Association Of Gene Sigle Nucleotide Polymorphisms With Susceptibility And Severity Of Mycoplasma Pneumoniae Pneumonia In Children

Background:Mycoplasma pneumoniae is a common cause of community-acquired pneumonia(CAP)and the clinical presentation of mycoplasma pneumoniae pneumonia(MPP)varies widely.A better understanding of what determines individual immune responses to M.pneumoniae is therefore crucial.It has been known that different pathogens,as well as variable virulence in different strains of a pathogen/or more pathogens are the most vital factors,while host genetic factors may affect the development and progression of many infectious diseases.Genetic variability affecting the host response may also influence the susceptibility to MPP,even the severity of infection.The relationship between genetic polymorphisms and M.pneumoniae infections has increasingly drawn public attention.Several studies have investigated the association between single nucleotide polymorphism(SNP)of some genes and the risks of CAP,however,the results were inconsistent.Genetic polymorphisms appear to be important in explaining variation in immune response to M.pneumoniae,and specific genes might influence susceptibility to M.pneumoniae and severity of MPP in Chinese children M.pneumoniae infections occur both endemically and epidemically worldwide,with epidemic peaks every 4 to 7 years.PCR-RFLP analys is the most common genotyping method for p1 gene,enabling separation of M.pneumoniae isolates into the two main strain subtypes(type 1 and type 2)that significantly differ in their sequences of the P1 gene.Clinical analysis of M.pneumoniae indicates that the prevalence of type 1 and 2 strains seems to shift in subsequent epidemic peaks.Typing of clinical isolates is practically important for understanding the epidemiology of M.pneumoniae infections and for analysis of endemic outbreaks.The pneumonia caused by M.pneumoniae is often serious and can consume significant resources,and even result in great economic losses and social consequences.Early diagnosis and timely intervention are the key to the management of MPP.So far,there are no reliable,economic,and reproducible screening tests that are available.Objective:The aim of this study was to study risk factors of MPP and whether genetic polymorphisms,typing of clinical isolates and cell factor levels associated with susceptibility and severity of mycoplasma pneumoniae pneumonia in children we investigated the association between genetic polymorphisms loci of 5 functional genes and the risks of MPP for all samples from the subjects of cases and controls,including ACE(rs4340),GSTM1(Ins/del),IL-6(rs1800795),NOS3(rs1799983),and CYP1A1(rs2606345),as well as the gene-gene interactions using the Multifactor Dimensionality Reduction(MDR),and cumulative genetic risk score approaches.The selection of the genes was based on the association with physiological and pathological processes probably involved in MPP,particularly in the immune and inflammatory response.In this study,we genotyped p1 gene from the M.pneumoniae-positive specimens collected from our hospital to get a better understanding of the complete natural process of M.pneumoniae outbreak in Shandong province,China.Our results will contribute to prevention of M.pneumoniae infection in the future.To assess the level of cell factor IL-6 and IL-27,as the new more effective prediction markers of and its recognition ability and early diagnosis of severe MPP.Methods:This study is divided into three parts.In the first part,A total of 715 subjects(415 cases,300 controls)were included in this study.The patients were classified into two levels of 205 severe mycoplasma pneumoniae pneumonia(SMPP)patients and 210 non-severe mycoplasma pneumoniae pneumonia(NSMPP)patients according to the criteria described below.The patients were also classified as single M.peumoniae infection(SMPP)and mixed M.peumoniae and other pathogens infection(MMPP).Five polymorphisms loci of in 5 genes of ACE(rs4340),GSTM1(Ins/del),IL-6(rs1800795),NOS3(rs1799983)and CYP1A1(rs2606345)were analyzed by using tetra-primer allele-specific PCR and Sanger sequencing.In the second part,a total of 516 bronchoalveolar lavage fluid(BALF)specimens collected from from children with CAP,hospitalized at Qilu Children’s Hospital of Shandong University.Quantitative real-time polymerase chain reaction(qRT-PCR)was first used for detecting M.pneumoniae in bronchoalveolar lavage fluid,and 240 specimens were confirmed positive.The positive specimens from 240 children hospitalized with MPP(118 boys and 120 girls,mean age 5.75±1.93 years)were rapidly collected and stored at-80℃ until DNA extract.DNA from clinical samples were extracted with the TIANamp bacteria DNA kit(Tiangen Biotech,Beijing,China),according to the manufacturer’s instructions.The DNA were measured photometrically for concentration and then were stored at-20℃ before PCR was done.The primer pairs used for initial screening for M.pneumoniae in clinical specimens and for optimizing thermal profiles in single-step and nested PCRs for detection and typing of M.pneumoniae.The first-step PCR product was amplified with primers P1-40 and MPAW2.For nested PCR,1-5μL of the first-step PCR product,diluted 1:1 000,were used as the template in the second-step PCR reaction systems.PCR products were analyzed by electrophoresis in 2.5%agarosegels stained with 0.5μL/mL of ethidium bromide.The positive clone was confirmed by restriction digestion and sequencing.In the third part,In order to investigate the relationship between the children mycoplasma pneumonia and alveolar filling cytokines in BALF,we adopt the interleukin 6(IL-6),interleukin 27(IL-7)as standard,selected from the clinical normal people and patients with bronchoalveolar lavage fluid as the control group and the experimental group to express enzyme immunoassay analysis(ELISA)technique for experimental study on the relationship between the above two,through the resulting data can be analyzed the relationship between Mycoplasma pneumoniae and Mycoplasma pneumoniae pneumonia in lavage fluid alveolar lavage.Results:In the first part,We demonstrated that ACErs4340,NOS3rs1799983 and CYP1A1rs2606345 were found three types,but only wild type and heterozygous were detected for IL-6rs1800795 and GSTM1(Ins/del).Three SNPs of ACE rs4340,IL-6 rs1800795,and NOS3 rsl799983 were significantly associated with the risks of MPP.There were no differences in genotype frequencies of GSTM1(Ins/del)and CYP1A1 rs2606345 between both groups.There were statistical significances in the genotypes distributions of ACErs4340 I/D(42.67%vs 54.22%,OR=1.81,95%CI:1.31-2,48,p<0.001),IL-6rs1800795 G/C(1.33%vs 5.78%,OR=4.54,95%CI:1.56-13.23,p=0.0025),NOS3rs1799983 T/T(3.20%vs 8.82%,OR=6.53,95%CI:1.95-21.87,p<0.001)as well as allele distribution of ACErs4340 I(OR=1.32,95%CI:1.06-1.65,p= 0.0137)/NOS3rs1799983 T(OR=6.53,95%CI:1.95-21.87,p=0.0005)/IL-6rs1800795 C(OR=4.44,95%CI:1.53-12.86,p= 0.0027)between the case and control groups(Table 2),indicating that ACErs4340 I/D,IL-6rs1800795 G/C and NOS3rs1799983 G/T were associated with increased risks of MPP.No significant differences in genotype or allele frequencies were found for GSTM1(Ins/del)and CYP1A1rs2606345 G/T between the two groups.We constructed possible allele combinations of ACErs4340,GSTM1(Ins/del),IL-6rs1800795,NOS3rs1799983 and CYP1A1 rs2606345 to analyze gene-gene interactions(Table 3).Several allele combination frequencies were indicated significant differences between cases and controls.When MPP patients were compared with controls,ACErs4340D/NOS3 rs1799983T/CYP1A1 rs2606345G(OR=3.08,95%CI:1.80-5.262);ACErs4340D/NOS3 rs1799983T(OR=3.44,95%CI:2.01-5.89).MDR analysis was used to reveal the SNP-SNP interactions in this cohort of individuals.As a result,ACErs4340,NOS3rsl799983 were also selected as the most potent interaction in the risks of MPP by MDR methods(CV consisitency=9/10).The combination frequencies of ACErs4340 and NOS3rs11799983 genetic polymorphisms in the MPP patients(n =415)were analyzed using the SAS software(version9.1.3 portable).The result showed significant difference between the cases and the controls(OR= 3.32,95%CI:1.88-5.85).The genotype results demonstrated that carriers of the ACErs4340 I allele had a 1.44-fold higher risk for severe MPP(OR=1.44,95%CI:1.05-1.97,p=0.024)compared with non-severe MPP,and carriers of the IL-6 rs1800795 C allele had a 6.9-fold higher risk for severe MPP(OR=6.9,95%CI:2.14-22.22,p= 0.001)compared with non-severe MPP.In the second part,the positive rate of M.pneumoniae in 240 samples was 46.5%(240/516).MPP occurred mostly in preschool and school—age children(63.58%,155/240),and the lowest positive rate was seen in children aged under 23months(10.14%,21/240).The positive rate showed no significant differences between sexes.228 specimens of 240 M.pneumoniae positive samples were M.pneumoniae P1-Ⅰ type,the others were M.pneumoniae P1-Ⅱ type,which were further confirmed by sequencing of some samples.In the third part,The IL-6 level of BALF can reflect the degree of pulmonary infection in patients with severe MP and non severe MP pneumonia,the higher the level of IL-6,the more severe lung infection.IL-27 levels do not reflect the degree of pulmonary infection.Conclusion:1.The polymorphism loci of ACE rs4340,IL-6 rs1800795 and NOS3 rs 1799983 contribute to the genetic susceptibility of MPP in Chinese children.2.The combinations of ACE rs4340D/NOS3 rs1799983T contribute to the genetic susceptibility of MPP in Chinese children.3.In Chinese MPP patients,the polymorphism loci of ACE rs4340,IL-6 rs1800795 and NOS3 rs1799983 allele variants were susceptibility risk factors for MPP,whether single or mixed MPP pathogens.Individuals with the ACE rs4340 I or the IL-6 rs1800795 C had a higher prevalence of severe MPP,might be associated with the severity of MPP.4.During the study period from 2014 to 2016 in Shandong,the M.pneumoniae infection rate is significantly correlated with age.The dominant genotype of M.pneumoniae in children is P1-Ⅰ type,and nor variant strains were identified.The significant differences between genotypes of epidemic strains of M.pneumoniae and serverity of pneumonia were observed in in our local Children(P<0.05).5.The IL-6 level of BALF can reflect the degree of pulmonary infection in patients with severe MPP and non severe MPP,the higher the level of IL-6,the more severe lung infection.IL-27 levels do not reflect the degree of pulmonary infection.