Genetic polymorphisms of rs9313422 G>C and rs41297579 G>A at the promoter of TIM‐1 gene contribute to the risk of community‐acquired pneumonia in children

Abstract Objective To investigate the association of genetic polymorphisms of rs9313422 G>C and rs41297579 G>A at the promoter of TIM‐1 gene with the risk of community‐acquired pneumonia (CAP) in children. Methods A total of 112 children with CAP were included as the case group. Another 120 healthy children were enrolled as the control group. Polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) was applied for the genotyping of rs9313422 G>C and rs41297579 G>A in the promoter region of TIM‐1. Results rs9313422 G>C was related to the risk of CAP in children under codominant model, dominant model, recessive model, and allele model. Besides, the A allele of rs41297579 G>A could increase the risk of CAP in children. Besides, the haplotype GA (rs9313422‐rs41297579) and GG reduced the risk of children CAP, while haplotype CA had an elevated risk. rs9313422 G>C and rs41297579 G>A polymorphisms were both associated with the severity of CAP in children, and the rs9313422 G>C was also related to the ICU admission rate. In addition, patients carried with the mutant homozygotes of rs9313422 G>C and rs41297579 G>A showed higher levels of white blood cell (WBC), procalcitonin (PCT), and C‐reactive protein (CRP) than the wild type and heterozygous genotypes carriers. Conclusion rs9313422 G>C and rs41297579 G>A polymorphisms in the promoter region of TIM‐1 could increase the risk of CAP in children and showed a relation with inflammatory responses and severity.

respiratory infections (ARI), approximately accounting for 80% of all deaths suffering from this disease, 5 which was more frequent in developing countries. 6 Moreover, the incidence rate of CAP in children was nearly 22%, and there were about 55,000 children deaths every year caused by CAP in China. 7 Although the pathological mechanism of CAP has not been clearly elucidated, many researchers have focused their attentions on genetic factors in CAP. 8 For instance, the GG genotype and G allele of −174 G/C in IL-6 (an anti-inflammatory cytokine) had a close relation to the susceptibility of CAP in Egyptian children, as suggested by Zidan et al 9 The human T-cell immunoglobulin mucin (TIM) gene family, located on the human chromosome 5q23-35, contains three members, including TIM-1, TIM-3, and TIM-4, 10 which was shown to be relevant to the immune dysfunction, consequently inducing various diseases, such as autoimmune diseases, allergic diseases, chronic viral infections (CVI), immune rejections, and even tumors. 11 As the first identified gene, TIM-1, also known as HAVCR1, was responsible for regulating immune responses and maintaining immune homeostasis, which had relations to several human inflammatory diseases, 12  A virus (HAV)-induced severe liver disease. 15 More importantly, the two common SNPs (−1454 G>A (rs41297579) and −416 G>C (rs9313422) within TIM-1 were associated with respiratory diseases, like asthma, 16,17 but it has not been explored whether they have something to do with the susceptibility to CAP. Given the above, 112 children with CAP and 120 healthy controls were recruited as the subjects of study, and polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) was used for the genotyping, to investigate the relationship between SNPs including rs9313422 G>C and rs41297579 G>A in the promoter region of TIM-1 and the risk of in children with CAP.

| Ethics statement
This study got the approval of the Ethics Committee in Maternal and Child Health Hospital of Jingzhou City and strictly followed the Helsinki declaration. 18 The guardians of all patients signed an informed consent prior to study and agreed to the information collection and blood collection in clinical trials.

| Subjects of study
From May 2015 to May 2017, a total of 112 children with CAP in Maternal and Child Health Hospital of Jingzhou City were included as case group. All CAP patients met the diagnostic criteria of CAP created by the pediatric infectious diseases society and the infectious diseases society of America 19 as follows: (1) newly acquired cough and sputum or pre-existed respiratory tract symptoms aggravating due to purulent sputum, with or without chest pain; (2) fever; (3) signs of pulmonary consolidation and/or moist rale; (4) white blood cell count > 10 × 10 9 /L or < 4 × 10 9 /L, with or without nucleus shift to left; and (5) invasive shadow in patches detected by chest X-ray, with or without pleural effusion. Clinical diagnosis can be identified when patients met one criteria of (1)-(4) and (5). And the pneumonia of patients can be identified as community-acquired if they had no history of hospitalization during the 2 weeks prior to admission. In addition, the control group consisted of another 120 healthy children who underwent physical examination during the same period in our hospital.

| Inclusion and exclusion criteria
All subjects were unrelated Chinese (Han nationality), who were newly diagnosed and treated. The exclusion criteria were as follows: patients who combined with respiratory diseases, such as tuberculosis, non-infectious interstitial lung diseases, pulmonary edema, pulmonary atelectasis, pulmonary embolism, pulmonary eosinophilia, and pulmonary vasculitis, and particularly asthma; patients who were with a history of surgery or operations, serious dysfunction of heart, liver, kidney, lung or other organs, immunodeficiency, tumors or a history of tumor, hematologic disorders, severe damage in central nervous system, diabetes mellitus, obesity, or other infectious diseases; patients who exhibited poor compliance in the blood collection process; and patients who were unsuitable for intravenous blood sampling.

| Isolation of genomic DNA
Fasting peripheral sample (3 mL) was extracted from peripheral venous blood of all subjects, which were anti-coagulated with the addition of EDTA and centrifuged for 10 minutes at the rate of 1500 g.
Next, DNA extraction kit (Takara Company) was used to extract the whole blood DNA followed by the determination for the purity by a DNA concentration detector (NANODROP2000, Thermo Company), with the A260/A280 ratio between 1.6 and 1.8. The DNA extracted was preserved at −20°C in a refrigerator.

| Statistical method
Data processing was performed with the statistical software SPSS 21.0 (SPSS Inc). Hardy-Weinberg equilibrium was applied to evaluate the representativeness of rs9313422 G>C and rs41297579 G>A in the Case and control groups. Logistic regression analysis was used to calculate the odd ratio (OR) and 95% confidence intervals (CI), so as to estimate the strength of association between the mutation of polymorphic loci and CAP in children. Enumeration data were presented by rate or ratio, which was compared by using Chi-Square (χ 2 ) test. Measurement data were presented by mean ± standard deviation (x ±s). Comparison between two groups was analyzed by Student's ttest. Comparison among multiple groups was conducted by using oneway ANOVA followed by using Tukey's post hoc test. A two-tailed P value < .05 indicated that the difference was of statistical significance.

| Baseline characteristics of subjects
The baseline characteristics of 112 CAP patients in the case group were shown in Table 1. There are 64 males and 48 females with the average age of 57.55 ± 37.17 months (3-118 months). In addition, healthy subjects in the control group were consisted of 62 males and 58 females with the average age of 57.00 ± 31.63 months (4-110 months).
Obviously, no statistical difference was found between the case group and the control group in age and gender (both P > .05).

| Genotyping and sequencing of rs9313422 G>C and rs41297579 G>A in the promoter region of TIM-1
The PCR amplification product of rs9313422 G>C was 879bp in length. The digestion products showed three genotypes, including CC (a single uncuttable fragment of 879bp), GG (two fragments of 488bp and 391bp), and GC (three fragments of 879bp, 488bp, and 391bp), as shown in Figure 1A. After DNA sequencing, it was confirmed that GG genotype had only one G peak, CC genotype had only one C peak, while GC genotype had both a G peak and a C peak ( Figure 1B-C). In addition, the PCR amplification product of rs41297579 G>A was 515bp, and MspI cut the 515bp PCR product into two fragments of 399bp and 116bp length. A single 515bp band exhibited the presence of AA genotype; fragments 399bp and 116bp indicated the presence of the GG genotype and three fragments of 515bp, 399bp, and 116bp displayed the presence the GA genotype ( Figure 2A). Meanwhile, DNA sequencing showed that GG genotype had only one G peak, AA genotype had only one A peak, while GA genotype had both a G peak and an A peak ( Figure 2B-C).

| Association of TIM-1 rs9313422 G>C and rs41297579 G>A polymorphisms with the risk of CAP in children
Hardy-Weinberg equilibrium (HWE) test showed that the genotype frequencies of TIM-1 rs9313422 G>C and rs41297579 G>A in the control group were in genetic equilibrium, indicating that the sample analyzed was representative of the population (Table 2). Besides, as illustrated in Table 3 (Table 4).

| Association of TIM-1 rs9313422 G>C and rs41297579 G>A polymorphisms with the clinical characteristics of CAP in children
Stratified analysis was carried out based on the clinical characteristics of CAP in children (Table 5), and the result demonstrated that under the dominant model, both rs9313422 G>C and rs41297579 G>A polymorphisms were related to the severity of CAP in children (all P < .05), but they had no correlations with gender, identified pathogens and mortality of children with CAP (all P > .05). In addition, rs9313422 G>C had an impact on the ICU admission of patients (P < .05).

| Relationship between TIM-1 rs9313422 G>C and rs41297579 G>A polymorphisms and inflammatory responses of CAP in children
As presented in Figure 3,

| D ISCUSS I ON
In recent years, a large body of studies has found that the genetic polymorphisms of TIM-1 were correlated with the risk of a variety of diseases, including asthma, 20 hepatitis C persistence, 21 childhood atopic dermatitis, 22 and systemic lupus erythematosus (SLE). 23 In this study, we analyzed two SNPs in the human promoter rs9313422 G>C may function as a predisposing factor for atopic asthma and childhood allergic asthma. 16,17 In the study by Xie et al, the rs9313422 CC genotype and rs41297579 AA genotype could greatly affect the susceptibility and prognosis of dilated cardiomyopathy, 11 indicating that the gene mutation of rs9313422 G>C and rs41297579 G>A might be a risk factor for multiple diseases along the above evidence, as well as in children with CAP. As we know, both of the two SNPs were located in the promoter region TA B L E 4 Haplotype association analysis to CAP children using the two SNPs (rs9313422 G>C -rs41297579 G>A) of TIM-1, 25  Furthermore, we used the online software SHEsis to carry out haplotype analysis, and the result demonstrated both haplotypes GA and GG could effectively reduce the risk of CAP in children.
As such, we hypothesized that the TIM-1 promoter haplotype together with T-cell receptor can induce the production of anti-inflammatory Th2 cytokines, and thus having a protective effect on CAP; while CA genotype can significantly increase the risk of childhood CAP.
As one of the most common community-acquired infections, CAP usually means the necessity of ICU admission. 27 Severe CAP is a major cause of patients' mortality, and about 12%-36% CAP patients in the ICU die within a short period despite the application of effective antibiotic therapies. 28  severe inflammatory responses, and consequently affecting the disease severity and prognosis of these children.
In summary, the C allele of rs9313422 G>C and A allele of rs41297579 G>A in the promoter region of TIM-1 could increase the risk of CAP in children, which were associated with the inflammatory responses and disease severity. Besides, the haplotypes GA (rs9313422-rs41297579) and GG could reduce the risk of CAP in children, while CA could elevate the risk.

ACK N OWLED G M ENTS
We would like to give our sincere appreciation to the reviewers for their helpful comments on this article.

AUTH O R CO NTR I B UTI O N
Yang Liu designed the study, analyzed the data, and wrote the manuscript. Hong-Bo Xu conducted the experiment, analyzed the data, and revised the manuscript. All of them approved the final manuscript.