Study subjects From January 2014 to December 2018, 95 laboratory confirmed PM patients, comprising 62 males and 33 females in Department of Infectious Diseases, Beijing Children’s Hospital, Beijing, China were enrolled as the case group. The median age was 19 months (range, 2 months to 13 years). All PM patients met the standards PM diagnosis as established by European Society of Clinical Microbiology and Infectious Diseases (ESCMID) . In addition, 330 healthy adults (168 males and 162 females) who attended annual medical examination in June 2015 were randomly selected as the control group. The age of the control group ranged from 21 to 60 years with a median age of 38 years. All study subjects were Chinese from different families and thus considered as unrelated individuals.
Clinical data collection Clinical characteristics of the study subjects were summarized in Table 1. According to electronic medical records (EMR), Chinese Han descendants were selected. Clinical information including fever, consciousness and seizures were obtained by an experienced pediatrician at the Department of Infectious Diseases, Beijing Children’s Hospital. Other clinical information on blood routine, cerebrospinal fluid (CSF) routine and biochemical examination, imaging examination, hospitalization time and other clinical information for treatment were all from EMR.
Sample collection and bacterial culture In the first three days of admission, peripheral blood and CSF samples were taken aseptically. To identify the pathogens, bacterial culture, Gram staining, acid fast staining and S. pneumoniae antigen test were performed in addition to the routine biochemical analysis. All pathogen classification and identification follow the standard laboratory identification method.
DNA extraction Genomic DNA was extracted from 200μL of whole blood samples in EDTA tubes using a DNA purification kit (QIAamp DNA Blood Mini Kit; Qiagen, Germantown, MD, USA) according to manufacturer’s instructions. The extracted DNA was kept at −20°C until use.
SNPs analysis The primers used in this study were designed using the Primer Premier 6.0 software. The forward primer used was 5’-TCATTCACCTCAGTGGGGGTA-3’
and the reverse primer was 5’-ATGGATGAGTTTGTGCCTGCT-3’. The product size was 967bp. The total reaction volume of 50μL contained 5μL of 10×buffer, 1μL of each primer, 2μL (15-20ng/μL) of genomic DNA, 1μL of dNTP mixture, 0.4μL of Pfu DNA Polymerase and 40.6μL of deionized water. Amplification conditions consisted of initial denaturation at 95°C for 2min followed by 42 cycles of 95°C for 30s, 54°C for 30s, and 72°C for 2min, with a final extension for 5min at 72°C. The amplified products were examined by agarose gel electrophoresis and sequencing. Both positive and negative controls were used in each PCR run. The amplified products were sequenced at the Tsingke Biological Technology Co., Ltd, Beijing, China. The SNPs in interests were determined using Bioedit sequence alignment editor version 7.2.1. In order to ensure accuracy, one out of every 10 samples was selected for PCR and sequencing for the second time.
Construction of luciferase reporter plasmids Three luciferase reporter plasmids were constructed to explore whether IL-17A polymorphisms had an effect on IL-17A gene expression. The constructs of a 1663bp DNA fragment corresponding to the upstream region of TFBS of IL-17A, which were amplified from individual homozygous templates and were cloned into the pGL3-basic luciferase vector (Promega, Madision, USA). The vectors were then sequenced to confirm that there were no nucleotide errors.
Transient transfections and luciferase assays HEK293T cells were seeded in 24-well plates, and each well was transfected with 0.5μg of the vector DNA containing haplotype A-C-G, A-C-A or A-T-G of rs4711998 (-877A>G), rs8193036 (-737C>T) and rs2275913 (-197G>A), and 0.05μg of pGL4.74 which contains the Renilla luciferase gene by Viafect (Promega, Madision, USA), according to the manufacture’s instruction. Cells were collected 24h after transfection, and luciferase activity was measured with a dual Luciferase reporter assay system (Promega, Madision, USA) and was normalized against the activity of the Renilla luciferase gene. Independent triplicate experiments were performed for each plasmid.
Serum cytokines measurement Of the 95 cases studied, 63 had sera available. The Luminext kit was used for measuring IL-17A and CXCL-1. The measurement run was done by Luminext MAGPIX system (Luminext) according to manufacturer’s instructions. Duplicate wells for each sample were did and a volume of 50μL was used per well. Samples were diluted to 1:2 with sample diluent provided by the manufacturer before added into wells. The detection limit of IL-17A and CXCL-1 were 2.3pg/mL and 3.0pg/mL, respectively.
Statistical analysis Allelic and genotypic frequencies of each SNP studied were calculated. All of the SNPs analyzed were found to be in HWE (P>0.05). Categorical variables were analyzed by χ2 test or Fisher’s exact test as appropriate. Univariate analysis was performed for continuous variables with Mann-Whitney U test or Student’s t-test, The Odds Ratio (OR) and 95% confidence interval (CI) were calculated using unconditional binary logistic regression. Genotypes found to be statistically significant by univariate analysis were further analyzed by logistic regression. A two-tailed P value less than 0.05 was considered as significant. Bonferroni correction was employed for multiple testing correction and P < 0.05/n (n =the number of comparisons) was considered significant. All the statistical analyses were conducted using the SPSS software, version 23.0 (IBM, Armonk, NY, USA). Estimation of haplotype frequencies and haplotype association analysis with the χ2 test were performed with SHEsis (http://analysis.bio-x.cn). The statistical analyses were carried out by using Graph-Pad Prism 6 (San Diego, CA).