3.1. Demographic profile and clinical characteristics of cases and controls
The demographic profile and clinical characteristics of the patients and controls were summarized in Table 1. Gender ratios (Males/Females) were 48.5/51.5, 57.5/42.5 and 72/28 in HC, T2DM and T2DN, respectively. The mean ages of HC, T2DM and T2DN were 57.57 ± 10.95, 61.64 ± 9.80 and 60.05±10.82 years, respectively. The mean age at onset of diabetes in T2DM was 48.48 ± 9.53 years, in T2DN groups was 46.64 ± 9.96 years. Age at onset of T2DM, BMI, HDL cholesterol, Urea, Creatinine and Urea/Creatinine ratio showed significant variation among the T2DN vs. T2DM group (t-test, p < 0.05).
3.2. Genotyping and Sequencing analysis
As shown in Fig 1, the normalized and temperature-shifted difference plots for rs3761548C/A and rs2294021C/T were performed. Wild genotype, heterozygous and homozygous mutations can be accurately identified on the difference plot curves. Direct sequencing results confirmed the high-resolution melting (HRM) analysis.
3.3. Genotype distributions and allelic frequencies
The results of PCR-HRM for genotype and allelic distributions were illustrated in Table 2. With respect to rs3761548C/A, the genotype distribution of CC, CA, AA showed 67%, 14%, 19% in patients (72%, 19%, 9% in T2DM, 62.5%, 9%, 28.5% in T2DN), and 72.5%, 18.5%, 9% in the controls. Statistical significance was observed regarding genotype frequencies for both HC vs. patients (χ2=10.452; p=0.005) and T2DM vs. T2DN (χ2=28.765; p=0.000). Furthermore, a significant correlation was noted in allele frequencies of C and A (74% and 26% in patients, 81.5% and 18.5% in T2DM, 67% and 33% in T2DN, 82% and 18% in the control group respectively), showing an odds ratio of 0.625 (95% CI=0.463-0.843; p=0.002) in HC vs. patients and 0.461 (95% CI=0.332-0.639; p=0.000) in T2DM vs. T2DN. The wild type CC vs. other genotypes presented an odds ratio of 0.530 (95% CI=0.354-0.791; p=0.002), 0.648(95% CI=0.425-0.987; p=0.043) and homozygous AA vs. others genotype exhibited an odds ratio of 4.897 (95% CI=2.398-10.003; p=0.000), 4.030 (95% CI=2.272-7.151; p=0.000) in HC vs. patients and T2DM vs. T2DN respectively. As for rs2294021, we obtained the genotype frequencies of CC, CT and TT were 41.5%, 17%, 41.5% in patients and 41%, 27.5%, 31.5% in controls respectively, showing a significant difference in HC vs. patients (χ2=10.672; p=0.005) while no statistical difference was observed in allelic distribution. Both genotype and allelic frequencies differed significantly from T2DM to T2DN subjects (P=0.002; P=0.002). Further analysis demonstrated participants with the heterozygote CT genotype (OR=0.483; 95% CI=0.281-0.831; p=0.009) were protected while those with homozygous TT genotype were at higher risk to develop T2DN (OR=1.946; 95% CI=1.299-2.914; p=0.001). Similar conclusions on the roles of CT and TT genotypes were drawn in HC vs. Patients group (OR=0.540; 95% CI=0.360-0.816; p=0.003; OR=1.543; 95% CI=1.078-2.208; p=0.018).
3.4. The gender-based evaluation
As FOXP3 gene is X-linked, three groups (T2DM, T2DN and HC) were stratified by gender and performed the statistical analysis. Further gender-based data was performed in Table 3. At position rs3761548C/A in females, genotypes did not show significant difference between female controls and patients, while allele frequencies C and A (82% and 18% in HC, 73% and 27% in patients) exhibited an odds ratio of 0.593 (95% CI=0.381-0.924; p=0.021). It can be found that higher AA (14% vs. 4%) and lower CA (27% vs. 42%) at T2DN compared to T2DM, and a statistical significance in genotype frequencies (χ2=9.503; p=0.009), independent of female allele frequency. Odds ratios of 3.168 (95% CI=1.027-9.777; p=0.045) and 4.556 (95% CI=1.153-17.997; p=0.031) were observed for homozygous AA vs. others in female HC vs. patients and T2DM vs. T2DN respectively. Concerning rs3761548C/A in male subjects, the frequencies of CC, CA, AA genotypes were 92%, 3%, 5% in the control group, 73%, 4%, 23% in patients, 85%, 2%, 13% in T2DM group, and 62%, 7%, 31% in T2DN, respectively. A significant difference between HC vs. patients (χ2=15.923; p=0.000) and T2DM vs. T2DN (χ2=15.944; p=0.000162) can be observed. The higher percentage of male patients and T2DN were A carriers compared to the male controls and T2DM respectively. Additionally, we noticed that CC vs. other genotypes with an odds ratio of 0.238 (95% CI=0.110-0.516; p=0.000), 0.307 (95% CI=0.166-0.569; p=0.000176) and homozygous AA vs. other genotypes with OR of 5.428(95% CI=2.108-13.975; p=0.000), 3.439 (95% CI=1.808-6.540; p=0.000166) in male HC vs. patients and T2DM vs. T2DN was noticed, respectively.
For rs2294021C/T polymorphism, neither genotypes nor allele significantly differed as a slight increase in patients with variant allele T compared with wild allele C in both female HC vs. patients and T2DM vs. T2DN. In male subjects, the statistical significance of allele frequencies was denoted between controls and patients (p=0.010), yet no variation in genotype frequencies (p > 0.05). Moreover, analysis of the genotype distribution (χ2=20.454; p=0.000) and allele frequencies (OR=0.621,95% CI=0.438-0.881; p=0.008) suggested there were significant correlations between T2DM and T2DN group. Meanwhile, we noticed that CT genotype may play a protective role in the progression of T2DM towards T2DN (OR=0.081; 95% CI=0.018-0.359; p=0.001), while the variant homozygote type did the opposite effect (OR=2.117; 95% CI=1.284-3.488; p=0.003).
3.5. The haplotype combination analysis
A comparison of combined genotype combinations of rs376548C/A and rs2294021C/T polymorphisms in patients and controls was shown in Fig 2. The AA-CC vs. others and AA-TT vs. others genotype combinations showed an odds ratio of 3.793 (95% CI = 1.584-9.082; p = 0.003) and 5.517 (95% CI = 1.666-18.273; p = 0.005) in HC vs. patients, 3.619 (95% CI = 1.727-7.584; p = 0.001) and 3.760 (95% CI = 1.581-8.942; p = 0.003) in T2DM vs. T2DN respectively, suggesting that both AA-CC and AA-TT genotypes could be risk combinations, whereas CC-CT genotype was considered as a protective combination as its OR value of 0.600 (95% CI = 0.365-0.986; p = 0.044) in HC vs. patients and 0.287 (95% CI = 0.137-0.603; p = 0.001) in T2DM vs. T2DN.
Regarding males, the AA-CC vs. others and AA-TT vs. others displayed an odds ratio of 2.970 (95% CI = 1.344-6.564; p = 0.007) and 2.930 (95% CI = 1.136-7.561; p = 0.026) in HC vs. patients, 5.046(95% CI = 2.035-12.512; p = 0.000) and 3.093 (95% CI = 1.460-6.554; p = 0.003) in T2DM vs. T2DN respectively (Fig 2. A&B), while the CC-CC genotype combinations showed an OR value of 0.418 (95% CI = 0.258-0.676; p = 0.000378) in male HC vs. patients, the CC-CC vs. others and CC-CT vs. others had OR value of 0.407 (95% CI = 0.234-0.708; p = 0.001) and 0.040 (95% CI = 0.005-0.308; p = 0.002) in male T2DM vs. T2DN respectively (Fig 2. C&D). Further evaluation of allele combinations of C-C vs. others (OR = 0.378, 95% CI =237-0.603; p = 0.00046), A-C vs. others (OR = 3.504, 95% CI =1.343-9.145; p = 0.010), and A-T vs. others (OR = 4.280, 95% CI = 1.277-14.344; p = 0.018) between male T2DM and T2DN groups were described (Fig 2. E&F). Pertaining to females, neither statistically significance in combined genotypes nor any differences in allele combinations among female individuals was observed.