Association of TLR3 L412F Polymorphism with Cytomegalovirus Infection in Children.

Intracellular Toll-like receptor 3 (TLR3) recognizes viral double-stranded RNA (dsRNA) and activates antiviral immune responses through the production of type I interferons (IFNs) and inflammatory cytokines. This receptor binds to dsRNA molecules produced during human cytomegalovirus (HCMV) replication. TLR7 senses viral single-stranded RNA (ssRNA) in endosomes, and it can interact with endogenous RNAs. We determined the genotype distribution of single-nucleotide polymorphisms (SNPs) within the TLR3 and TLR7 genes in children with HCMV infection and the relationship between TLR polymorphisms and viral infection. We genotyped 59 children with symptomatic HCMV infection and 78 healthy individuals for SNPs in the TLR3 (rs3775290, c.1377C>T, F459F; rs3775291, c.1234C>T, L412F; rs3775296, c.-7C>A) and TLR7 (rs179008, c.32A>T, Q11L; rs5741880, c.3+1716G>T) genes. SNP genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and capillary electrophoresis. The HCMV DNA load was quantified by real-time PCR. We found an increased frequency of the heterozygous genotype TLR3 L412F in children with HCMV infection compared with uninfected cases. In individuals with a mutation present in at least one allele of the L412F SNP, an increased risk of HCMV disease was found, and this result remained highly significant after Bonferroni's correction for multiple testing (Pc < 0.001). The heterozygous genotype of this SNP was associated with the increased risk of HCMV disease in an adjusted model that included the HCMV DNA copy number in whole blood and urine (P < 0.001 and P = 0.008, respectively). Moreover, those with a heterozygous genotype of rs3775296 showed an increased relative risk of HCMV infection (P = 0.042), but this association did not reach statistical significance after correction for multiple testing. In contrast, the rs3775290 SNP of TLR3 and TLR7 SNPs were not related to viral infection. A moderate linkage disequilibrium (LD) was observed between the SNPs rs3775291 and rs3775296 (r2 = 0.514). We suggest that the L412F polymorphism in the TLR3 gene could be a genetic risk factor for the development of HCMV disease

TLR9 -1486T/C and 2848C/T SNPs Are Associated with Human Cytomegalovirus Infection in Infants.

Toll-like receptor 9 (TLR9) recognizes non-methylated viral CpG-containing DNA and serves as a pattern recognition receptor that signals the presence of human cytomegalovirus (HCMV). Here, we present the genotype distribution of single-nucleotide polymorphisms (SNPs) of the TLR9 gene in infants and the relationship between TLR9 polymorphisms and HCMV infection. Four polymorphisms (-1237T/C, rs5743836; -1486T/C, rs187084; 1174G/A, rs352139; and 2848C/T, rs352140) in the TLR9 gene were genotyped in 72 infants with symptomatic HCMV infection and 70 healthy individuals. SNP genotyping was performed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Digested fragments were separated and identified by capillary electrophoresis. The HCMV DNA copy number was measured by a quantitative real-time PCR assay. We found an increased frequency of heterozygous genotypes TLR9 -1486T/C and 2848C/T in infants with HCMV infection compared with uninfected cases. Heterozygous variants of these two SNPs increased the risk of HCMV disease in children (P = 0.044 and P = 0.029, respectively). In infants with a mutation present in at least one allele of -1486T/C and 2848C/T SNPs, a trend towards increased risk of cytomegaly was confirmed after Bonferroni's correction for multiple testing (Pc = 0.063). The rs352139 GG genotype showed a significantly reduced relative risk for HCMV infection (Pc = 0.006). In contrast, the -1237T/C SNP was not related to viral infection. We found no evidence for linkage disequilibrium with the four examined TLR9 SNPs. The findings suggest that the TLR9 -1486T/C and 2848C/T polymorphisms could be a genetic risk factor for the development of HCMV disease

Frequency and distribution of <i>TLR</i> genotypes and their association with HCMV infection.

<p>Frequency and distribution of <i>TLR</i> genotypes and their association with HCMV infection.</p

Restriction enzymes and length of the restriction fragments.

<p>Restriction enzymes and length of the restriction fragments.</p

Demographic and clinical characteristics of study subjects with HCMV infection.

<p>Demographic and clinical characteristics of study subjects with HCMV infection.</p

Visualization of PCR-RFLP products for <i>TLR3</i> SNP (A) and <i>TLR7</i> SNP (B) genotyping.

<p>Gel image: A) rs3775290: 1. wild type CC, 2. heterozygous CT, 3. homozygous recessive TT; rs3775291: 4. wild type CC, 5. heterozygous CT, 6. homozygous recessive TT; rs3775296: 7. wild type CC, 8. heterozygous CA, 9. homozygous recessive AA. B) rs179008: 1. wild type AA, 2. heterozygous AT, 3. homozygous recessive TT; rs5741880: 4. wild type GG, 5. heterozygous GT, 6. homozygous recessive TT. Alignment markers (15 bp, 1 kbp).</p

Selected single-nucleotide polymorphisms in the <i>TLR3</i> and <i>TLR7</i> genes.

<p>Selected single-nucleotide polymorphisms in the <i>TLR3</i> and <i>TLR7</i> genes.</p

The distribution of allele frequencies of TLR SNPs in infants with and without HCMV infection.

<p>The distribution of allele frequencies of TLR SNPs in infants with and without HCMV infection.</p

Haplotype analysis of <i>TLR3</i> SNPs in children with and without HCMV infection.

<p>Haplotype analysis of <i>TLR3</i> SNPs in children with and without HCMV infection.</p