Clinical characteristics of IS patients and controls
There was no statistically significant difference between the IS patients and the healthy control patients with respect to age, sex, and BMI (P > 0.05, Table 1). However, IS patients’ levels of hypertension and smoking (P < 0.001) diabetes (P < 0.003), drinking (P < 0.005), and dyslipidemia (P < 0.026) were significantly higher than those of the controls (Table 1).
Table 1
Clinical characteristics of study subjects.
Characteristics | Cases (n = 114) | Controls (n = 114) | p |
Male, n (%) | 68 (59.64) | 68 (59.64) | 0.999a |
Female, n (%) | 46 (40.35) | 46 (40.35) |
Age (years) | 65.88 ± 14.44 | 65.88 ± 14.44 | 0.999b |
BMI (kg/m2) | 25.80 ± 5.21 | 26.21 ± 4.51 | 0.523 |
NIHSS at admission ≤ 6 ≥ 7 | 63 (55.26) 51 (44.73) | - | |
Toast of classification cases n (%) | | | |
LAA | 34(29.82) | | |
SVO | 26(22.8) | | |
CE | 26(22.8) | | |
UD | 28(24.5) | | |
Vascular risk factors | | | |
Hypertension, n (%) | 65 (57.02) | 31 (27.20) | < 0.001a |
Diabetes, n (%) | 35 (30.7) | 16 (14.04) | 0.003a |
Smoking, n (%) | 34 (29.82) | 8 (7.02) | < 0.001a |
Drinking, n (%) | 16 (14.03) | 3 (2.63) | 0.005a |
Dyslipidemia | 39 (34.21) | 24(21.05) | 0.026a |
NIHSS: National institutes of health stroke scale; LAA: large-artery atherosclerosis; SVO: small vessel occlusion CE: cardio embolism; and UD: undetermined etiology; BMI: Body Mass Index; |
Data were shown as mean ± standard deviation (SD) or as n (%).aChi-square Test; bIndependent two-sample T-test. P–value of < 0.05 was regarded as statistically significant |
H19 rs217727 polymorphism has a positive association with the risk of IS
We did not find any significant deviations from the Hardy-Weinberg equilibrium for rs217727 polymorphism in the control group. The results confirmed the association between the IS and rs217727polymorphism in Iranian individuals. We found that the C allele of rs217727 was a significant protective factor against IS in the Iranian populations (C vs. T: OR = 0.62, 95% CI: 0.39–0.99, P = 0.04). Furthermore, the frequency of TT genotypes of H19 rs217727 was significantly higher in IS patients than in healthy controls (11. 4 vs. 4.38%). Moreover, the TT genotype was associated with a 2.92-fold increase in IS risk in the co-dominant model (OR = 2.92, 95% CI = 0.91–10.92, P = 0.04). Also, H19 rs217727 polymorphism was related to a 2.80-fold increase in the risk of IS in the recessive model (TT vs. CC + CT genotypes) (Table 2).
Table 2
Allele and genotype frequencies of H19 rs217727 for IS patients and the control group.
Inheritance | rs217727 | Cases | Controls | | |
model | Polymorphism | (%) | (%) | OR (95% CI) | P |
Codominanta | Genotype | | | | |
| CC | 73 (64.03) | 82 (71.92) | 1 | |
| CT | 28 (24.56) | 27 (23.68) | 1.16 (0.62–2.15) | 0.62 |
| TT | 13 (11.40) | 5 (4.38) | 2.92 (0.91–10.92) | 0.04 |
Dominantb | CC | 73 (64.03) | 82 (71.92) | 1 | |
| CT + TT | 41 (35.96) | 32 (28.07) | 1.43 (0.82–2.51) | 0.20 |
Recessivec | CC + CT | 101 (88.59) | 109 (95.61) | 1 | |
| TT | 13 (11.40) | 5 (4.38) | 2.80 (0.96–8.15) | 0.04 |
Over-dominantd | CC + TT | 86 (75.43) | 87 (76.31) | 1 | |
| CT | 28 (24.56) | 27 (23.68) | 1.04 (0.57–1.92) | 0.87 |
| Allele | | | | |
| T | 54 (23.68) | 37 (16.22) | 1 | |
| C | 174 (76.31) | 191 (83.77) | 0.62 (0.39–0.99) | 0.04 |
OR, odd ratio; CI, confidence interval. |
aCo-dominant; major allele homozygotes vs. heterozygotes. |
bDominant; major allele homozygotes vs. heterozygotes + minor allele homozygotes. |
cRecessive; major allelehomozygotes + heterozygotes vs. minor allele homozygotes. |
dOver dominant; major allele homozygotes + minor allele homozygotes vs. heterozygotes. |
H19 expression was significantly upregulated in the peripheral blood of IS Patients
We found that H19 expression was significantly upregulated in IS cases when compared to controls (Fig. 1, A, P < 0.001). Further analysis indicated that H19 expression was found more often in IS patients than in the controls 0–24, 24–48, and 48–72 hours after the onset of stroke (P < 0.001, P < 0.05, P < 0.01, respectively). An independent t-test was used to compare the relative H19 expression in peripheral blood of IS cases at each time point with age- and sex-matched controls (Figs. 1B-1D). H19 expression levels in IS patients were compare to levels of controls 0–24 hours (4.51 ± 0.88 vs 0.96 ± 0.12), 24–48 hours (7.12 ± 1.53 vs 2.73 ± 1.19), and 48–72 hours (6.38 ± 1.36 vs 1.84 ± 0.36) after stroke. Interestingly, we observed that upregulated H19 remained high 72 hours after a stroke (Fig. 1E).
H19 expression was associated with TOAST subtypes of IS patients
Patients were assigned to four subtypes according to TOAST classification (Table 1). We found a significant elevation in H19 expression in patients with LAA, SVO, and UD when compared to controls (ANOVA, P < 0.01). IS patients with CE showed the lowest levels of H19 without a significant difference with healthy controls (Fig. 2) (P > 0.05).
Multiple logistic regression analysis was done to detect the association between TOAST subtypes and H19 expression after adjusting for several confounders (Table 3). We showed that H19 expression levels were significantly associated with TOAST subtypes. H19 expression levels in SVO and LAA patients were 3.74 and 3.34 times higher than the UD subtype, respectively [OR = 3.74 95% CL (1.14–12.27) P = 0.030 and OR = 3.34 95% CL (1.13–9.85) P = 0.029].
Table 3
The results of univariate and multiple logistic regression analyses
Characteristics | Univariate | | Multiplevariate * |
OR | 95% CI | P-Value | | OR | 95% CI | P-Value |
Male sex | 1.26 | 0.59–2.67 | 0.542 | | 1.36 | 0.59–3.14 | 0.466 |
Age (year) | 1.00 | 0.97–1.02 | 0.835 | | 0.99 | 0.96–1.02 | 0.567 |
Hypertension | 1.80 | 0.85–3.81 | 0.125 | | 1.82 | 0.80–4.11 | 0.151 |
Diabetes mellitus | 1.14 | 0.52–2.53 | 0.743 | | 0.99 | 0.40–2.47 | 0.988 |
Dyslipidemia | 1.33 | 0.61–2.89 | 0.477 | | 1.35 | 0.56–3.22 | 0.502 |
Cigarette Smoking | 1.05 | 0.47–2.35 | 0.903 | | | | |
NIHSS score | 1.01 | 0.95–1.07 | 0.727 | | 1.02 | 0.96–1.09 | 0.505 |
Alcohol use | 1.04 | 0.36–2.99 | 0.940 | | | | |
Type of IS UD | 1 | | | | | | |
| CE | 0.95 | 0.31–2.92 | 0.933 | | 1.24 | 0.38–4.06 | 0.725 |
| SVO | 2.88 | 0.95–8.69 | 0.061 | | 3.74 | 1.14–12.27 | 0.030 |
| LAA | 2.91 | 1.03–8.20 | 0.044 | | 3.34 | 1.13–9.85 | 0.029 |
BMI | 1.02 | 0.94–1.10 | 0.664 | | | | |
*The OR was adjusted for all predictors with ≤ 0.3 of the univariate logistic regression analysis and for the most important clinical factors. NIHSS: National institutes of health stroke scale, BMI: Body Mass Index; LAA: large-artery atherosclerosis; SVO: small vessel occlusion CE: cardio embolism; and UD: undetermined etiology. P–value < 0.05 was regarded as statistically significant. |
H19 expression levels in different genotypes of rs217727 polymorphism in IS patients
Further analysis revealed that there were no significant differences in relative H19 expression levels among the three groups of rs217727 (CC, CT and TT) genotypes in case (F = 0.00; df = 2, P = 0.999) and control (F = 0.14; df = 2, P = 0.865) (Fig. 3).
H19 expression level in peripheral blood might serve as a diagnostic biomarker
The ROC curve analysis showed that H19 is a potential biomarker for IS diagnosis cases (Table 4). For the period 0–24 hours after IS, AUC was 0.875 ± 0.039 (95% CI, 0.798–0.952; Fig. 4A); the sensitivity and specificity were 79.49% and 80.00%, respectively. For the period 24–48 hours after IS, AUC was 0.813 ± 0.049 (95% CI, 0.715–0.910; Fig. 4B), and the sensitivity and specificity were 77.5% and 75.00%, respectively. For the period 48–72 hours after IS, AUC was 0.788 ± 0.052 (95% CI, 0.685–0.891; Fig. 4C), and the sensitivity and specificity were 75.68% and 67.57%, respectively. Circulating H19 levels within the first 24 hours after the onset of a stroke indicated high sensitivity and specificity for the early diagnosis of acute stroke.
Table 4
Prediction of diagnostic value
Parameter | AUC ± SE | 95% CI | P | Se (%) | Sp (%) |
Diagnostic value | | | | | |
H19 expression in 0–24 hours | 0.875 ± 0.039 | 0.798–0.952 | < 0.0001 | 79.49% | 80.00% |
H19 expression in 24–48 hours | 0.813 ± 0.049 | 0.715–0.910 | < 0.0001 | 77.5% | 75.00% |
H19 expression in 48–72 hours | 0.788 ± 0.052 | 0.685–0.891 | < 0.0001 | 75.68% | 67.57% |
Abbreviations: AUC; Area Under the Curve; CI, Confidence Interval; Se, sensitivity; Sp, specificity P < 0.05 was considered statistically significant |