A Polymorphism in Toll-like Receptor 2 Gene Is Associated with Occurrence of Bacterial Infections in Sickle Cell Disease Patients

Background: Sickle cell disease (SCD) patients (pts) are at increased risk of infection. This susceptibility is partially explained by the loss of spleen function, but other factors such as inflammatory status might also play a role. Despite adequate immunization and antimicrobial prophylaxis, bacterial infection remains a major cause of morbidity and mortality in SCD pts. Toll-like receptors (TLR), transmembrane proteins that recognize a wide range of pathogen molecular patterns, are key receptors in innate immune response. The association among TLR and infections in SCD has scarcely been explored.

Objective: To analyze if functional polymorphisms in TLR genes can modulate the susceptibility to bacterial infections in SCD pts.

Methods: Case-control retrospective study; 430 SCD pts followed in university hospitals in Brazil (n=196), France (n=180, mainly from Subsaharan Africa and French West Indies) and Senegal (n=54) were included. Sites of bacterial infection were lower respiratory system, abdominal, upper urinary system, bone/joints, central nervous system, blood stream, or any site for tuberculosis. For pts who underwent hematopoietic stem cell transplant (HSCT), only bacterial infections before HSCT were considered. All pts were tested for 7 single nucleotide polymorphisms (SNP) in TLR-1(rs4833095), TLR-2 (rs4696480, rs3804099, rs3804100), TLR-6 (rs5743810) and TLR-10 (rs11466653, rs11096957) using TaqMan 5'-nuclease assay. Univariate analysis was performed with chi-square or Mann-Whitney for categorical and continuous variables respectively. Associations were measured by odds ratio (OR). For SNP associations, significant P-value corrected by Bonferroni (cP) was 0.007; for genetic models, cP was 0.01.

Results: Median age of cohort was 30 years (range: 2-70) and 87% were ≥18 years; 57% of pts were female. SCD genotype was SS in 81%, SC in 11%, SB in 7% and other in 1%. Fifty-eight percent received hydroxyurea and 79% received red blood cell transfusions; seven pts underwent HSCT. Eleven pts died during follow-up, mostly from acute chest syndrome and hemorrhagic stroke. All pts had access to immunization against encapsulated bacteria and to penicillin prophylaxis. One hundred ninety-five pts (45%) did not present any bacterial infection, whereas 235 (55%) pts presented at least one episode of bacterial infection, of which 20% had ≥ 3 episodes. Bacterial infections were mainly respiratory (51%), bone/joints (23%) and urinary (20%). In 106 cases, etiological agents were identified; most common agents were Escherichia coli, Streptococcuspneumoniae, Mycobacterium tuberculosis and Salmonella spp. In univariate analysis, there was no association among occurrence of infections and gender, origin and age distribution. Gene analysis: all tested SNPs had a typing rate >90% and minimum allele frequency >1%. The TLR-2 SNP rs4696480 (n=425) showed significant association with infection (p<0.001). Next, we tested genetic models for this association. Distribution of rs4696480 genotypes was: infected - AA=35%, TA=50%, TT=15%; non infected - AA=28%, TA=67%, TT=5%. In the overdominant model, the heterozygous genotype (TA) occurred significantly more in non infected (67%) than in infected pts (50%) compared with TT+AA (OR 0.5, 95%CI 0.34-0.75, P<0.001). Also, in the recessive model, genotype TT occurred significantly more in infected (15%) than in non infected pts (5%) compared with TA+AA (OR 3.18, 95%CI 1.53-6.61, P<0.001). Furthermore, in comparison with an African/African-American population described in the 1000 genomes database (n=661), TA genotype was more frequent in SCD pts, and TT genotype was significantly less frequent. Other TLR SNPs genotype and haplotype analysis did not show any significant association with occurrence of infections.

Discussion: In SCD pts, TLR-2 rs4696480 TA genotype might be protective against bacterial infections, whereas TT genotype might increase risk of such infections. Previous reports demonstrated higher secretion of inflammatory factors in cells from AA individuals, lower occurrence and severity of immune diseases in T carriers. TA genotype might stand between deleterious effects of over inflammatory response (AA genotype) and under response (TT genotype) to infectious agents.