A functional polymorphism at the transcriptional initiation site
in b
2
-glycoprotein I (apolipoprotein H) associated with reduced
gene expression and lower plasma levels of b
2
-glycoprotein I
Haider Mehdi
1
, Susan Manzi
2
, Purnima Desai
1
, Qi Chen
1
, Cara Nestlerode
1
, Franklin Bontempo
2
,
Stephen C. Strom
3
, Reza Zarnegar
3
and M. Ilyas Kamboh
1
1
Department of Human Genetics, Graduate School of Public Health,
2
Department of Medicine and
3

reporter (luciferase) gene expression by twofold. Electro-
phoretic gel mobility shift assay (EMSA) revealed that the
)1CfiA mutation disrupts the binding for crude hepatic
nuclear extracts and purified TFIID. These results suggest
that the substitution of C with A at the b
2
GPI transcriptional
initiation site is a causative mutation that affects its gene
expression at the transcriptional level and ultimately b
2
GPI
plasma levels and the occurrence of anti-phospholipid anti-
bodies.
Keywords: b
2
-glycoprotein I; apolipoprotein H; anti-phos-
pholipid antibodies; polymorphism; lupus.
Human b
2
-glycoprotein I (b
2
GPI), also known as apolipo-
protein H, is a plasma glycoprotein of approximately
50 kDa [1], which is primarily expressed in liver and is
associated with very low-density lipoproteins, high-density
lipoproteins, and chylomicrons and it also exists in lipid-free
form in plasma [2,3]. The gene organization of b
2
GPI has
been characterized, which consists of eight exons, spanning

GPI-
deficiency in thrombosis is controversial [18–20]. Recently,
b
2
GPI has become the subject of extensive study because
of its central role in the production of APA in sera of
patients with primary anti-phospholipid syndrome and
lupus. Originally, it was thought that APA in sera are
produced against simple anionic phospholipid molecules,
however, subsequent data showed that APA are produced
against a complex antigen consisting of both b
2
GPI and
anionic phospholipid [21–25].
There is a wide range of interindividual variation in b
2
GPI
plasma levels, ranging from immunologically undetectable to
as high as 35 mgÆdL
)1
, with a mean value of 20 mgÆdL
)1
in
whitepeopleand15 mgÆdL
)1
in black people [26–29]. Based
on family data, two autosomal codominant alleles b
2
GPI*N
(normal) and b

(Received 26 July 2002, accepted 20 November 2002)
Eur. J. Biochem. 270, 230–238 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03379.x
The variation in b
2
GPI plasma levels is thought to be
under genetic control, but its molecular basis is
unknown. Therefore, it is critical to delineate the genetic
determinants of b
2
GPI variation. In our genetic associ-
ation studies of population-based [30] and lupus patient
[31] samples, we have shown that two polymorphisms in
the b
2
GPI gene, Cys306Gly and Trp316Ser, independ-
ently affect variation in b
2
GPI plasma levels. However,
our in vitro mutagenesis and expression studies revealed
that these mutations were not associated with altered
expression or secretion of recombinant b
2
GPI (rb
2
GPI)
[32]. We hypothesized that the Cys306Gly and
Trp316Ser mutations are in linkage disequilibrium with
two different functional mutations. There are three main
plausible regions in which mutations can directly affect
plasma protein levels: the promoter region, splice

to 5.4 mgÆdL
)1
,
from our two previous studies [30,31], were subjected to
denaturing high performance liquid chromatography
(DHPLC) and DNA sequencing. For association with the
new mutation, we used 232 lupus women (mean age
45.11 ± 11.28 years) in which we have previously reported
b
2
GPI plasma levels, APA (anticardiolipin and lupus
anticoagulant), anti-b
2
GPI and b
2
GPI codons 306 and
codon 316 genotypes [31]. Normal human liver tissues
(n ¼ 50) were obtained from the NIH-funded program:
Liver Tissue Procurement and Distribution System
(LTPADS) at the University of Pittsburgh. The study was
approved by the Institutional Review Board.
Mutation detection by denaturing high performance
liquid chromatography (DHPLC) and DNA sequencing
Themutationdetectioninthe5¢ flanking region of the
b
2
GPI gene in seven selected subjects with lower b
2
GPI
plasma levels was performed by DHPLC. Briefly, a set of

+2 and )2, and the eluted fragments
were detected by a UV detector. The PCR products
showing the DHPLC variant patterns were then sub-
cloned into a pCR II-TOPO cloning vector (Invitrogen,
Carlsbad, CA, USA) using the supplier’s standard
procedure. The positive clones with a full-length DNA
insert were subjected to DNA sequencing using Thermo
Sequenase Cy 5.5 Terminator Cycle Sequencing kit
(Amersham Pharmacia Biotech Inc., Piscataway, NJ,
USA). The sequencing reactions were then analyzed by
OpenGene Automatic DNA Sequencer System (Visible
Genetics, Suwanee, GA, USA) for mutation detection.
Genotyping for the -1CÔA mutation
The genomic DNA was isolated from buffy coats and
liver tissues using the QIAamp Blood kit (Qiagen,
Valencia, CA, USA). Genotyping for the )1CfiAmuta-
tion was performed by using a forward mismatch primer
starting at nucleotide )22 (5¢-GTCTTTTTAGCAGACG
AAA
GC-3¢; the mismatch base is underlined), which
creates the CviJ1 restriction site at nucleotide )1, in
combination with a reverse primer as described above to
PCR-amplify the genomic DNA. The amplified fragment
of 96 bp was digested with CviJ1 (Molecular Biology
Resources, Milwaukee, WI, USA) at 37 °Covernight
followed by electrophoresis on 6% (w/v) polyacrylamide
gel. The homozygous wild type (CC) yielded two
fragments of 22 bp and 74 bp, while the homozygous
mutant type (AA) remained uncut (96 bp).
Northern blotting

X-100, 0.1% (w/v) SDS] containing 1 m
M
phenyl-
methanesulfonyl fluoride followed by centrifugation at
1500 g for 15 min to remove the cellular debris. b
2
GPI levels
were measured by capture ELISA after diluting the lysates
(50, 100 and 200-fold) in NaCl/P
i
(0.137
M
NaCl, 2.7 m
M
KCl, 4.3 m
M
Na
2
HPO
4
.7H
2
O, 1.4 m
M
KH
2
PO
4
,pH7.3)
containing 1% (w/v) bovine serum albumin as described

the full-length wild ()1C) and mutant ()1A) type fragments
were identified by restriction analysis and DNA sequencing.
Transient transfection and dual-luciferase assay
The wild ()1C) and mutant ()1A) type chimeric-firefly Luc
constructs (4 lg) were used to transiently cotransfect COS-1
cells along with Renila Luc control vector (pRL-CMV)
(1 lg) (Promega, Madison, WI, USA) using the DEAE-
dextran method as described earlier [34]. The transfection
control dish (mock transfected) received only DEAE-
dextran, but no-DNA and Luc-control dishes received only
pGL3-basic or pRL-CMV vector. After 48 h of transfection,
cells were washed twice with NaCl/P
i
and lysed in the lysis
buffer (Promega, Madison, WI, USA) followed by meas-
urement of Luc activity by TD-20/20 Luminometer (Turner
Design, Sunnyvale, CA, USA) using the dual luciferase
assay system (Promega, Madison, WI, USA), as described
elsewhere [33]. Actual Luc activity was calculated as the ratio
of firefly to Renila Luc activity for each experiment.
Preparation of nuclear extracts and electrophoretic gel
mobility shift assay (EMSA)
The nuclear extracts from mouse liver tissues were prepared
as reported earlier [35,36]. Purified TFIID was purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA,
USA). The double-stranded wild ()1C) and mutant ()1A)
type oligonucleotides were labeled with [a
32
P] dCTP
(3000 CiÆmmol

GPI levels among different genotype groups adjusted by
age. Pair-wise measure of linkage disequilibrium between
markers in the b
2
GPI gene was estimated by ID’I calcula-
tion [37]. Comparison of genotype and allele frequencies
between antibody positive and antibody negative groups
was made by 2 · 2 v
2
test and Z-test, respectively. The
b
2
GPI concentration in liver samples was calculated as
lg b
2
GPIÆmg
)1
total liver protein where average optical
density of the mixture of 11 samples is taken as standard for
b
2
GPI concentration (5 lg b
2
GPIÆmg
)1
total liver protein).
The luciferase activity of each construct was calculated as a
mean ± SD value of three experiments in triplicate after
adjusting the transfection efficiency by normalizing them
with the Rluc control value.

2
GPI plasma levels 0.2 mgÆdL
)1
was
homozygous for the Cys306Gly mutation and the other
with b
2
GPI plasma levels 0.8 mgÆdL
)1
was wild type at all
three sites. We subsequently genotyped 232 subjects for the
)1CfiA mutation, and its distribution stratified by the
Cys306Gly and Trp316Ser polymorphisms is presented in
Table 1. The distribution of all polymorphisms was in the
Hardy–Weinberg equilibrium. The frequency of the )1A
allele was 0.0625 with a carrier frequency of 12.1%. The
carrier frequencies of the Gly306 and Ser316 alleles were
7.1% and 9.1%, respectively. There was a nearly complete
linkage disequilibrium between the )1CfiA and Trp316Ser
sites (P < 0.0001). Of the 21 individuals with the Trp316Ser
mutation, 20 had the )1CfiA mutation. On the other hand,
of the 16 individuals with the Cys306Gly mutation, only one
had the )1CfiA mutation, strongly indicating that these
two sites are in linkage equilibrium, as also reflected in the
pair-wise measure of linkage disequilibrium (Table 2).
Impact of the -1CÔA mutation on b
2
GPI
plasma levels
Table 3 presents the age-adjusted b

b
2
GPI (data not shown). However, both )1CfiAand
Fig. 1. Identification of the -1CÔA mutation.
(A) DHPLC profile of the )1CfiAmutation
where the heterozygous PCR fragments were
separated into two peaks (retention times
5.05 and 5.32) while the wild type PCR frag-
ment gave a single peak (retention time 5.30).
(B) Sequence differences between wild type
()1C) and mutant type ()1A) alleles. (C)
Genotyping for the )1CfiAmutationwhere
CviJI restriction pattern of homozygous wild
type (CC, 74 bp), homozygous mutant type
(AA, 96 bp), and heterozygous type (CA,
96 bp and 74 bp), were separated on 6%
polyacrylamide gel.
Ó FEBS 2003 Functional polymorphism in b
2
GPI (Eur. J. Biochem. 270) 233
Trp316Ser polymorphisms showed significant association
with APA (Table 4). The frequencies of the )1A (2.4% vs.
8.7%; P ¼ 0.0034) and Ser316 (1.6% vs. 6.9%;
P ¼ 0.0045) alleles were significantly lower in the APA-
positive group than the APA-negative group. For the
nucleotide )1 site, the age-adjusted odds ratio between
CA + AA and CC genotypes was 0.25 (95% CI ¼
0.07–0.86; P ¼ 0.028) and for the codon 316 site, the odds
ratio between Trp/Ser + Ser/Ser and Trp/Trp genotypes
was 0.22 (95% CI ¼ 0.05–0.94; P ¼ 0.042).

)1CfiA
CC CA AA Total
Trp316Trp
Trp/Trp 203 8 0 211
Trp/Ser 1 19 0 20
Ser/Ser 0 0 1 1
Total 204 27 1 232
Cys306Gly
Cys/Cys 183 26 1 210
Cys/Gly 14 1 0 15
Gly/Gly 1 0 0 1
Total 198 27 1 226
a
a
6 individuals with wild type genotypes at the )1CfiA and
Trp316Ser sites could not be genotyped for the Cys306Gly site due
to technical problems.
Table 2. Pair-wise measure of linkage disequilibrium between b
2
GPI
polymorphisms.
Pair-wise comparison P-value*
Nucleotide )1 vs. codon 316 < 0.0001
Nucleotide )1 vs. codon 306 0.294
codon 316 vs. codon 306 0.368
*P-values were obtained by v
2
–tests.
Table 3. Mean b
2

Total 63 144
A allele 0.024 0.087*
Trp316Ser
Trp/Trp 61 96.83% 125 86.81%
Trp/Ser 2 3.17% 18 12.50%
Ser/Ser 0 0.00% 1 0.69%
Total 63 144
Ser allele 0.016 0.069**
Cys306Gly
Cys/Cys 59 93.65% 133 92.36%
Cys/Gly 4 6.35% 10 6.94%
Gly/Gly 0 0.00% 1 0.69%
Total 63 144
Gly allele 0.032 0.042***
*P ¼ 0.0034, **P ¼ 0.0045, ***P ¼ 0.61 between APA-positive
and APA-negative groups.
234 H. Mehdi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Effect of the )1CfiA mutation on the
in vivo
level
of b
2
GPI transcripts
To examine if the )1CfiA mutation affects b
2
GPI
transcription that eventually determines low b
2
GPI plasma
levels, we screened 50 human liver tissues for the )1CfiA

2
GPIÆmg
)1
total liver protein) along
with three wild types (CC genotype) having normal b
2
GPI
protein levels (9.8, 8.2 and 9.1 lg b
2
GPIÆmg
)1
total liver
protein). While the level of GADPH mRNA (a house
keeping gene) was constant in each lane, b
2
GPI mRNA
level was significantly lower in the CA genotype (lane 3)
than the CC genotype (lanes 1, 2 and 4).
Effect of the )1CfiA mutation on reporter gene
expression
To further confirm that the )1CfiA mutation is associated
with low expression of the b
2
GPI gene, we performed
in vitro reporter gene expression assays. We constructed a
chimeric b
2
GPI-Luc vector to evaluate the promoter activity
of b
2

-acting factors
As the )1CfiA mutation disrupts the consensus sequence
for the b
2
GPI transcriptional initiation site and TFIID
(Fig. 2A), we designed double-stranded wild ()1C) and
mutant ()1A) type oligonucleotides as probes for EMSA to
evaluate the effect of this mutation on the binding of trans-
acting factors, using mouse liver nuclear extracts and
TFIID. We used mouse nuclear extracts because they were
easily available, and more importantly the consensus
sequence of the transcriptional initiation site is conserved
among human and mouse b
2
GPI (Fig. 2). EMSA of the
wild type ()1C) probe revealed two prominent and specific
bands of DNA-binding complexes (Fig. 5; lane 2), while the
mutant type ()1A) probe showed little or no binding to liver
nuclear proteins (Fig. 5; lane 3). We also found that the
purified TFIID bound weakly to the mutant type ()1A)
Fig. 3. Northern blot analysis to determine b
2
GPI mRNA levels and
capture ELISA to determine the b
2
GPI protein levels in human liver
samples carrying the wild (-1C) or mutant (-1A) type allele. Total RNA
was isolated from frozen human liver samples using TRIzol reagent
and 10 lg of total RNA was loaded in each lane for Northern blotting.
The corresponding liver samples were lysed in radioimmunoprecipi-

2
GPI sequence at its transcriptional initiation site. These
results also confirm the location of the TFIID consensus
sequence at the b
2
GPI transcriptional initiation site, which is
disrupted by the )1CfiA mutation that would ultimately
affect the b
2
GPI gene expression.
Discussion
Human b
2
GPI, also known as apolipoprotein H, is a
plasma glycoprotein that has been implicated in a variety of
physiological pathways, including blood coagulation,
thrombosis, and the production of autoantibodies (APA).
b
2
GPI plasma levels vary significantly among individuals,
ranging from immunologically undetectable to as high as
35 mgÆdL
)1
, and family data indicate that this variation is
under genetic control [26–28,38]. We have recently deter-
mined the heritability of b
2
GPI plasma levels to be 66%
(Kamboh et al. unpublished data). In addition to the b
2

not rule out the possibility that these mutations might affect
the stability of b
2
GPI in vivo, we hypothesized that they are
in linkage disequilibrium with two different functional
mutations, as their effects on b
2
GPI plasma levels were
independent. To search for the functional mutations that
are associated with altered gene expression and b
2
GPI
plasma levels, we focused on a 626-bp fragment in the
5¢ flanking region of b
2
GPI that has been characterized
recently [4].
Here, we report a new point mutation ()1CfiA) at the
b
2
GPI transcriptional initiation site (Fig. 2A), which is
associated with low b
2
GPI plasma and mRNA levels as well
as a twofold reduced expression of the tagged-Luc gene. The
)1CfiA mutation was also in strong linkage disequilibrium
with the Trp316Ser mutation. In the univariate analysis,
both sites showed significant association with b
2
GPI plasma

2
GPI plasma levels observed in nine individuals with the
)1CfiA mutation. Alternatively, other genetic or non-
genetic factors modulate the effect of the )1CfiAmutation
on b
2
GPI plasma levels. We also found that the )1CfiA
mutation cannot explain the independent effect of
Cys306Gly on b
2
GPI plasma levels. This indicates that
another functional mutation is responsible for the lowering
effect of Cys306Gly on b
2
GPI plasma levels. Another
subject with only 0.8 mgÆdL
)1
b
2
GPI plasma levels was wild
type homozygous at nucleotide )1, codon 306 and 316 sites
and thus must be a carrier of a yet to be discovered
functional mutation. These data suggest that multiple
functional mutations in the b
2
GPI gene affect b
2
GPI plasma
levels.
In our earlier work, we found a protective effect of the

mutation, which is also associated with lower b
2
GPI
plasma levels, was not associated with protection from the
presence of APA. Furthermore, although the )1CfiAand
Trp316Ser mutations were associated with protection
against APA, there were three (4.8%) individuals with
these two mutations, who were positive for APA but had
lower b
2
GPI plasma levels (3.7, 4.3 and 7.3 mgÆdL
)1
). This
indicates that the genetic basis of APA is complex and
other genetic and/or biological factors are involved in the
occurrence of APA.
The structural organization of the b
2
GPI gene, including
626 bp sequence in the 5¢ flanking region has been reported
together with the transcriptional initiation site 31 bp
upstream of the translation start codon [4], which com-
pletely agrees with the consensus for an initiator element,
PyPyA
+1
N(TA)PyPy known to sustain transcriptional
initiation [40]. The computer analysis for transcriptional
elements within this region did not reveal any TATA box or
CG rich region close to the transcriptional initiation site
(nucleotide +1) but a TFIID binding sequence was

ses b
2
GPI gene expression by twofold. The twofold
difference observed between the )1A and )1C alleles in
the reporter gene assay is similar to that seen in the plasma
level difference between the AA (9.4 mgÆdL
)1
)andCC
(18.5 mgÆdL
)1
) genotypes (Table 3). As the effect of the
)1CfiAmutationonb
2
GPI gene expression was moderate,
this does not preclude the possibility that other sequence
variationinthe5¢ region of b
2
GPI might also have an effect
on the regulation of b
2
GPI expression. The functional
characterization of the b
2
GPI promoter would enable the
targeting of regulatory regions for mutation detection.
Further evidence that the )1CfiA mutation is functional
comes from our EMSA data that demonstrate an allele-
specific binding of nuclear factors and TFIID to the
mutation containing sequence; )1A has less affinity than
)1C. Our novel data demonstrate that the )1CfiA

Acknowledgements
This study was supported by a National Heart, Lung and Blood
Institute of Health grant HL 54900 and Central Research Development
Fund award by the University of Pittsburgh.
References
1. Schultz, H.E., Heide, K. & Haupt, H. (1961) Uber ein bisher
unbekanntes neidermolekul es b
2
Globulin des Humanserums.
Naturwissenschaften 48,719.
2. Polz, E. & Kostner, G.M. (1979) The binding of b
2
-glycoprotein I
to human serum lipoproteins: Distribution among density frac-
tions. FEBS Lett. 102, 183–186.
3. Polz, E. & Kostner, G.M. (1979) Binding of b
2
-glycoprotein I to
intralipid: Distribution of the dissociation constant. Biochem.
Biophys. Res. Commun. 90, 1305–1312.
4. Okkels, H., Rasmussen, T.E., Sanghera, D.K., Kamboh, M.I. &
Kristensen, T. (1999) Structure of the b
2
-glycoprotein I (apolipo-
protein H) gene. Eur. J. Biochem. 259, 435–440.
5. Lozier, J., Takahashi, N. & Putnam, F.W. (1984) Complete amino
acid sequence of human plasmid b
2
-glycoprotein I. Proc. Natl
Acad. Sci. USA 81, 3640–3364.

13. Schousboe, I. (1985) b
2
-glycoprotein I: a plasma inhibitor of the
contact activation of the intrinsic blood coagulation pathway.
Blood 66, 1086–1091.
14. Nimpf, J., Bevers, E.M., Bomans, P.H.H., Till, U., Wurm, H.,
Kostner, G.M. & Zwaal, R.F.A. (1986) Prothrombinase activity
of human platelets is inhibited by b
2
-glycoprotein I. Biochim.
Biophys. Acta 884, 142–149.
15. Schousboe, I. (1988) In vitro activation of the contact activa-
tion system (Hageman factor system) in plasma by acidic
Ó FEBS 2003 Functional polymorphism in b
2
GPI (Eur. J. Biochem. 270) 237
phospholipids, and the inhibitory effect of b
2
-glycoprotein I on the
activation. Int. J. Biochem. 20, 309–315.
16. Schousboe, I. & Rasnussen, M.S. (1988) The effect of b
2
-glyco-
protein I on the dextran sulfate and sulfatide activation of the
contact system (Hageman factor system) in the blood coagulation.
Int. J. Biochem. 20, 787–792.
17. Halkier, T. & Magnussen, S. (1988) Contact activation of blood
coagulation is inhibited by plasma factor XIIIb-chain. Thromb.
Res. 51, 313–324.
18. Bancsi, L.F.J.M.M., van der Linden, I.K. & Bertina, R.M. (1992)

phospholipid antibodies required b
2
-glycoprotein I (apolipopro-
tein H) as cofactor. J. Rhematol. 19, 1397–1402.
24. Roubey,R.A.,Pratt,C.W.,Buyon,J.P.&Winfield,J.B.(1992)
Lupus anticoagulant activity of autoimmune anti-phospholipid
antibodies is dependent upon b
2
-glycoprotein I. J. Clin. Invest. 90,
1100–1104.
25. Cabral, A.R., Cabiedes, J. & Alarcon-Segovia, D. (1995) Anti-
bodies to phospholipid-free b
2
-glycoprotein I in patients with
primary anti-phospholipid syndrome. J. Rheumatol. 22, 1894–1898.
26. Cleve, H. (1968) Genetic studies on the deficiency of b
2
-glyco-
protein I of human serum. Hum. Genet. 5, 294–304.
27. Koppe, A.L., Walter, H., Chopra, V.P. & Bajatzadeh, M. (1970)
Investigations on the genetics and population genetics of the
b
2
-glycoprotein I polymorphism. Hum. Genet. 9, 164–171.
28. Propert, D.N. (1978) The relationship of sex, smoking status, birth
rank and parental age to b
2
-glycoprotein I levels and phenotypes
in a sample of Australian Caucasian adults. Hum. Genet. 43, 281–
288.

at CUG, GUG, and ACG codons in mammalian cells. Gene 91,
173–178.
35. Liu, Y., Beedle, A.B., Lin, L., Bell, A.W. & Zarnegar, R. (1994)
Identification of a cell-type-specific transcriptional repressor in the
promoter region of the mouse hepatocyte growth factor gene.
Mol. Cell. Biol. 14, 7046–7058.
36. Jiang, J.G., Bell, A., Liu, Y. & Zarnegar, R. (1997) Transcriptional
regulation of the HGF gene by COUP-TF and estrogen receptor.
J. Biol. Chem. 272, 3928–3934.
37. Lewontin, R.C. (1964) The interaction of selection and linkage. I.
General considerations; heterotic models. Genetics 49, 49–67.
38. Eiberg, H., Nielsen, L.S. & Mohr, J. (1989) Exclusion mapping of
apolipoprotein H (APOH) and relationship between electro-
phorative polymorphism. Cytogenet. Cell. Genet. 51, 994.
39. Kamboh, M.I., Ferrell, R.E. & Sephernia, B. (1988) Genetic stu-
dies of human apolipoproteins. IV. Structural heterogeneity of
apolipoprotein H (b
2
-glycoprotein I). Am.J.Hum.Genet.42, 452–
457.
40. Lo, K. & Smale, S.T. (1996) Generality of a functional initiator
consensus sequence. Gene 182, 13–22.
238 H. Mehdi et al.(Eur. J. Biochem. 270) Ó FEBS 2003