Investigation of the Association Between Toll-like Receptor 9 Gene Polymorphisms and Sarcoidosis in Japanese Patients

Sarcoidosis is a chronic multisystemic inflammatory disorder, affecting the lungs, skin, eyes, lymph nodes, central and peripheral nervous system, and heart. The disease is characterized by noncaseating granulomas and an exaggerated cellular immune response caused by increased inflammatory activity. The etiology of sarcoidosis is still unclear, but racial differences in incidence rates and disease clustering in families support the belief that genetics contribute to sarcoidosis etiology.1 Many studies have reported genetic associations between the human leukocyte antigen (HLA) region and sarcoidosis.2,3 Outside the HLA, a genome-wide association study has recently reported the annexin A11 gene as a new susceptibility gene.4

It has been suggested that sarcoidosis results from exposure of genetically susceptible hosts to specific but unidentified environmental agents. It is expected that the granuloma in sarcoidosis develops as a response to persistent weakly virulent antigens that induce a local helper T-cell type 1 (Th1) immune response. Propionibacterium acnes is one of the candidate causative agents in sarcoidosis, and detection of P. acnes DNA in bronchoalveolar lavage cells was significantly more common in the patients with confirmed sarcoidosis.5

Toll-like receptors (TLRs) are transmembrane proteins that function as pattern-recognition receptors in innate immunity. TLRs also provide a link to the activation of adaptive immunity by recognizing microbial components and inducing the production of cytokines, such as interleukin (IL)-12 and IL-18 driving naive T cells, to differentiate into Th1 cells.6 Among TLR family members, it is assumed that TLR2 and TLR4 play an important role in the innate immune response against candidate causative agents in sarcoidosis such as P. acnes and Mycobacterium tuberculosis. A previous study reported that two nonsynonymous TLR4 SNPs (Asp299Gly and Thr399Ile) are associated with an increased susceptibility to chronic sarcoidosis in a German population.7 However, other studies could not replicate the association in Greek and Dutch populations.8,9 Our previous study showed the two nonsynonymous TLR4 SNPs are monomorphic in a Japanese population and other TLR4 SNPs have no association with Japanese sarcoidosis.10 Veltkamp et al. showed an association between a TLR2 promoter polymorphism (rs4696480) and sarcoidosis in a Dutch Caucasian population, although they could not confirm the association in their validation cohort.11 In our recent study showed there was no significant association between TLR2 SNPs and sarcoidosis in a Japanese population; this study did not examine rs4696480 due to its monomorphism in a Japanese population.12 More recently, this group reported that absence of the common haplotype in the TLR10-TLR1-TLR6 gene cluster increases the risk of developing chronic disease in sarcoidosis patients and suggested that aberrant TLR-2 function is important in the pathogenesis of sarcoidosis since TLR-10, TLR-1, and TLR-6 act as co-receptors for TLR-2.13

TLR9 is activated in response to DNA, in particular DNA containing unmethylated CpG motifs that are more prevalent in microbial than mammalian DNA. By detecting foreign DNA signatures TLR9 can sense the presence of certain viruses or bacteria inside the cell and mount an immune response.14 Therefore, TLR9 is also important in the innate immune response against P. acnes and M. tuberculosis. The aim of this study was to clarify the role of TLR9 gene polymorphisms in the development of sarcoidosis by assessing the association of TLR9 polymorphisms with sarcoidosis in Japanese patients.

This study included 239 Japanese patients with sarcoidosis and 268 healthy Japanese from Yokohama City University, Hokkaido University, Fujita Health University, Tokyo University, Keio University, and Kumamoto City Hospital. All 239 patients had chronic sarcoidosis. The diagnosis of sarcoidosis was established according to the criteria developed by the Japanese Society of Sarcoidosis and Other Granulomatous Disorders previously described.10 All subjects had the same ethnic background and resided in the same urban area. The study was approved by the ethics committee of each participating institute, and complied with the guidelines of the Declaration of Helsinki. All study details were explained to the patients and controls before obtaining informed consent for genetic analysis. Peripheral blood lymphocytes were collected, and genomic DNA was extracted from peripheral blood cells using QIAamp DNA Blood Maxi Kit (QIAGEN).

The TLR9 gene is located on chromosome 3p21. All three single-nucleotide polymorphisms (SNPs) with minor allele frequency ≥5% on the gene region were selected from HapMap Japanese data for genotyping (rs352140, rs352139, and rs187084). We also selected five potentially functional SNPs: four nonsynonymous SNP (rs5743846, rs5743845, rs5743843, and rs5743842) and a promoter SNP (rs5743836). Genotyping of these SNPs was performed with the TaqMan 5′ exonuclease assay using primers supplied by Applied Biosystems. The probe emitted a fluorescent signal and was incorporated during the TaqMan Assay for Real-Time PCR (7500 Real Time PCR System; Applied Biosystems), following the manufacturer’s instructions. Allele frequencies of all detected SNPs were tested for Hardy-Weinberg equilibrium (HWE). Differences in allele frequencies between patients and controls were assessed by the χ² test. p values less than 0.05 were considered statistically significant. To obtain a measure of significance corrected for multiple testing bias, we ran 10,000 permutations to compute p values using the Haploview 4.1 program. The Haploview 4.1 program (www.broad.mit.edu/mpg/haploview/) was also used to estimate linkage disequilibrium (LD) by measuring pairwise D′ and r² value defining LD blocks.

Eight SNPs in TLR9 were genotyped. Genotype distributions for all SNPs were in HWE in both patients and controls, and no genetic bias was observed for each SNP. The allele frequencies and the pairwise LD of the SNPs are shown in Table 1 and Figure 1, respectively. Three of them, rs187084, rs352139, and rs352140, were common variants and two of them, rs5743836 and rs5743845, were rare variants, whereas rs5743846, rs5743843, and rs5743842 were monomorphic. The magnitude of LD between the two rare SNPs was extremely high (D′ = 1, r²= 1) and the three common SNPs were also in strong LD (D′ ≥ 0.99, r²≥ 0.92). No statistically significant association was observed for any of the SNPs between patients and controls, whereas the two rare SNPs, rs5743836 and rs5743845, showed marginal significance with sarcoidosis (p = 0.058, p_c = 0.199). Stratification analysis according to lesion locations, including the eye, lungs, bilateral hilar lymphadenopathy, skin, heart, and nerves, did not show any association of TLR9 polymorphisms with these parameters.

FIGURE 1.  Linkage disequilibrium plot of five polymorphic TLR9 SNPs in 507 study participants. A schematic of the TLR9 gene is shown as a black line with black boxes representing the coding region and white boxes representing the untranslated region. The locations of the genotyped SNPs are indicated by the dotted line. The D′ value and r² value (in parentheses) corresponding to each SNP pair are expressed as a percentage and shown within the respective square. Color scheme is based on D′ and logarithm of the odds (LOD) score values: blue, D′ = 1 and LOD < 2; shades of pink/red, D′ < 1 and LOD ≥ 2; bright red, D′ = 1 and LOD ≥ 2.

TABLE 1. Allele frequencies of SNPs of the TLR9 gene among sarcoidosis patients and controls.

					Minor allele frequency, n (%)
SNP	Position (build 37.1)	Gene location	Amino acid change	Alleles (1/2)	Patients (n = 239)	Controls (n = 268)	p	pc
rs5743846	52,255,688	Exon 2	Ala to Thr	G/A	0 (0.0)	0 (0.0)	–
rs5743845	52,255,744	Exon 2	Arg to Gln	G/A	0 (0.0)	4 (0.7)	0.058	0.199
rs352140	52,256,697	Exon 2	Synonymous	T/C	240 (50.2)	266 (49.6)	0.853
rs5743843	52,258,095	Exon 2	His to Gln	T/G	0 (0.0)	0 (0.0)	–
rs5743842	52,258,319	Exon 2	Arg to Cys	C/T	0 (0.0)	0 (0.0)	–
rs352139	52,258,372	Intron 1		G/A	238 (49.8)	262 (48.9)	0.772
rs5743836	52,260,782	5′ UTR		T/C	0 (0.0)	4 (0.7)	0.058	0.199
rs187084	52,261,031	5′ UTR		C/T	234 (49.0)	256 (47.8)	0.704

1, major allele; 2, minor allele. Position is distance from the short arm telomere of chromosome 3. p values were calculated by χ² test 2 × 2 contingency table. We corrected these p values (p_c) for multiple testing by Haploview program using 10,000 permutations.

The chromosome 3p21, which harbors TLR9, was identified as a region related to the susceptibility to sarcoidosis by genome-wide microsatellite linkage analysis in a German population,3 and therefore TLR9 may be considered a candidate gene for sarcoidosis. In this study, we investigated whether there were any associations between TLR9 polymorphisms and sarcoidosis and found no significant association of the TLR9 SNPs analyzed here with our Japanese patients. Our results were in line with the recent study in a Dutch population reported by Veltkamp et al.15

In conclusion, the present study showed a lack of association between TLR9 polymorphisms and Japanese sarcoidosis, suggesting that TLR9 polymorphisms do not play an important role in the etiology of sarcoidosis.

Declaration of Interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan, and grants from the Ministry of Health, Labour, and Welfare of Japan.