Polymorphisms of the toll-like receptor 9 (TLR9) gene with systemic lupus erythematosus in Chinese

M. W. Ng, C. S. Lau1, T. M. Chan1, W. H. S. Wong and Y. L. Lau

Department of Paediatrics and Adolescent Medicine and 1 Department of Medicine, Queen Mary Hospital, University of Hong Kong, Pokfulam and Genome Research Centre, University of Hong Kong, Hong Kong SAR, China

Correspondence to: Y. L. Lau, Department of Paediatrics and Adolescent Medicine, Queen Mary Hospital, University of Hong Kong, Pokfulam, Hong Kong SAR, China.E-mail: lauylung{at}hkucc.hku.hk

SIR, Systemic lupus erythematosus (SLE) patients and murine lupus models of SLE are characterized by the presence of autoantibodies that recognize DNA and nucleosomes. It has been shown that interaction of bacterial CpG DNA with toll-like receptor 9 (TLR9) expressed on B cells can stimulate production of antibodies [1]. In the lupus mouse model and SLE patients, DNA fragments isolated from plasma are hypomethylated and they may mimic microbial DNA and trigger TLR9 signalling, leading to the production of autoantibodies against these DNA fragments [2]. This hypothesis has been confirmed in MRLlpr/lpr mice, in which exogenous CpG oligodeoxynucleotides activate B cells through the cross-linking of MyD88-dependent TLR9 and B-cell receptors by IgG2a–chromatin immune complexes. As a result, expression of renal CCL2/MCP-1 and CCL5/RANTES was enhanced and this led to crescentic glomerulonephritis, interstitial fibrosis and heavy proteinuria in MRLlpr/lpr mice [3].

Production of IFN-{alpha}, which is a proposed target for therapy in SLE, by plasmacytoid dendritic cells also requires the TLR9 pathway. Blanco et al. [4] demonstrated that circulating IFN-{alpha} induced monocytes to differentiate into dendritic cells, which then captured apoptotic cells and nucleosomes in SLE patients’ blood and presented them to CD4+ T cells. This was followed by differentiation of autoantibody-producing B cells and formation of immune complexes, which could potentially sustain IFN-{alpha} production via TLR9 signalling and further activate B cells by dual engagement of immunoglobulin M and TLR9 to produce autoantibodies [5].

The TLR9 gene is located on chromosome 3p21.3, one of the susceptibility regions for SLE [6]. Twenty single-nucleotide polymorphisms (SNPs) have been identified using three self-identified US ethnic groups, and four of the SNPs are common [7]. Two of them are located in the promoter, namely –1486T/C and –1237T/C, one in intron 1, IVS1-44A/G, and one in exon 2, 1635G/A (P545P).

As there is increasing evidence showing that TLR9 may be involved in the pathogenesis of SLE, we hypothesized that the four common SNPs of the TLR9 gene may be associated with susceptibility to and/or the severity of SLE in our population. Our study included 799 Hong Kong Chinese healthy blood donors and 467 SLE patients [8], and was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (HKU/HA HKW IRB). All SLE patients met the revised American College of Rheumatology criteria for SLE [9] and gave informed consent. The four common SNPs of the TLR9 gene were genotyped using the Sequenom MassArray service provided by the Genome Research Centre of the University of Hong Kong. The mean age of the patients was 39.2 ± 11.9 yr and that of the controls was 29.7 ± 9.4 yr, while the male to female ratio of the SLE patients was 1:9 and that of the controls was 6:4.

The genotype frequencies of the four SNPs were similar in both the SLE patients and controls (Table 1). Haplotypes were constructed using the expectation-maximization (EM) algorithm with permutation. As the –1237T/C is non-polymorphic in our population, it was excluded in the haplotype analysis and other analyses. The haplotype frequencies were also similar between the SLE patients and the controls (Table 1). Clinical symptoms such as skin and oral disorders, arthritis, serositis and internal organ disorders, and the autoantibody profile, including ANA, anti-double-stranded DNA, anti-Sm, anti-Ro, anti-La and anti-nRNP, were also analysed for association with the genotypes. Although no significant association was found, the TT genotype of TLR9 –1486 tended to be over-represented in patients with serositis (61.1%) compared with those without serositis (44.1%) (P = 0.056) (data not shown).


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TABLE 1. Frequencies of genotypes and haplotypes of TLR9 in Hong Kong Chinese SLE patients and controls

 
Recently, polymorphisms of the TLR9 gene were studied in a Korean population [10]. The group found that none of the four SNPs was associated with susceptibility to SLE and related phenotypes. The genotype frequencies of the four SNPs in the Koreans studied were similar to those of our Chinese population.

Although the present study showed no association of TLR9 gene polymorphisms with SLE, we cannot entirely exclude the role of TLR9 as a candidate gene for SLE. Other SNPs of the TLR9 gene that were not analysed in our study may be associated with SLE, and further studies are needed to elucidate the role of TLR9 in the pathogenesis of SLE.

The authors have declared no conflicts of interest.

References

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  4. Blanco P, Karolina Palucka A, Gill M, Pascual V, Banchereau J. Induction of dendritic cell differentiation by IFN-{alpha} in systemic lupus erythematosus. Science 2001;294:1540–2.[Abstract/Free Full Text]
  5. Magnusson M, Magusson S, Vallin H, Ronnbiom L, Alm GV. Importance of CpG dinucleotides in activation of natural IFN-alpha-producing cells by a lupus-related oligodeoxynucleotides. Scand J Immunol 2001;54:543–50.[CrossRef][ISI][Medline]
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  7. Lazarus R, Klimecki WT, Raby BA et al. Single-nucleotide polymorphisms in the Toll-like receptor 9 gene (TLR9): frequencies, pairwise linkage disequilibrium, and haplotypes in three US ethnic groups and exploratory case-control disease association studies. Genomics 2003;81:85–91.[CrossRef][ISI][Medline]
  8. Chong WP, Ip WK, Wong WHS, Lau CS, Chan TM, Lau YL. Association of interleukin-10 promoter polymorphisms with systemic lupus erythematosus. Genes Immun 2004;5:484–92.[CrossRef][ISI][Medline]
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Accepted 12 August 2005





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