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 Department of Pathology, University of Pittsburgh, USA Human b 2 -glycoprotein I (b 2 GPI), also known as apolipo- protein H, has been implicated in haemostasis and the pro- duction of anti-phospholipid antibodies. There is a wide range of interindividual variation in b 2 GPI plasma levels that is thought to be under genetic control, but its molecular basis remains unknown. To understand the genetic basis of b 2 GPI variation, we analyzed the 5¢ flanking region of the b 2 GPI gene 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 b 2 GPI plasma levels (P < 0.0001) and low occurrence of anti-phospholipid antibodies in lupus patients (4.8% antibody-positive group vs. 16.6% in the antibody-negative group; P ¼ 0.019). Northern blot analysis confirmed that the )1CfiAmutation 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 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 18 kb on chromosome 17q23–24 [4]. b 2 GPI is a single chain polypeptide of 326 amino acids [5–8] that shows extensive internal homology within its five consecutive homologous segments of approximately 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]. b 2 GPI has been implicated in a variety of physiological pathways, including blood coagulation, haemostasis and the production of anti-phospholipid antibodies (APA). b 2 GPI inhibits the contact activation of the 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, b 2 GPI- deficient plasma is unable to inhibit the contact activation of blood coagulation [17] and therefore raises the possi- bility that persons deficient in b 2 GPI may be more susceptible to thrombosis. However, the role of b 2 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 2 GPI*D (deficient), have been proposed to control the expression of three quantitative phenotypes, NN (normal), ND (intermediate) and DD (deficient). Homozy- gous NN individuals have a b 2 GPI 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 )1 are classified as DD homozygotes. Correspondence to H. Mehdi or M. I. Kamboh, Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, 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) 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 junctions and the coding region. In our extensive survey of the 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 b 2 GPI harbors functional mutations that determine the interindividual variation in b 2 GPI plasma levels. Here we report the existence of a mutation at position )1(CfiA) in the 5¢ flanking region of b 2 GPI, which is in linkage disequilibrium with the Trp316Ser mutation and is associated with significantly lower b 2 GPI plasma and mRNA levels and the low occurrence of APA. Furthermore, this mutation reduces the expression of the 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 b 2 GPI 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 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 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 of PfuTurbo DNA polymerase (Stratagene, La Jolla, CA, USA) to eliminate the possibility of PCR- induced mutations. The reactions were then denatured at 95 °C for 4 min and gradually reannealed by decreasing the temperature to 25 °C over a period of 45 min, which allowed the PCR-amplified DNA to form hetero- and homo-duplexes. The PCR products were then analyzed on the WAVE TM DNA Fragment Analysis System (Transgenomic Inc., San Jose, CA, USA) using a linear acetonitrile gradient. The melting temperature (T m )and acetonitrile gradient were determined by the size and GC content of the 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 of the PCR fragments at temperatures, T m +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 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 lgoftotalRNAon formaldehyde containing 1% (w/v) agarose gels and transferring them to Zeta probe membranes (Bio-Rad Laboratories, Hercules, CA, USA). Blots were hybridized with 32 P-labeled cDNA probes for b 2 GPI as described elsewhere [6] or GAPDH using the manufacturer’s protocol (Ambion, Inc., Austin, TX, USA). Ó FEBS 2003 Functional polymorphism in b 2 GPI (Eur. J. Biochem. 270) 231 b 2 GPI quantification Liver tissues were homogenized in radioimmunoprecipita- tion buffer [20 m M Tris/HCl, pH 7.5, 0.15 m M NaCl, 2 m M EDTA, 1% (w/v) sodium deoxycholate, 1% (v/v) Triton 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 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 of genomic 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¢)andthe 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 of PCR 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 of the DNA insert and fidelity of DNA polymerase was confirmed by restriction analysis and DNA sequencing. The wild ()1C) and mutant ()1A) type fragments were then inserted upstream of the 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/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 )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 of poly(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 acrylamide/bis acrylamide) gels. Gels were run in 0.5· TBE buffer (45 m M Trisborate and 1 m M EDTA) at a constant voltage of 190 V, then dried and autoradio- graphed using intensifying screens. The concentration of nuclear protein extracts used in each reaction was 2 lgand that of the 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 v 2 good- ness-of-fit test. One-way ANOVA wasusedtotestformean b 2 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. Results Identification of the -1CÔA mutation To enhance the likelihood of identifying functional muta- tions associated with b 2 GPI-deficiency, DNA samples from seven individuals previously identified with low b 2 GPI 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 of the b 2 GPI gene 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 of the seven samples with low b 2 GPI plasma levels (3.4, 3.7, 4.3, 5.2 and 5.4 mgÆdL )1 )hadthe)1CfiA 232 H. Mehdi et al.(Eur. J. Biochem. 270) Ó FEBS 2003 mutation (CA genotype, Fig. 1C) and they were also heterozygous for the Trp316Ser mutation. Of the remaining two subjects, one with b 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 2 GPI plasma levels among the )1CfiA genotype along with the Trp316Ser and Cys306Gly genotypes. All three polymorphisms were signi- ficantly associated with b 2 GPIplasmalevels(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 (P ¼ 0.29), but it remained significant for the )1CfiA (P ¼ 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 of the 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 of the Trp316Ser polymorphism with the occurrence of APA (anticardiolipin or lupus anticoagulant), but not with anti- b 2 GPI 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 )1CfiAandanti- 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). Fig. 2. Partial 5¢ flanking region of the b 2 GPI gene and EMSA probes. (A) A partial 5¢ flanking sequence of human b 2 GPI (in lower case) showing the transcriptional initiation site (nucleotide + 1; arrow) as well as portion of exon 1 (upper case) including the 5¢ untranslated region (UTR) upstream of the translation start site (ATG, nucleo- tide +32) and a TFIID consensus sequence (underlined) [4]. A partial 5¢ flanking sequence of mouse 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 of the human b 2 GPI gene, which were labeled with 32 P and used for EMSA. The mutant nucleotide at )1 position is indicated by lower case. Table 1. Distribution of the b 2 GPI -1CÔA polymorphism in relation to the Trp316Ser and Cys306Gly polymorphisms. )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 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 CC 60 95.24% 120 83.33% CA 3 4.76% 23 15.97% AA 0 0.00% 1 0.69% 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 mutation followed by determination of b 2 GPI plasma and b 2 GPI mRNA levels in selected samples. We identified three liver samples with low b 2 GPI levels (1.0, 1.3 and 2.1 lg b 2 GPIÆmg )1 total liver protein), but only two of them 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 b 2 GPI levels, not all b 2 GPI-deficient samples have this mutation. We then performed Northern blot analysis on selected liver samples with the mutant and wild type genotypes of the )1CfiA mutation. Figure 3 shows the results of Northern blot for one heterozygous (CA genotype) having a low b 2 GPI protein level (1.0 lg b 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 GPI within the 626 bp sequence in the 5¢ flanking region and tested the effect of the )1CfiAmutationon 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 of each 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 half of the b 2 GPI 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, if not all, promoter activity and that the )1CfiA mutation down regulates b 2 GPI gene expression. Effect of the )1CfiA mutation on binding of trans -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- 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 GPImRNAineachlane.(B)Northernautoradi- ograph displaying levels of GAPDH mRNA in each lane. (C) b 2 GPI levels (lgÆmg )1 of total liver protein) for the corresponding liver samples in each lane. The genotypes at nucleotide )1areindicated beneath each lane. Fig. 4. Effect of the -1CÔA mutation on reporter (Luc) gene expres- sion. The effect of )1C (wild type) and )1A (mutant type) alleles was measured as the mean of the 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. Ó FEBS 2003 Functional polymorphism in b 2 GPI (Eur. J. Biochem. 270) 235 probe (Fig. 5; lane 5) as compared to the wild type ()1C) probe (Fig. 5; lane 4). These results demonstrate a sequence- specific binding of liver nuclear extracts and TFIID to b 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 GPI quantitative polymorphisms, we have originally described a common protein polymorphism in the b 2 GPI gene [39] and both polymorphisms were found to be tightly linked [38]. Thus, the family, heritability, and linkage data provide strong support that b 2 GPI plasma variation is under genetic control and that genetic variation in b 2 GPI is a major determinant of this variation. In our attempt to deter- mine the molecular basis of b 2 GPI plasma variation, we conducted association studies and found that two of the b 2 GPI mutations, Trp316Ser and Cys306Gly, were signifi- cantly associated with b 2 GPI plasma levels and their effects were independent of each other [30,31]. However, our in vitro mutagenesis and expression study did not link these mutations to an altered b 2 GPI gene expression [32]. Although, in vitro mutagenesis and expression study do 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 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 b 2 GPI plasma levels between 4.3 mgÆdL )1 and 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 ,andtwowith 20.5 mgÆdL )1 and 20.6 mgÆdL )1 . Although the bulk of b 2 GPI plasma variation is under genetic control (66% heritability) other nongenetic factors also influence this variation [26,29] and this may explain higher than expected b 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 Trp316Ser polymorphism against the occurrence of APA 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 b 2 GPI plasma levels and consequently lower risk of developing APA. Paradoxically, however, the Cys306Gly Fig. 5. Effect of the -1CÔA mutation on the binding of crude nuclear factors and purified TFIID using EMSA. The EMSA was performed using 32 P-labeled 23 bp oligonucleotide ()12 to +11 nucleotides of the 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. 236 H. Mehdi et al.(Eur. J. Biochem. 270) Ó FEBS 2003 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 identified between nucleotides )2 and +5 (CCACTTT) that is disrupted by the )1CfiA mutation. Thus, we predicted that lower b 2 GPI plasma levels associated with the )1CfiA mutation might be due to its direct impact on b 2 GPI 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 b 2 GPI transcription, we examined its effect on b 2 GPI gene expression using tagged-Luc constructs expressed in COS-1 cells. Although the promoter of b 2 GPI is not yet characterized, we cloned the reported 5¢ flanking region of the b 2 GPI gene (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, if not all, the promoter activity and the )1CfiA mutation is a functional substitution that suppres- 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 mutation at the transcriptional initiation site is causative, which regulates b 2 GPI gene expression at the transcriptional level that ultimately affects b 2 GPI plasma levels. In summary, we have identified a new polymorphism at the transcriptional initiation site of the b 2 GPI gene that is associated with less binding with a putative transcriptional factor, lower gene expression, lower b 2 GPI plasma levels, lower b 2 GPI mRNA levels and protection from the occurrence of APA in lupus patients. Our data also indicate that the molecular basis of plasma b 2 GPI deficiency is heterogenous. 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The mutation was associated with significantly lower b 2 GPI plasma levels (P. 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