Báo cáo khoa học: A functional polymorphism at the transcriptional initiation site in b2-glycoprotein I (apolipoprotein H) associated with reduced gene expression and lower plasma levels of b2-glycoprotein I docx

To understand the genetic basis of b2GPI variation, we analyzed the 5¢ flanking region ofthe b2GPIgene for mutation detection by DHPLC and identi-fied a point mutation at the transcription

A functional polymorphism at the transcriptional initiation site

Haider Mehdi1, Susan Manzi2, Purnima Desai1, Qi Chen1, Cara Nestlerode1, Franklin Bontempo2,

Stephen C Strom3, Reza Zarnegar3and M Ilyas Kamboh1

1

Department of Human Genetics, Graduate School of Public Health,2Department of Medicine and3Department of Pathology, University of Pittsburgh, USA

Human b2-glycoprotein I (b2GPI), also known as

apolipo-protein H, has been implicated in haemostasis and the

pro-duction ofanti-phospholipid antibodies There is a wide

range ofinterindividual variation in b2GPI plasma levels

that is thought to be under genetic control, but its molecular

basis remains unknown To understand the genetic basis of

b2GPI variation, we analyzed the 5¢ flanking region ofthe

b2GPIgene for mutation detection by DHPLC and

identi-fied a point mutation at the transcriptional initiation site

()1CfiA) with a carrier frequency of 12.1% The mutation

was associated with significantly lower b2GPI plasma levels

(P < 0.0001) and low occurrence ofanti-phospholipid

antibodies in lupus patients (4.8% antibody-positive group

vs 16.6% in the antibody-negative group; P¼ 0.019)

Northern blot analysis confirmed that the)1CfiA mutation was associated with lower mRNA levels and it reduced the 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 ofC with A at the b2GPItranscriptional initiation site is a causative mutation that affects its gene expression at the transcriptional level and ultimately b2GPI plasma levels and the occurrence ofphospholipid anti-bodies

Keywords: b2-glycoprotein I; apolipoprotein H; anti-phos-pholipid antibodies; polymorphism; lupus

Human b2-glycoprotein I (b2GPI), also known as

apolipo-protein H, is a plasma glycoapolipo-protein ofapproximately

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 b2GPIhas

been characterized, which consists ofeight exons, spanning

18 kb on chromosome 17q23–24 [4] b2GPI is a single chain

polypeptide of326 amino acids [5–8] that shows extensive

internal homology within its five consecutive homologous

segments ofapproximately 60 amino acid each These

segments are referred to variously as GP-I domains [9], sushi

domains [10], short consensus repeats (SCR) or complement

control protein (CCP) repeats [5,11,12]

b2GPI has been implicated in a variety ofphysiological

pathways, including blood coagulation, haemostasis and

the production ofanti-phospholipid antibodies (APA)

b2GPI inhibits the contact activation ofthe intrinsic pathway by binding to and neutralizing negatively charged macromolecules that might enter the blood stream and therefore diminishes inappropriate activation of the blood coagulation pathway [13–16] In in vitro studies, b2 GPI-deficient plasma is unable to inhibit the contact activation ofblood coagulation [17] and therefore raises the possi-bility that persons deficient in b2GPI may be more susceptible to thrombosis However, the role of b2 GPI-deficiency in thrombosis is controversial [18–20] Recently,

b2GPI has become the subject ofextensive study because ofits central role in the production ofAPA 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 ofboth b2GPI and anionic phospholipid [21–25]

There is a wide range ofinterindividual variation in b2GPI plasma levels, ranging from immunologically undetectable to

as high as 35 mgÆdL)1, with a mean value of20 mgÆdL)1in white people and 15 mgÆdL)1in black people [26–29] Based

on family data, two autosomal codominant alleles b2GPI*N (normal) and b2GPI*D (deficient), have been proposed to control the expression ofthree quantitative phenotypes, NN (normal), ND (intermediate) and DD (deficient) Homozy-gous NN individuals have a b2GPI plasma concentration between 16 and 35 mgÆdL)1, heterozygous ND individuals between 6 and 15 mgÆdL)1, and individuals having values less than 6 mgÆdL)1are classified as DD homozygotes

Correspondence to H Mehdi or M I Kamboh, Department of

Human Genetics, Graduate School ofPublic Health,

University ofPittsburgh, Pittsburgh, PA 15261, USA.

Fax: + 1 412 383 7844, Tel.: + 1 412 383 7193,

E-mail: haider.mehdi@mail.hgen.pitt.edu

or ilyas.kamboh@mail hgen.pitt.edu

Abbreviations: b 2 GPI, b 2 -glycoprotein I; APA, anti-phospholipid

antibodies; EMSA, electrophoretic gel mobility shift assay;

Luc, luciferase; DHPLC, denaturing high performance

liquid chromatography.

(Received 26 July 2002, accepted 20 November 2002)

The variation in b2GPI 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 b2GPI variation In our genetic

associ-ation studies ofpopulassoci-ation-based [30] and lupus patient

[31] samples, we have shown that two polymorphisms in

the b2GPI gene, Cys306Gly and Trp316Ser,

independ-ently affect variation in b2GPI plasma levels However,

our in vitro mutagenesis and expression studies revealed

that these mutations were not associated with altered

expression or secretion ofrecombinant b2GPI (rb2GPI)

[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

junctions and the coding region In our extensive survey

ofthe coding region and the intron–exon boundaries,

we have not found causative mutations, other than the

Cys306Gly and Trp316Ser We therefore hypothesized

that the 5¢ flanking region of b2GPI harbors functional

mutations that determine the interindividual variation in

b2GPI plasma levels Here we report the existence ofa

mutation at position)1 (CfiA) in the 5¢ flanking region

of b2GPI, which is in linkage disequilibrium with the

Trp316Ser mutation and is associated with significantly

lower b2GPI plasma and mRNA levels and the low

occurrence ofAPA Furthermore, this mutation reduces

the expression ofthe luciferase (Luc) reporter gene by

twofold and disrupts the binding of nuclear trans-acting

factors

Experimental procedures

Human subjects

For mutation detection, seven individuals with low b2GPI

plasma levels, ranging from 0.2 mgÆdL)1 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

b2GPI plasma levels, APA (anticardiolipin and lupus

anticoagulant), anti-b2GPI and b2GPI codons 306 and

codon 316 genotypes [31] Normal human liver tissues

(n¼ 50) were obtained from the NIH-funded program:

Liver Tissue Procurement and Distribution System

(LTPADS) at the University ofPittsburgh The study was

approved by the Institutional Review Board

Mutation detection by denaturing high performance

liquid chromatography (DHPLC) and DNA sequencing

The mutation detection in the 5¢ flanking region ofthe

b2GPI gene in seven selected subjects with lower b2GPI

plasma levels was performed by DHPLC Briefly, a set of

PCR primers between nucleotides)132 and +74 (forward:

5¢-GAATGTGGGTCTCAGAGTTCC-3¢ and reverse:

5¢-GGCAGAGAAAACTCGAGAAC-3¢) were designed

to generate a 206 bp 5¢ flanking DNA fragment For

DHPLC analysis, PCR was performed under oil-free

conditions using AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA) mixed with

9 : 1 ratio ofPfuTurbo DNA polymerase (Stratagene, La Jolla, CA, USA) to eliminate the possibility ofPCR-induced mutations The reactions were then denatured at

95C for 4 min and gradually reannealed by decreasing the temperature to 25C over a period of45 min, which allowed the PCR-amplified DNA to form hetero- and homo-duplexes The PCR products were then analyzed

on the WAVETM DNA Fragment Analysis System (Transgenomic Inc., San Jose, CA, USA) using a linear acetonitrile gradient The melting temperature (Tm) and acetonitrile gradient were determined by the size and GC content ofthe DNA fragments using WAVEMAKER 4.0 software (Transgenomic Inc., San Jose, CA, USA) The optimal mutation detection conditions were standardized

by analyzing the elution profiles ofthe PCR fragments at temperatures, Tm+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)1CfiA muta-tion was performed by using a forward mismatch primer starting at nucleotide )22 (5¢-GTCTTTTTAGCAGACG AAAGC-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 of96 bp was digested with CviJ1 (Molecular Biology Resources, Milwaukee, WI, USA) at 37C overnight 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 Total RNA was isolated from human liver tissues using TRIZol solution (Gibco/BRL/Life Technologies, Inc., Rockville, MD, USA) according to the manufacturer’s instructions Total RNA concentrations were determined

by measuring the optical density at 260 nm Northern blots were prepared by separating 10 lg of total RNA on formaldehyde containing 1% (w/v) agarose gels and transferring them to Zeta probe membranes (Bio-Rad Laboratories, Hercules, CA, USA) Blots were hybridized with 32P-labeled cDNA probes for b2GPI as described elsewhere [6] or GAPDH using the manufacturer’s protocol (Ambion, Inc., Austin, TX, USA)

b2GPI quantification

Liver tissues were homogenized in

radioimmunoprecipita-tion buffer [20 mMTris/HCl, pH 7.5, 0.15 mMNaCl, 2 mM

EDTA, 1% (w/v) sodium deoxycholate, 1% (v/v) Triton

X-100, 0.1% (w/v) SDS] containing 1 mM

phenyl-methanesulfonyl fluoride followed by centrifugation at

1500 g for 15 min to remove the cellular debris b2GPI levels

were measured by capture ELISA after diluting the lysates

(50, 100 and 200-fold) in NaCl/Pi(0.137MNaCl, 2.7 mM

KCl, 4.3 mMNa2HPO4.7H2O, 1.4 mMKH2PO4, pH 7.3)

containing 1% (w/v) bovine serum albumin as described

elsewhere [30,31] Total protein in liver lysates was measured

using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories,

Hercules, CA, USA) according to the manufacturer’s

instructions Bovine serum albumin was used as standard

to determine the protein concentrations in the liver lysates

Plasmid DNA constructs

The 5¢ flanking region ofgenomic DNA containing a

696-bp long DNA fragment (from nucleotide)622 to +74)

from heterozygous subjects (CA genotype) was PCR

amplified using a forward primer starting at nucleotide

)622 (5¢-CCAAGACATACTAAGAATGG-3¢) and the

same reverse primer ending at nucleotide +74 (5¢-GGCA

GAGAAAACTCGAGAAC-3¢) as described above The

696 bp long PCR amplified fragment ()1C or )1A allele)

was then ligated to the pCR II-TOPO cloning vector

(Invitrogen, Carlsbad, CA, USA) using the supplier’s

standard procedure For PCR amplification, we used Pfu

DNA polymerase (Strategene, La Jolla, CA, USA) to reduce

the chances ofPCR induced mutation followed by five

minute extension with AmpliTaq Gold DNA polymerase

(Applied Biosystems, Foster City, CA, USA) to facilitate TA

cloning into the pCR II-TOPO vector The size ofthe DNA

insert and fidelity ofDNA polymerase was confirmed by

restriction analysis and DNA sequencing The wild ()1C)

and mutant ()1A) type fragments were then inserted

upstream ofthe firefly Luc reporter gene into the

promoter-less pGL3-basic vector (Promega, Madison, WI, USA) using

appropriate restriction enzymes followed by ligation with

T4DNA ligase (New England Biolabs, Inc., Beverly, MA,

USA), as described elsewhere [33] The positive clones with

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/Piand lysed in the lysis

buffer (Promega, Madison, WI, USA) followed by

meas-urement ofLuc 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 offirefly 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 [a32P] dCTP (3000 CiÆmmol)1) (NEN, Boston, MA, USA) by filling in and blunt ending with Klenow enzyme (Gibco BRL/Life Technologies, Inc., Rockville, MD, USA) The labeled probes were then gel purified and used in EMSA, as described earlier [35,36] Two micrograms ofpoly(dI-dC) (Amersham Pharmacia Biotech, Piscataway, NJ, USA) were used as the nonspecific competitor in the binding reactions that were carried out at room temperature for

20 min before loading onto 5% nondenaturing polyacryl-amide (19 : 1 acrylpolyacryl-amide/bis acrylpolyacryl-amide) gels Gels were run

in 0.5· TBE buffer (45 mMTrisborate and 1 mMEDTA) at

a constant voltage of190 V, then dried and autoradio-graphed using intensifying screens The concentration of nuclear protein extracts used in each reaction was 2 lg and that ofthe labeled probe was between 0.2 and 0.4 ng Statistical analysis

Allele frequencies were calculated by allele counting Hardy–Weinberg equilibrium was tested by a v2 good-ness-of-fit test One-wayANOVAwas used to test f or mean

b2GPI levels among different genotype groups adjusted by age Pair-wise measure oflinkage disequilibrium between markers in the b2GPIgene was estimated by ID’I calcula-tion [37] Comparison ofgenotype and allele frequencies between antibody positive and antibody negative groups was made by 2· 2 v2 test and Z-test, respectively The

b2GPI concentration in liver samples was calculated as

lg b2GPIÆmg)1 total liver protein where average optical density ofthe mixture of11 samples is taken as standard for

b2GPI concentration (5 lg b2GPIÆmg)1total liver protein) The luciferase activity of each construct was calculated as a mean ± SD value ofthree experiments in triplicate after adjusting the transfection efficiency by normalizing them with the Rluc control value

Results Identification of the -1CÔA mutation

To enhance the likelihood ofidentifying functional muta-tions associated with b2GPI-deficiency, DNA samples from seven individuals previously identified with low b2GPI plasma levels, ranging from 0.2 mgÆdL)1 to 5.4 mgÆdL)1 [30,31] were screened by DHPLC for mutation detection in the 5¢ flanking region ofthe b2GPIgene between nucleo-tides)132 and +74 A point mutation was identified at position)1 (CfiA) (Fig 1A,B), which affects the consensus sequence for transcriptional initiation site and TFIID (Fig 2A) Five ofthe seven samples with low b2GPI plasma levels (3.4, 3.7, 4.3, 5.2 and 5.4 mgÆdL)1) had the)1CfiA

mutation (CA genotype, Fig 1C) and they were also

heterozygous for the Trp316Ser mutation Of the remaining

two subjects, one with b2GPI plasma levels 0.2 mgÆdL)1was

homozygous for the Cys306Gly mutation and the other

with b2GPI plasma levels 0.8 mgÆdL)1was 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 ofall 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) Ofthe 21 individuals with the Trp316Ser

mutation, 20 had the)1CfiA mutation On the other hand,

ofthe 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 oflinkage disequilibrium (Table 2)

Impact of the -1CÔA mutation on b2GPI

plasma levels

Table 3 presents the age-adjusted b2GPI plasma levels

among the)1CfiA genotype along with the Trp316Ser and

Cys306Gly genotypes All three polymorphisms were signi-ficantly associated with b2GPI plasma levels (P < 0.0001)

In all cases the less common genotypes were associated with lower levels as compared to the homozygous wild type genotypes and the effect was gene-dosage related When all polymorphisms were included in the regression model, the significant effect of the Trp316Ser polymorphism was lost (P¼ 0.29), but it remained significant for the )1CfiA (P¼ 0.035) and Cys306Gly (P < 0.001) polymorphisms This confirms our hypothesis that the effect of the Trp316Ser polymorphism was due to its non-random association with the)1CfiA mutation and that the effect ofthe Cys306Gly is independent, which is perhaps mediated

by a yet to be discovered mutation

Impact of the)1CfiA mutation on the occurrence

of anti-phospholipid antibodies (APA) Previously we have reported the association ofthe Trp316Ser polymorphism with the occurrence ofAPA (anticardiolipin or lupus anticoagulant), but not with

anti-b2GPI in this sample [31] As the Trp316Ser polymorphism

is tightly linked to the )1CfiA mutation, we predicted a similar association with the)1CfiA mutation As expected,

no association was observed between )1CfiA and

anti-bGPI (data not shown) However, both )1CfiA and

Fig 1 Identification of the -1CÔA mutation.

(A) DHPLC profile ofthe )1CfiA mutation

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 )1CfiA mutation where

CviJI restriction pattern ofhomozygous 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.

Trp316Ser polymorphisms showed significant association

with APA (Table 4) The frequencies of the)1A (2.4% vs

8.7%; P¼ 0.0034) and Ser316 (1.6% vs 6.9%;

P¼ 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% CI¼

0.07–0.86; P¼ 0.028) and for the codon 316 site, the odds ratio between Trp/Ser + Ser/Ser and Trp/Trp genotypes was 0.22 (95% CI¼ 0.05–0.94; P ¼ 0.042)

Fig 2 Partial 5¢ flanking region of the b 2 GPI gene and EMSAprobes.

(A) A partial 5¢ flanking sequence ofhuman b 2 GPI (in lower case)

showing the transcriptional initiation site (nucleotide + 1; arrow) as

well as portion ofexon 1 (upper case) including the 5¢ untranslated

region (UTR) upstream ofthe translation start site (ATG,

nucleo-tide +32) and a TFIID consensus sequence (underlined) [4] A partial

5¢ flanking sequence ofmouse b 2 GPI (italic) is aligned with the human

b 2 GPI sequence where the TFIID sequences at the transcriptional

initiation site are conserved between the two species, and are

under-lined and marked with the parallel bars (B) Synthetic oligonucleotides

(wild and mutant types) corresponding to the 23 bp DNA fragment

from nucleotides )12 to +11 ofthe human b 2 GPI gene, which were

labeled with32P and used for EMSA The mutant nucleotide at )1

position is indicated by lower case.

Table 1 Distribution of the b 2 GPI -1CÔApolymorphism in relation to

the Trp316Ser and Cys306Gly polymorphisms.

)1CfiA

Trp316Trp

Cys306Gly

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 v2–tests.

Table 3 Mean b 2 GPI plasma levels among b 2 GPI genotypes Polymorphic site/genotype b 2 GPI levels (mgÆdL)1) P-value )1CfiA

CC (n ¼ 204) 18.45 ± 3.90

CA (n ¼ 27) 14.21 ± 4.22

AA (n ¼ 1) 9.40 < 0.0001 Trp316Ser

Trp/Trp (n ¼ 211) 18.39 ± 3.88 Trp/Ser (n ¼ 20) 13.37 ± 4.33 Ser/Ser (n ¼ 1) 9.40 < 0.0001 Cys306Gly

Cys/Cys (n ¼ 210) 18.41 ± 3.54 Cys/Gly (n ¼ 15) 10.49 ± 2.56 Gly/Gly (n ¼ 1) 0.20 < 0.0001

Table 4 Distribution of the b 2 GPI polymorphisms in APA-positive and APA-negative groups.

Anti-phospholipid antibodies Genotype Positive Negative )1CfiA

A allele 0.024 0.087*

Trp316Ser Trp/Trp 61 96.83% 125 86.81%

Ser allele 0.016 0.069**

Cys306Gly Cys/Cys 59 93.65% 133 92.36%

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.

Effect of the)1CfiA mutation on the in vivo level

of b2GPI transcripts

To examine ifthe )1CfiA mutation affects b2GPI

transcription that eventually determines low b2GPI plasma

levels, we screened 50 human liver tissues for the)1CfiA

mutation followed by determination of b2GPI plasma and

b2GPI mRNA levels in selected samples We identified three

liver samples with low b2GPI levels (1.0, 1.3 and 2.1 lg

b2GPIÆmg)1total liver protein), but only two ofthem had

the )1CfiA mutation (CA genotype), while the third

sample was wild type (CC genotype) These results are

consistent with our findings in plasma samples, i.e although

the)1CfiA mutation is associated with lower b2GPI levels,

not all b2GPI-deficient samples have this mutation We then

performed Northern blot analysis on selected liver samples

with the mutant and wild type genotypes ofthe)1CfiA

mutation Figure 3 shows the results ofNorthern blot for

one heterozygous (CA genotype) having a low b2GPI

protein level (1.0 lg b2GPIÆmg)1total liver protein) along

with three wild types (CC genotype) having normal b2GPI

protein levels (9.8, 8.2 and 9.1 lg b2GPIÆmg)1 total liver

protein) While the level ofGADPH mRNA (a house

keeping gene) was constant in each lane, b2GPI 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 ofthe b2GPI gene, we performed

in vitroreporter gene expression assays We constructed a chimeric b2GPI-Luc vector to evaluate the promoter activity

of b2GPI within the 626 bp sequence in the 5¢ flanking region and tested the effect of the )1CfiA mutation on reporter (Luc) gene expression Wild ()1C) and mutant ()1A) type constructs were subsequently tested for promo-ter activity by cotransfecting COS-1 cells along with the Renila Luc control vector (pRl-CMV) that was used to adjust the transfection efficiency within different sets of experiments The promoter activity ofeach vector was determined by the dual luciferase assay system that revealed

a twofold decrease in the promoter activity associated with the mutant type allele ()1A) as compared to the wild type allele ()1C) (Fig 4) These results are similar to those seen

in the association studies (Table 3), in which the homozy-gous AA mutant had almost one halfofthe b2GPI plasma level (9.4 mgÆdL)1) than that observed in the CC wild type homozygotes (18.45 mgÆdL)1) These results demonstrate that the 626 bp 5¢ flanking region has some, ifnot all, promoter activity and that the )1CfiA mutation down regulates b2GPIgene expression

Effect of the)1CfiA mutation on binding

oftrans-acting factors

As the)1CfiA mutation disrupts the consensus sequence for the b2GPI 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 ofthe transcriptional initiation site is conserved among human and mouse b2GPI (Fig 2) EMSA ofthe wild type ()1C) probe revealed two prominent and specific bands ofDNA-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 mRNAlevels and

capture ELISAto 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 oftotal RNA was loaded in each lane for Northern blotting.

The corresponding liver samples were lysed in

radioimmunoprecipi-tation lysis buffer and b 2 GPI levels were determined by capture

ELI-SA Total liver protein was estimated by Bio-Rad protein assay kit

where BSA was used as standard (A) Northern autoradiograph

dis-playing levels of b 2 GPI mRNA in each lane (B) Northern

autoradi-ograph displaying levels ofGAPDH mRNA in each lane (C) b 2 GPI

levels (lgÆmg)1 oftotal liver protein) f or the corresponding liver

samples in each lane The genotypes at nucleotide )1 are indicated

beneath each lane.

Fig 4 Effect of the -1CÔAmutation on reporter (Luc) gene expres-sion The effect of )1C (wild type) and )1A (mutant type) alleles was measured as the mean ofthe firefly Luc levels, which were adjusted with the Renila Luc levels which served as the reference for the transfection efficiency The results presented are from three inde-pendent clones for each construct in triplicate, and each error bar represents the standard error.

probe (Fig 5; lane 5) as compared to the wild type ()1C)

probe (Fig 5; lane 4) These results demonstrate a

sequence-specific binding ofliver nuclear extracts and TFIID to

b2GPI sequence at its transcriptional initiation site These

results also confirm the location ofthe TFIID consensus

sequence at the b2GPItranscriptional initiation site, which is

disrupted by the)1CfiA mutation that would ultimately

affect the b2GPIgene expression

Discussion

Human b2GPI, 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 ofautoantibodies (APA)

b2GPI 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 b2GPI plasma levels to be 66%

(Kamboh et al unpublished data) In addition to the b2GPI

quantitative polymorphisms, we have originally described a

common protein polymorphism in the b2GPIgene [39] and

both polymorphisms were found to be tightly linked [38]

Thus, the family, heritability, and linkage data provide

strong support that b2GPI plasma variation is under genetic

control and that genetic variation in b2GPI is a major

determinant ofthis variation In our attempt to

deter-mine the molecular basis of bGPI plasma variation, we

conducted association studies and found that two of the

b2GPImutations, Trp316Ser and Cys306Gly, were signifi-cantly associated with b2GPI plasma levels and their effects were independent ofeach other [30,31] However, our

in vitromutagenesis and expression study did not link these mutations to an altered b2GPI gene expression [32] Although, in vitro mutagenesis and expression study do not rule out the possibility that these mutations might affect the stability of b2GPI in vivo, we hypothesized that they are

in linkage disequilibrium with two different functional mutations, as their effects on b2GPI plasma levels were independent To search for the functional mutations that are associated with altered gene expression and b2GPI plasma levels, we focused on a 626-bp fragment in the 5¢ flanking region of b2GPI that has been characterized recently [4]

Here, we report a new point mutation ()1CfiA) at the

b2GPI transcriptional initiation site (Fig 2A), which is associated with low b2GPI 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 b2GPI plasma levels However, in multivariate analysis, the effect of Trp316Ser was no longer significant, indicating that the )1CfiA is the functional mutation Of the 27 individuals in the CA genotype group (Table 3), 18 had b2GPI plasma levels between 4.3 mgÆdL)1and 15.9 mgÆdL)1, which would fall in the heterozygous category (ND) based on the quantitative polymorphism The remaining nine individuals fell in the normal (NN) category; seven in a narrow range between 16.2 mgÆdL)1 and 18.4 mgÆdL)1, and two with 20.5 mgÆdL)1 and 20.6 mgÆdL)1 Although the bulk of

b2GPI plasma variation is under genetic control (66% heritability) other nongenetic factors also influence this variation [26,29] and this may explain higher than expected

b2GPI plasma levels observed in nine individuals with the )1CfiA mutation Alternatively, other genetic or non-genetic factors modulate the effect of the)1CfiA mutation

on b2GPI plasma levels We also found that the)1CfiA mutation cannot explain the independent effect of Cys306Gly on b2GPI plasma levels This indicates that another functional mutation is responsible for the lowering effect of Cys306Gly on b2GPI plasma levels Another subject with only 0.8 mgÆdL)1b2GPI plasma levels was wild type homozygous at nucleotide)1, codon 306 and 316 sites and thus must be a carrier ofa yet to be discovered functional mutation These data suggest that multiple functional mutations in the b2GPIgene affect b2GPI plasma levels

In our earlier work, we found a protective effect of the Trp316Ser polymorphism against the occurrence ofAPA

in the lupus sample [31] In this study, the )1CfiA mutation also showed a significant protective effect The carrier frequency of the )1A allele was almost fourfold lower in the APA-positive group than the APA-negative group (4.8% vs 16.6%) As the Trp316Ser polymorphism

is in almost complete linkage disequilibrium with the )1CfiA mutation, our data link the protective effect directly to the)1CfiA mutation, as this is associated with lower b2GPI plasma levels and consequently lower risk of developing APA Paradoxically, however, the Cys306Gly

Fig 5 Effect of the -1CÔAmutation on the binding of crude nuclear

factors and purified TFIID using EMSA The EMSA was performed

using32P-labeled 23 bp oligonucleotide ( )12 to +11 nucleotides ofthe

b 2 GPI gene) carrying the wild (CC) or mutant (AA) type sequence at

nucleotide )1 followed by binding with crude nuclear extracts from

mouse liver or purified TFIID The binding reactions were performed

at room temperature for 20 min in the presence of the nonspecific

competitor poly (dI–dC) Lanes 1 and 6 are wild and mutant type

probes without nuclear extracts or TFIID, respectively Lanes 2 and 3

are wild and mutant type probes incubated with nuclear extracts,

respectively Lanes 4 and 5 are wild and mutant type probes incubated

with TFIID, respectively.

mutation, which is also associated with lower b2GPI

plasma levels, was not associated with protection from the

presence ofAPA Furthermore, although the)1CfiA and

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 b2GPI plasma levels (3.7, 4.3 and 7.3 mgÆdL)1) This

indicates that the genetic basis ofAPA is complex and

other genetic and/or biological factors are involved in the

occurrence ofAPA

The structural organization ofthe b2GPIgene, including

626 bp sequence in the 5¢ flanking region has been reported

together with the transcriptional initiation site 31 bp

upstream ofthe translation start codon [4], which

com-pletely agrees with the consensus for an initiator element,

PyPyA+1N(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

identified between nucleotides )2 and +5 (CCACTTT)

that is disrupted by the )1CfiA mutation Thus, we

predicted that lower b2GPI plasma levels associated with the

)1CfiA mutation might be due to its direct impact on

b2GPI transcription Indeed, our Northern blot analysis on

liver samples containing the)1CfiA mutation confirmed

this prediction in which all samples containing the CA

genotype had lower mRNA levels (Fig 3)

As Northern blot analysis revealed that the )1CfiA

mutation affects b2GPI transcription, we examined its effect

on b2GPI gene expression using tagged-Luc constructs

expressed in COS-1 cells Although the promoter of b2GPI

is not yet characterized, we cloned the reported 5¢ flanking

region ofthe b2GPIgene (from nucleotide)622 to +74) in

front of the Luc gene for in vitro functional studies The

reporter gene assay revealed that the 626 bp 5¢ flanking

region had some, ifnot all, the promoter activity and the

)1CfiA mutation is a functional substitution that

suppres-ses b2GPI 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) and CC

(18.5 mgÆdL)1) genotypes (Table 3) As the effect of the

)1CfiA mutation on b2GPIgene expression was moderate,

this does not preclude the possibility that other sequence

variation in the 5¢ region of b2GPImight also have an effect

on the regulation of b2GPI expression The functional

characterization ofthe b2GPIpromoter would enable the

targeting ofregulatory regions for mutation detection

Further evidence that the)1CfiA mutation is functional

comes from our EMSA data that demonstrate an

allele-specific binding ofnuclear factors and TFIID to the

mutation containing sequence;)1A has less affinity than

)1C Our novel data demonstrate that the )1CfiA

mutation at the transcriptional initiation site is causative,

which regulates b2GPIgene expression at the transcriptional

level that ultimately affects b2GPI plasma levels

In summary, we have identified a new polymorphism at

the transcriptional initiation site ofthe b2GPIgene that is

associated with less binding with a putative transcriptional

factor, lower gene expression, lower b2GPI plasma levels,

lower bGPI mRNA levels and protection from the

occurrence ofAPA in lupus patients Our data also indicate that the molecular basis ofplasma b2GPI deficiency is heterogenous The characterization offunctional b2GPI promoter and identification ofsequence variation in these regulatory elements may help to further delineate the molecular basis of b2GPI deficiency

Acknowledgements This study was supported by a National Heart, Lung and Blood Institute ofHealth grant HL 54900 and Central Research Development Fund award by the University ofPittsburgh.

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