Possible role of TLR4 and TLR9 SNPs in protection against congenital toxoplasmosis.

The purpose of this investigation was the determination of the distribution of genotypes at single nucleotide polymorphisms (SNPs) of the toll-like receptor 4 (TLR4) and the toll-like receptor 9 (TLR9) in fetuses and newborns congenitally infected with Toxoplasma gondii and the identification of genetic changes predisposing to infection development. The study involved 20 fetuses and newborns with congenital toxoplasmosis and 50 uninfected controls. The levels of IgG and IgM antibodies against T. gondii, as well as IgG avidity, were estimated by enzyme-linked fluorescent assay (ELFA) tests. T. gondii DNA loads in amniotic fluids were assayed by the real-time (RT) quantitative polymerase chain reaction (Q PCR) technique for parasitic B1 gene. TLR4 and TLR9 SNPs were identified using a self-designed multiplex nested PCR-restriction fragment length polymorphism (RFLP) assay. Randomly selected genotypes at SNPs were confirmed by sequencing. All the genotypes were tested for Hardy-Weinberg equilibrium and TLR4 genotypes were analyzed for linkage disequilibrium. A correlation was studied between the genotypes or haplotypes and the development of congenital toxoplasmosis using a logistic regression model. Single SNP analysis showed no statistically significant differences in the distribution of distinct genotypes at the analyzed TLR4 and TLR9 SNPs between T. gondii-infected fetuses and newborns and the controls. Taking into account the prevalence of alleles residing within polymorphic sites, similar prevalence rates were observed in both of the studied groups. The multiple SNP analysis indicated GTG variants at the TLR4 and TLR9 SNPs to be significantly less frequent in offspring with congenital toxoplasmosis than in uninfected offspring (p ≤ 0.0001). TLR4 and TLR9 SNPs seem to be involved in protection against congenital toxoplasmosis.