www.elsevier.com/locate/jhep

Journal of Hepatology 46 (2007) 198–205

Polymorphisms of type I interferon receptor 1 promoter and theireffects on chronic hepatitis B virus infection

Jie Zhou1, Liwei Lu2,*, Man-Fung Yuen3, Ting-Wa Lam4, Chi-Ping Chung4,Chun-Lit Lam4, Bin Zhang5, Song Wang5, Yu Chen6, Sharon HW Wu1,

Vincent KM Poon1, Fai Ng1, Chris CS Chan1, Shibo Jiang7,Kwok-Yung Yuen1, Bo-Jian Zheng1,*

1Department of Microbiology, The University of Hong Kong, Hong Kong2Department of Pathology, The University of Hong Kong, Hong Kong

3Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong4Department of Medicine, Queen Elizabeth Hospital, Hong Kong

5Dong-Hu Hospital, Shenzhen, China6You-An Hospital, Affiliated Capital University of Medical Science, Beijing, China

7Lindsley F. Kimball Research Institute, The New York Blood Center, New York, USA

Background/Aims: Exposure to HBV leads to a distinct clinical course which is partially pertained to host genetic

variability. We aimed to study polymorphisms of type I interferon receptor 1 (IFNAR1) promoter and their potential

effects on chronic HBV infection.

Methods: Polymorphisms of IFNAR1 promoter were identified in 320 chronic hepatitis B patients, 148 spontaneously

recovered individuals, 148 healthy Chinese donors and 114 Caucasians. Their functional capability in driving reporter gene

expression was analyzed.

Results: Four polymorphic alleles were identified at loci �568, �408, �77 and �3. Association analysis revealed thatcarriers of alleles �568G, �408C and their related haplotype I were less susceptible to chronic HBV infection whereas

those of alleles �568C, �408T and related haplotype III were significantly associated with higher risk to chronic hepatitis

B (P < 0.01). In a reporter-driven system, the promoter variants with alleles �408C and �3C could drive higher expression

of the reporter gene than those with alleles �408T and �3T (P < 0.01). Interestingly, an allele with 9 GT repeats at �77

that was rarely found in Chinese but prevalent in Caucasian exhibited the highest transcriptional ability.

Conclusions: Our results showed that polymorphisms of IFNAR1 promoter may affect, at least in part, the outcomes of

HBV infection.

� 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Keywords: IFNAR1 promoter polymorphisms; Haplotypes; Transcriptional activity; Chronic HBV infection

0168-8278/$32.00 � 2006 European Association for the Study of the Liver.

doi:10.1016/j.jhep.2006.08.017

Received 19 May 2006; received in revised form 21 July 2006; accepted 5

August 2006; available online 17 October 2006* Corresponding authors. Tel.: +852 2855 4870; fax: +852 2872 5197

(L. Lu); Tel.: +852 2855 4383; fax: +852 2855 1241 (B.-J. Zheng).E-mail addresses: liweilu@hkucc.hku.hk (L. Lu), bzheng@hkucc.

hku.hk (B.-J. Zheng).

1. Introduction

The World Health Organization has estimated thatabout 400 million people worldwide are chronicallyinfected with hepatitis B virus (HBV). The incidence ofHBV infection in the Asia-Pacific region is among thehighest in the world. Chronic infection by HBV in par-ticular has a high prevalence (>10%) in most of the

Published by Elsevier B.V. All rights reserved.

J. Zhou et al. / Journal of Hepatology 46 (2007) 198–205 199

countries within this region (http://www.who.int/en/).Exposure to HBV can cause variable clinical conditionsincluding spontaneous recovery from acute hepatitis,asymptomatic carrier, chronic hepatitis, liver cirrhosisand hepatocellular carcinoma. It is acknowledged thatthe diversity of the disease spectrum was attributed tothe host immunological and genetic factors [1–5]. Hostgenetics such as single nucleotide polymorphisms(SNPs) of a variety of genes have been implicated inthe diversity of HBV clinical course [6–9].

The endogenous production of interferons (IFN)serves as an initial defense mechanism against viralinfection, inhibits the replication of a broad range ofRNA and DNA animal viruses in animal models andcell cultures [10,11]. The treatment of IFN-a/b in hepa-tocyte cell lines can effectively suppress HBV replication[12]. Injections of exogenous IFN-a/b and induction ofendogenous IFN-a/b may inhibit HBV replication intransgenic mice [13–15]. In clinical practice, interferon-a treatment remains a mainstay administration in activehepatitis B [16].

One scenario, which was posed to be operative inanti-HBV effect of type I IFNs, is that some patientsmight generate less IFNs after they were exposed tothe virus. However, it has been found that serum levelsof interferon-a did not show a significant differencebetween patients who subsequently cleared the virusand those who progressed to persistent infection [17].Another possibility is that the variation in type Iinterferon receptor, consisting of interferon receptor 1(IFNAR1) and interferon receptor 2 (IFNAR2)[18–20], might contribute to the susceptibility to chronicHBV infection. Both IFNAR1 and IFNAR2 arerequired for high-affinity binding, maintenance of spe-cies specificity and signal transduction [21–23]. Antibod-ies against human IFNAR1 can block the binding of alltype I IFNs to human cells, indicating that IFNAR1 isessential for the binding of type I IFNs [24,25]. In mice,the homozygous deletion of IFNAR1 resulted inincreased susceptibility to the infection by several virus-es and abolished anti-proliferative activity of IFN-a andIFN-b [26,27]. Several polymorphisms in IFNAR1 pro-moter region have been reported [28–30], but it remains

Table 1

Demographic and serological profiles of chronic hepatitis B patients and contr

Groups Number Male/female HBsAg Anti-HBs

CP 226 138/88 + �94 50/44 + �

SR 140 65/77 � +8 5/3 � �

HBD 113 47/66 � +35a 20/15 � �

CP, chronic hepatitis B patient who had not progressed to cirrhosis or hepahealthy blood donors; NA, not applicable.

a We cannot exclude persons with occult HBV infection or non-responders oblood donors.

unclear whether and how these polymorphisms mightaffect the susceptibility to chronic HBV infection. There-fore, this study aimed to genotype the IFNAR1 promot-er region and to validate the association between geneticvariations of IFNAR1 promoter and the HBV persis-tence or clearance.

2. Patients and methods

2.1. Study subjects and sample collections

The study subjects consisted of 616 Chinese adults, including 320patients with chronic HBV infection (CP group), 148 spontaneouslyrecovered blood donors (SR group), 148 healthy blood donors(HBD group), and 114 healthy Caucasians (HC group). The criteriafor diagnosis of chronic HBV patients and information of their con-trols are shown in Table 1. After ethical approvals were obtained,blood samples of CP group were, respectively, collected from QueenMary Hospital, Elizabeth Hospital (both in Hong Kong SAR), DongHu Hospital (Shenzhen, China) and You An Hospital (Beijing, China),while blood samples of SR and HBD groups were obtained from RedCross Blood Bank of Hong Kong and those of HC group were fromthe New York Blood Center (New York, USA).

2.2. Detection of HBV antigens and antibodies

Serum HBsAg, HBeAg, anti-HBs, anti-HBe and anti-HBc wereassayed by ELISA kits (BIOKIT, Spain), according to the manufactur-er’s instruction.

2.3. Detection of mutations in IFNAR1 promoter region

Genomic DNA was extracted from blood samples of study subjectsusing QIAamp DNA blood mini kits (Qiagen, Valencia, CA). The 755-bp segment covering IFN-AR1 minimal promoter region was ampli-fied using a pair of primers: forward 5 0-TCT CTC CAA GGG ACCATC TCG-3 0 and reverse 5 0-AGG AGG ACG ACC ATC ATC TG-3 0 and PCR kit (Applied Biosystems, Foster City, CA). PurifiedPCR products were detected by automated sequencing using the sameprimers for PCR and Big-Dye Teminator version 2 (Applied Biosys-tems). Each mutation was confirmed by two directions of sequencing.All acquired sequences were aligned to sequence of X60459 (GenBank)for the comparison.

2.4. Determination of allelic linkage

Allelic linkage between loci �408 and �3 in a heterozygote wasdetermined by PCR using two pairs of allele-specific primers, �408Cforward 5 0-AAG AGC GCC GGG CCG CGA CC-3 0, �408T forward

ol subjects

HBeAg Anti-HBe Anti-HBc ALT normal/elevated

+ � + 34/192� + + 11/83� � + NA� � + NA� � � NA� � � NA

tocellular carcinoma; SR, spontaneously recovered individuals; HBD,

f HBV vaccination in these 35 HBV antigen/antibody negative healthy

Exon1 2 3 4 5 6 7 8 9 10 11

–568 G/C

–408FC/FT

IFNAR1-F

–3RC/RT

–408C/T –77(GT)n –3C/T

IFNAR1-R

Fig. 1. Schematic representation of IFNAR1 gene and single nucleotide

polymorphisms located in promoter and open reading frame. Promoter

was amplified and sequenced using primers IFNAR1-F/R. Allele-specific

primers, �408FC/FT, and �3RC/RT were used to confirmed allelic

linkage between �408 and �3. Eleven exons are represented as black

boxes and 5 0 and 3 0-UTR as white boxes.

Table 2

Frequency of genetic markers in Chinese and Caucasian study subjects

Polymorphisms Chinese Caucasian P valuea

Genotypes

�568G/G 0.1802 0.1316 0.2070�568G/C 0.4545 0.4825 0.5828�568C/C 0.3653 0.3860 0.6738�408C/C 0.6071 0.5526 0.2754�408C/T 0.3182 0.3947 0.1103

200 J. Zhou et al. / Journal of Hepatology 46 (2007) 198–205

5 0-AAG AGC GCC GGG CCG CGA CT-3 0, �3C reverse 5 0-AAGTCA CAC CGC CCC TCT GCG-3 0, and �3T reverse 5 0-TAA GTCACA CCG CCC CTC TGC A-3 0. PCR was performed by combina-tion of these two pairs of primers. PCRs were carried out using GC-RICH PCR system (Roche Diagnostics, USA).

2.5. Construction of IFNAR1 promoter-luciferasereporter vectors

Five naturally occurring haplotypes of IFNAR1 promoter variantswere amplified by PCR using a pair of primers forward 5 0-AATACGCGT GGG CCC GTG GCT GTT CTC T-3 0 and reverse 5 0-GC AGATCT CTA AGT CAC ACC GCC CCT CT-30, whichcontained MluI and BglII cleavage sites (nucleotides underlined).Amplified fragments were cloned into T vector (Invitrogen) and theninserted into MluI and BglII sites of pGL3-Basic plasmid (Promega,Madison, WI) using T4 ligase (Roche) to generate IFNAR1 promot-er-luciferase reporter vectors. The inserts were further confirmed bysequencing.

2.6. Transfection and luciferase assay

Five pGL3-IFNAR1 promoter vectors and blank pGL3-Basic plas-mid (negative control) (225 ng/well), together with the internal controlpRL-CMV (25 ng/well; Promega,), were transfected to 293T cell cul-tures in 96-well plate in quadruplicate using calcium phosphate precip-itation methods as previously described [31,32]. The transfected cellswere harvested after further cultured for 48 h and assayed for fireflyand Renilla luciferase activities using Dual-Glo luciferase kit (Pro-mega). All experiments were independently repeated for three times.Luciferase indices (LI = Firefly luminescence/Renilla luminescence)were calculated for each transfectant and compared with that of blankpGL3-Basic transfected cultures.

2.7. Statistical analysis

Comparisons were made by v2 test and independent samples T test.For each haplotype, allele and genotype, odds ratios (ORs) were deter-mined by binary logistic regression (SPSS, version 13.0). The oddsratio herein was used to reflect the likelihood of carrying a specificmarker if persistently infected. A genetic marker with OR value loweror higher than 1 signifies that the marker is resistant or susceptible tochronic HBV infection, respectively. For these analyses, a P value lessthan 0.05 was regarded as significant.

�408T/T 0.0747 0.0526 0.4006

Alleles

�568G 0.4075 0.3728 0.3268�568C 0.5925 0.6272 0.3268�408C 0.7662 0.7500 0.5961�408T 0.2338 0.2500 0.5961

(GT)n

5 0.5946 0.5526 0.23216 0.0070 0.0044 0.66658 0.0007 0.0833 <0.0001

9 0.0014 0.1360 <0.0001

10 0.0000 0.0088 0.0003

12 0.0007 0.0044 0.131113 0.0593 0.0395 0.228414 0.1431 0.0921 0.0371

15 0.1389 0.0482 0.0002

16 0.0258 0.0263 0.932617 0.0195 0.0044 0.109618 0.0077 0.0000 0.188319 0.0007 0.0000 0.692521 0.0007 0.0000 0.6925

a v2 test was applied for comparison. Statistically significant valuesare in bold.

3. Results

3.1. Identification of polymorphisms in IFNAR1 promoter

Four polymorphisms of IFNAR1 promoter wereidentified in study subjects. They were G to C transitionat �568, C to T transition at �408 and �3, and GTrepeat microsatellite at �77 relative to transcriptionalstart site (Fig. 1). The novel finding of allele �568C inthis study was indeed a prevalent mutation identifiedin Chinese study subjects. The polymorphism of GTrepeats at �77 was found to vary considerably in thestudy subjects (Table 2). The 5GT repeats was the mostprevalent allele found in Chinese participants, followedby 14 and 15GT repeats. The 9GT allele published inGenBank was rarely found in the Chinese subjects.

In Caucasian population, the 5GT allele is also themost common one, followed by the 9, 14 and 8GT

alleles. Notably, frequencies of �77 alleles were differentbetween Chinese and Caucasian. The 8, 9 and 10GTrepeats were highly over-represented in Caucasian, whilethe 14 and 15GT repeats were found to be greatlyincreased in Chinese. Frequencies of alleles andgenotypes at the other three loci were not significantlydifferent between the two ethnic groups.

J. Zhou et al. / Journal of Hepatology 46 (2007) 198–205 201

3.2. Linkage disequilibrium between polymorphic sites and

identification of haplotypes

The sequencing results revealed two linkages betweenthe four polymorphic sites. �568 SNP was found to beassociated with �77GT repeat microsatellite. The�568C was linked to less (L) GT repeats (namely, 5,6), �568G to more (M) GT repeats ranging from 9 to21. The only exception was an allele of 8GT repeats,in which both �568G/�77GT8 and �568C/�77GT8

were identified. A complete linkage was also identifiedbetween polymorphic sites of �408 and �3 in homozy-gous carriers by sequencing, in which �408T was linkedto �3T, while �408C was associated with �3C. Thelinkages were further confirmed by PCR analysis usingallele-specific primers (Fig. 2) in heterozygous carriers.All of the SNPs met the Hardy–Weinberg equilibrium.�568 alleles and �408 alleles were in strong linkage dis-equilibrium. Based on the disequilibrium between thesetwo loci, three prevailing haplotype patterns wereidentified. They were haplotype I (�568G/�408C/�3C/�77GTM, or GCCM), haplotype II (�568C/�408C/�3C/�77GTL, or CCCL) and haplotype III(�568C/�408T/�3T/�77GTL, or CTTL).

3.3. Effects of polymorphisms of IFNAR1 promoter on

susceptibility of chronic HBV infection

Distributions of IFNAR1 promoter haplotypes,alleles and genotypes in three study groups are shownin Table 3. Haplotype I (GCCM) was found more fre-quently in SR group (OR = 0.687, 95% CI 0.520–0.909, P = 0.009) while haplotype III (CTTL) wasover-represented in CP group (OR = 1.794, 95% CI

Fig. 2. PCR confirmation of allelic linkage between loci �408 and �3 in

a heterozygote (genotype C/T). PCR performed with a pair of allelic-

specific primers �408FC/�3RC and �408FT/�3RT showed strong

bands which correspond to a 446 bp amplicon. PCRs using other primer

combinations, �408FC/�3RT and �408FT/�3RC, did not show prod-

ucts. The 100 bp marker was used as reference. NC, negative control.

1.269–2.535, P = 0.001). Haplotype II (CCCL) exhibitedno significant association with chronic HBV infection.To determine which polymorphism(s) in haplotypes Iand III would play a key role in affecting the susceptibil-ity to chronic HBV infection, we further compareddistributions of the haplotype-tagging alleles and geno-types between CP and SR groups. Chronic HBVpatients had significantly higher frequencies of �568C(OR = 1.455, 95% CI 1.100–1.924, P = 0.009) and�408T (OR = 1.794 95% CI 1.269–2.535, P = 0.001)alleles, while �588G (OR = 0.687, 95% CI 0.520–0.909, P = 0.009) and �408C (OR = 0.557, 95% CI0.394–0.788, P = 0.001) alleles were significantly moreoften to be detected in SR group. Genotype �568G/Gwas in strong association with viral resolution(OR = 0.522, 95% CI 0.320–0.851, P = 0.009), whereas�568 C/C was trended for the association with viral per-sistence (OR = 1.419, 95% CI 0.943–2.134, P = 0.098).The �408 T/T genotype was in borderline associationwith viral persistence (OR = 2.448, 95% CI 0.996–6.017, P = 0.051). The association was more significantin heterozygotes (C/T genotype, OR = 1.556, 95% CI1.014–2.388, P = 0.043) than in homozygotes (T/Tgenotype), which may be due to relatively small portionof T/T homozygotes in population. It also suggestedthat �408T was a dominant negative allele. Frequenciesof all haplotypes, alleles and genotypes in healthy con-trol group, except genotype �568C/C, lay between CPand SR group. Furthermore, there was no significantdifference in frequencies of haplotypes, alleles and geno-types between subgroups of chronic hepatitis B patientswith positive and negative HBeAg or with normal andabnormal ALT levels (Table 4).

Taken together, these results indicated that mutantallele �568C (and �77GTL), �408T (and �3T), andhaplotype III (�568C/�408T/�3T/�77GTL) may con-tribute to the susceptibility to chronic HBV infection,whereas carriers of wild type allele �568G (and�77GTM), �408C (and �3C) and haplotype I(�568G/�408C/�3C/�77GTM) were associated withviral resolution.

3.4. Evaluation of transcriptional activity in different

haplotypes of IFNAR1 promoter

To evaluate the functional significance of promoterpolymorphisms and the aforementioned genetic associa-tions, transcriptional activity of distinct IFNAR1promoter variants was tested by an IFNAR1 promot-er-luciferase reporter assay (Fig. 3). All pGL3-Basic-IFNAR1 promoter constructs displayed strongerpromoter activity than pGL3-Basic vector with incre-ment ranging from 33- to 65-fold. Promoter vectors ofhaplotype Ia (�568G/�408C/�3C/�77GT15) andhaplotype II (�568C/�408C/�3C/�77GT5) showedsignificantly stronger transcriptional activity than

Table 3

Distribution of genetic markers in Chinese patients and controls

Polymorphisms CP SR HBD CP versus SR CP versus HBD

N = 320 N = 148 N = 148 OR 95% CI P OR 95% CI P

Haplotypes

I (GCCM) 0.366 0.456 0.449 0.687 0.520–0.909 0.009 0.706 0.534–0.934 0.015

Ia (GCC15) 0.125 0.151 0.156 0.803 0.541–1.191 0.276 0.772 0.515–1.157 0.210Ib (GCC14) 0.128 0.178 0.141 0.679 0.466–0.990 0.044 0.893 0.590–1.352 0.593Ic (GCC9) 0.003 0 0 5,064,184 0 0.995 5,064,184 0 0.995II (CCCL) 0.358 0.368 0.351 0.956 0.718–1.273 0.757 1.029 0.771–1.372 0.848III (CTTL) 0.277 0.176 0.199 1.794 1.269–2.535 0.001 1.536 1.100–2.144 0.012

Alleles

�568G 0.366 0.456 0.449 0.687 0.520–0.909 0.009 0.706 0.534–0.934 0.015

�568C 0.634 0.544 0.551 1.455 1.100–1.924 0.009 1.416 1.070–1.873 0.015

�408C 0.723 0.824 0.801 0.557 0.394–0.788 0.001 0.651 0.466–0.909 0.012

�408T 0.277 0.176 0.199 1.794 1.269–2.535 0.001 1.536 1.100–2.144 0.012

Genotypes

�568G/G 0.144 0.243 0.196 0.522 0.320–0.851 0.009 0.737 0.438–1.242 0.252�568G/C 0.444 0.426 0.507 1.076 0.726–1.595 0.714 0.776 0.525–1.147 0.204�568C/C 0.413 0.331 0.297 1.419 0.943–2.134 0.093 1.660 1.094–2.518 0.017

�408C/C 0.541 0.689 0.669 0.531 0.352–0.801 0.003 0.582 0.388–0.875 0.009

�408C/T 0.366 0.270 0.264 1.556 1.014–2.388 0.043 1.611 1.047–2.478 0.030

�408T/T 0.094 0.041 0.068 2.448 0.996–6.017 0.051 1.428 0.679–3.004 0.348

Binary logistic regression models were applied for calculating the odds ratios, 95% confidential interval and corresponding P values. Significantassociations are in bold. N is the total number of participants in each group. Denotation of haplotype accorded to the allelic variants in order of�568, �408, �3 and �77. Due to the complete linkage between �408 and �3 alleles, frequencies and statistic of �3 alleles were represented by thoseof �408. So does �77 alleles. Two subtypes of Haplotype I, Ia (GCC15) and Ic (GCC9), did not show significant association with viral clearance,which may be due to reduced sample numbers in these subgroups. Abbreviations: CP, chronic HBV patients; SR, spontaneously recovered indi-viduals; HBD, healthy blood donors.

202 J. Zhou et al. / Journal of Hepatology 46 (2007) 198–205

that of haplotype III (�568C/�408T/�3T/�77GT5)(P 6 0.011). However, haplotype Ia and II promotervectors were not significantly different in driving theexpression of reporter gene (P > 0.05).

Table 4

Correlation of genetic markers and the patients with HBe+/� or ALT normal

Polymorphisms Frequencies

HBeAg-negative N = 94 HBeAg-positive N = 226

Genotypes

�568G/G 0.096 0.155�568G/C 0.482 0.425�568C/C 0.422 0.42�408C/C 0.446 0.56�408C/T 0.458 0.35�408T/T 0.096 0.09

Alleles

�568G 0.337 0.368�568C 0.663 0.633�408C 0.675 0.735�408T 0.325 0.265

Haplotypes

I (GCCM) 0.337 0.368II (CCCL) 0.337 0.368III (CTTL) 0.325 0.265

Frequencies of genotypes, alleles and haplotypes in chronic hepatitis B patienttest was applied to analyze the potential distribution differences of genetic madifferentially distributed in HBeAg negative and positive groups, as well as

To determine if variable numbers of �77GT repeatsmay also affect the IFNAR1 promoter function, we con-structed the other two subtypes of haplotype I, haplo-type Ib with 14GT repeats and haplotype Ic with 9GT

/elevated

P Frequencies P

ALT normal N = 45 ALT elevated N = 275

0.1928 0.103 0.169 0.37460.3800 0.517 0.433 0.39490.9791 0.379 0.399 0.84170.0798 0.483 0.596 0.25400.0891 0.414 0.309 0.26330.8655 0.103 0.096 0.8932

0.4959 0.362 0.387 0.74070.4959 0.638 0.619 0.74070.1469 0.690 0.750 0.33030.1469 0.310 0.250 0.3303

0.4959 0.362 0.387 0.74070.1469 0.328 0.363 0.58030.1469 0.310 0.250 0.3303

s with HBeAg negative/positive and ALT normal/elevated are listed. v2

rkers in the patients. It is indicated that none of the genetic markers isALT normal and elevated groups.

Fig. 3. Comparison of transcriptional activity among IFNAR1 promoter

variants in 293T cell. Promoter variants haplotype Ia (�568G/�408C/

�3C/�77GT15), haplotype Ib (�568G/�408C/�3C/�77GT14), haplo-

type Ic (�568G/�408C/�3C/�77GT9), haplotype II (�568G/�408C/

�3C/�77GT5), haplotype III (�568C/�408T/�3T/�77GT5) were

cloned into pGL3 to construct vectors for evaluation of their transcrip-

tional activity. Luciferase indexes (LI) were compared among promoters

with haplotype variants. LI = Firefly luminescence/Renilla luminescence.

Significant P values are shown.

J. Zhou et al. / Journal of Hepatology 46 (2007) 198–205 203

repeats. Compared to haplotype Ia, haplotype Ic, whichwas very rare in Chinese population, displayed signifi-cantly higher transcriptional activity (P = 0.0018), buthaplotype Ib showed a comparable level of promoterfunction.

These results demonstrated that �408 and/or �3SNP and microsatellite instability at loci �77 conferreddifferential functional effects to the promoter activity.Genotypes �408C/C and �77 9/9GT repeats bestowedhigher transcriptional activity to IFNAR1 promoterthan their counterparts. The �408 and/or �3 SNPmight play a predominant role in IFNAR1 promoterfunction. In contrast, �568G/C nucleotide changeseemed to have no apparent impact on the transcription-al activity.

4. Discussion

Chronic HBV infection is endemic in Asia and Africabut rarely occurs in Caucasian population. Several pro-inflammatory cytokines and immunoregulatory genesinvolved in the host immune response to HBV infection[33–35] have been shown to exhibit substantial differenc-es among ethnic populations [36]. Therefore, it was pos-tulated that susceptibility to chronic HBV infectionmight stem from the genetic variations of host immunityto the virus in various ethnic populations. It has beendemonstrated that type I IFNs may effectively inhibitHBV replication. The antiviral effects of IFNs areelicited upon binding of these cytokines to two subunitsof their cognate receptor, IFNAR1 and IFNAR2.Polymorphisms in the IFNAR1 promoter region havebeen reported in UK Caucasian and other ethnic

population, but not yet in Chinese population. Current-ly, it remains largely unknown whether polymorphismsin the IFNAR1 promoter would affect the progressionof HBV infection. In this study, we recruited 616 Chi-nese adults, including 320 chronic hepatitis B patients,148 spontaneously recovered blood donors and 148healthy blood donors, and 114 healthy Caucasians.Four polymorphisms of IFNAR1 promoter have beenidentified, including a novel mutation at position�568, two reported SNPs at position �408, �3 andmicrosatellite instability at �77. We further demonstrat-ed two allelic linkages between position �408 and �3,and �568 and �77.

Analysis of distributions of IFNAR1 promoteralleles, haplotypes and genotypes in three Chinesegroups has revealed an association between IFNAR1promoter polymorphisms and the susceptibility tochronic HBV infection. Carriers of mutant allele�568C (and less GT repeat), �408T (and �3T) and hap-lotype III (�568C/�408T/�3T/�77GTL) are more sus-ceptible to chronic HBV infection. Conversely,individuals bearing wild-type allele �568G (and moreGT repeat), �408C (and �3C) and haplotype I(�568G/�408C/�3C/�77GTM) are predisposed torecover from HBV infection.

Using an IFNAR1 promoter-driven luciferase report-er system, we demonstrate that �408 and/or �3 SNPencoded functional polymorphism. The C to T transi-tion significantly reduced IFNAR1 transcriptional activ-ity, consistent with the genetic association analysis.Clinically, �408T/�3T alleles are more prevalent inchronic hepatitis B patients than in spontaneouslyrecovered persons. It has been reported that the expres-sion level of IFNAR1 on cell surface can affect the effi-ciency of IFN-a through influencing the sensitivity ofthe cytokine binding to human cells [37]. Our resultsthus suggest that the mechanism underlying the associa-tion between genetic polymorphisms and variable phe-notypes the disease is to modulate the expression ofIFNAR1 receptor, which in turn influences the antiviraleffects of type I IFN.

Although our analysis suggested an association of�568 SNP with clinical course of HBV infection,IFNAR1 promoter-luciferase reporter assay showedinsignificant difference between promoter variants of�568 wild type G/G and its mutant genotype C/C.Therefore, �568 SNP seems to have no apparent alle-lic-specific effect on IFNAR1 transcriptional regula-tion. Its association with persistence or clearance ofHBV infection might be due to its disequilibrium link-age to other causative factor(s). In fact, in a parallelstudy of IFNAR1 open reading frame, we found thata SNP was in strong disequilibrium with �568 SNP.Further studies are warranted to determine the func-tional implication of the SNPs in IFNAR1 codingsequence.

204 J. Zhou et al. / Journal of Hepatology 46 (2007) 198–205

Since GT repeat 9, 14 and 15 are uniquely linked to�568G allele, they are classified as the same allele group(designated M) for statistical analysis. In order to under-stand whether subtypes of haplotype I with differentnumber of GT repeats are indeed indistinguishable fortranscriptional activity of IFNAR1 promoter, vectorsof promoter variants with 9, 14 and 15 were constructedand their promoter function was compared. We haverevealed that 14 and 15 GT repeats do not significantlyalter the activity of IFNAR1 promoter. We concludethat �77 alleles denoted M is functionally homogeneousand appropriate to be classified into one group for dataanalysis in Chinese subjects.

Notably, allele �77GT9 has been found in only twoheterozygous carriers (5/9 and 9/14) in 616 Chinesestudy subjects (f = 0.0014), but it is much prevalent inCaucasian study cohort (f = 0.136). Although its fre-quency is too low in Chinese study subjects for meaning-ful statistical analysis to be performed, the ex vivo studyhas shown that the promoter variant carrying �77GT9

endows higher transcriptional activity than other alleliccounterparts carrying 5, 14 and 15GT repeats. Thismight result in more IFNAR1 molecules expressed oncell membrane and subsequently mediate stronger anti-viral effects. Thus, the promoter variant that carries�77GT9 may be one of the elements contributing tomuch higher proportion of chronic HBV infection inChinese than in Caucasian.

It is conceivable that HBV chronicity has a multi-factorial background. The host immunological basisfor viral persistence is not well understood. Deficit inantigen presenting cells, helper T cells, and CTLs iscontributable in progressing to chronic HBV infectionstage [38–40]. Rapid advances in human genomics offergreat opportunities to correlate genetic variations torisks of diseases. Several case-control studies in largecohort have been performed and unraveled a wide rangeof genetic determinants of chronic HBV infection.Nevertheless, our study provides strong evidence thatpolymorphisms of IFNAR1 promoter are one of thegenetic elements in affecting the susceptibility to chronicHBV infection.

Acknowledgements

This work was supported in part by grants fromResearch Grants Council of Hong Kong SAR andUniversity Development Fund of the University ofHong Kong.

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