Association Of Pattern Recognition Receptor Gene Polymorphisms With The Severity Of Infectious Diseases

Objective: To establish a molecular diagnostic method for genotyping 11 single nucleotide polymorphisms（SNPs）of pattern recognition receptors and analyze the correlation between 11 SNPs and severity of infectious diseases.Methods: In this study,primers of 11 SNPs including rs606231248,rs3211938,rs3775291,rs4986790,rs4986791,rs2072493,rs5744174,rs5744168,rs3796508,rs5743845 and rs5743842 were designed using online primer-BLAST software.A PCR-HRM molecular diagnostic detection system was established,and the Sanger sequencing method was used as the gold standard to evaluate the detection performance（sensitivity,specificity,repeatability and reproducibility）of the self-built system,which was used to genotype 113 patients with severe infection and 287 patients with mild infection during the same period.The patients with mild infection were used as controls.Infected patients were deeply stratified according to inflammatory indicators（CRP,PCT,IL-6,NLR,PLR and LMR）,and the association of each SNP with infectious disease progression in Lanzhou was analyzed.The chi-square test and binary logistic regression of SPSS software were used to for comparison between groups and correlation analysis.The online SHEsis software was used to Linkage Disequilibrium（LD）and haplotype analysis.Results:（1）The PCR-HRM method successfully tested 11 SNPs in 400 patients with infectious diseases.The accuracy,sensitivity and specificity of the self-built method reached 100% with repeatability and reproducibility after Sanger sequencing verification.（2）The patients were divided into groups according to the infection severity,and 11 SNPs were analyzed between groups.It was found that the genotype and allele frequencies of rs3775291 and rs606231248 between the mild and severe infection groups was statistically different（P<0.05）.（3）The analysis of the correlation between inflammatory indicators and the degree of infection found that the levels of CRP,PCT,IL-6 and NLR were positively correlated with the degree of infection,and the level of PLR was negatively correlated with the degree of infection（P<0.05）.（4）The case group was stratified according to the inflammatory indicators CRP,PCT,NLR,PLR,IL-6 and LMR,and it was found that the allele frequency of rs2072493 was significantly different between high and normal CRP groups,between high and normal PCT groups.The genotype and allele frequency of rs606231248 were significantly different between high and normal PCT groups,between high and normal IL-6 groups.The genotype and allele frequency of rs3775291 were significantly different between high and low NLR groups,between high and low LMR groups.The genotype and allele frequency of rs3796508 was significantly different between high and low LMR groups（P<0.05）.（5）The genetic risk model analysis of infectious disease showed that the homozygous mutant TT and heterozygous CT of rs3775291 and the wild genotype GG of rs606231248 increased the risk of developing severe infection and inflammation,while the wild genotype CC of rs3775291,the homozygous mutant AA of rs606231248 reduced the risk of developing infection and inflammation.（6）The results of LD and haplotype analysis showed that there was no linkage disequilibrium relationship between rs3775291 and rs3796508 located on chromosome 4,their haplotype CC was a protective factor for the progression of infection,and haplotypes TC was a risk factor for the progression of infection.Conclusions: The self-built PCR-HRM molecular diagnostic method in this study has good detection performance and can be used for routine detection of clinical specimens from patients with infectious diseases.The correlation analysis shows that rs3775291,rs2072493,rs606231248 and rs3796508 affect the infection severity and inflammation of patients with infectious diseases in Lanzhou.