
containing G (–772) carried eight TTTTA repeats, while those
containing A (–772) had a far higher proportion carrying nine or
10 repeats. For convenience, alleles will be identified by the
pattern of variants, reading from the 5′ end.
The apo(a) phenotype was determined for the members of 17
of the families, comprising 186 individuals. By tracking the
isoforms through the families, it was possible to establish the
number of Kringle 4 repeats contained by 156 of the alleles for
which variants at the polymorphic sites were known. In assessing
whether different variants were associated with alleles of
different size, these alleles were augmented with those from the
white Caucasian subjects used previously who were homozygous
at the site of interest. The frequency of alleles containing different
numbers of Kringle 4 repeats is given for individual variants in
Figure 4. The frequency profiles for alleles with the G or A
variants at position –772 were similar, as were those for the N–
or N+ variants in Kringle 4-37. In contrast, there was a greater
proportion of large and a smaller proportion of small alleles
among those carrying the A variant at position +121. The T (+45)
variant and nine TTTTA repeats were mainly on middle sized
alleles, whereas a high proportion of the 10 TTTTA repeats were
on small alleles.
Table 1. Allele frequencies at five variable sites in the apo(a) gene and its 5′ region
Polymorphism
Position
Allele
White Caucasians
Frequency ± SE
Heterozygosity
Asian-Indians
Frequency ± SE
Heterozygosity
G/A
–772
G
0.586 ± 0.020
0.485
0.429 ± 0.038**
0.489
C/T
+93
C
0.867 ± 0.013
0.231
0.765 ± 0.033**
0.359
G/A
+121
G
0.844 ± 0.014
0.263
0.765 ± 0.033*
0.359
NcoI
‘Kr37’
N–
0.697 ± 0.018
0.422
0.582 ± 0.038**
(TTTTA)n
–1231
0.485
0.486
0.552
n =7
0.005 ± 0.003
0.006 ± 0.005
n=8
0.687 ± 0.018
0.612 ± 0.037
n=9
0.144 ± 0.014
0.206 ± 0.031*
n = 10
0.149 ± 0.014
0.176 ± 0.029
n = 11
0.016 ± 0.005
0
Nucleotide positions are expressed relative to the transcription start site. The NcoI site represents a C/T polymorphism at position 12 605 of the coding sequence,
in Kringle 4-37, as described by McLean et al. (3). Values are shown for a population of 319 white Caucasians and a population of 85 Asian-Indians.
Significant difference between populations (χ2 test); *P 0.05, **P <0.001.
1101
Human
Genetics,
1997,
6, No.
NucleicMolecular
Acids Research,
1994,
Vol. Vol.
22, No.
1 7
1101
Table 2. Frequency of alleles containing different haplotypes at the apo(a) gene locus in white Caucasian families
Variant at position
772
+93
No. of alleles
+121
‘Kr37’
Row
No. of TTTTA repeats
Total (%)
7
8
9
10
11
G
G
G
G
C
C
C
C
G
G
A
A
N–
N+
N–
N+
0
0
0
0
59
44
1
3
4
1
1
0
4
1
0
0
2
0
0
0
69 (35%)
46 (23%)
2 (1%)
3 (2%)
G
T
G/A
N–/N+
0
0
0
0
0
0 (0%)
A
A
A
A
A
A
A
A
C
C
C
C
T
T
T
T
G
G
A
A
G
G
A
A
N–
N+
N–
N+
N–
N+
N–
N+
Column total
0
0
1
0
0
0
0
0
5
2
12
8
2
0
0
0
136
2
1
0
0
14
0
2
0
25
13
1
3
1
7
1
2
0
33
0
0
0
0
0
0
0
0
20 (10%)
4 (2%)
16 (8%)
9 (5%)
23 (12%)
1 (1%)
4 (2%)
0 (0%)
1
2
197
Haplotypes were constructed from the segregation of variants at five sites through 27 Caucasian families that included 269 individuals. The full pedigrees contained 134
unrelated individuals, of whom 91 were available for study. A total of 231 independent alleles could be identified, of which full haplotypes could be constructed for 197.
Figure 1. Diagram of the apo(a) gene showing the relative positions of the
polymorphisms studied. Exons are shown as boxes with the translated regions
shaded. The 5′-flanking region (homology to plasminogen hatched) and exons
are approximately to scale. The introns (lines) are not to scale and are greatly
shortened. The positions of the polymorphisms (arrows) and of the various
types of Kringle 4 repeat are illustrated.
Relationship to plasma Lp(a) concentrations
Our sample of normal, unrelated white Caucasian subjects
showed the typically skewed distribution of plasma Lp(a)
concentrations. The mean, after log transformation, was 10.1 ±
4.7 (SD) mg/dl, with a range of 0.2–129 mg/dl and a median of
13.7 mg/dl. To establish the relationship between L(a)
concentration and apo(a) isoform size, the total Lp(a)
concentration for each subject was apportioned between the two
Lp(a) species as described in Materials and Methods. This gives
an estimate of the Lp(a) concentration associated with each apo(a)
isoform, with an error of ∼12% (SD) (8). As observed before for
hyperlipidaemic subjects (8), there was a general inverse
non-linear relationship between Lp(a) concentration and the
number of Kringle 4 repeats in the apo(a) protein, with a large, up
to 500-fold, range of values for each of the different-sized
isoforms (Fig. 5).
The plasma Lp(a) concentration was apportioned between the
Lp(a) species for each member of the 17 families for whom the
apo(a) phenotype had been determined. There were a few instances,
such as those shown in Figure 6, where families contained alleles of
the same or similar size with the same, most common, haplotype. In
the RW family, the alleles with 25 Kringle 4 repeats inherited from
different mothers had the same haplotype but were associated with
markedly different Lp(a) concentrations. In the NL family, the three
middle siblings expressed alleles containing 28, 29 or 30 Kringle 4
repeats with the same haplotype, which were associated with very
different Lp(a) concentrations, even when two of them were
expressed in the same individual. Thus it is clear that major
differences in Lp(a) concentrations occur without any changes in the
polymorphisms that we are studying. However, this does not
necessarily imply that the changes themselves have no effect on the
rate of transcription. To check this, the Lp(a) concentration
associated with each independent allele in the families was
estimated. Each value was then corrected for the effect of allele size
using the exponential line of best fit for the white Caucasian
population shown in Figure 5 (see Materials and Methods), allowing
a direct comparison between values for all alleles (Fig. 7). After
correcting for size, there was still a vast range of Lp(a)
concentrations, although there was a suggestion from the spread of
the points, seen most clearly in the column for the G (+121) variant,
that the values fell into distinct groups. Overall there was no
significant association between Lp(a) concentration and the variants
at the G/A (–772), C/T (+93) or N–/N+ (Kr 4-37) sites. The A
variant at position +121 was associated with a significantly higher
mean Lp(a) concentration than the G variant, due mainly to an
absence of very low values. Alleles containing 10 TTTTA repeats
were associated with a significantly lower mean concentration than
those carrying nine repeats.
Haplotype relationships
There were 136 alleles for which the whole haplotype and the
associated Lp(a) concentration were known. Over 90% of these
had one of six haplotypes. Figure 8 shows the size and Lp(a)
concentrations, corrected for size, for the alleles with each of
these haplotypes. Alleles with 8GCG haplotypes were the most
1102 Human Molecular Genetics, 1997, Vol. 6, No. 7
Figure 2. Values for pairwise disequilibrium (∆) and relative linkage disequilibrium (Rel. D) between polymorphic sites in the apo(a) gene. The variable TTTTA site
was recoded as a diallelic system of eight or ‘not eight’ repeats. Results were obtained for 319 unrelated normal white Caucasians and 85 Asian-Indians living in and
around London. *Significant value of ∆ taken at the level of P < 0.005, giving an overall significance level for the 10 tests of 0.05.
between the 9ATGN–, 10ACGN– and 8ACAN– alleles and the
8ACAN– and 8ACAN+ alleles were all statistically significant (P
<0.05, Student’s t-test).
DISCUSSION
Figure 3. Frequency of apo(a) Kringle 4 repeats. Apo(a) phenotype was
determined for 170 unrelated normal white Caucasians. Isoform sizes were
converted to Kringle 4 content using the relationship derived previously (8).
abundant and were associated with a wide range of sizes and
concentrations. A quarter of 8GCG alleles with the N– variant,
but none of those with the N+ variant, contained <20 Kringle 4
repeats and were associated mostly with high concentrations (Fig.
8a). There were less alleles with haplotypes containing the A
variant at position –772, but these fell into more clearly defined
groups (Fig. 8b). All but one of the 9ATGN– alleles contained
27–29 Kringle 4 repeats (26.9 ± 1.2 Kringles, mean ± SE) and all
were associated with corrected Lp(a) concentrations within the
100–300% range (189.5 ± 21.4%). Similarly, all the 10ACGN–
haplotypes were confined to alleles containing 21–23 Kringle 4
repeats (22.4 ± 0.2 Kringles) and all but one were associated with
corrected Lp(a) concentrations of between 15 and 50% (48.5 ±
23.0%). Points for the 8ACAN– haplotypes were more
widespread, but were confined to alleles with 27 or more Kringle
4 repeats (32.8 ± 1.1 Kringles) associated with corrected
concentrations of ∼200% or less (115.1 ± 19.8%). In contrast,
8ACAN+ haplotypes were on alleles with a greater spread of
sizes (25.8 ± 3.2 Kringles), the majority of which were associated
with particularly high Lp(a) concentrations (332.5 ± 74%).
Differences in both Kringle number and corrected concentration
The C/T (+93) and G/A (+121) polymorphisms studied here are
in the first exon of the apo(a) gene. The G/A (–772) and variable
TTTTA sites are further upstream, beyond the region that has
homology with the plasminogen gene (Fig. 1). The
disequilibrium statistic was statistically significant between all
these sites except C/T (+93) and G/A (+121). However, the
disequilibrium between these two sites was of the negative type
and both polymorphic frequencies were low, so the probability of
detecting disequilibrium with this sample size was small (19).
Since the rel. D values were high, the results indicate strong
linkage disequilibrium throughout the region. Linkage
disequilibrium was also observed between the 5′-flanking region
and the NcoI polymorphism in Kringle 4–37. If the expansion of
the variable Kringle 4 region had involved a crossing-over
mechanism, these sites on either side would have reached
equilibrium rapidly. Lackner et al. (20) have reported a case
where the number of repeats had altered from one generation to
the next with no exchange of flanking polymorphic markers,
suggesting that the new apo(a) allele of different length had arisen
without any crossing-over of homologous chromosomes. The
disequilibrium described here, together with that observed
previously for a DraIII polymorphic pattern exhibited by a subset
of the variable Kringle 4 units (21), is consistent with their
proposal (20) that much of the variation in Kringle 4 number
results from sister chromatid exchange or complex gene
conversion events.
The disequilibrium between the G/A (–772) and NcoI sites in
the Caucasians arises because a high proportion (82%) of the A
(–772) variants are on N– alleles. Since there is no disequilibrium
and a far higher frequency of A (–772) variants in the Indian
population, it seems that there has been an accumulation of G
(–772)N+ alleles in the Caucasians. Similarly, although we did
not have enough families to confirm this, the data suggest that
there has been an accumulation of A (+121)N+ alleles in the
Indian group. In both populations, the disequilibrium between the
1103
Human
Genetics,
1997,
6, No.
NucleicMolecular
Acids Research,
1994,
Vol. Vol.
22, No.
1 7
Figure 4. Size distribution of apo(a) isoforms coded by alleles containing
different polymorphic variants. Isoform size and variants at polymorphic sites
in the apo(a) gene were determined for 156 independent alleles from white
Caucasian subjects by following their segregation through families. These were
augmented by alleles from individuals in the Caucasian ‘population’ who were
homozygous at the site of interest. Sizes are shown for 116 alleles with the G
variant and 80 alleles with the A variant at position –772, for 22 alleles with nine
TTTTA repeats and 26 with 10 TTTTA repeats, for 20 alleles with the T variant
at position 93, 38 alleles with the A variant at position +121 (A*), 151 alleles
with the N– variant and 69 alleles with the N+ variant in Kringle 4-37.
Distributions for alleles with eight TTTTA repeats, C (+93) and G (+121) were
similar to that shown in Figure 3 and are not shown.
Figure 5. Lp(a) apoB concentrations associated with different-sized apo(a)
isoforms. Total plasma Lp(a) concentration was apportioned between the two
Lp(a) species (8) for the unrelated subjects described in Figure 3. Values are
shown for 323 white Caucasian isoforms with the exponential line of best fit.
1103
NcoI site and the C/T site or the variable TTTTA region was
probably a reflection of the relatively recent occurrence of the
C→T mutation and of the latest addition to the repeats. Most
(89%) of the variants containing nine or 10 TTTTA repeats and
all but one of the T (+93) variants were present on an N– allele.
Indeed, the T variant and the expansion to nine or 10 repeats
probably all arose on A (–772)G (+121)N– alleles, which, with
C (+93) and eight TTTTA repeats, is the most likely original
haplotype.
Two apo(a) isoforms were identified in 91% of the individuals
studied. This is close to the number expected from studies of the
gene (22) and justifies the use of polyacrylamide–agarose gels,
which are difficult to perfect but are sensitive and give clear, sharp
bands. By determining the sizes of the isoforms coded by alleles
containing the different variants in the families, it was clear that
alleles with the T variant were associated mainly with middle
sized isoforms and those with the A (+121) variant with middle
and large isoforms. This approach also revealed that alleles with
nine TTTTA repeats were mostly confined to the middle sizes and
that those with 10 TTTTA repeats apparently fell into two groups,
one of small sizes and one of middle sizes. A similar association
of TTTTA repeat number with allele size, although with a greater
spread of sizes than observed here, has been reported previously
for another Caucasian population (15). The variants at position
–772 were not associated with alleles of any particular size and
we did not detect in our Caucasian subjects the high frequency of
alleles with 28 Kringle 4 repeats among N+ alleles that was
observed in a Tyrolean population (18).
As the apo(a) alleles were further subdivided through the
construction of detailed haplotypes, association with allele size
became more clearly defined. Nearly all of the nine TTTTA
variants and most of those with T at +93 were on the same alleles
(9ATGN–) with a very restricted range of sizes (27–29 Kringle 4
units). Similarly, 10 TTTTA repeats with ACGN– haplotypes
were restricted to alleles with 21–23 Kringle 4 units. Although the
range of sizes was greater, other relationships could also be
demonstrated. For instance, all of the 8ACAN– alleles were large
and most of the very small alleles of 20 Kringle 4 units or less
were 8GCGN–. Thus there appear to be subgroups of apo(a)
alleles of the same haplotype associated with a limited range of
sizes, the spread of which presumably reflects the time that has
elapsed since the last mutation.
Having used segregation analysis to define apo(a) alleles by
haplotype, it was clearly of interest to discover if they were
associated with different Lp(a) concentrations. By apportioning
the total according to the proportions shown on the apo(a)
phenotyping blots, it was possible to calculate the concentration
of the separate species in individual subjects and so to estimate the
Lp(a) concentration associated with each of the defined alleles in
the families. There were clearly major differences in expression
of alleles of similar size within families and, in numerous
instances such as that in Figure 6, within the same individual.
Thus large differences in the expression of apo(a) alleles cannot
be explained by the effects of environment or other genes, and it
is justifiable when examining these differences to treat each
independent allele as being phenotypically distinct (15,21,23). If
this assumption were made, it became apparent that some
subgroups of apo(a) alleles defined by haplotype were associated
with narrow ranges of Lp(a) concentrations. For instance,
9ATGN– alleles were all associated with moderately high
concentrations, while most 10ACGN– alleles were associated
1104 Human Molecular Genetics, 1997, Vol. 6, No. 7
Figure 6. Pedigree diagrams illustrating the segregation of variants and their relationship to Lp(a) concentration. The apo(a) isoforms for each subject, given as the
number of repeated Kringle 4 units, are shown in italics immediately below the symbols. The apportioned Lp(a) apoB concentration (µg/ml) is shown in bold under
the appropriate isoform, with the variants carried by the allele beneath (reading 5′ to 3′, top to bottom). Alleles of interest are shown in bold type.
Figure 7. Lp(a) concentrations associated with alleles containing different
polymorphic variants. The Lp(a) apoB concentration associated with each of
the white Caucasian alleles from Figure 4 was estimated and corrected for
isoform size as described in Materials and Methods. Each value is given as a
percentage of the average for an isoform of the same size. Results are shown
for 218 alleles with either eight, nine or 10 TTTTA repeats at position –1231,
196 alleles with either the G or A variant at –772, 241 alleles with either C or
T at +93, 259 alleles with G or A at +121 and 228 alleles with the N– or N+
variant in Kringle 4-37. Mean values are shown by the solid bar. There were
significant differences (P <0.05, Student’s t-test) between the mean values for
the G and A variants at +121 and mean values for alleles with nine and 10
TTTTA repeats.
with fairly low concentrations. These haplotypes were the two
that were restricted to alleles with a size spread of only three
Kringle 4 units. Generally, as the spread of allele sizes in the
subgroup increased, the range of associated concentrations also
increased. Thus it appears that after the defining mutation,
features that regulate the gene gradually exchange and equilibrate
at a rate similar to the changes in Kringle 4 number.
At various times, it has been suggested that the polymorphisms
observed in the 5′ region could directly influence the expression
of the apo(a) gene (11,14,16). It is also possible that the NcoI site
in Kringle 4-37 is linked to changes in lysine binding ability that
could affect Lp(a) assembly. Since the overall variation in
expression of a given allele within families has been estimated to
be 21% (SD), the present analysis would not be able to confirm
relatively small effects of less than ∼2-fold (8). However, within
this limitation, there was no evidence that variation at any of the
polymorphic sites studied had any direct effect on plasma Lp(a)
concentration. Our studies confirm the association of low
concentrations with alleles containing 10 TTTTA repeats observed by Trommsdorff et al. (16), but indicate that this is because
a large proportion are in a subgroup (10ACGN–) linked to a
relatively inactive regulatory element rather than through a direct
effect on transcription. Similarly the higher concentrations
associated with the A (+121) variant can be attributed to the fact
that the majority were in the 8ACAN– or 8ACAN+ subgroups,
which were not linked to very low activity elements. These results
provide physiological support for in vitro assays that have shown
no difference in the transcriptional activity of constructs with
different variants at the G/A (–772) and variable TTTTA sites
(15,24). Unfortunately, the T (+93) variant was present mainly in
only two subgroups, so it was not possible to evaluate the effect
of the reduced transcriptional activity observed in vitro (14).
The Lp(a) concentration associated with different apo(a) alleles
of the same size can differ >100-fold. The data presented here
indicate that it is unlikely that common sequence changes in the
immediate 5′-flanking region of the gene could be responsible for
such large differences in expression. The results also suggest that
apo(a) alleles can be divided into subgroups which accumulate to
a greater or lesser extent in different populations and that these
subgroups can be associated with a limited range of Lp(a)
concentrations, presumably reflecting those of their precursor
alleles. They would predict that there are regulatory elements of
different activities in the population, situated far enough away
from the gene to allow exchange between alleles. The three
liver-specific DNase-hypersensitive sites in the 40 kb region
between the apo(a) and plasminogen genes (25) are obvious
1105
Human
Genetics,
1997,
6, No.
NucleicMolecular
Acids Research,
1994,
Vol. Vol.
22, No.
1 7
1105
Figure 8. Size and associated Lp(a) concentrations of apo(a) alleles carrying different haplotypes. Haplotypes at five polymorphic sites were constructed from the
segregation of variants through 17 white Caucasian families. Whole haplotypes were obtained for 136 independent alleles for which the allele size and associated Lp(a)
concentration had been determined (see Materials and Methods and text). These alleles were augmented by those from 11 independent white subjects who were
homozygous at each polymorphic site. Each value is given as a percentage of the average for an isoform of the same size (see Materials and Methods). (a) Values for
8GCGN– () alleles and 8GCGN+ () alleles, (b) values for 8ACAN– alleles (), 8ACAN+ alleles (∆), 9ATGN+ alleles () and 10ACGN– alleles ().
candidates for the location of these elements, and experiments to
characterize them currently are being pursued.
MATERIALS AND METHODS
Materials and general methods
Restriction endonucleases were obtained from BoehringerMannheim, Lewes, UK and Biotaq polymerase from Bioline UK
Ltd, London, UK. Agarose (ultra pure) and DNA size markers
were supplied by Gibco-BRL, Paisley, UK and MetaPhor agarose
by FMC Bio Products, Rockland ME. Hybond N nylon
membrane (0.45 mm) was obtained from Amersham International plc, Little Chalfont, UK.
General methods and the definition and composition of
solutions used are given in Sambrook et al. (26).
Subjects
The subjects in this study comprised 269 members of 27
Caucasian families and 356 unrelated white Caucasian and 85
unrelated Asian-Indian individuals living in and around London.
The unrelated subjects were apparently normal individuals from
screening projects, together with laboratory volunteers. Both
sampled populations were heterogeneous within their basic racial
groups. Venous blood was collected into tubes containing EDTA
and either immediately frozen or separated by centrifugation
within 2 h. Whole blood and separated plasma and blood cells
were stored at –70C.
Lp(a) and apo(a) analysis
Plasma Lp(a) concentration was assayed using a TintElize
immunoassay kit (Biopool AB, Umea, Sweden) and the apo(a)
phenotype determined by immunoblotting (8). To estimate the
Lp(a) concentration associated with each of the apo(a) isoforms
in a sample, the phenotyping blot was scanned and the total Lp(a)
concentration was apportioned between the two constituent Lp(a)
species containing the different apo(a) isoforms, as described
previously (8). Values from individual family members were used
to estimate the Lp(a) concentration associated with each of the
independent alleles. Seven of the 17 families used for these
studies contained members with familial hypercholesterolaemia,
and values from these subjects were excluded from the estimations. Where there was more than one normolipidaemic subject
with the same apo(a) allele, which occurred in ∼50% of cases, the
values were averaged. Individual values never varied by >40% of
the average, which was small compared with the variation in
values between different alleles.
Apportioned values of Lp(a) concentration were corrected for
the effect of apo(a) allele size using the plot of isoform size
against associated Lp(a) concentration for unrelated white
Caucasian individuals shown in Figure 5. The points were best
described by an exponential line, which was used to give an
estimate of the average concentration associated with each size of
apo(a) allele. Each individual Lp(a) concentration was then
expressed as a percentage of the estimated average concentration
associated with an allele of the same size.
DNA analysis
DNA was isolated from frozen whole blood or packed blood cells
as described previously (27) or by a rapid small scale method (28).
A 1458 bp fragment, encompassing the region +170 to –1288
relative to the transcription start site of the apo(a) gene, was
amplified by the polymerase chain reaction (PCR), carried out in
a final volume of 50 µl containing ∼150 ng of genomic DNA,
1.25 mM MgCl2, 250 ng of each primer, 0.125 mM of each
deoxynucleotide triphosphate and 1.25 U of Taq polymerase in
the ‘ammonium’ buffer provided by the manufacturer. Samples
were overlaid with mineral oil and heated in a thermal cycler for
1 min at 95C, followed by three cycles of 30 s at 97C, 30 s at
50C and 2.5 min at 65C, and then by 30 cycles of 30 s at 94C,
30 s at 50C and 2.5 min at 65C, ending with 7 min at 72C. A
smaller, ∼100 bp fragment encompassing the TTTTA repeats 5′
1106 Human Molecular Genetics, 1997, Vol. 6, No. 7
to position –1230 was amplified under similar conditions only
using 100 ng of each primer and 48C for the annealing reaction.
The primers used were 1458 bp fragment: 5′-primer, 5′-GAA
AGA TTG ATA CTA TGC-3′; 3′-primer, 5′-AGT AGA AGA
ACC ACT TC-3′; 100 bp fragment: 5′-primer, 5′-GCG GAA
AGA TTG ATA CT-3′; 3′-primer, 5′-ACG TCA GTG CAC TTC
AA-3′.
The authenticity of the 1458 bp fragment was verified by
restriction enzyme digestion with HindIII, which cuts at one site
to give fragments of 986 and 472 bp. G and A variants at position
–772 were detected by digestion with TaqI followed by electrophoresis on 1.5% agarose. The G variant contains a cutting site for
TaqI and produces a fragment of 942 bp, whereas the non-cutting
A variant produces an equivalent fragment of 1139 bp. The C and
T variants at position +93 and the G and A variants at position
+121 were detected by annealing with allele-specific oligonucleotides. Samples (10 µl) of PCR reaction mixture were
diluted with 200 µl of 15× SSC, heated at 100C for 10 min and
100 µl were added to wells of a slot-blot apparatus containing a
nylon membrane pre-soaked in 15× SSC. After washing with 15×
SSC, the nucleic acids were fixed to the membrane with UV light.
The membrane was pre-hybridized for 10 min at 40C in 5×
SSPE (0.75 M NaCl), 5× Denhardt’s solution and 0.5% SDS [see
(26) for solution composition] and hybridized for 3 h at 40C in
the same solution containing ∼1×106 c.p.m./ml of the appropriate
oligonucleotide, end-labelled with [γ-32P]ATP using standard
methods (26). Oligonucleotides employed were 5′CAA CAA
CGT CCT GG3′ (coding strand) for the C variant, 5′CCA GGA
CAT TGT TGA3′ (non-coding strand) for the T variant, 5′TTC
TGG GCA CTG CT3′ (coding strand) for the G (+121) variant
and 5′AGC AGT GTC CAG AAA3′ (non-coding strand) for the
A (+121) variant. The membrane was rinsed once and washed for
30 min at 48C with 5× SSPE, 0.1% SDS before autoradiography.
The number of TTTTA repeats was determined by size
fractionation of the smaller PCR product on non-denaturing 8%
polyacrylamide gels (20 cm × 20 cm × 1 mm, 250 V for 5 h)
visualized by silver staining, or on 4% MetaPhor agarose
visualized with ethidium bromide. Sizes were estimated by
comparison with a 10 bp DNA size ladder. A fragment of 96 bp
contained eight TTTTA repeats.
The DNA encoding the lysine-binding region of Kringle 4-37
was amplified by PCR after BamHI digestion as described by
Pfaffinger et al. (29). The product was incubated with the
restriction enzyme NcoI and the fragments separated on 8%
polyacrylamide gels as described above. In the presence of the
cutting site, the enzyme cut the 182 bp PCR product into
fragments of 122 and 60 bp.
Statistical methods
Departures of genotype distributions from Hardy–Weinberg
equilibrium were tested for significance by the χ2 test (1 d.f., P
<0.05). Allele frequencies for the polymorphic sites were determined by allele counting of the independent chromosomes, and
standard errors and heterozygosity indices calculated by standard
procedures (30). Non-random association between a pair of sites
was measured by the standardized disequilibrium statistic (∆),
calculated according to Chakravarti et al. (30) after haplotype
frequencies had been estimated using the maximum-likelihood
procedure outlined by Hill (31) with the assumption for each pair
of Hardy–Weinberg equilibrium, selective neutrality and co-dominance. Statistical significance was tested using N∆2, where N is the
number of genotypes observed, which is distributed as a χ2 random
variable with 1 d.f. Since it cannot be assumed that the pairwise
comparisons are independent, statistical significance was assessed
at a level for each ∆ value that gave an overall significance level
of P <0.05. Relative linkage disequilibrium was estimated as
described by Thompson et al. (19).
ACKNOWLEDGEMENTS
We thank Dr G. Lindahl, Dr B. Zysow and Mr Y.F.N. Perombelon
who established the conditions for amplifying the 1.5 kb fragment
and determining the C/T (+93) variants. We are indebted to Dr G.
Thompson (Hammersmith), Dr M. Seed (Charing Cross) and Dr
A. Hale (BUPA) for access to clinical material. This work was
supported in part by a grant from the British Heart Foundation
(PG 94142).
REFERENCES
1. Scanu, A.M. and Fless, G.M. (1990) Lipoprotein(a). Heterogeneity and
biological relevance. J. Clin. Invest., 85, 1709–1715.
2. Eaton, D.L., Fless, G.M., Kohr, W.J., McLean, J.W., Xu, Q.T., Miller, C.G.,
Lawn, R.M. and Scanu, A.M. (1987) Partial amino acid sequence of
apolipoprotein(a) shows that it is homologous to plasminogen. Proc. Natl
Acad. Sci. USA, 84, 3224–3228.
3. McLean, J.W., Tomlinson, J.E., Kuang, W.J., Eaton, D.L., Chen, E.Y., Fless,
G.M., Scanu, A.M. and Lawn, R.M. (1987) cDNA sequence of human
apolipoprotein(a) is homologous to plasminogen. Nature, 330, 132–137.
4. Boerwinkle, E., Leffert, C.C., Lin, J., Lackner, C., Chiesa, G. and Hobbs,
H.H. (1992) Apolipoprotein(a) gene accounts for greater than 90% of the
variation in plasma lipoprotein(a) concentrations. J. Clin. Invest., 90, 52–60.
5. Kraft, H.G., Kochl, S., Menzel, H.J., Sandholzer, C. and Utermann, G. (1992)
The apolipoprotein(a) gene: a transcribed hypervariable locus controlling
plasma lipoprotein(a) concentration. Hum. Genet., 90, 220–230.
6. DeMeester, C.A., Bu, X., Gray, R.J., Lusis, A.J. and Rotter, J.I. (1995)
Genetic variation in lipoprotein(a) levels in families enriched for coronary
artery disease is determined almost entirely by the apolipoprotein(a) gene
locus. Am. J. Hum. Genet., 56, 287–293.
7. Utermann, G., Menzel, H.J., Kraft, H.G., Duba, H.C., Kemmler, H.G. and
Seitz, C. (1987) Lp(a) glycoprotein phenotypes. Inheritance and relation to
Lp(a)-lipoprotein concentrations in plasma. J. Clin. Invest., 80, 458–465.
8. Perombelon, Y.F., Soutar, A.K. and Knight, B.L. (1994) Variation in
lipoprotein(a) concentration associated with different apolipoprotein(a)
alleles. J. Clin. Invest., 93, 1481–1492.
9. Azrolan, N., Gavish, D. and Breslow, J.L. (1991) Plasma lipoprotein(a)
concentration is controlled by apolipoprotein(a) (apo(a)) protein size and the
abundance of hepatic apo(a) mRNA in a cynomolgus monkey model. J. Biol.
Chem., 266, 13866–13872.
10. Wade, D.P., Knight, B.L., Harders-Spengel, K. and Soutar, A.K. (1991)
Detection and quantitation of apolipoprotein(a) mRNA in human liver and its
relationship with plasma lipoprotein(a) concentration. Atherosclerosis, 91,
63–72.
11. Wade, D.P., Clarke, J.G., Lindahl, G.E., Liu, A.C., Zysow, B.R., Meer, K.,
Schwartz, K. and Lawn, R.M. (1993) 5′ control regions of the apolipoprotein(a) gene and members of the related plasminogen gene family. Proc. Natl
Acad. Sci. USA, 90, 1369–1373.
12. Malgaretti, N., Acquati, F., Magnaghi, P., Bruno, L., Pontoglio, M., Rocchi,
M., Saccone, S., Della Valle, G., D’Urso, M., LePaslier, D. et al. (1992)
Characterization by yeast artificial chromosome cloning of the linked
apolipoprotein(a) and plasminogen genes and identification of the apolipoprotein(a) 5′ flanking region (published erratum appears in Proc. Natl Acad.
Sci. USA, 1993, 90, 1634). Proc. Natl Acad. Sci. USA, 89, 11584–11588.
13. Cohen, J.C., Chiesa, G. and Hobbs, H.H. (1993) Sequence polymorphisms in
the apolipoprotein(a) gene. J. Clin. Invest., 91, 1630–1636.
14. Zysow, B.R., Lindahl, G.E., Wade, D.P., Knight, B.L. and Lawn, R.M. (1995)
C/T polymorphism in the 5′ untranslated region of the apolipoprotein(a) gene
introduces an upstream ATG and reduces in vitro translation. Arterioscler.
Thromb. Vasc. Biol., 15, 58–64.
1107
Human
Genetics,
1997,
6, No.
NucleicMolecular
Acids Research,
1994,
Vol. Vol.
22, No.
1 7
15. Mooser, V., Mancini, F.P., Bopp, S., Petho Schramm, A., Guerra, R.,
Boerwinkle, E., Muller, H.J. and Hobbs, H.H. (1995) Sequence polymorphisms in the apo(a) gene associated with specific levels of Lp(a) in plasma.
Hum. Mol. Genet., 4, 173–181.
16. Trommsdorff, M., Kochl, S., Lingenhel, A., Kronenberg, F., Delport, R.,
Vermaak, H., Lemming, L., Klausen, I.C., Faergeman, O., Utermann, G. et al.
(1995) A pentanucleotide repeat polymorphism in the 5′ control region of the
apolipoprotein(a) gene is associated with lipoprotein(a) plasma concentrations in Caucasians. J. Clin. Invest., 96, 150–157.
17. van der Hoek, Y.Y., Wittekoek, M.E., Beisiegel, U., Kastelein, J.J. and
Koschinsky, M.L. (1993) The apolipoprotein(a) kringle IV repeats which
differ from the major repeat kringle are present in variably-sized isoforms.
Hum. Mol. Genet., 2, 361–366.
18. Kraft, H.G., Haibach, C., Lingenhel, A., Brunner, C., Trommsdorff, M.,
Kronenberg, F., Muller, H.J. and Utermann, G. (1995) Sequence polymorphism in kringle IV 37 in linkage disequilibrium with the apolipoprotein(a) size
polymorphism. Hum. Genet., 95, 275–282.
19. Thompson, E.A., Deeb, S., Walker, D. and Motulsky, A.G. (1988) The
detection of linkage disequilibrium between closely linked markers: RFLPs
at the AI-CIII apolipoprotein genes. Am. J. Hum. Genet., 42, 113–124.
20. Lackner, C., Cohen, J.C. and Hobbs, H.H. (1993) Molecular definition of the
extreme size polymorphism in apolipoprotein(a). Hum. Mol. Genet., 2,
933–940.
21. Mancini, F.P., Mooser, V., Guerra, R. and Hobbs, H.H. (1995) Sequence
microheterogeneity in apolipoprotein(a) gene repeats and the relationship to
plasma Lp(a) levels. Hum. Mol. Genet., 4, 1535–1542.
22. Gaw, A., Boerwinkle, E., Cohen, J.C. and Hobbs, H.H. (1994) Comparative
analysis of the apo(a) gene, apo(a) glycoprotein, and plasma concentrations
of Lp(a) in three ethnic groups. Evidence for no common ‘null’ allele at the
apo(a) locus. J. Clin. Invest., 93, 2526–2534.
23. Rainwater, D.L. (1995) Genetic basis for multimodal relationship between
apolipoprotein (a) size and lipoprotein (a) concentration in Mexican-Americans. Atherosclerosis, 115, 165–171.
1107
24. Bopp, S., Kochl, S., Acquati, F., Magnaghi, P., Petho Schramm, A., Kraft,
H.G., Utermann, G., Muller, H.J. and Taramelli, R. (1995) Ten allelic
apolipoprotein(a) 5′ flanking fragments exhibit comparable promoter
activities in HepG2 cells. J. Lipid Res., 36, 1721–1728.
25. Magnaghi, P., Mihalich, A. and Taramelli, R. (1994) Several liver specific
DNAse hypersensitive sites are present in the intergenic region separating
human plasminogen and apoprotein(A) genes. Biochem. Biophys. Res.
Commun., 205, 930–935.
26. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
27. Soutar, A.K., McCarthy, S.N., Seed, M. and Knight, B.L. (1991) Relationship
between apolipoprotein(a) phenotype, lipoprotein(a) concentration in
plasma, and low density lipoprotein receptor function in a large kindred with
familial hypercholesterolemia due to the pro664→leu mutation in the LDL
receptor gene. J. Clin. Invest., 88, 483–492.
28. Talmud P., Tybjaerg Hansen A., Bhatnagar, D., Mbewu, A., Miller, J.P.,
Durrington, P. and Humphries, S. (1991) Rapid screening for specific
mutations in patients with a clinical diagnosis of familial hypercholesterolaemia. Atherosclerosis, 89, 137–141.
29. Pfaffinger, D., McLean, J. and Scanu, A.M. (1993) Amplification of human
APO(a) kringle 4-37 from blood lymphocyte DNA (published erratum
appears in Biochim. Biophys. Acta, 1995; 1270: 101). Biochim. Biophys.
Acta, 1225, 107–109.
30. Chakravarti, A., Buetow, K.H., Antonarakis, S.E., Waber, P.G., Boehm, C.D.
and Kazazian, H.H. (1984) Nonuniform recombination within the human
beta-globin gene cluster. Am. J. Hum. Genet., 36, 1239–1258.
31. Hill, W.G. (1974) Estimation of linkage disequilibrium in randomly mating
populations. Heredity Edinburgh, 33, 229–239.