Báo cáo khoa học: Functional and genetic characterization of the promoter region of apolipoprotein H (b2-glycoprotein I) ppt

We examined the individual effects of 12 APOH promoter single nucleotide polymorphisms in the 5¢ flanking region of APOH 1.4 kb on luciferase activity in COS-1 cells and HepG2 cells and

region of apolipoprotein H (b2-glycoprotein I)

Sangita Suresh1, F Yesim Demirci1, Iliya Lefterov2, Candace M Kammerer1, Rosalind

Ramsey-Goldman3, Susan Manzi4and M Ilyas Kamboh1

1 Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA

2 Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA

3 Northwestern University, Feinberg School of Medicine, Division of Rheumatology, Chicago, IL, USA

4 Division of Rheumatology and Clinical Immunology, Lupus Center of Excellence, University of Pittsburgh, Pittsburgh, PA, USA

Introduction

Human apolipoprotein H (APOH), also known as

b2-glycoprotein I (b2GPI) (here we will use APOH to

refer to the gene as used in human genome databases

and b2GPI to refer to the protein as commonly used

in rheumatology literature) is a major autoantigen recognized by predominant anti-phospholipid antibod-ies found in sera of many autoimmune diseases, such

as primary anti-phospholipid syndrome and systemic

Keywords

APOH; association; polymorphisms;

promoter; b2-glycoprotein I

Correspondence

M I Kamboh, Department of Human

Genetics, Graduate School of Public Health,

Pittsburgh, PA 15261, USA

Fax: + 1 412 383 7844

Tel: + 1 412 624 3066

E-mail: kamboh@pitt.edu

(Received 2 September 2009, revised 1

December 2009, accepted 7 December

2009)

doi:10.1111/j.1742-4658.2009.07538.x

This study characterized the human apolipoprotein H [APOH; b2 -glycopro-tein I (b2GPI)] promoter and its variants by in vitro functional experiments and investigated their relationship with human plasma b2GPI levels We examined the individual effects of 12 APOH promoter single nucleotide polymorphisms in the 5¢ flanking region of APOH ( 1.4 kb) on luciferase activity in COS-1 cells and HepG2 cells and their impact on plasma b2GPI levels in 799 American White people, the DNA binding properties of the APOH promoter using an electrophoretic mobility shift assay in HepG2 cells, the effects of serial deletion analysis of the APOH 5¢ flanking region

in COS-1 and HepG2 cells and cross-species conservation of the APOH promoter sequence The variant alleles of three single nucleotide poly-morphisms ()1219G>A, )643T>C and )32C>A) showed significantly lower luciferase expression (51, 40 and 37%, respectively) as compared with the wild-type allele The electrophoretic mobility shift assay demonstrated that these three variants specifically bind with protein(s) from HepG2 cell nuclear extracts Three-site haplotype analysis ()1219G>A, )643T>C and )32C>A) revealed one haplotype carrying )32A (allele fre-quency = 0.075) to be significantly associated with decreased plasma

b2GPI levels (P < 0.001) Deletion analysis localized the core APOH pro-moter to  160 bp upstream of ATG codon with the presence of critical cis-acting elements between)166 and )65 Cross-species conservation anal-ysis of the APOH promoters of seven species indicated that basic promoter elements are highly conserved across species In conclusion, we have char-acterized the functional promoter of APOH and identified functional vari-ants that affect the transcriptional activity of the APOH promoter

Abbreviations

APOH, apolipoprotein H; EMSA, electrophoretic mobility shift assay; LD, linkage disequilibrium; SLE, systemic lupus erythematosus; SNPs, single-nucleotide polymorphisms; b2GPI, b2-glycoprotein I.

lupus erythematosus (SLE) [1,2] APOH spans 18 kb

on chromosome 17q23-24 [3] and encodes for a mature

protein of 326 amino acid residues b2GPI is a 50 kDa

single-chain plasma glycoprotein exhibiting internal

homology comprised of four contiguous homologous

regions of 60 amino acid residues, and an additional

variable fifth C-terminal domain The variable

configu-ration of the fifth domain is essential for the binding

of b2GPI to anionic phospholipids [4–6] Primer

exten-sions determined alternate transcription start sites at

31 and 21 bp upstream of the APOH translation start

codon [3] A transcription start site 31 bp upstream

agreed completely with the consensus for an initiator

element (Inr) known to sustain transcription initiation

Previously [7], an atypical TATA box and HNF-1a

cis-elements have been identified to be critical for

APOHcell type-specific transcriptional regulation

lead-ing to differential expression of APOH in humans

b2GPI is primarily expressed in the liver and

sporadi-cally in intestinal cell lines and tissues [8] The plasma

concentration of b2GPI is  20 mgÆdL)1, of which a

small portion is bound to lipoproteins and the rest exists

in lipid free form [9–11] There is a wide range of

inter-individual variation in b2GPI plasma levels, ranging

from immunologically undetectable to as high as

35 mgÆdL)1, with a mean value of 20 mgÆdL)1 in the

White population and 15 mgÆdL)1 in African

Ameri-cans [12], which may have clinical relevance in b2

GPI-related pathways Family and heritability data have

provided strong support for the genetic basis of b2GPI

plasma variation, but the exact molecular basis of this

variation remains largely unknown b2GPI is suggested

to regulate thrombin inactivation by heparin cofactor II

[13] and thus variation in plasma b2GPI may affect

pro-thrombic tendency in PAP patients Thus, it is

impor-tant to determine the molecular basis of b2GPI plasma

variation Previously we have shown that two single

nucleotide polymorphisms (SNPs) in coding regions

(Cys306Gly, Trp316Ser) [12,14] and one SNP in the

promoter ()32C>A) [15] region of APOH have a

sig-nificant impact on b2GPI plasma variation Since then

we have characterized the complete DNA sequence

vari-ation in APOH and identified 150 SNPs, including 13

SNPs and one deletion ()742delT) in the 5¢ region [16]

Variations in the promoter DNA sequence may

potentially alter the affinities of existing protein–DNA

interactions or recruit new proteins to bind to the

DNA, altering the specificity and kinetics of the

tran-scriptional process Given the importance of promoters

in harboring functionally relevant SNPs that regulate

gene expression and phenotypic variation, it is

impor-tant to examine the role of promoter SNPs in relation

to disease, gene expression and corresponding plasma

levels Recently we have reported associations of APOH promoter SNPs with SLE risk and carotid pla-que formation in SLE patients [17]

The objectives of this study were: to characterize a

 1.4 kb (1418 bp) genomic fragment in the 5¢ region

of human APOH to identify the functional promoter;

to examine the impact of all 13 reported APOH pro-moter SNPs in the White population ()1284C>G, )1219G>A, )1190G>C, )759 A>G, )700C>A, )643T>C, )38G>A and )32C>A) and African Americans ()1076G>A, )1055T>G, )627A>C, )581A>C and )363C>T) on APOH gene expression;

to determine the association of eight promoter SNPs

in a White population on b2GPI levels among Ameri-can White people; and to determine the cross-species conservation of the APOH promoter sequence

Results Identification and characterization of the APOH promoter region

In order to localize the active promoter region and to identify regions that are important for the regulation of human APOH expression, the wild-type 1418 bp 5¢ flanking region of APOH was amplified from genomic DNA and used as a template to create a series of five different deletion constructs containing 5¢ truncated fragments of APOH promoter fused upstream to a pro-moterless firefly luciferase gene of the pGL3-basic reporter vector The sequence of each construct was verified by sequencing (data not shown) Figure 1A shows the expression of deletion mutants in COS-1 cells 5¢ deletions of the promoter sequence to )815 (Del mutant 1, )815 ⁄ +43) and )575 (Del mutant 2, )575 ⁄ +43) increased promoter activity slightly com-pared with the wild-type, but the difference was not sig-nificant (wild-type versus Del mutant 1; P = 0.260, wild-type versus Del mutant 2; P = 0.135) Successive removal of nucleotides from )575 (Del mutant 2, )575 ⁄ +43) to )325 (Del mutant 3, )325 ⁄ +43) enhanced promoter activity appreciably (wild-type ver-sus Del mutant 3; P = 0.019), suggesting the possibil-ity of negative regulatory elements within the )575 ⁄ )325 regions The Del mutant 3 construct ()325 ⁄ +43) conferred maximum luciferase activity in COS-1 cells A slight decrease in promoter activity was observed after further deletion of a sequence from)325

to )166 (Del mutant 4, )166 ⁄ +43; P = 0.04) How-ever, when the sequence from )166 to )65 was removed (Del mutant 5, )65 ⁄ +43), promoter activity dropped significantly (P < 0.001) compared with the wild-type This suggests the presence of a critical

element in the region extending between)166 and )65.

We replicated the deletion analysis using the human

HepG2 cell line, as liver is a major site of synthesis of

b2GPI and found an overall similar trend as seen in

COS-1 cells, with Del mutant 3 ()325 ⁄ +43) showing

the highest and Del mutant 5 ()65 ⁄ +43) showing the

lowest (P < 0.001) promoter activity (Fig 1B) A

slight difference in trend was observed for the

wild-type, mutant 1 ()815 ⁄ +43) and mutant 2 ()575 ⁄ +43)

constructs, wherein mutant 1 was lower than the

wild-type for HepG2, but not in COS-1 cells Thus, using

both COS-1 and HepG2 cell lines, we identified the

region  166 bp upstream of the translation start site

as the basal promoter of human APOH containing key

cis-acting elements that regulate APOH expression

Functional characterization of APOH promoter SNPs

In order to investigate the differential allele-specific effect on promoter activity, pGL3-basic–APOH promoter constructs harboring individual point mutations for 12 of 14 APOH promoter sequence vari-ants identified earlier [16] ()1284C>G, )1219G>A, )1190G>C, )1076G>A, )1055T>G, )759A>G, )700C>A, )643T>C, )627A>C, )363C>T, )38G>

A and)32C>A) were generated The relative luciferase activity assessed in three independent experiments per-formed in triplicate for all the above APOH promoter SNPs is listed in Table 1 The insertion⁄ deletion polymorphism ()742delT) could not be characterized

Luc

B

0.00 20.00 40.00 60.00 80.00 100.00 Del mutant 5

Del mutant 4 Del mutant 3 Del mutant 2 Del mutant 1 Wild-type pGL3-B

*

Del mutant 5 Del mutant 4 Del mutant 3 Del mutant 2 Del mutant 1 Wild-type pGL3-B

*

Fig 1 (A) Dual luciferase reporter gene expression of APOH promoter deletion mutants in COS-1 cells Left panel, schematic representation

of 5¢ deleted fragments of the APOH promoter in conjunction with the luciferase gene in pGL3-basic vector The nucleotides are numbered from the translation start site (ATG) The effect of the wild-type and mutants was measured as the mean of the firefly luciferase levels nor-malized by the Renilla luciferase activity, which served as the reference for the transfection efficiency The results presented are from one

of three independent experiments pGL3-B indicates the promoterless vector The asterisk indicates that Del mutant 5 had significantly lower luciferase activity than the wild-type (P < 0.001) (B) Dual luciferase reporter gene expression of APOH promoter deletion mutants in HepG2 cells Left panel, schematic representation of 5¢ deleted fragments of the APOH promoter in conjunction with the luciferase gene in pGL3-basic vector The nucleotides are numbered from the translation start site The effect of the wild-type and mutants was measured as the mean of the firefly luciferase levels normalized by the Renilla luciferase activity, which served as the reference for the transfection effi-ciency The results presented are from one of two independent experiments pGL3-B indicates the promoterless vector The asterisk indi-cates that Del mutant 5 had significantly lower luciferase activity than the wild-type (P < 0.001).

due to repetitive sequences in the surrounding region.

Similarly, the )581A>C mutant construct was not

successful

Three SNPs were found to be significantly associated with differential gene expression (36% or higher differ-ence at P < 0.001), including two previously reported, )643T>C [17] and )32C>A [15] An additional SNP, )1219G>A, showed a significant difference of  51%

in luciferase gene expression between wild-type and mutant alleles (Fig 2) An electrophoretic mobility shift assay (EMSA) was performed in order to deter-mine whether the APOH promoter )1219G>A SNP affects the binding activity of nuclear factors Upon incubation of radiolabeled oligonucleotides specific for wild-type ()1219G) and mutant ()1219A) alleles with HepG2 nuclear extracts, DNA–protein complexes were observed, indicating the presence of nuclear factor(s) (Fig 3) Competition assays using increasing amounts

of unlabeled wild-type oligonucleotides confirmed the specificity of the binding

Potential liver-specific transcription factor binding sites for the three promoter SNPs that showed differential gene expression ()1219G>A, )643T>C and )32C>A) were sought by using the matin-spector program (http://www.genomatix.de/products/ MatInspector/index.html) [18], which matches by comparing DNA sequences with weighted matrix descriptions of functional binding sites, based on the TRANSFAC database (http://www.biobase.de) Fig-ure 4 shows the locations of these three functional SNPs relative to potential binding sites, together with all other SNPs detected in the 5¢ flanking region The list of all the predicted transcription factors, including their consensus sequences and specific binding sites, is given in Table S1 The program identified binding sites for the )1219G>A and )643T>C SNPs (Fig 4) Although the binding site for HNF1 was observed adjacent to the )1219G>A SNP site, the )643T>C SNP region showed binding to CLOX and CLOX homology CCAAT displacement protein fac-tors EMSA results previously reported by us [15]

Table 1 Dual luciferase results of each APOH promoter construct

in COS-1 cells.

SNPs

Wildtype

allele

(Mean ± SD)

Variant allele (Mean ± SD)

% decrease P-value

)1284C>G 5.06 ± 0.10 4.16 ± 0.36 17.79 0.014

)1219G>A 2.86 ± 0.05 1.40 ± 0.01 51.05 < 0.001

4.10 ± 0.21 2.06 ± 0.16 49.76 < 0.001

3.70 ± 0.12 1.81 ± 0.08 51.08 < 0.001

)1190G>C 3.01 ± 0.19 2.16 ± 0.03 28.24 < 0.01

2.79 ± 0.19 1.98 ± 0.23 29.03 < 0.01

3.93 ± 0.50 2.84 ± 0.08 27.74 < 0.01

)1076G>A 10.01 ± 0.38 9.13 ± 0.86 8.79 0.178

)1055T>G 4.66 ± 0.18 3.44 ± 0.17 26.18 < 0.01

7.66 ± 0.53 6.13 ± 0.04 19.97 < 0.01

3.49 ± 0.09 2.53 ± 0.14 27.51 < 0.01

)700C>A 4.65 ± 0.05 4.31 ± 0.10 7.31 < 0.01

)643T>C 19.91 ± 1.68 11.94 ± 0.15 40.03 0.001

5.73 ± 0.07 3.20 ± 0.24 44.15 < 0.001

)363 C>T 3.82 ± 0.34 3.34 ± 0.25 12.57 0.117

3.81 ± 0.09 3.16 ± 0.03 17.06 < 0.001

)32C>A 18.91 ± 0.38 11.92 ± 0.39 36.96 < 0.001

15.79 ± 1.03 10.32 ± 0.17 34.64 < 0.001

16.71 ± 0.92 10.56 ± 0.06 36.8 < 0.001

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

1.00

Experiment I

pGL3-Basic

2.86

1.40*

1.

Wild-type (–1219G)

.00 4.10

Experiment II

2.06*

1.00 3

Experiment III

Mutant (–1219A) 3.70

1.81*

Fig 2 Dual luciferase reporter gene expression of the APOH promoter )1219G>A SNP (*P < 0.0001) The results of three independent experiments are shown.

have revealed that the )32C>A SNP disrupts the

binding of crude mouse hepatic nuclear extracts and

purified transcription factor II D, which is part of the

RNA polymerase II preinitiation complex, indicating

its functional role in the transcriptional regulation of

APOH promoter However, in silico analysis using the

matinspector program for liver-specific factors did

not identify any liver-specific transcription factor to

bind to the region including the )32C>A SNP

In order to determine the cross-species conservation

of the APOH promoter sequence, we used the ECR

Browser (http://ecrbrowser.dcode.org/) to visualize the

conservation profile of the 5¢ region of APOH

(1418 bp; )1375 ⁄ +43 nucleotides from the translation

initiation codon ATG) to identify the evolutionary

conserved regions Figure 5 shows the graphical

dis-play of the pairwise alignments and comparisons of

sequences from six other species (monkey, dog, cow,

mouse, rat, opossum) with that of human (base

gen-ome) Consistent with our deletion analyses, which

indicated the presence of critical promoter elements

in the region spanning between )166 and )65, the

evolutionary conserved region extending from the 5¢

end of the gene (exon + UTR) to the immediately

upstream region ( 250 bp upstream of the ATG

start codon) was highly conserved across all seven

species

APOH promoter SNPs and plasma b2GPI levels The distribution of plasma b2GPI levels showed only

a modest difference (17.90 ± 4.15 mgÆdL)1 versus 18.72 ± 4.68; P = 0.054) in mean plasma b2GPI lev-els between cases (n = 241) and controls (n = 206) Therefore, the association analyses were carried out using the combined case + control cohort data Step-wise regression analysis revealed that age, body mass index and ever smoking were the significant determi-nants of plasma b2GPI levels Only the)32C>A SNP showed significant associations with the adjusted mean plasma b2GPI levels in both single-site (P < 0.001) and multiple regression (P < 0.001) analyses Mean plasma b2GPI levels were higher in homozygotes

of the wild-type allele, CC (mean = 18.62 mgÆdL)1), compared with both the heterozygotes, CA (mean = 16.24 mgÆdL)1), and homozygotes of the less common allele, AA (mean = 13.90 mgÆdL)1) An eight-site hap-lotype analysis including six APOH promoter SNPs (present in White people) and two coding SNPs identi-fied a total of 11 haplotypes with a frequency of

> 1% (Table 2) Because data for plasma b2GPI levels were only available for the White population, we excluded the five SNPs present in the Black popula-tion Out of the eight SNPs present in White people, the)1284C>G SNP was excluded due to its rare pres-ence (MAF < 0.01) and the )700C>A SNP was excluded because of its high linkage disequilibrium (LD) to )759A>G as shown previously [17] Three haplotypes (H5, H6, H10) showed a significant associa-tion with plasma b2GPI levels (P < 0.001) The haplo-type H5 harbored minor alleles for the )1190G>C, )32C>A and Trp316Ser SNPs The other two signifi-cant haplotypes were predominantly defined by the minor alleles of the two coding polymorphisms (H6: Cys306Gly; H10: Trp316Ser) that are already known

to be major determinants of plasma b2GPI levels Although the)32C>A SNP was significant in the sin-gle-site analysis, the other haplotype (H7) defined by minor alleles only at the )1190G>C and )32C>A SNPs and not for Trp316Ser did not show significant association, suggesting that the effect of the )1190G>C and )32C>A SNPs is dependent upon Trp316Ser polymorphism None of the individual hapl-otypes harboring less common alleles for the )643T>C (H2 and H9) and )1219G>A SNPs (H4) that significantly decrease gene expression in vitro showed significant impact on plasma b2GPI levels A three-site haplotype analysis (data not shown) with the functionally relevant (based on dual luciferase and EMSA data) )1219G>A, )643T>C and )32C>A SNPs was consistent with the individual SNP results

–1219 A (Mutant) –1219 G (Wild–type)

_

Competitor – – 1x 5x 20x 50x 100x 1x 5x 20x 50x 100x

Fig 3 EMSA result for )1219G>A polymorphism Each sample

contained a mixture of 5 lg of nuclear extract derived from human

HepG2 cell nuclear extract and 30xmer [ 32 P]-labeled wild-type

oligo-nucleotide containing G allele The arrow indicates a specific DNA–

protein complex associated with the )1219G>A polymorphic site.

Lane 1, labeled oligonucleotide without nuclear extract from HepG2

cells; lane 2, labeled oligonucleotide with nuclear extracts Lanes

3–7 had increasing amounts of G oligonucleotide competitor (1, 5,

20, 50, 100·, respectively); lanes 8–12 had increasing amounts of

A oligonucleotide competitor (1, 5, 20, 50, 100·, respectively).

Fig 4 MATINSPECTOR results for the liver-specific transcription factor binding sites of the APOH promoter The transcription factors are shown in green together with the exact binding position marked by a dotted line; the APOH promoter SNPs are in red The ATG start codon is highlighted

in grey Bases in purple indicate the repeat region, those in green indicate the untranslated region (UTR), and those in dark blue indicate Exon 1.

That is, only the haplotype carrying )32A was

signifi-cantly associated with decreased plasma b2GPI levels

(P < 0.001)

Discussion

The aims of this study were: (a) to clone and

charac-terize a 1418 bp fragment of the 5¢ region of APOH;

(b) to functionally characterize the APOH promoter

SNPs present in the 1418 bp fragment; (c) to examine

the effect of the APOH promoter SNPs on plasma

b2GPI levels; and (d) to determine the cross-species

conservation of the APOH promoter sequence

To identify regions of the APOH promoter that

affect its basal transcription, several 5¢ promoter

dele-tion mutants were linked to the luciferase reporter gene

and assayed Promoter constructs containing either

)1375 ⁄ +43 (wild-type) or )166 ⁄ +43 (Del mutant 4)

of the upstream sequence had similar high levels of

basal transcriptional activity when transfected into

either COS-1 or HepG2 cell lines These results

indi-cate that all of the necessary machinery for driving

basal APOH expression is localized in this )166 ⁄ +43

sequence Further deletion from)166 to )65 revealed

regions within the APOH promoter that are important

for its function This deletion resulted in an  60%

decrease in transcriptional activity in COS-1 cells and

an even more pronounced ( 98%) decrease in HepG2

cells, indicating the presence of an activator motif(s)

within this sequence These results are consistent with

the previous deletion analysis [7] that identified the

proximal promoter region necessary for hepatic-specific APOH expression The smallest APOH 5¢ deletion mutant ()65 ⁄ +43) used in this study differed from the previous study [7] as it lacked both the critical cis-ele-ments (TATTA and HNF-1a) identified within this region, whereas the smallest deletion mutant used in the previous study [7] lacked only the TATTA element Despite this difference, our study replicated the key findings in which the smallest 5¢ deletion mutant almost completely abolished luciferase activity by

 98% (present study) and  91% [7] in HepG2 cells, emphasizing the vital role of the TATTA cis-element

in APOH transcription Our cross-species conservation analysis of APOH promoters from different species indicates that basic promoter elements are highly con-served across the seven species examined

Approximately one-third of promoter variants exert

a functional effect on gene expression [19] The func-tional importance of the APOH promoter SNPs was predicted by allelic differences in expression of the luciferase reporter gene In this study, we ‘functionally’ validated SNPs in the APOH promoter based on two experimental approaches (reporter assays and EMSA) For this purpose, we tested 12 of the 14 sequence vari-ants located within the 1418 bp of the 5¢ flanking region of APOH for allele-specific regulatory effects on the expression of the dual luciferase reporter gene and

by EMSA for SNPs within transcription factor binding sites Of the 12 SNPs examined, three SNPs at posi-tions )1219G>A, )643T>C and )32C>A showed a significant decrease in luciferase expression ( 50%,

Fig 5 ECR Browser conservation profile of the 5¢ region of APOH (1418 bp; )1375 ⁄ +43 nucleotides from the translation initiation codon ATG) Sequence elements of significant length (‡ 100 nucleotides) that are conserved above a certain level of sequence identity (‡ 65%) between the two compared genomes are highlighted as evolutionary conserved regions (pink rectangles at the top of the graphs) The hori-zontal axis represents positions in the base genome (human) and the vertical axis represents the percentage identity between the base and aligned genomes (monkey, dog, cow, mouse, rat and opossum) The color-coding used by ECR Browser is: blue for coding exons, yellow for UTRs, red for intergenic regions, and green for transposable elements and simple repeats.

b2

rs8178819 ()

rs3760290 ()1190G>C) rs817820 ()759A>G)

rs3760292 ()

rs8178822 ()32C>A) rs1801689 (Cys306Gly) rs1801690 (Trp316Ser)

Base haplotype

Rare haplotype

 40% and  36%, respectively) in COS-1 cells The

)32C>A SNP is a part of the core APOH promoter

region ()166 bp upstream from ATG) identified in this

study and has been previously shown to play a key

role in the transcription initiation process by serving as

a site for the binding of transcription factor II D [15]

Although 5¢ serial deletion of the APOH promoter

identified the basal transcriptional activity restricted to

the region  160 bp upstream of ATG codon, it does

not eliminate the possibility of the functional roles of

the )643T>C and )1219G>A SNPs as part of the

extended APOH promoter transcriptional machinery

To further substantiate the functional relevance of the

three APOH promoter SNPs ()1219G>A, )643T>C

and)32C>A), EMSA revealed strong in vitro protein

binding for both wild-type and mutant-type

oligo-nucleotides for each SNP using nuclear extracts of

HepG2 cells However, no significant differential

binding was observed for the two alleles for all SNPs

In silico analysis using the matinspector program

for the prediction of liver-specific transcription factor

binding sites revealed potential binding sites for the

)1219G>A and )643T>C SNPs (Fig 4) Binding of

an important liver-enriched transcription factor, HNF1,

was observed adjacent to the)1219G>A polymorphic

site, which could explain the functional relevance of

this SNP HNF1 plays a prominent role in regulating

genes that are expressed in hepatocytes [20] The

)643T>C SNP region binds to CLOX and CLOX

homology CCAAT displacement protein factors, which

have been previously reported as transcriptional

repres-sors [21] This could probably explain the decrease in

reporter gene expression observed by the mutant allele

In addition to characterizing the basal APOH

pro-moter and its functional variants, the effect of the

APOH promoter SNPs on plasma b2GPI levels was

examined for a subgroup of the Pittsburgh White

pop-ulation (SLE cases, n = 241; controls, n = 206) In

univariate analysis, only the previously reported

)32C>A SNP showed a significant effect after

adjust-ment for covariates None of the other APOH

pro-moter SNPs used in this study had a significant effect

on plasma b2GPI levels Our previous report [17]

sug-gested a role for the)643T>C polymorphism

protect-ing against carotid plaque formation in

autoimmune-mediated atherosclerosis in SLE patients and the

)1219G>A SNP showed a moderate effect on lupus

nephritis A functional role for the two SNPs was

established using promoter gene assays and EMSA

Despite the functional effects of the )1219G>A and

)643T>C SNPs on gene expression, their lack of

association with plasma b2GPI levels is interesting

Although in vitro luciferase assays measuring promoter

activity suggest that the two polymorphisms show an effect on gene expression, this may not be an entirely true reflection of the complexity of regulation that occurs in vivo The regulation of human gene expres-sion is a critical, highly coordinated and complex pro-cess The core promoter is generally within 50 bp of the transcription start site, where the preinitiation complex forms and the general transcription machinery assembles [22] The extended promoter can contain specific regulatory sequences that control spatial and temporal expression of the downstream gene The tran-scription machinery, which consists of interconnected coregulatory protein complexes in a regulatory net-work, is responsible for mRNA synthesis from a given promoter Control of gene regulation could occur at various stages, including the level of transcription, post-transcriptional regulation, alternative splicing, translation, post-translational modification and secre-tion of b2GPI, all of which may have an effect on the quantitative measure of plasma b2GPI levels Alterna-tively, it is also possible that a change in promoter activity does not necessarily result in a quantitative change at the protein level Whether the APOH pro-moter SNPs ()643T>C and )1219G>A) could influ-ence the promoter activity by either the former or latter methods is beyond the scope of in vitro experi-ments Further studies will be needed to explore the mechanism for these associations

APOH promoter SNPs explain a small proportion

of the variance in APOH expression Therefore, the ability of these SNPs to influence plasma b2GPI levels may be obscured by the strong effects of other factors (undefined promoter elements that are in strong LD with the promoter SNPs and other regulatory factors that affect in vivo gene expression) in aggregate How-ever, given the reporter gene expression data on pro-moter activity and EMSA results indicating possible binding to transcription factors, there is clearly a func-tional effect of the two polymorphisms on APOH reg-ulation that is worthy of further investigation However, haplotype analysis including APOH pro-moter SNPs alone or in conjunction with previously known coding SNPs affecting plasma b2GPI levels (Cys306Gly and Trp316Ser, Table 1) gave us no new insights into determining the genetic basis of plasma

b2GPI levels The significant haplotypes were defined predominantly by the minor alleles at the coding SNPs, which are already known to have a major effect

on b2GPI levels Consistent with the univariate data, none of the haplotypes defined by the minor alleles at the APOH promoter SNPs reached significance Although the )32C>A SNP was significant in the univariate analysis, the individual haplotype (H7)

harboring the minor allele )32A was not significant,

indicating that the effect of the )32C>A SNP is

dependent upon the presence of the Trp316Ser coding

SNP, which is in strong LD with the )32C>A SNP,

as shown in haplotype H5 A three-site haplotype

anal-ysis with only the APOH promoter functionally

rele-vant SNPs ()643T>C, )1219G>A and )32C>A)

showed a highly significant effect for the haplotype

defined by the )32A allele and also a moderate effect

for the )1219A allele Another questionable

mecha-nism for the lack of association of APOH promoter

SNPs on plasma b2GPI levels in this study is the

modi-fied capture-ELISA method that was used to determine

the plasma b2GPI levels, wherein the analyzed

anti-bodies could have been targeted against only a small

number of the antigenic sites in b2GPI Therefore,

given both the method and also the small sample size,

further studies are warranted in larger cohorts using

improvised methods (antibody titers measured against

other⁄ additional b2GPI sites) that will help to delineate

better the molecular basis of plasma b2GPI levels

Materials and methods

Construction of APOH promoter luciferase

reporter gene vector (wild-type and individual

mutant constructs)

A 1418 bp fragment of the human APOH 5¢ region

()1375 ⁄ +43 nucleotides from the translation

initia-tion codon ATG) containing the promoter and the first

untranslated exon was PCR amplified using

for-ward (5¢-TGGCAGCACACTCTTCTTAT-3¢) and reverse

APOH promoter fragment was amplified from an

individ-ual who had wild-type alleles for all 13 SNPs ()1284C>G,

)1219G>A, )1076G>A, )1055T>G, )1190G>C,

)759A>G, )700C>A, )643T>C, )627A>C, )581A>C,

)363C>T, )38G>A, )32C>A) and no deletion at )742

site The PCR condition consisted of denaturation at 95C

for 2 min, followed by 35 cycles of denaturing at 95C for

30 s, annealing at 55C for 30 s and extension at 72 C for

1 min, before a final extension at 72C for 10 min The

PCR-generated fragment was cloned into the

pCR-2.1-TOPO vector (Invitrogen Corporation, Carlsbad, CA,

USA) using the supplier’s standard protocol The size and

orientation of the DNA insert were confirmed by restriction

analysis (HindIII and SacI) The promoter fragment was

then excised out of the TOPO vector using enzymes KpnI

and EcoRV and ligated into the KpnI–SmaI restricted

pGL3-basic firefly luciferase reporter plasmid and

trans-formed into top 10 chemically competent cells (Invitrogen)

Following transformation, the positive clones were

con-firmed by sequencing

Constructs bearing mutant⁄ minor alleles for each APOH promoter SNP were generated by PCR using the wild-type APOH promoter⁄ luciferase reporter construct ( 1.4 kb 5¢ region of APOH promoter inserted into the pGL3-basic luciferase reporter vector) as a template using the Quick-Change II Site-directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s protocol

Construction of APOH promoter deletion mutants

A series of 5¢ deletion mutants of the  1.4 bp APOH promoter fragment were subcloned into a new luciferase reporter vector (pGL3-basic) For this purpose, the original wild-type construct carrying the 1418 bp APOH promoter fragment served as a parental template for designing PCR primers to amplify several truncated APOH promoter frag-ments We designed five APOH deletion mutant constructs differing in  200 bp between each fragment: APOH dele-tion fragment 1 (APOH del FR #1) is the largest (858 bp)

of all five fragments The position of this region with respect to the translational start site is +43 to )815 APOH deletion fragment 2 (APOH del FR #2) contains

618 bp The location of this deletion mutant from the translational start site is +43 to )575 APOH deletion fragment 3 (APOH del FR #3) is the third fragment (368 bp) The location of this fragment with respect to the translational start site is +43 to )325 APOH deletion fragment 4 (APOH del FR #4) is the fourth fragment It is further truncated to position)166 and is 209 bp

APOH deletion fragment 5 (APOH del FR #5) is the smallest of all five fragments (109 bp) The position of this region with respect to the translational start site is +43 to )65

primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/ primer3.cgi) was used to design PCR primers contain-ing linker sites for the restriction enzymes KpnI and BamHI at the 5¢ and 3¢ ends of each deleted fragment, respectively The PCR products were gel purified (Qiagen, Valencia, CA, USA) and then digested with KpnI and BamHI restriction enzymes The digested fragments were again gel purified The promoterless pGL3-basic vector (Promega Corporation, Madison,

WI, USA) was digested with KpnI and BglII, gel puri-fied and calf intestinal alkaline phosphatase treated

in order to prevent self-ligation of the empty vector The APOH–PCR DNA was then ligated to the gel-purified and calf intestinal alkaline phosphatase-treated pGL3-basic vector by T4 DNA ligase to generate the fusion vector construct carrying the APOH upstream truncated sequence fused to the inframe luciferase reporter gene The ligated product was then transformed into competent Escherichia coli, fol-lowed by screening of recombinant plasmids using a colony PCR technique The positive clones were