Characteristics of Study Population
Baseline demographic and clinical features were shown in Table 1. A total of 477 IS (316 males and 161 females) patients and 480 healthy controls (313 males and 167 females) were recruited for the study. The mean age was 64.13 ± 10.82 years for IS patients and 63.69 ± 6.69 years for the control subjects. The distribution of age and gender was no significant differences between two groups (p = 0.443 and p = 0.735, respectively). There were no significant differences between cases and controls in levels of triglyceride, HDL-C, hemoglobin, PLT; but significant differences for the levels of total protein, total bilirubin, total cholesterol, LDL-C and TLC were found. Cases included 340 patients (71.3%) with hypertension and 103 patients (21.6%) with coronary heart disease.
Table 1
Characteristics of patients with IS patients and controls
Variable | Cases (n = 477) | Controls ( n = 480) | p |
Age, year (mean ± SD) | 64.13 ± 10.82 | 63.69 ± 6.69 | 0.443 |
Gender,(male/female), n | 316/161 | 313/167 | 0.735 |
Total protein (g/L) | 53.50 ± 24.36 | 66.40 ± 18.33 | < 0.001 |
Total bilirubin (µmol/L) | 12.35 ± 7.55 | 14.85 ± 6.99 | 0.001 |
Triglyceride (mmol/L) | 1.61 ± 0.96 | 1.80 ± 1.40 | 0.080 |
Total cholesterol (mmol/L) | 3.94 ± 1.02 | 5.50 ± 7.90 | 0.020 |
HDL-C (mmol/L) | 1.18 ± 0.32 | 1.19 ± 0.30 | 0.696 |
LDL-C (mmol/L) | 1.87 ± 0.60 | 2.69 ± 0.73 | < 0.001 |
Hemoglobin (g/L) | 122.87 ± 45.41 | 129.79 ± 37.40 | 0.073 |
TLC (109/L) | 7.84 ± 8.27 | 5.72 ± 1.40 | < 0.001 |
PLT (109/L) | 152.89 ± 85.51 | 150.18 ± 94.77 | 0.763 |
Hypertension, n | 340 (71.3%) | | |
Coronary heart disease, n | 103 (21.6%) | | |
IS; ischemic stroke; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TLC, total leukocyte count; PLT, platelets. |
Bold indicate that p < 0.05 indicates statistical significance. |
Association of ZSCAN25 and CYP2E1 polymorphism with IS risk.
Three selected SNPs were successfully genotyped, and the success rate of genotyping was > 99.2%. All genotype distribution of the studied SNPs in controls was in HWE (p > 0.05, Supplementary Table 2). The allelic and genotype frequencies for selected SNPs between cases and controls were shown in Table 2. The results of association analysis showed that ZSCAN25 rs10242455 was a protect factor for IS susceptibility; while rs2070672 and rs2515641 in CYP2E1 conferred to the increased risk for IS occurrence. For rs10242455, G allele (G vs A: OR = 0.81, 95% CI: 0.66–0.99, p = 0.036; and AA + AG + GG: OR = 0.80, 95% CI: 0.66–0.98, p = 0.033) and GG genotype (GG vs AA: OR = 0.56, 95% CI: 0.34–0.93, p = 0.024; and GG vs AA-AG: OR = 0.60, 95% CI: 0.36–0.97, p = 0.039) were related to the reduced IS risk. The significant association of rs2070672 polymorphism with IS susceptibility was found under the allele (OR = 1.40, 95% CI: 1.12–1.75, p = 0.003), genotype (OR = 2.81, 95% CI: 1.37–5.77, p = 0.005), dominant (OR = 1.38, 95% CI: 1.06–1.80, p = 0.017), recessive (OR = 2.58, 95% CI: 1.26–5.27, p = 0.009), additive (OR = 1.41, 95% CI: 1.12–1.77, p = 0.003) models. In addition, we also found that rs2515641 was increased IS occurrence (T vs C: OR = 1.27, 95% CI: 1.00–1.62, p = 0.046; and CC + CT + TT: OR = 1.29, 95% CI: 1.01–1.64, p = 0.041).
Table 2
Association between ZSCAN25 and CYP2E1 polymorphisms and IS risk
SNP ID | Model | Genotype | Case | Control | Adjusted by age and gender |
OR (95%CI) | p |
ZSCAN25 rs10242455 | Allele | A | 705 | 668 | 1 | |
| G | 249 | 292 | 0.81 (0.66–0.99) | 0.036 |
Genotype | AA | 256 | 233 | 1 | |
| AG | 193 | 202 | 0.87 (0.67–1.14) | 0.305 |
| GG | 28 | 45 | 0.56 (0.34–0.93) | 0.024 |
Dominant | AA | 256 | 233 | 1 | |
| AG-GG | 221 | 247 | 0.81 (0.63–1.05) | 0.112 |
Recessive | AA-AG | 449 | 435 | 1 | |
| GG | 28 | 45 | 0.60 (0.36–0.97) | 0.039 |
Log-additive | --- | --- | --- | 0.80 (0.66–0.98) | 0.033 |
CYP2E1 rs2070672 | Allele | A | 734 | 787 | 1 | |
| G | 220 | 169 | 1.40 (1.12–1.75) | 0.003 |
Genotype | AA | 284 | 320 | 1 | |
| AG | 166 | 147 | 1.28 (0.97–1.68) | 0.082 |
| GG | 27 | 11 | 2.81 (1.37–5.77) | 0.005 |
Dominant | AA | 284 | 320 | 1 | |
| AG-GG | 193 | 125 | 1.38 (1.06–1.80) | 0.017 |
Recessive | AA-AG | 450 | 467 | 1 | |
| GG | 27 | 11 | 2.58 (1.26–5.27) | 0.009 |
Log-additive | --- | --- | --- | 1.41 (1.12–1.77) | 0.003 |
CYP2E1 rs2515641 | Allele | C | 762 | 805 | 1 | |
| T | 182 | 151 | 1.27 (1.00–1.62) | 0.046 |
Genotype | CC | 307 | 336 | 1 | |
| CT | 148 | 133 | 1.22 (0.92–1.62) | 0.164 |
| TT | 17 | 9 | 2.10 (0.92–4.79) | 0.078 |
Dominant | CC | 307 | 336 | 1 | |
| CT-TT | 165 | 142 | 1.28 (0.97–1.68) | 0.080 |
Recessive | CC-CT | 455 | 469 | 1 | |
| TT | 17 | 9 | 1.98 (0.87–4.49) | 0.103 |
Log-additive | --- | --- | --- | 1.29 (1.01–1.64) | 0.041 |
IS; ischemic stroke; SNP, single nucleotide polymorphism; OR, odds ratio; 95% CI, 95% confidence interval. |
p values were calculated by logistic regression analysis with adjustments for age and gender. |
Bold indicate that p < 0.05 means the data is statistically significant. |
Stratification analysis by age and sex for the association of ZSCAN25 and CYP2E1 variants with IS risk
Considering age and sex as a potential risk factor for IS, stratification analysis by age and sex was performed to estimate potential effect of age and gender on the association between ZSCAN25 and CYP2E1 variants and IS risk (Table 3). Among the subgroup with age > 64 years, CYP2E1 rs2070672 GG genotype was observed to correlate with the risk of IS compared with AA genotype (OR = 4.06, 95% CI: 1.08–15.26, p = 0.038) or AA-AG genotype (OR = 3.90, 95% CI: 1.05–14.55, p = 0.043). In the subgroup at age ≤ 64 years, ZSCAN25 rs10242455 also had a protective effect on IS susceptibility (G vs A: OR = 0.75, 95% CI: 0.57–0.98, p = 0.035; and AA + AG + GG: OR = 0.74, 95% CI: 0.55–0.99, p = 0.045). In addition, we observed an increased IS risk for CYP2E1 rs2070672 under the allele (OR = 1.41, 95% CI: 1.05–1.90, p = 0.023), dominant (OR = 1.49, 95% CI: 1.02–2.17, p = 0.038), and additive (OR = 1.46, 95% CI: 1.06–2.02, p = 0.021) models at age ≤ 64 years.
Table 3
Association between ZSCAN25 and CYP2E1 polymorphisms and IS risk according to the stratification by gender and age
SNP ID | Model | Genotype | Case | Control | OR (95%CI) | p | Case | Control | OR (95%CI) | p |
Age | | | > 64 years | ≤ 64 years |
ZSCAN25 rs10242455 | Allele | A | 330 | 270 | 1 | | 375 | 398 | 1 | |
| G | 128 | 120 | 0.87 (0.65–1.17) | 0.368 | 121 | 172 | 0.75 (0.57–0.98) | 0.035 |
Genotype | AA | 118 | 96 | 1 | | 138 | 137 | 1 | |
| AG | 94 | 78 | 0.90 (0.59–1.38) | 0.639 | 99 | 142 | 0.74 (0.50–1.08) | 0.113 |
| GG | 17 | 21 | 0.58 (0.28–1.21) | 0.148 | 11 | 24 | 0.54 (0.25–1.18) | 0.125 |
Dominant | AA | 118 | 96 | 1 | | 138 | 137 | 1 | |
| AG-GG | 111 | 99 | 0.83 (0.56–1.25) | 0.376 | 110 | 148 | 0.71 (0.49–1.02) | 0.062 |
Recessive | AA-AG | 212 | 174 | 1 | | 237 | 261 | 1 | |
| GG | 17 | 21 | 0.61 (0.30–1.24) | 0.170 | 11 | 24 | 0.62 (0.29–1.33) | 0.221 |
Log-additive | --- | --- | --- | 0.82 (0.60–1.11) | 0.199 | --- | --- | 0.74 (0.55–0.99) | 0.045 |
CYP2E1 rs2070672 | Allele | A | 355 | 321 | 1.00 | | 379 | 466 | 1 | |
| G | 103 | 67 | 1.39 (0.99–1.96) | 0.059 | 117 | 102 | 1.41 (1.05–1.90) | 0.023 |
Genotype | AA | 139 | 130 | 1 | | 145 | 190 | 1 | |
| AG | 77 | 61 | 1.13 (0.73–1.75) | 0.586 | 89 | 86 | 1.41 (0.95–2.08) | 0.086 |
| GG | 13 | 3 | 4.06 (1.08–15.26) | 0.038 | 14 | 8 | 2.43 (0.93–6.32) | 0.070 |
Dominant | AA | 139 | 130 | 1 | | 145 | 190 | 1 | |
| AG-GG | 90 | 64 | 1.27 (0.83–1.93) | 0.275 | 103 | 94 | 1.49 (1.02–2.17) | 0.038 |
Recessive | AA-AG | 216 | 191 | 1 | | 234 | 276 | 1 | |
| GG | 13 | 3 | 3.90 (1.05–14.55) | 0.043 | 14 | 8 | 2.15 (0.83–5.55) | 0.113 |
Log-additive | --- | --- | --- | 1.36 (0.94–1.97) | 0.098 | --- | --- | 1.46 (1.06–2.02) | 0.021 |
Sex | | | Male | Female |
ZSCAN25 rs10242455 | Allele | A | 474 | 446 | 1 | | 231 | 222 | 1 | |
| G | 158 | 180 | 0.83 (0.64–1.06) | 0.133 | 91 | 112 | 0.78 (0.56–1.09) | 0.144 |
Genotype | AA | 172 | 162 | 1 | | 84 | 71 | 1 | |
| AG | 130 | 122 | 1.00 (0.72–1.39) | 0.983 | 63 | 80 | 0.66 (0.42–1.05) | 0.077 |
| GG | 14 | 29 | 0.45 (0.23–0.89) | 0.021 | 14 | 16 | 0.71 (0.32–1.56) | 0.391 |
Dominant | AA | 172 | 162 | 1 | | 84 | 71 | 1 | |
| AG-GG | 144 | 151 | 0.9 (0.66–1.23) | 0.501 | 77 | 96 | 0.67 (0.43–1.04) | 0.072 |
Recessive | AA-AG | 302 | 284 | 1 | | 147 | 151 | 1 | |
| GG | 14 | 29 | 0.45 (0.23–0.87) | 0.018 | 14 | 16 | 0.86 (0.41–1.84) | 0.705 |
Log-additive | --- | --- | --- | 0.82 (0.64–1.06) | 0.129 | --- | --- | 0.76 (0.54–1.07) | 0.120 |
CYP2E1 rs2070672 | Allele | A | 495 | 518 | 1.00 | | 239 | 269 | 1 | |
| G | 137 | 104 | 1.38 (1.04–1.83) | 0.026 | 83 | 65 | 1.44 (0.99–2.08) | 0.053 |
Genotype | AA | 194 | 212 | 1 | | 90 | 108 | 1 | |
| AG | 107 | 94 | 1.24 (0.89–1.75) | 0.207 | 59 | 53 | 1.33 (0.84–2.13) | 0.225 |
| GG | 15 | 5 | 3.28 (1.17–9.21) | 0.024 | 12 | 6 | 2.37 (0.85–6.59) | 0.098 |
Dominant | AA | 194 | 212 | 1 | | 80 | 108 | 1 | |
| AG-GG | 122 | 99 | 1.35 (0.97–1.87) | 0.076 | 71 | 59 | 1.44 (0.92–2.25) | 0.109 |
Recessive | AA-AG | 301 | 306 | 1 | | 149 | 161 | 1 | |
| GG | 15 | 5 | 3.05 (1.10–8.51) | 0.033 | 12 | 6 | 2.14 (0.78–5.85) | 0.140 |
Log-additive | --- | --- | --- | 1.40 (1.05–1.86) | 0.024 | --- | --- | 1.42 (0.98–2.06) | 0.060 |
IS; ischemic stroke; SNP, single nucleotide polymorphism; OR, odds ratio; 95% CI, 95% confidence interval. |
p values were calculated by logistic regression analysis with adjustments for age and gender. |
Bold indicate that p < 0.05 means the data is statistically significant. |
Stratified by sex, ZSCAN25 rs10242455 GG genotype had the protective effect on IS risk in males compared with AA genotype (OR = 0.45, 95% CI: 0.23–0.89, p = 0.021) or AA-AG genotype (OR = 0.45, 95% CI: 0.23–0.87, p = 0.018), but not females. The contribution of CYP2E1 rs2070672 to the increased IS occurrence was observed for various genetic models (G vs A: OR = 1.38, 95% CI: 1.04–1.83, p = 0.026; GG vs AA: OR = 3.28, 95% CI: 1.17–9.21, p = 0.024; GG vs AA-AG: OR = 3.05, 95% CI: 1.10–8.51, p = 0.033; and AA + AG + GG: OR = 1.40, 95% CI: 1.05–1.86, p = 0.024) in males.