Toll-like receptor 4 polymorphisms and their haplotypes modulate the risk of developing diabetic retinopathy in type 2 diabetes patients

Abstract

Purpose: Persistent inflammation and impaired neovascularization in type 2 diabetes mellitus (T2DM) patients may lead to development of macro- and microvascular complications. Diabetic retinopathy (DR) is one of the secondary microvascular complications of T2DM. Improper activation of the innate immune system may be an important contributor in the pathophysiology of DR. Toll-like receptor 4 (TLR4) is an important mediator of innate immunity, and genetic alterations in TLR4 support inflammation in the hyperglycemic condition. The present work was designed to investigate whether the TLR4 single nucleotide polymorphisms (SNPs) rs4986790, rs4986791, rs10759931, rs1927911, and rs1927914 are associated with DR in a north Indian population.

Methods: The study group of 698 individuals (128 DR, 250 T2DM, 320 controls) was genotyped by PCR-RFLP. Haplotype and linkage disequilibrium between SNPs were determined using Haploview software.

Results: Combined risk genotypes of TLR4 SNPs rs10759931 (odds ratio [OR] 1.50, p = 0.05) and rs1927914 (OR 1.48, p = 0.05) were found to be significantly associated with pathogenesis of DR. A total of 14 haplotypes with frequency >1% were obtained using Haploview software. Haplotypes ACATC (37.5%) and ACATT (14.8%) were the two most common haplotypes obtained.

Conclusions: Results of the present case-control study that included 698 north Indian subjects suggested that TLR4 SNPs rs10759931 and rs1927914 modulate the risk of DR in T2DM cases. Association analysis using haplotypes showed none of the haplotypes were associated with either susceptibility or resistance to DR in a north Indian population.

Figure 1

Gels showing PCR-RFLP analysis of different SNPs of the TLR4 gene. The amplified products of the SNPs rs4986790, rs4986791, rs10759931, rs1927911, and rs1927914 were digested with restriction enzymes BccI, BslI, KpnI, StyI and SphI, respectively. The restricted products were separated on 3% agarose gel, according to our previous report [10]. A: For genotyping rs4986790, the 140-bp PCR product was digested with BccI. The A allele is not cut by the enzyme, whereas the G allele yields 77 and 63 bp products. B: For genotyping rs4986791, the 110-bp PCR product was digested with BslI. The T allele is not cut by the enzyme, whereas the C allele yields 89 and 21 bp products. C: For genotyping rs1927911, the 203-bp PCR product was digested with StyI. The T allele is not cut by the enzyme, whereas the C allele yields 178 and 25 bp products. D: For genotyping rs10759931, the 241-bp PCR product was digested with KpnI. The A allele is not cut by the enzyme, whereas the G allele yields 190 and 51 bp products. E: For genotyping rs1927914, the 157-bp PCR product was digested with SphI. The T allele is not cut by the enzyme, whereas the C allele yields 90 and 67 bp products.

Figure 2

Linkage disequilibrium plot. Haplotype frequencies and LD were calculated using Haploview software (version 4.2). The LD parameter D is represented by the specific value in each cell. The cells are color graduated representing the strength of LD between the two markers. The rs numbers are SNP IDs extracted from the Ensembl database. The loci rs10759931, rs1927911, and rs1927914 are in intermediate LD.