Introduction
Toll-like receptors (TLRs) play a central role in detecting microbial pathogens. TLRs initiate innate immune responses and also induce adaptive immune responses [
1]. Recently, TLRs have been strongly implicated in autoimmune diseases [
2]. The
TLR7 and
TLR9 genes, which are expressed intracellularly in plasmacytoid dendritic cells (pDCs) and B cells, recognize single-stranded RNA and DNA containing cytidine-phosphate-guanosine motifs, respectively. Activation of pDCs by TLR7 and TLR9 induces a large amount of type I interferon (IFN). It has become evident that RNA- and DNA-containing immune complexes, which often exist in sera of patients with systemic lupus erythematosus (SLE), can activate TLR7 and TLR9 signaling [
2].
Several lines of evidence support a role of TLR7 in SLE pathogenesis [
2]. Male BXSB mice bearing the Y chromosome-linked autoimmune accelerator (
Yaa) gene develop severe SLE. It has been revealed that
Yaa mutation is caused by a translocation of a portion of the X chromosome containing
TLR7 onto the Y chromosome [
3,
4].
Yaa-bearing mice have been demonstrated to have twofold overexpression of TLR7 protein and mRNA [
3,
4]. In contrast, lupus-prone MRL/Mp
lpr/lprmice lacking
TLR7 showed impaired production of antibodies to RNA-containing antigens, such as anti-Smith (anti-Sm) antibodies, and developed less severe disease [
5]. Furthermore, upregulated expression of
TLR7 mRNA in peripheral blood mononuclear cells (PBMNCs) was observed in human SLE [
6].
Recently, a multicenter collaborative study including our group reported an association of
TLR7, located in Xp22.3, with SLE in combined East Asian populations [
7]. In a discovery panel consisting mainly of Chinese and Korean populations, the association of 27 single-nucleotide polymorphisms (SNPs) in the
TLR7-
TLR8 region with SLE was examined, and a significant association of the
TLR7 3' untranslated region (3' UTR) SNP, rs3853839, was identified. Subsequently, the association of rs3853839 was replicated in two independent Chinese and Japanese case-control sets. The association was prominent in males with SLE. In addition, rs3853839 was associated with elevated expression of
TLR7. The study also revealed some differences in the association of rs3853839 and other SNPs among Chinese, Korean and Japanese populations [
7], indicating that systematic SNP screening should be performed in each population.
In this study, we examined the association of eight TLR7 tag SNPs with SLE in Japanese women and discovered a newly identified association of two intronic SNPs, rs179019 and rs179010, with SLE. These SNPs and the 3'UTR rs3853839 were found to independently contribute to the genetic risk for SLE.
Materials and methods
Patients and controls
Three hundred forty-four Japanese female patients with SLE (mean age ± SD, 42.9 ± 13.8 years) and 274 healthy female controls (mean age ± SD, 31.3 ± 8.9 years) were recruited at University of Tsukuba, Juntendo University, Sagamihara National Hospital, and at the University of Tokyo. Among them, 296 SLE patients and 250 healthy controls were also examined in a previous study to replicate the association of rs3853839 with SLE in Japanese, but other SNPs were not investigated in that study [
7]. All patients and healthy individuals were native Japanese living in the central part of Japan. All patients with SLE fulfilled the American College of Rheumatology criteria for SLE [
8].
This study was carried out in compliance with the Declaration of Helsinki. The study was reviewed and approved by the research ethics committees of University of Tsukuba, Sagamihara National Hospital, the University of Tokyo and Juntendo University. Informed consent was obtained from all study participants.
Genotyping
Eight tag SNPs in the
TLR7 region were selected on the basis of the HapMap Phase II JPT (Japanese in Tokyo) data obtained from the HapMap database [
9] with the criteria of minor allele frequency >0.1 and an
r2 threshold of 0.9. Genotyping of the tag SNPs was carried out using the TaqMan genotyping assay on the Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions. Thermal cycling conditions consisted of initial denaturation at 95°C for 10 minutes, followed by 50 cycles at 95°C for 15 seconds each and at 60°C for one minute each. TaqMan probes used in this study were as follows: Assay ID: C__15757400_10 (rs2302267), C___2259585_10 (rs179019), C___7625717_10 (rs1634322), C___2259582_10 (rs179016), C___2259578_10 (rs179012), C___2259576_10 (rs179010), C___2259575_10 (rs179009), and C___2259573_10 (rs3853839).
Expression analysis by real-time quantitative reverse transcription polymerase chain reaction assay
Total RNA was extracted from PBMNCs of 18 females with SLE using the RNeasy Mini Kit (QIAGEN, Hilden, Germany), reverse transcribed into cDNA and used for real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) assay. Expression of TLR7 was analyzed using the TaqMan Gene Expression Assay (Applied Biosystems), Hs00152971_m1. Amplification of cDNA was conducted using the Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems) under the following conditions: 50°C for 2 minutes and 95°C for 10 minutes, and 50 cycles at 95°C for 15 seconds and at 60°C for 1 minute, and then the cycle threshold (CT) value for each sample was obtained using Applied Biosystems 7300 System SDS version 1.4 software (Applied Biosystems). Relative quantitative levels were calculated on the basis of the CT value by a standard curve method and were normalized to β-actin (ACTB) expression (Hs99999903_m1). The experiments were done in triplicate for each sample.
Statistical analysis
Differences in allele and genotype frequencies between SLE patients and healthy controls were analyzed by using a χ2 test with 2 × 2 contingency tables. When one or more of the variables in the contingency tables was 20 or less, Fisher's exact test was employed. Linkage disequilibrium (LD) was analyzed using HaploView version 4.0 software (Broad Institute, Cambridge, MA, USA). Pairwise r2 values were calculated on the basis of the genotypes of 274 healthy controls. Estimation of haplotype frequencies and association tests were performed using HaploView version 4.0 software.
To examine whether each SNP independently contributes to susceptibility to SLE, conditional logistic regression analysis was employed. Dominant, codominant and recessive models were tested for each SNP, and the model that provided the lowest P value was selected as the best fit model. As a result, the following were used as independent variables: rs3853839, C/C = 0, G/C = 1 and G/G = 2 under the codominant model for the G allele; rs179019, C/C = 0, C/A = 0, A/A = 1 under the recessive model for the A allele; rs179010, C/C = 0, C/T = 0, and T/T = 1 under the recessive model for the T allele.
The association of TLR7 SNPs with TLR7 mRNA expression was assessed by using the Kruskal-Wallis test.
Discussion
In the recently reported multicenter study, an association of rs3853839 was originally found by screening the
TLR7-
TLR8 region in Chinese and Korean populations and was subsequently replicated in Chinese and Japanese populations [
7]. In the process of the study, some population difference was noted for rs3853839 and other SNPs, even among these East Asian populations. Because association between
TLR7 and SLE had not been examined in a systematic manner in a Japanese population, we thought that
TLR7 SNPs other than rs3853839 might also contribute to SLE.
To explore such a possibility, we analyzed the association of eight tag SNPs in TLR7 and the newly detected association of two SNPs in intron 2, rs179019 and rs179010. Conditional logistic regression analysis indicated that the association of the intronic SNPs cannot be explained by LD with rs3853839. In agreement with these results, the association of the intronic SNPs remained significant after excluding the effect of the 3'UTR SNP by testing the association only among individuals carrying the 3'UTR risk allele. Furthermore, haplotype analysis showed significant association of the haplotype containing all of the three SLE risk alleles, but not of the other haplotypes. All of these results support the possibility that the possession of both the 3'UTR and intronic risk alleles may confer further risk for SLE.
Although rs179019 and rs179010 were also investigated in the Discovery Panel in the previous study, the majority of whom were Chinese and Korean participants, no significant association was detected [
7]. The Japanese patients and controls analyzed in this study were not included in the Discovery Panel. Population difference was also observed for rs3853839 between the Chinese and Korean populations, as this SNP was strongly associated with SLE in Chinese, but not in Koreans [
7], suggesting that the genetic background with respect to
TLR7 association with SLE might be somewhat different, even among the closely related East Asian populations. Minor allele frequencies of rs179019 and rs179010 in the HapMap CHB (Han Chinese in Beijing) samples (rs179019: 30.9%, rs179010: 37.3%) available in the International HapMap database [
9] are similar to those in the Japanese observed in this study (rs179019: 28.5%, rs179010: 35.2%). Thus, the difference in the association cannot be explained by differences in the minor allele frequencies. We cannot rule out the possibility that another SNP tagged by rs179019 and rs179010 in Japanese, but not in Chinese or Koreans because of difference in the LD status, might play a causative role. Such a possibility would be addressed by resequencing the entire
TLR7 region.
There is growing evidence to support involvement of type I IFN in the development of SLE. TLR7 is crucial for the production of type I IFN. Thus, the most plausible role of
TLR7 SNPs in SLE pathogenesis is likely to be explained by elevated type I IFN production. The sera of SLE patients displayed elevated levels of type I IFN, and expression of IFN-inducible genes in PBMNCs was also upregulated in SLE [
10]. Occasional occurrence of SLE symptoms following treatment with IFNα in patients with cancer or hepatitis underscored the relevance of type I IFN [
10]. Type I IFN is thought to be a potential therapeutic target for SLE, and clinical trials of anti-IFNα antibodies in SLE are currently underway [
11].
Recent genetic studies have identified an association of type I IFN pathway-related genes, IFN regulatory factor 5 (
IRF5) and
STAT4, with SLE in various populations [
10,
12‐
16]. An
IRF5 SLE risk haplotype has been shown to be associated with high serum IFNα activity in SLE patients [
17], whereas the
STAT4 SLE risk variant was associated with increased sensitivity to IFNα
in vivo [
18]. These observations, as well as the previous study on
TLR7 showing upregulation of TLR7 in the risk genotype [
7], suggest that SLE-associated alleles in the type I IFN pathway are gain-of-function alleles in nature.
Another potential role of
TLR7 polymorphisms may be related to the induction of proinflammatory cytokines. IRF5 is activated by TLR7 signaling and regulates the expression of many genes, including type I IFN and proinflammatory cytokines [
19]. STAT4 is activated by type I IFN as well as interleukin 12 and plays a role in Th1 differentiation [
20]. In view of these observations, the association between
TLR7 SNPs and SLE might also be explained by overproduction of proinflammatory cytokines in addition to type I IFN.
There are conflicting reports about copy number variation (CNV) of
TLR7. Initially, the existence of CNV was reported by Kelley
et al. [
21]. They showed that, although common CNV was observed in Caucasians and African-Americans, no association with SLE was detected [
21]. Recently, García-Ortiz
et al. [
22] reported an association of CNV with childhood-onset SLE in a Mexican population. In contrast to these observations, Shen
et al. [
7] did not find common
TLR7 CNV in multiple populations, including Asians. The latter observation is consistent with the fact that no CNV was registered in the Database of Genomic Variants [
23], which includes results derived from the HapMap JPT (Japanese in Tokyo) samples.
Although our observation in the expression analysis supported the previous report that indicated the association between the risk allele of the 3'UTR SNP and elevated expression of
TLR7 [
7], evidence for the association of the intronic SNPs with levels of
TLR7 mRNA was not observed, and therefore the molecular mechanism of the intronic SNPs requires further study. TLR7 is mainly expressed in pDCs and B cells. pDCs represent the major source of type I IFN, but constitute less than 1% of PBMNCs. If the intronic SNPs have a regulatory role in a cell type-specific fashion and influence the expression level of
TLR7 in pDCs but not in other white blood cells, such an effect may not have been detected in the analysis of total PBMNCs. In addition, the sample size of this study may not have been large enough for us to conclude that the intronic SNPs have no effect on the expression of
TLR7.
Because we focused only on the Japanese population, the sample size of this study was limited and the observed statistical association was modest. Therefore, the association of the intronic SNPs should be confirmed in future independent studies.
This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AK participated in the study design; carried out all genotyping, expression analysis and statistical analyses; and wrote the manuscript. HF, YK, SI, TH, MK, IM, ST, YT, HH and TS recruited the patients and controls and collected clinical information. NT designed and coordinated the study and helped in the manuscript preparation. All authors read and approved the final manuscript.