Association between miRNA‐196a2 rs11614913 T>C polymorphism and Kawasaki disease susceptibility in southern Chinese children

Abstract Background miRNAs play important roles in a variety of diseases. Thus, the association between miRNA‐196a2 rs11614913 T>C polymorphism and Kawasaki disease susceptibility is still unknown. Methods We included 532 children with Kawasaki disease and 623 healthy children from South China, and their DNA was extracted for genotyping by TaqMan methodology. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the strength of association. Results No significant associations were observed between the miRNA‐196a2 rs11614913 T>C polymorphisms and Kawasaki disease risk (TC vs TT: adjusted OR = 1.04, 95% CI = 0.79‐1.37; CC vs TT: adjusted OR = 0.87, 95% CI = 0.63‐1.21; dominant model: adjusted OR = 0.99, 95% CI = 0.76‐1.27; and recessive model: adjusted OR = 0.85, 95% CI = 0.64‐1.13). There was also no significant correlation found in stratified analyses. Conclusion This study suggests that miRNA‐196a2 rs11614913 T>C may not be associated with Kawasaki disease susceptibility in a southern Chinese population. Larger, multicenter studies are needed to confirm our conclusions.


| Study subjects
A total of 532 unrelated Chinese children who went to the Guangzhou Women and Children's Medical Center were diagnosed with Kawasaki disease from January 2012 to January 2017. The control group was composed of 623 healthy Chinese children who went to the hospital for physical examination in the same period. [16][17][18] Peripheral venous blood was taken from each child to extract DNA.
In addition, personal information such as gender, age, and coronary diameter of cases and controls was collected. This study obtained the consent of the Guangzhou Women and Children Medical Center Ethics Committee and the guardian of each child (ethics number: 2014073009).

| DNA extracting and genotyping
We used a Qiagen kit (Qiagen Inc) to extract genomic DNA from blood and stored in a −80°C freezer and genotyped this SNP using the TaqMan real-time PCR method with an ABI-Q6 instrument (Applied Biosystems), as described previously. [21][22][23] For accuracy and reliability, each plate was provided with an eight-well blank control, including equal amounts of distilled water but no DNA.

| Statistical analysis
The chi-square test was used to assess significant differences between the case group and the control group, the risk factors, and the distribution of this SNP in the control and case groups, including additive, dominant, and recessive models. Logistic regression analysis was used to calculate the original odds ratio (OR) value and 95% confidence interval (CI). Stratified analysis was used to analyze subgroups of data, such as gender, age, and coronary artery outcomes.
Data were analyzed using SAS 9.1 software, and P values <0.05 were considered indicative of statistical significance.

| Research population characteristics
The basic information of the case group and the control group is shown in Table S1. The study included 532 KD children and 623 healthy children matched by age (P = 0.602) and gender (P = 0.143).
The incidence of coronary artery aneurysm and coronary artery lesions was 9.59% and 31.58%, respectively. Table 1 shows the distribution of genotypes in the case and control groups. We can see that the genotype distribution in the two groups is equivalent, so there is no significant difference (TT, TC, and CC in the two groups are 28

| Stratification analysis
The results for the stratified analysis are shown in Table 2. No significant difference was found in the stratified analyses of age, gender, and coronary artery outcome.

| DISCUSS IONS
In this study, we found that the miRNA-196a2 rs11614913 T>C may not be associated with Kawasaki disease susceptibility in a southern Chinese population. Over the past 50 years, researchers have tried to find the cause of Kawasaki disease, but it has not been revealed so far. The genetic susceptibility of Kawasaki disease has driven us to study the relationship between miRNA polymorphisms and Kawasaki disease. Our previous study reported that miRNA-146a rs2910164 C>G may not be associated with Kawasaki disease susceptibility in children from South China. 18 We found that miRNA-137 rs1625579 T>G is associated with the onset of Kawasaki disease in the same population in children younger than 1 year old. 16 To our knowledge, this report is the first on the association of miRNA-196a2 rs11614913 T>C with Kawasaki disease susceptibility among children in South China.
Due to the high conservation and tissue specificity of miRNAs, many studies report them as diagnostic markers of diseases such as cancer, 24,25 nervous system disease, 26 immune disease, 27 and cardiovascular disease. 28 miRNA-196a was identified more than a decade ago. 29 SNPs can alter miRNA function by activating miRNA transcription and processing miRNA precursors to alter miRNA expression. 30  lial cell proliferation and prostaglandin production, and plays a protective role in the anti-inflammatory process. 39,40 Annexin A1 is expressed by inflammation-related cells such as white blood cells, mast cells, and vascular endothelial cells. 41 In neutrophils, Annexin A1 is mainly distributed in the cytoplasm. 42 The expression of Annexin A1 in circulating neutrophils was higher in inflammatory cells than that in neutrophils, indicating that Annexin A1 was lost during neutrophil adhesion and migration. 43 In vitro, neutrophils interact with endothelial cells to stimulate Annexin A1 externalization through intercellular adhesion molecules and platelet adhesion molecules. 43 Externalized Annexin A1 binds to formyl peptide receptors to exert anti-inflammatory effects, 44  In our study, it was revealed that miRNA-196a2 rs11614913 T>C may not be associated with Kawasaki disease susceptibility in a southern Chinese population and that there was no significant association in the stratified analysis. The advantage of this study is that it is the first report involving miRNA-196a2 rs11614913 T>C and Kawasaki disease susceptibility. Due to the relatively large number of subjects, the effectiveness of the experimental results is guaranteed. There are also some limitations. First, due to a lack of data, our study examined the incidence of coronary lesions only in children with Kawasaki disease during hospitalization and failed to have long-term follow-up. In the future, we will continue to study and consider these patients. Second, this is a single-center, single-ethnicity study, and as explained in the Discussion, the heterogeneity of the population may produce different results.
Overall, we failed to find any significant association between miRNA-196a2 rs11614913 T>C polymorphism and Kawasaki disease susceptibility in a southern Chinese population. Larger multicenter, multiethnic research is needed to validate our results.

ACK N OWLED G M ENTS
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