Analysis of the genetic diversity in TLR4 3'-untranslated region among Asian populations and the biological effects of altofrequent SNPs

Abstract

Background: Numerous case-control studies have demonstrated that single nucleotide polymorphism (SNP) loci in the TLR4 3'-untranslated region (UTR) are associated with multiple inflammatory disorders in various populations, particularly in Asians. However, the distribution frequency of polymorphisms in different Asian races and its effects on TLR4 expression remain unclear. Here, we extracted variants from the 1000 Genomes project database and analyzed the SNPs and haplotypes genetic diversity of TLR4 3'-UTR in 10 Asian populations, and then assessed the biological effects of the altofrequent polymorphic loci on TLR4 expression by luciferase assay. Results: Total 6 SNPs were identified as true polymorphic loci with minor allele frequencies (MAFs) ≥ 1% in Asian populations, including rs41426344, rs7869402, rs11536889, rs7873784, rs11536891, and rs11536896. Considering 4 Tag SNPs, we inferred 5 haplotypes that present a Asiatic frequency higher than 1%. It is noteworthy that rs41426344 is unique to East Asian populations, and the H-5 haplotype frequency was reduced when analyzing pooled data from East and South Asian populations. The MAFs of rs7869402 and rs11536889, and H-2 and H-4 frequencies significantly differed between the populations (P < 0.001). We constructed a pGL3-3494-3UTR luciferase plasmid to simulate TLR4 gene structure in vivo, and used PCR-mediated site-directed mutagenesis to construct a series of mutant luciferase constructs corresponding to the six SNPs. In addition, we identified that TLR4 mRNA was selectively expressed in SiHa and THP-1 cell lines, but not in C33A, Hela, and 293T cell lines. The luciferase activity of constructs containing rs7869402 T allele and rs11536889 C allele increased significantly upon LPS or IL-6 stimulation in THP-1 and SiHa cells. Conclusions: The distributions of SNPs and haplotypes in TLR4 3'-UTR were significantly different among Asian populations. The biological effects of rs7869402 and rs11536889 on TLR4 are significant clues that revealing its critical role in harmful TLR4-mediated responses. These results provided a reminder for future investigation on TLR4 related inflammatory diseases susceptibility.