Clinical characteristics and biochemical analysis
The clinical characteristics and biochemical analysis of the Slovenian subjects with T2DM are listed in Table 1. Cases (335 subjects with MI) had lower BMI, lesser waist circumference and better-controlled hypertension. Additionally, they had a higher total and LDL cholesterol, triglycerides, and lower HDL cholesterol. Moreover, cases had longer duration of T2DM. The two groups of subjects were well matched with regard to age, gender, fasting glucose, HbA1c, hsCRP level and concomitant history of cerebrovascular insult (CVI) or transitory ischaemic attack (TIA).
Table 1
Demographic and clinical characteristics of cases and controls in Slovenian subjects with T2DM
| Cases | Controls | |
| (Myocardial infarction) | (Without CAD) | p-value |
Number | 335 | 737 | |
Age (years) | 64.33 ± 9.79 | 64.12 ± 9.08 | 0.75 |
Male gender (%) | 200 (59.7) | 399 (54.1) | 0.09 |
BMI (kg/m2) | 29.64 ± 4.14 | 30.69 ± 4.59 | < 0.001 |
Waist circumference (cm) | 104.99 ± 11.42 | 107.86 ± 12.73 | 0.02 |
Systolic blood pressure (mm Hg) | 148.06 ± 19.75 | 150.78 ± 19.74 | 0.05 |
Diastolic blood pressure (mm Hg) | 82.11 ± 10.59 | 84.63 ± 11.52 | < 0.001 |
DM duration (years) | 15 (10–23) | 13 (9–18) | < 0.001 |
Fasting glucose (mmol/l) | 8.87 ± 2.,90 | 8.60 ± 2.54 | 0.25 |
HbA1c (%) | 7.88 ± 1.34 | 7.50 ± 1.9 | 0.64 |
Total cholesterol (mmol/l) | 5.15 ± 1.45 | 4.64 ± 1.12 | < 0.001 |
HDL cholesterol (mmol/l) | 1.14 ± 0.30 | 1.24 ± 0.35 | < 0.001 |
LDL cholesterol (mmol/l) | 2.94 (2.22–3.76) | 2.50 (2.02–3.10) | < 0.001 |
Triglycerides (mmol/l) | 1.90 (1.34–2.70) | 1.60 (1.10–2.43) | < 0.001 |
Smoking prevalence (%) | 43 (22.8) | 66 (9.0) | 0.05 |
CVI (%) | 27 (8.1) | 44 (6.0) | 0.20 |
TIA (%) | 17 (5.1) | 21 (2.8) | 0.07 |
hsCRP (mg/l) | 2.40 (1.28–4.80) | 2.40 (1.30–3.90) | 0.15 |
Abbreviations: BMI: Body mass index; DM: Diabetes mellitus; HbA1c: Glycated haemoglobin A1c; CVI: Cerebrovascular insult; TIA: Transitory ischaemic attack; hsCRP: high-sensitivity C-reactive protein. Values in bold indicate statistically significance. |
Genotyping analysis
The genotype and allele frequencies of the ROMO-1 rs6060566 polymorphism are shown in Table 2. Genotype distributions for both cases (subjects with MI) and controls (subjects without CAD) did not miss HWE (cases: p = 0.70; controls: p = 0.83, Pearson χ2 test; respectively). Moreover, in each of the studied group genotype (cases: p = 0.40 and controls: p = 0.21, Pearson χ2 test) and allele (cases: p = 0.24 and controls: p = 0.1, Pearson χ2 test) frequencies were not significantly different from those reported for the datasets in the 1000 Genomes Project Phase 3 European population.
Table 2
Genotype and allele frequencies distribution of the rs6060566
| | Cases (%) (N = 335) | Controls (%) (N = 737) | p-value |
Genotypes | CC | 10 (3.0%) | 21 (2.8%) | 0.98 |
CT | 90 (26.9%) | 202 (27.4%) |
TT | 235 (70.1%) | 514 (69.7%) |
Alleles | C allele (%)* | 110 (16.4%) | 244 (16.6%) | 0.94 |
T allele (%) | 569 (83.6%) | 1230 (83.4%) |
p (HWE) | | 0.70 | 0.83 | |
Abbreviations: HWE: Hardy-Weinberg equilibrium; * MAF: Minor allele frequency. |
Association analyses
Further, binary logistic regression analyses for different genetic models found no significant associations between different genotypes or alleles of the rs6060566 polymorphism and the risk of MI in Slovenian subjects with T2DM. Estimates of odds ratio (OR)s were adjusted (AOR)s (Table 3) for the variables (BMI, waist circumference, diastolic blood pressure, total cholesterol, HDL and LDL cholesterols, triglycerides, duration of DM in years) that were significant in the univariate analyses (Table 1).
Table 3
Binary logistic regression analyses for the association between rs6060566 of the ROMO-1 and MI in Slovenian subjects with T2DM
Genetic Model | Cases/Controls | AOR (95% CI) | p-value |
Co-dominant |
CC vs. TT* | 10/21 vs. 235/514 | 1.12 (0.12–10.83) | 0.92 |
CT vs. TT* | 90/202 vs. 235/514 | 1.68 (0.84–3.36) | 0.14 |
Dominant |
[CC + CT] vs. TT* | 100/223 vs. 235/514 | 1.64 (0.83–3.22) | 0.15 |
Recessive |
CC vs. [CT + TT]* | 10/21 vs. 325/716 | 0.96 (0.10–9.17) | 0.97 |
* The reference; OR: Odds ratio; AOR: Adjusted OR for BMI, waist circumference, diastolic blood pressure, total cholesterol, HDL and LDL cholesterols, triglycerides, duration of DM in years; CI: Confidence interval. |
No significant differences in genotype and allele frequency distribution for the rs6060566 polymorphism were observed among subjects with different distribution and extent of CAD, which was defined by coronary CT angiography (Table 4).
Table 4
Genotype and allele frequency distributions of the rs6060566 polymorphism in 128 subjects with T2DM who underwent coronary CT angiography
| Number of diseased vessels | Percentage of the cross-sectional area stenosis |
| None | 1 VD | 2 VD | 3 VD | p-value | < 50% | ≥ 50% ≤ 75% | > 75% | p-value |
| N (%) | N (%) | N (%) | N (%) | | N (%) | N (%) | N (%) | |
CC | 2 (4.2) | 0 | 0 | 0 | 0.895* | 2 (2.1) | 1 (3.6) | 0 | 0.262* |
CT | 14 (29.2) | 4 (18.2) | 10 (25) | 4 (25) | 25 (25.8) | 12 (42.9) | 1 (33.3) |
TT | 33 (66.7) | 18 (81.8) | 31 (75) | 12 (75) | 70 (72.2) | 15 (53.6) | 2 (66.7) |
C | 18 (18.4) | 4 (9.1) | 10 (12.2) | 4 (12.5) | 0.44† | 29 (14.9) | 14 (15) | 1 (16.7) | 0.213† |
T | 80 (81.6) | 40 (90.9) | 72 (87.8) | 28 (87.5) | 165 (85.1) | 42 (75) | 5 (83.3) |
* The p-value was obtained with Fischer̕ s Exact test |
† The p-value was obtained with Pearson χ2 test |
Abbreviations: VD: Vessel disease. |
Finally, we performed multinomial logistic regression analysis to evaluate the association of the rs6060566 polymorphism with CAD. Because of the low frequency of the minor C allele (Table 4) the analyses were performed assuming the dominant genetic model ([CC + CT] vs. TT). The final model is shown in Table 5. The dependent variables describing the severity of CAD were the number of diseased and extent of stenosis (none diseased vessel and stenosis < 50% were used as references, respectively). Independent variables included in the model were dominant genetic model (TT genotype was used as reference), age, gender, lipid parameters and duration of T2DM in years. We did not observe any interactions between dominant genetic model and CAD without adjustment for the possible confounders (Table 5). Nevertheless, when well-known CAD risk factors (age, gender, lipid parameters and duration of T2DM in years) were fixed in the model the association between carriers of the [CC + CT] genotypes and ≥ 50%≤75% cross-sectional area stenosis became statistically significant (p = 0.025, multinomial logistic regression). The carriers of the C allele of the ROMO-1 rs6060566 had a threefold increased likelihood of having coronary artery stenosis (AOR = 3.27, 95% CI 1.16–9.20, Table 5).
Table 5
Multinomial logistic regression analyses for the association between dominant genetic model ([CC + CT] vs. TT*) of the rs6060566 polymorphism and CAD
| OR (95% CI) | p-value | AOR (95% CI) | p-value |
≥ 50% ≤ 75% | 2.25 (0.95–5.34) | 0.067 | 3.27 (1.16–9.20) | 0.025 |
> 75% | 1.3 (0.11–14.89) | 0.835 | 0.94 (0.03–25.8) | 0.969 |
1 VD | 0.44 (0.08–2.56) | 0.364 | 0.25 (0.02–2.58) | 0.245 |
2 VD | 0.67 (0.18–2.5) | 0.547 | 0.88 (0.12–6.39) | 0.899 |
3 VD | 0.66 (0.11–4.08) | 0.661 | 0.85 (0.03–2.78) | 0.928 |
Abbreviations: OR: Odds ratio; AOR: Adjusted ORs. ORs were adjusted for age, gender, lipid parameters and duration of T2DM in years. Statistically significant result (p-value < 0.05) is highlighted in bold. |
* The reference |
Coronary computed tomography angiography
A total of 128 subjects with T2DM underwent coronary angiography (Fig. 1). A single vessel disease (1 VD) was observed in 22 (17%) subjects, two-vessel disease (2 VD) in 41 (32%) and three-vessel disease (3 VD) in 16 (13%) subjects. Further, 49 (38%) subjects had normal all major epicardial coronary arteries (LMCA, LAD, LCx and RCA) on CT angiograms (Fig. 1a). Moreover, 97 (76%) subjects had nonobstructive CAD (cross-sectional area stenosis of < 50%), in 28 (22%) subjects a cross-sectional area stenosis of ≥ 50%≤75% was detected while only 3 subjects (2%) had stenosis of > 75% (Fig. 1a). As can be seen in Fig. 1a, subjects with 2 VD (14/41, 34.1%) and nonobstructive CAD (11/97, 11.3%) suffered from nonfatal MI more often than other subjects in both comparative groups (number of diseased vessels and percentage of the cross-sectional area stenosis). Of note, there was a statistically significant difference (p = 0.0096, Pearson χ2 test) in frequency distribution between subgroups with and without MI with regard to the extent of the CAD (Fig. 1a). In contrast, no difference (p = 0.283; Fischer̕ s Exact test) was observed between subgroups with regard to the coronary cross-sectional area stenosis (Fig. 1a). Regarding the number of the involved vessels, a significantly higher frequency (p = 0.013; Pearson χ2 test) of MI was found in subjects with 2 VD. Interestingly, subjects with two affected coronary arteries showed a 3.72 fold risk for MI (OR = 3.72, 95% CI 1.27–10.84, Fig. 1a).
Coronary angiography revealed that of the 128 subjects more than 50% of them had developed CAD in LAD (Fig. 1b), while the remainder of the coronary arteries were spared of atherosclerotic disease more frequently. Atherosclerotic changes were noticed in LMCA in 39 subjects (30.5%), while a slightly higher percentage of atherosclerotic disease was seen in LCx and RCA (41.4%) (Fig. 1b). Furthermore, the relationship between presence or absence of CAD and coronary arteries was statistically significant (p = 0.0055, Pearson χ2 test; Fig. 1b).
As depicted in Fig. 1c, subjects with CAD (CAD+) in LMCA or LAD had about 3.5-fold higher risk of experiencing MI (p = 0.007 for LMCA and p = 0.01 for LAD, Pearson χ2 test; respectively) compared with subjects without CAD (CAD-). However, in subjects with diseased LCx and RCA MI occurred more frequently than in subjects with disease-free coronary arteries, although the difference was not statistically significant (p = 1.0 for LCx and p = 0.9 for RCA, Pearson χ2 test; respectively).
Immunohistochemistry
At the end of this study, the coronary artery segments, which were obtained by endarterectomy from subjects with advanced atherosclerosis, were examined with immunohistochemical staining. A statistically significantly higher numerical areal density of ROMO-1 positive cells was found in 17 subjects with the C allele (Fig. 2) in comparison with 23 subjects with ROMO-1 TT genotype (wild type) (835 ± 215/mm2 versus 412 ± 153/mm2; p < 0.001, Student̕ s t test).