Single Nucleotide Polymorphisms of Toll-Like Receptor 7

in Hepatitis C Virus Infection Patients from a High-Risk

Chinese Population

Xing-xin Xue,

1,2

Jian-ming Gong,

1,2

Shai-di Tang,

1,2

Chun-fang Gao,

Jia-jia Wang,

1,2

Li Cai,

1,2

Jie Wang,

Rong-bin Yu,

Zhi-hang Peng,

Nai-jun Fan,

Chang-jun Wang,

Jin Zhu,

and Yun Zhang

1,2,5

Abstract—Hepatitis C virus (HCV) infection varies in the outcomes depending on both viral and host factors.

This study aims to investigate the associations of three single nucleotide polymorphisms (SNPs) of T oll-

like receptor 7 (TLR7), rs179016, rs5743733, and rs1634323, with susceptibility to HCV infection and

clearance. The three SNPs were genotyped in a high-risk Chinese population, including 444 HCV

spontaneous clearance cases, 732 persistent infection cases, and 1 107 healthy controls . The G allele of

rs1634323 was related to the protection from persistent infection among females (dominant model: odds

ratio (OR)=0.558, 95 % confidence interval (CI)=0.348–0.894, P=0.015). This protective effect was

more evident in blood donation and HCV non-1 genotype-infecte d subgroups (all P<0.05). The carriage

of rs179016 C allele was more prone to develop persistent infection (OR=1.444, 95 % CI=1.096–1.903,

P=0.009) in males, and the risk effect remained significant among older (>50 years), hemodialysis (HD),

and HCV-1 and HCV non-1 genotypes-infected subjects (all P<0.05). Haplotype analyses showed that

CCA haplotype among females was correlated with the elevated risk of HCV susceptibility while the

carriage of GGA was more prone to be infected with HCV and CCA was more likely to develop

persistent infection (all P<0.05) among males. Our results first demonstrated that the carriage of rs179016

C allele had a negative effect on spontaneous clearance of HCV among males while rs1634323 G allele

conferred a protective effect against persistent infection among female subjects.

KEY WORDS: Toll-like receptor 7; hepatitis C virus; polymorphism; infection; susceptibility; persistence.

INTRODUCTION

Hepatitis C virus (HCV), a single-stranded

RNA (ssRNA) virus, is one of the most common

blood-borne viruses that cause ac ute and chronic

infection and inflammation of the liver [1]. To date,

HCV infection has become a major public health

problem, with approximately 170 million people

getting infected globally [2] and over 3.5 million

new sufferers occurring annually [3]. China is con-

sidered a relatively high endemic area of HCV in-

fection, and its estimated preval ence rate in the

general population is 3.2 % [4]. The severity of

hepatitis C ranges from mild, short-term symptoms

to a complicated, life-long liver disease. According

to WHO, around 75–8 5 % of n ewly infected persons

develop chronic infection and 60– 70 % of

Xing-xin Xue and Jian-ming Gongcontributed equally to this work and

should be considered co-first authors.

Electronic supplementary material The online version of this article

(doi:10.1007/s10753 -014- 0016-x) contains supplementary material,

which is available to authorized users.

Department of Epidemiology and Biostatistics, School of Public Health,

Nanjing Medical University, No. 140 Hanzhong Road, Nanjing, 210029

Jiangsu, China

Institute of Epidemiology and Microbiology, Huadong Research Institute

for Medicine and Biotechnics, No. 293 Zhongshan East Road, Nanj-

ing, 210002 Jiangsu, China

Department of Anal-Colorectal Surgery, The 150th Central Hospital of

Chinese People’s Liberation Army, No. 2 Huaxia We st Road,

Luoyang, 471031 Henan, China

Department of General Practice, Kangda College, Nanjing Medical

University, No. 140 Hanzhong Road, Nanjing, 210029 Jiangsu, China

To whom correspondence should be addressed at Institute of Epidemi-

ology and Microbiology, Huadong Research Institute for Medicine and

Biotechnics, No. 293 Zhongshan East Road, Nanjing, 210002 Jiangsu,

China. E-mail: zhangyunvip@126.com

0360-3997/15/0100-0142/0

2014 Springer Science+Business Media New York

Inflammation, Vol. 38, No. 1, February 2015 (

2014)

DOI: 10.1007/s10753-014-0016-x

142

chronically infected people develop chronic liver

disease, including cirrhosis and hepatocellular carci-

noma (HCC) (http://www.who.int/mediacentre/fact-

sheets/fs164/en/index.html) . However, the mechanism

of this dramatically variable among individuals has not

been clearly clarified yet.

Toll-like receptors (TLRs) are a family of highly

conserved receptors called pattern-recognition recep-

tors (PRRs) and play a critical role in both innate

and adaptive immunity. Currently, thirteen TLRs

(TLR1-13) ha ve b een identif ied i n m amma l ian s pe-

cies, including 11 in humans [5]. Different T LRs

have their own sp ecific ligands a nd TLR7 senses

unmethylated viral ssRNA [6 ], for example, the hep-

atitis C virus. There are two lines of evidence sup-

porting the role of TLR7 in H CV infection. First, a

clinical study observed that once-daily 7-day treat-

ment with isatoribine, an agonist of TLR7, could

cause a significant reduction of viral load i n patients

with chronic HCV infection [7]. Second, the activa-

tion of TLR7 on hepatocytes could exert an antiviral

effect. When exposed to a TLR7 ligand [SM360320

(9-benzyl-8-hydroxy-2-(2-methoxyethoxy)adenine)],

several antiviral genes (eg, interferon regulatory

factor-7) we re induce d and HCV replication was

inhibited in hepatocytes [8]. Thus, as both immune cells

and hepatocytes are involved in its antiviral response,

TLR7 is a promising candidate for an immune mediator

in HCV infection.

ThegeneofTLR7islocatedonthe4-MbregionofX

chromosome, spanning three exons [9]. Recent studies

have shown that genetic variations of TLR7 are associated

with enhanced susceptibility and severity of various infec-

tious and immune diseases [10], such as acquired immune

deficiency syndrome (AIDS) [11], systemic lupus erythe-

matosus (SLE) [12, 13], and allergic rhinitis (AR) [14]. In

the last decades, a few studies had been done to evaluate

the potential role of TLR7 variation in the pathogenic

process of HCV infection. However, many of them drew

incompatible conclusions. To further test the hypothesis

that TLR7 gene variants are correlated with the outcomes

of HCV infection, we systematically selected three single

nucleotide polymorphisms [SNPs (rs179016, rs5743733,

and rs1634323)] in the TLR7 gene and genotyped these

three SNPs in 444 HCV spontaneous clearance cases, 732

persistent infection cases, and 1107 healthy controls in a

high-risk Chinese population. By assessing the frequencies

of the SNPs mentioned above, we may first explicate the

mechanism of TLR7 polymorphisms in the pathogenesis

of HCV infection.

MATERIALS AND METHODS

Study Population

A total of 2283 subjects were enrolled in the present

study, including 442 drug users recruited from Nanjing

compulsory detoxification center, 791 hemodialysis (HD)

subjects recruited from nine hospital hemodialysis centers in

southern China, and 1050 paid blood donors recruited from

two villages in Danyang City and six villages in Zhenjiang

City from October 2008 to January 2013. Patients coinfected

with any other virus (such as HBV, HIV), with other types of

liver diseases (e.g., autoimmune, alcoholic or metabolic liver

diseases), or treated with any antiviral medications during

the trial were excluded. All subjects were categorized into

three groups for analysis. The healthy control group (group

A) included 1107 uninfected subjects (175 drug users, 610

HD patients, and 322 paid blood donors), who were tested

anti-HCV negative. The spontaneous clearance group

(group B) consisted of 444 spontaneous viral clearance cases

(142 drug users, 99 HD patients, and 203 paid blood

donors), who were anti-HCV seropositive with persistently

normal ALT levels (<40 U/L) for all three tests and clear of

viremia, as demonstrated by ≥2instancesseparatedbya

minimum of 6 months, in which HCV-RNA was detected

negative. The persistent HCV infection group (group C) was

composed of 732 persistent HCV infection cases (125 drug

users, 82 HD patients, and 525 paid blood donors), who

were anti-HCV seropositive and HCV-RNA positive with

persistently normal or elevated ALT levels for at least three

biochemical tests within consecutive 6 months during fol-

low-up.

This study was approved by the Ethics Committee of

Nanjing Medical University, and all subjects provided

voluntary informed consent to participate in the study. An

interview was scheduled for each participant, and a stan-

dardized questionnaire was administered by well-trained

interviewers to collect information on demographic data

and environmental exposure history. The quality assurance

procedures of epidemiological investigation were estab-

lished to ensure the quality of the data.

Viral Testing

An approximately 5-mL venous blood sample was

collected from each participant. The serum and white blood

cells were isolated by centrifugation and stored at −80 °C

until assay. The antibody to HCV was tested by the third

generation enzyme-linked immunosorbent assay (Diagnos-

tic Kit for Antibody to HCV 3.0 ELISA, InTec Products

Inc, Xiamen, China). The HCV-RNA was extracted from a

143Single Nucleotide Polymorphisms of Toll-Like Receptor 7

serum by Trizol LS Reagent (Takara Biotech, Tokyo, Ja-

pan), and a reverse transcription-polymerase chain reaction

(RT-PCR) kit (Takara Biotech, Tokyo, Japan) was used for

a10-μL HCV-RNA extracting solution. The products of

RT-PCR were detected by a 2 % agarose gel (Biowest

Agarose) stained with ethidium bromide, and the HCV

genotype determi nation was performed by PCR with

type-specific primers from the 5′ non-coding region (5′-

NCR) [15]. For sera with non-detectable HCV-RNA, sero-

typing using Murex HCV Serotyping 1-6 Assay ELISA

Kit (Abbott, Wiesbaden, Germany) was conducted to de-

termine the type-specific antibodies to various HCV gen-

otypes [16].

Polymorphisms Selection

We searched the SNPs from NCBI dbSNP database

(http://www.ncbi.nlm.nih.gov/SNP) and public HapMap

SNP database (http://www.hapmap.org) and selected SNPs

with minor allele frequency (MAF) >5 % in Chinese Han

population. SNPs meeting the following criteria were prior

to consideration: (1) the reported SNPs from previous

studies associated with other diseases, especially liver or

immune-related disorders, and (2) the role of the SNPs in

the pathogenic process of hepatitis C infection have not

been evaluated yet, especially in Chinese Han population.

As reported, rs179016 was associated with skin prick test

(SPT) res ponse for Dermatopha goides pteronyssinus

among allergic rhinitis patients and the controls [14]. The

distribution of the rs5743733 alleles was significantly dif-

ferent between patients with Behcet’s disease (BD) and the

controls [17]. The G allele of rs1634323 was associated

with an elevated risk of systemic lupus erythematosus

(SLE) among Chinese females [18]. Therefore, SNPs at

positions -14241G>C (rs179016), -9507C>G (rs5743733),

and -7926A>G (rs1634323) were selected as candidates

for the present study. The positions and regions of these

selected SNPs were represented in Table S2.

Genotyping Assays

Genomic DNA was extracted from leucocytes in pe-

ripheral blood by sodium dodecyl sulfate lysis and protease

K digestion and followed by phenol-chloroform extraction

and ethanol precipitation. Genotyping of the three SNPs

was performed by the TaqMan allelic discrimination assay

on the ABI PRISM 7900HT Sequence Detection System

(SDS) (Applied Biosystems, Foster City, CA, USA). The

primers and probe sequences for the selected SNPs were

listed in Table S2. Reactions were carried out in a 384-well

format with a final volume of 5 μL, containing 1 μL

genomic DNA (10 ng/μL), 2.5 μL TaqMan Master Mix,

0.225 μL forward and reverse primer (20 pmol/μL) with a

final concentration of 900 nM, 0.125 μL VIC and FAM (10

pmol/μL) with a final concentration of 250 nM, and 0.8 μL

sterile double distilled H

O(ddH

O). All the genotyping

was performed without knowing the s ubjects’ case or

control status. Each plate consisted of three blank samples

as negative controls and two samples from the same indi-

vidual as positive controls for the confirmation of genotyp-

ing quality. The accordance rate of each SNP was 100 %

for the repeated experiments of 10 % random samples.

Statistical Analysis

Student’s t test, χ

test, and Kruskal-Wallis test were

used to evaluate the distributions of the general demograph-

ic, clinical, and virological features between cases and con-

trols when appropriate. Hardy-Weinberg equilibrium

(HWE) test for each SNP among females was conducted

by using a goodness-of-fit χ

test. PHASE 2.0 software was

used to estimate the haplotype frequencies of the three

polymor phism s [19]. The associations between HCV

infection and SNPs were determined by computing the

odds ratios (ORs) and 95 % confidence intervals (CIs) from

unconditional logistic regression analysis with the adjust-

ment for age, the source of infection, and/or viral genotypes.

A two-tailed P value less than 0.05 was considered statis-

tically significant. For multiple comparisons among geno-

types, we applied the Bonferroni correction and the P value

was adjusted to 0.017 (0.05/3). All statistical analyses in the

present study were performed with Statistical Package for

the Social Sciences (version 17.0, SPSS Institute, Chicago,

IL, USA) and Statistical Analysis System software (version

9.1.3, SAS Institute, Cary, NC, USA).

RESULTS

All analyses in this study were performed with groups

subdivided according to gender since TLR7 gene is located

on the X chromosome. Distributions of genotypes in wom-

en were in accordance with the Hardy-Weinberg equilibri-

um (all P>0.05). The minor allele frequencies (MAFs)

were found to be closed to that given for Chinese Han in

Beijing (CHB) in public databases.

Basic Characteristics of the Study Population

The distributions of selected characteristics among

1107 healthy controls, 444 spontaneous clearance patients,

and 732 persistent infection patients were summarized in

144 Xue, Gong, Tang, Gao, Wang, Cai, Wang, Yu, Peng, Fan, Wang, Zhu, and Zhang

Table 1. All subjects recruited were Chinese Han people.

Significant differences were observed in the distributions

of ALT level and HCV genotype and the likely source of

infection among three groups in both females and males

(all P<0.05). No differences were observed in the distri-

butio n of age (all P>0.05). In females, the clearance/

persistence rates were 13.6 %/7.8 % in hemodialysis

(HD) subjects, 34.0 %/27.1 % in drug users, and 20.6 %/

49.9 % in paid blood donors. In males, the rates were

11.9 %/11.9 %, 31.2 %/28.9 %, and 15.2 %/50.2 % in

HD subjects, drug users, and paid blood donors, respec-

tively (data not shown). The rates of clearance and chro-

nicity according to SNP genotypes were shown in Table 1.

Association Analyses of TLR7 Genetic Polymorphisms

(rs179016, rs5743733, and rs1634323)

with the Susceptibility to HCV Infection

The genotype distributions of rs179016, rs5743733,

and rs1634323 in TLR7 among healthy control group,

spontaneous clearance group, and persistent HCV infection

group were depicted in Table 2 and Table 3.Asshown,no

significant associations between these three SNPs and

HCV infection susceptibility were observed in the overall

analysis in both female and male individuals (all P>0.05).

In Table 4 and Table 5, further stratification analyses

in females indicated that a significant increased risk of

HCV infection was found in the carriage of rs179016 C

allele in the drug use subgroup (OR=2.567, 95 % CI=

1.232–5.348, P=0.012). Subjects carrying rs1634323 G

allele was observed to be related to an increased risk of

HCV susceptibility in the blood donation subgroup (OR=

1.634, 95 % CI=1.051–2.542, P=0.029). In males, logistic

regression analysis revealed that subjects with rs1634323

G allele were less prone to be infected with the HCV in the

younger subg roup (≤50 years, OR=0.619, 9 5 % CI=

0.386–0.994, P=0.047) and drug use subgroup (OR=

0.324, 95 % CI=0.167–0.627, P=0.001), but more liable

in HD subgroup (OR=3.100, 95 % CI=1.380–6.963, P=

0.006). In other strata, there was no significant association

between the three SNPs and risk of HCV infection (all

P>0.05).

Tabl e 1. Demographic and Biochemical Characteristics Among Healthy Control, Spontaneous Clearance, and Persistent Infection Groups

Variables Group A (%)

Group B (%)

Group C (%)

P value

N 526/581 255/189 465/267

Age [median (P

, P

)] 51(42, 61)/49(38, 61) 53(44, 61)/48(44, 59.5) 53(47.5, 59)/51(37.9, 59) 0.736

/0.522

ALT [median (P

, P

)] 16(8, 23)/12(8, 21) 30(19, 40)/26(16, 39) 37(25, 53)/32(20, 51) <0.001

/<0.001

HCV genotype <0.001

/0.007

Genotype-1 – 171(67.1)/124(65.6) 271(58.3)/163(61.0)

Non-1 – 35(13.7)/29(15.3) 131(28.2)/71(26.6)

Mixed – 49(19.2)/36(19.0) 63(13.5)/33(12.4)

Likely source of infection <0.001

/<0.001

Drug use 56(10.6)/119(20.5) 49(19.2)/93(49.2) 39(8.4)/86(32.2)

HD 232(44.1)/378(65.1) 40(15.7)/59(31.2) 23(4.9)/59(22.1)

Paid blood donation 238(45.2)/84(14.5) 166(65.1)/37(19.6) 403(86.7)/122(45.7)

rs179016 0.181

/0.036

GG 386(43.7)/483(56.2) 184(20.8)/166(19.3) 313(35.4)/210(24.4)

GC 125(38.8) 60(18.6) 137(42.5)

CC 15(36.6)/98(55.1) 11(26.8)/23(12.9) 15(36.6)/57(32.0)

rs5743733 0.806

/0.727

CC 454(42.5)/533(56.2) 213(19.9)/170(17.9) 402(37.6)/245(25.8)

CG 67(41.1) 38(23.3) 58(35.6)

GG 5(35.7)/48(53.9) 4(28.6)/19(21.3) 5(35.7)/22(24.7)

rs1634323 0.220

/0.638

AA 467(42.4)/556(56.2) 219(19.9)/178(18.0) 416(37.7)/256(25.9)

AG 55(44.0) 30(24.0) 40(32.0)

GG 4(21.1)/25(53.2) 6(31.6)/11(23.4) 9(47.4)/11(23.4)

Group A: Healthy controls; Group B: Spontaneous clearance subjects; Group C: Persistent infection patients; Non-1: genotypes 2 and 3; Mixed: genotypes

1/2, 1/3, and 2/3

HD hemodialysis

Female/male

test

Kruskal-Wallis test

145Single Nucleotide Polymorphisms of Toll-Like Receptor 7

Association Analyses of TLR7 Genetic Polymorphisms

(rs179016, rs5743733, and rs1634323)

with the Clearance of HCV Infection

Compared with rs1634323 AA wild-type genotype,

the carriage of G allele appeared to be related to the

protection from persistent HCV infection in female indi-

viduals (dominant model: OR=0.558, 95 % CI=0.348–

0.894, P=0.015). In males, logistic regression analysis

revealed that the carriage of rs179016 C allele seemed to

be more prone to develop persistent infection (OR=1.444,

95 % CI=1.096–1.903, P=0.009). However, no significant

correlation was observed between rs5743733 and the clear-

ance of HCV (P>0.05).

In female individuals, the stratified analysis showed

that rs1634323 carriage of G allele seemed to favor spon-

taneous HCV clearance in the terms of blood donation

(OR=0.668, 95 % CI=0.452–0.988, P=0.044) and HCV

Tabl e 2. Genotype and Allele Distributions of the TLR7 rs179016, rs5743733, and rs1634323 Among Female Healthy Control, Spontaneous Clearance, and

Persistent Infection Groups

SNPs Allele

Group A

Group B

Group C

Group C/Group B Group (B + C)/Group A

(n=526) (n=255) (n=465) OR(95 % CI)

P value

OR(95 % CI)

P value

rs179016 G/C 386/125/15 184/60/11 313/137/15

G/C(%) 73.4/23.8/2.9 72.2/23.5/4.3 67.3/29.5/3.2

Dominant 1.332(0.939–1.891) 0.108 1.186(0.901–1.562) 0.223

Additive 1.209(0.899–1.627) 0.210 1.184(0.936–1.499) 0.159

rs5743733 C/G 454/67/5 213/38/4 402/58/5

C/G(%) 86.3/12.7/1.0 83.5/14.9/1.6 86.5/12.5/1.1

Dominant 0.721(0.466–1.117) 0.143 1.036(0.724–1.481) 0.848

Additive 0.737(0.501–1.083) 0.120 1.043(0.757–1.439) 0.795

rs1634323 A/G 467/55/4 219/30/6 416/40/9

A/G(%) 88.8/10.5/0.8 85.9/11.8/2.4 89.5/8.6/1.9

Dominant 0.558(0.348–0.894) 0.015 0.938(0.636–1.384) 0.748

Additive 0.648(0.444–0.945) 0.024 1.051(0.754–1.465) 0.770

Group A: Healthy controls; Group B: Spontaneous clearance subjects; Group C: Persistent infection patients; Group (B + C): Infected individuals. For

multiple comparisons among genotypes, we applied the Bonferroni correction and the P value was adjusted to 0.017 (0.05/3). Boldfaced values indicate

statistically significant results

Major/minor allele

Major homozygote/heterozygote/rare homozygote among three groups

Logistic regression model, adjusted by age, likely source of infection and/or viral genotypes

Tabl e 3 . Genotype and Allele Distributions of the TLR7 rs179016, rs5743733, and rs1634323 Among Male Healthy Control, Spontaneous Clearance, and

Persistent Infection Groups

SNPs Allele

Group A

Group B

Group C

Group C/Group B Group (B + C)/Group A

(n=581) (n=189) (n=267) OR(95 % CI)

P value

OR(95 % CI)

P value

rs179016 G/C 483/98 166/23 210/57 1.444(1.096–1.903) 0.009 1.020(0.856–1.217) 0.822

G/C(%) 83.1/16.9 87.8/12.2 78.7/21.3

rs5743733 C/G 533/48 170/19 245/22 0.868(0.623–1.209) 0.403 1.004(0.795–1.269) 0.970

C/G(%) 91.7/8.3 89.9/10.1 91.8/8.2

rs1634323 A/G 556/25 178/11 256/11 0.807(0.515–1.266) 0.352 0.799(0.583–1.095) 0.163

A/G(%) 95.7/4.3 94.2/5.8 95.9/4.1

Group A: Healthy controls; Group B: Spontaneous clearance subjects; Group C: Persistent infection patients; Group (B + C): Infected individuals. For

multiple comparisons among genotypes, we applied the Bonferroni correction and the P value was adjusted to 0.017 (0.05/3). Boldfaced values indicate

statistically significant results

Major/minor allele

Logistic regression model, adjusted by age, likely source of infection and/or viral genotypes

146 Xue, Gong, Tang, Gao, Wang, Cai, Wang, Yu, Peng, Fan, Wang, Zhu, and Zhang

non-1 genotype-infected (OR=0.340, 95 % CI=0.130–

0.890, P=0.028) subgroups. While in males, as stratifica-

tion analyses showed, an increased risk of persistent HCV

infection was found to be associated with rs179016 C

variant in older (>50 years, OR=1.750, 95 % CI=1.117–

2.742, P=0.015), HD (OR=1.755, 95 % CI=1.029–2.995,

P=0.039), and HCV-1 (OR=1.407, 95 % CI=1.020–

1.940, P=0.038) and HCV non-1 genotypes-infected

(OR=3.603, 95 % CI=1.112–11.670, P=0.033) sub-

groups. No significant effects of rs179016, rs5743733,

and rs1634323 were found in other subgroups (all

P>0.05).

Haplotype Analyses Among the Three Groups

As shown in Table 6, haplotypes were reconstructed

using PHASE 2.0 software. The three-locus haplotypes

were consisted of rs17 9016 G/C, rs5743733 C/G, and

rs1634323 A/G variant alleles. Compared with the most

frequent GCA haplotype, CCA haplotype was correlated

with the elevated risk of the susceptibility to HCV infection

in females (OR=1.358, 95 % CI=1.046–1.762, P=0.021).

In males, compared with the most frequent GCA

haplotype, a significant association was found between

GGA and t he increased risk of HCV infection (OR=

1.795, 95 % CI=1.214–2.653, P=0.003) while CCA was

correlated with the increased risk of persistent HCV infec-

tion (OR=2.120, 95 % CI=1.391–3.233, P<0.001).

DISCUSSION

TLR7, a receptor mainly expressing in the endosome-

lysosome m embranes of plasmacytoid dendritic cells

(pDCs, including hepatic pDCs), B lymphocytes, and he-

patic natural killer cells [20, 21], is considered to be re-

sponsible for antigen recognition and pathogen clearance.

Upon sensing unmethylated viral ssRNA, it can activate

the nuclear factor-κB(NF-κB) and induce the gene tran-

scription of IFN-α and several proinflammatory cytokines

[20, 22, 23], thus regulating the response of these cells.

Both in vivo and in vitro studies support that HCV, as

an ssRNA virus, can be recognized by TLR7, resulting in

the altered susceptibility to HCV and capacity of viral

clearance. In a previous study, Abe et al.[24]established

mouse macrophage cell lines stably expressing HCV pro-

teins and found that the immune cells expressing NS3,

NS3/4A, NS4B, or NS5A could inhibit the activation of

TLR7 signaling pathway. In turn, activation of TLR7

might exert an antiviral effect. For instance, Lee et al.[8]

Table 4. Stratified Analysis of the TLR7 rs179016, rs5743733, and rs1634323 Polymorphisms Among Female Healthy Control, Spontaneous Clearance, and Persistent Infection Groups

SNPs Allele Subgroups Group A (%) Group B (%) Group C (%) Group C/Group B Group (B + C)

/Group A

(1/2) 11

/12/22 11

/12/22 OR(95 % CI) P value

OR(95 % CI) P value

rs179016 G/C Route of infection

Drug use 44(78.6)/11(19.6)/1(1.8) 31(63.3)/16(32.7)/2(4.1) 18(46.2)/21(53.8)/0(0.0) 1.609(0.725–3.571) 0.242 2.567(1.232–5.348) 0.012

HD 174(75.0)/51(22.0)/7(3.0) 28(70.0)/8(20.0) / 4(10.0) 16(69.6)/6(26.1)/1(4.3) 0.916(0.390–2.155) 0.841 1.379(0.851–2.236) 0.192

Blood donation 168(70.6)/63(26.5)/7(2.9) 125(75.3)/36(21.7)/5(3.0) 279(69.2)/110(27.3)/

14(3.5)

1.259(0.881–1.799) 0.207 0.964(0.724–1.283) 0.800

rs1634323 A/G Route of infection

Drug use 49(87.5)/7(12.5)/0(0) 45(91.8)/4(8.2)/0(0.0) 39(100.0)/0(0.0)/0(0.0) ––0.331(0.091–1.207) 0.094

HD 204(87.9)/24(10.3)/4(1.7) 40(100.0)/0(0.0)/0(0.0) 23(100.0)/0(0.0)/0(0.0) ––––

Blood donation 214(89.9)/24(10.1)/0(0) 134(80.7)/26(15.7)/6(3.6) 354(87.8)/40(9.9)/9(2.2) 0.668(0.452–0.988) 0.044 1.634(1.051–2.542) 0.029

Viral genotype

Genotype-1 – 146(85.4)/23(13.5)/2(1.2) 242(89.3)/22(8.1)/7(2.6) 0.732(0.455–1.178) 0.199 ––

Non-1 – 31(88.6)/2(5.7)/2(5.7) 119(90.8)/11(8.4)/1(0.8) 0.340(0.130–0.890) 0.028 ––

Mixed – 42(85.7)/5(10.2)/2(4.1) 55(87.3)/7(11.1)/1(1.6) 0.687(0.284–1.663) 0.405 ––

Group A: Healthy controls; Group B: Spontaneous clearance subjects; Group C: Persistent infection patients; Group (B + C): Infected individuals. Boldfaced values indicate statistically significant

results

The subjects with 11 (homozygote) genotype was used as the reference in the stratified analysis

Derived from additive model, adjusted by age, likely source of infection and/or viral genotypes (the stratified factor in each stratum was excluded)

147Single Nucleotide Polymorphisms of Toll-Like Receptor 7

Table 5. Stratified Analysis of the TLR7 rs179016, rs5743733, and rs1634323 Among Male Healthy Control, Spontaneous Clearance, and Persistent Infection Groups

SNPs Allele Subgroups Group A (%) Group B (%) Group C (%) Group C/Group B Group (B + C)

/Group A

(1/2) 1/2 1/2 1/2 OR(95 % CI)

P value

OR(95 % CI)

P value

rs179016 G/C Age

≤50 263(83.2)/53(16.8) 112(87.5)/16(12.5) 110(84.6)/20(15.4) 1.081(0.754–1.550) 0.672 0.922(0.717–1.185) 0.525

>50 220(83.0)/45(17.0) 54(88.5)/7(1 1.5) 100(73.0)/37(27.0) 1.750(1.117–2.742) 0.015 1.184(0.919–1.525) 0.192

Route of infection

Drug use 104(87.4)/15(12.6) 81(87.1)/12(12.9) 75(87.2)/11(12.8) 1.018(0.560–1.850) 0.953 1.005(0.685–1.475) 0.978

HD 309(81.7)/69(18.3) 52(88.1)/7(1 1.9) 44(74.6)/15(25.4) 1.755 (1.029–2.995) 0.039 1.011(0.774–1.319) 0.937

Blood donation 70(83.3)/14(16.7) 33(89.2)/4(10.8) 91(74.6)/31(25.4) 1.751(0.987–3.106) 0.055 1.152(0.814–1.630) 0.426

Viral genotype

Genotype-1 – 107(86.3)/17(13.7) 126(77.3)/37(22.7) 1.407(1.020–1.940) 0.038 ––

Non-1 – 28(96.6)/1(3.4) 57(80.3)/14(19.7) 3.6 03 (1.1 12–11.670) 0.033 ––

Mixed – 31(86.1)/5(13.9) 27(81.8)/6(18.2) 1.274(0.407–3.983) 0.678 ––

rs1634323 A/G Age

≤50 302(95.6)/14(4.4) 124(96.9)/4(3.1) 126(96.9)/4(3.1) 0.884(0.432–1.813) 0.737 0.619(0.386–0.994) 0.047

>50 254(95.8)/11(4.2) 54(88.5)/7(1 1.5) 130(94.9)/7(5.1) 0.624(0.354–1.100) 0.103 1.010(0.654–1.559) 0.964

Route of infection

Drug use 106(89.1)/13(10.9) 90(96.8)/3(3.2) 85(98.8)/1(1.2) 0.458(0.028–7.522) 0.584 0.324(0.167–0.627) 0.001

HD 376(99.5)/2(0.5) 56(94.9)/3(5.1) 56(94.9)/3(5.1) 0.841(0.359–1.969) 0.690 3.100

(1.380–6.963) 0.006

Blood donation 74(88.1)/10(11.9) 32(86.5)/5(13.5) 115(94.3)/7(5.7) 0.551(0.292–1.041) 0.066 0.789(0.505–1.233) 0.299

Group A: Healthy controls; Group B: Spontaneous clearance subjects; Group C: Persistent infection patients; Group (B + C): Infected individuals. Boldfaced values indicate statistically significant

results

Derived from additive model, adjusted by age, likely source of infection and/or viral genotypes (the stratified factor in each stratum was excluded)

148 Xue, Gong, Tang, Gao, Wang, Cai, Wang, Yu, Peng, Fan, Wang, Zhu, and Zhang

showed that a TLR7 ligand SM360320 could inhibit HCV

replication in hepatocytes via a type I IFN-independent

mechanism. Also, a clinical study revealed that twice-

weekly 4-week exposure with PF-04878691, a TLR7 ago-

nist, could significantly reduce the HCV-RNA viral load

[25]. Furthermore, studies [26–28] which applied other

TLR7 agonists (e.g., isatoribine) also reached the similar

conclusion. In a word, the activation of TLR7 signaling

pathway is involved in the anti-HCV immunity and very

likely to suppress the replication of HCV.

SNPs, the most common form of genetic variants in

the human genome, have been reported to have potential

functional effect on the susceptibility to human disease

including hepatitis C [29, 30]. Up to now, several studies

have evaluated the association of TLR7 gene polymor-

phisms with HCV infection susceptibility and viral clear-

ance. For example, Wang et al.[31] observed a significant

difference in the distribution of TLR7 rs179009 among

HCV-infected patients from Taiwan, China. Similarly,

Wei et al.[32] demonstrated that rs179009 G/A was a risk

factor for HCV susceptibility in Chinese female Han pop-

ulation. Moreover, in the German population, one study

[22] found no significant association between rs179008

and HCV-related inflammation activity or fibrosis progres-

sion. But in another study [9], c.1-120T>G, c.2403G>A

(rs864058), as well as c.32A>T (rs179008) were investi-

gated. The melting curve analysis indicated that only the

distribution of c.1-120T>G was associated with the grade

of inflammation. Nevertheless, the later investigation sug-

gested that c.2403G>A (rs864058) and c.32A>T

(rs179008) were both associated with chronic HCV infec-

tion [33]. Since the controversial conclusions had been

reached in different studies, verifying the functional effects

of these polymorphisms has been focused on recently.

Wang et al.[31] analyzed TLR7 mRNA expression and

the production of cytokines induced ex vivo by TLR7-

specific agonists using whole blood of subjects with dif-

ferent genotypes and found that individuals with rs179009

A allele had higher IFN-α production, less amount of

proinflammatory cytokines. Hence, such functional assays

validate and extend the findings of genotype analyses.

In the present study, we investigated the effects of

TLR7 polymorphisms on HCV-infected patients and

healthy controls in a high-risk Chinese population. No

significant associations were observed between the three

SNPs (rs179016, rs5743733, or rs1634323) and HCV

infection susceptibility in the overall analysis of both fe-

male and male individuals. Howe ver, the c arriage of

rs1634323 G allele was related to the protection from

persistent HCV infection in female individuals, and the

carriage of rs179016 C allele seemed to be more prone to

develop persistent infection in males. As shown in Table 2,

the OR was 0.558 (95 % CI=0.348–0.894) for rs1634323

(AG + GG vs. AA) carriers in females. This G allele had

already been associated with the elevated risk of SLE in

females [18]. In Table 3, the OR was 1.444 (95 % CI=

1.096–1.903) for rs179016 C allele carriers. Thus far, there

were two studies exploring the association of rs179016

with immune-related disorders. Sada et al.[17] analyzed

the frequency of rs179016 in 200 patients with Behcet’s

disease (BD) and 102 randomized controls coming from

Japan but did not find any statistical difference. But in

Nilsson et al.’s study [14], rs179016 was found to be

associated with skin prick test (SPT) response for

Table 6. Haplotypes Frequencies Constituted with TLR7 SNPs (rs179016, rs5743733, and Rs1634323) Among Healthy Control, Spontaneous Clearance,

and Persistent Infection Groups

Haplotype

Sex Group A (%) Group B (%) Group C (%) Group C/Group B Group (B + C)/Group A

OR(95 % CI)

P value

OR(95 % CI)

P value

N Female 526 255 465

Male 581 189 267

GCA Female 74.8 72.5 72.3 ––––

Male 74.9 76.7 70.8 ––––

CCA Female 12.6 14.1 15.9 1.235(0.893–1.708) 0.202 1.358(1.046–1.762) 0.021

Male 15.7 10.1 18.7 2.120(1.391–3.233)<0.001 0.997(0.764–1.300) 0.981

GGA Female 6.7 4.9 5.4 1.084(0.646–1.818) 0.761 0.833(0.573–1.212) 0.340

Male 5.2 7.4 6.4 0.965(0.562–1.657) 0.898 1.795(1.214–2.653) 0.003

Group A: Healthy controls; Group B: Spontaneous clearance subjects; Group C: Persistent infection patients; Group (B + C): Infected individuals; N: number

of subjects. Boldfaced values indicate statistically significant results

Order of single nucleotide polymorphisms comprising the TLR7 haplotypes: rs179016 G/C, rs5743733 C/G, and rs1634323 A/G

Logistic regression model, adjusted by age, likely source of infection and/or viral genotypes

149Single Nucleotide Polymorphisms of Toll-Like Receptor 7

D. pteronyssinus among allergic rhinitis patients and the

controls. Since conflicting conclusions had been drawn in

various investigations, further functional studies on the

effects of these SNPs are warranted.

The Institute of Medicine declared that the differ-

ences in disease exposure should also be taken into

account in the assessment of the differences in incidence

of infectious diseases between males and females [32].

For HCV infection, both pathogen and host character-

istics can influence the development and progress of the

disease. Previous studies had demonstrated that young

subjects had a higher spontaneous clearance rate of

HCV infection [34], so we further conducted an analysis

stratified by age. As was shown in Table 5, in male

subjects, rs179016 C allele was a risk factor for HCV

persistence in the elders (aged >50), and rs1634323 car-

riage of G allele might decrease the susceptibility to HCV

infection in the younger subgroup (aged ≤50). In this

regard, age has a pivotal effect on the HCV infection.

We also conducted additional stratified analyses accord-

ing to the likely source of infection and HCV genotype.

Interestingly, in some subgroups, we detected statistical

significances between TLR7 variants and the outcomes of

HCV infection, while not in the overall analyses. These

results suggested a complex relationship between age,

viral genotype, and genomic factors.

It is well known that multiple-linked SNPs have the

potential to provide more power to genetic analysis than

individual SNPs. In the present study , haplotype CCA in

females and haplotype GGA in males were associated with

elevated risk of the HCV susceptibility, whereas haplotype

CCA was associated with 2.120-fold increase in the risk of

developing into persistent HCV infection in male subjects.

These results suggested the possible interactions of the

three SNPs during the course of HCV infection.

In summary, this study presented the gender-

dependent association of TLR7 rs179016, rs574373 3,

and rs1634323 with susceptibility to HCV infection and

the clearance of the virus in a high-risk Chinese population.

Although this conclusion needs to be verified by similar

studies on other populations, our study is the first in this

category. Since the subjects were of high risk and the

selection bias was inescapable, we applied many strategies

to control and minimize the potential confounding factors,

such as matching the age and residential area and taking the

likely source of infection and viral genotype into consider-

ation. In face of the accumulating data on the association of

genetic polymorphisms and HCV infection, it is suggested

that TLR7 variants may be involved in the etiology of this

disease and these genetic markers might be useful for

providing some usable information for creating the indi-

vidual risk profiles.

ACKNOWLEDGMENTS

This work was supported by the National Natural

Science Foundation of China (Grant Numbers:

81273146, 81102165). We also thank Peng Huang and Li

Dong et al. for the sample collection work.

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151Single Nucleotide Polymorphisms of Toll-Like Receptor 7