Angiotensin-converting enzyme insertion/deletion (rs106180) and angiotensin type 1 receptor A1166C (rs106165) genotypes and psoriasis: Correlation with cellular immunity, lipid profile, and oxidative stress markers
Abstract
Psoriasis is a chronic inflammatory skin condition and angiotensin-converting enzyme (ACE) is a key circulating enzyme converting angiotensin (Ang) I to the vasoactive peptide Ang II. The exact role of ACE insertion (I)/deletion (D) polymorphism (rs106180) in psoriasis is not clear. We aimed to examine whether the ACE I/D and Ang II type 1 receptor (AT1R) A1166C-polymorphisms (rs106165), lipid profile, and stress oxidative are associated with susceptibility to psoriasis. One hundred patients with psoriasis and 100 sex- and age-matched unrelated healthy controls were recruited for this case-control study. ACE I/D and AT1R A1166C polymorphisms were identified by the polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism, respectively, malondialdehyde (MDA) was detected by the high-performance liquid chromatography, serum arylesterase (ARE) activity of paraoxonase and catalase activities were detected by the spectrophotometry, superoxide dismutase (SOD) activity and vascular adhesion protein (VAP)-1 were measured by ELISA. The presence of C allele of AT1R A1166C and I allele of ACE considerably increased the risk of psoriasis by 6.42-fold (P < 0.001). The distribution of II-genotype of ACE was significantly higher in psoriasis patients than in control group and increased the risk of disease by 3.11-times (P = 0.023). The higher levels of MDA in patients and the higher activity of SOD, ARE, and CAT was observed in healthy controls with I/D+I/I-genotype of ACE I/D. This study for the first time demonstrated that the ACE I/D and AT1R A 1166C genes polymorphisms robustly increases the risk of developing psoriasis in population from west of Iran. In addition, these individuals had significantly higher VAP-1 and MDA concentration and lower enzymatic and nonenzymatic antioxidant-status, suggesting that psoriatic patients carrying C allele of AT1R1166 polymorphism may be more susceptible to cardiovascular disease and myocardial infarction compared with A allele.
1 INTRODUCTION
Psoriasis, a common chronic autoimmune and multisystem disease is characterized by red, scaly, well-demarcated plaques, and increased blood flow.1, 2 Its prevalence is approximately 2% of the world population.3 Psoriasis may begin at any age, although it generally develops in adulthood and it is uncommon in children.4 It is a complex and multifactorial disease with idiopathic etiology, although a significant genetic association has been demonstrated by family and twin studies.5-7 Moreover, immunologic and environmental factors all may contribute in triggering or development of the disease.8 Angiotensin-converting enzyme (ACE) gene is mapped on chromosome 17q23 and consist of 26 exons and 25 introns converting angiotensin (Ang) I into Ang II.2, 9 Ang II not only increases blood pressure, but also is a potent proinflammatory modulator. Ang II through activation of NAD(P)H oxidase and production of reactive oxygen species (ROS) can induce inflammation and tissue damage.2, 10, 11 Insertion (I) and deletion (D) polymorphism of the ACE (rs4646994) affects serum levels of ACE. Individuals with DD genotype have the highest levels of serum ACE compared with careers of the II-genotype.12, 13 ACE is expressed in various tissues including skin, vascular endothelium, and immune cells.14 Results of Huskić and Alendar15 study revealed that the tissue ACE activity was significantly higher in patients with psoriasis than in healthy individuals. In addition, the greatest increase in tissue ACE activity was observed in patients with erythrodermic psoriasis, followed by those with multiple disseminated and solitary psoriatic lesions. Because almost all physiological functions of Ang II are correlated with the Ang II type 1 receptor (AT1R),16 it can be proposed that A1166C polymorphism at the 3′-untranslated region of the AT1R gene may be associated with susceptibility of psoriasis.
One of the complications in psoriasis is oxidative stress that through lipid peroxidation, DNA modification, and secretion of inflammatory cytokines exacerbate the disease, and this condition may be correlated with AT1R gene expression.17, 18 Vascular adhesion protein (VAP)-1 is an endothelial glycoprotein with an enzymatic activity that catalyzes the oxidation of amines to hydrogen peroxide, aldehyde, and ammonium.19, 20 This adhesion receptor is expressed by the endothelial cells of the vascular wall in the vast majority of organs such as skin, brain, lung, liver, and heart under both normal and inflammation conditions.21 Also, VAP-1 is involved in lymphocyte interactions, adhesion, and transmigration in chronic inflammatory and autoimmune diseases.22 Evidence show that increased oxidative stress has a crucial role in development of psoriasis and malondialdehyde (MDA) is a final product of lipid peroxidation.23 Previous studies demonstrated that the plasma MDA concentrations were significantly elevated in psoriatic patients compared with healthy controls.5, 24
In this study, we examined the association of ACE I/D and AT1R A1166C variants with the risk of psoriasis and also to find the effects of polymorphisms on lipid profile, VAP-1, lipoproteins, and MDA levels in psoriasis patients in a population from Iran. We have shown for the first time that ACE I/D and AT1R A1166C genes variants and lipid profiles, MDA and VAP-1 are associated with psoriasis susceptibility in Kurdish population from west of Iran.
2 MATERIALS AND METHODS
2.1 Subjects
The human subject study protocol was approved by the Ethics Committee of the Kermanshah University of Medical Sciences (KUMS) and was in accordance with the principles of the Declaration of Helsinki II and all subjects were provided with written informed consent. In total, 100 patients with psoriasis, mean age 35.3 ± 10.9 years; range 15 to 65 years; 57 females and 43 males, were selected from the Dermatology Unit of Haj Daiei clinic of the KUMS.
Baseline information including the age, sex, duration, and severity of the disease were collected by face to face interviewing. The severity of the psoriasis was determined by the Psoriasis Area and Severity Index score, and the patients with psoriasis were grouped based on the severity of their psoriasis (group III, mild <20; group II, moderate 20-50; or group I, severe >50).
Patients with body mass index (BMI) higher than 30 kg/m2, cigarette smokers, and individuals with diseases that can cause secondary hyperlipidemia, such as hypothyroidism, diabetes mellitus, nephrotic syndrome, chronic renal insufficiency, obstructive liver disease, and connective tissue diseases were excluded from the study. All patients had been treated only with topical agents, such as corticosteroids or vitamin D analogues for 6 months and they did not use any drugs, even topical agents for 3 months before the study.
The control subjects (mean age, 35.7 ± 13.2 years; range, 15-64 years; 56 females and 44 males) were healthy individuals who were referred to Hospitals of the KUMS for their annual checkup. The patients and control groups were matched according to the sex, age, and race. All healthy donors were interviewed and examined for psoriasis, coronary heart disease, liver disease, hyperlipidemia, and rheumatoid arthritis. Their family histories were obtained and no history of an autoimmune disease or a specific disease at medical checkup was identified.
2.2 Chemical analyses
The serum arylesterase (ARE) activity of paraoxonase was measured spectrophotometrically using phenyl acetate as substrate according to the protocol described previously.5
Plasma MDA concentration was measured by an Agilent Technologies 1200 Series HPLC (high-performance liquid chromatography) system (Agilent Corp, Germany) using EC 250/4.6 Nucleodur 100-5 C18ec column (Macherey-Nagel, Duren, Germany) as previously described.18
Serum VAP-1 concentration was determined by ELISA (Med System Diagnostic GmbH, Vienna, Austria) according to the manufacturer's instructions. Total serum cholesterol (TC), triacylglycerol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), APOA1, APOB, LP(a), bilirubin (total and direct), and uric acid levels were measured by the standard methods (Pars Azmoon kit, Iran), using an automated Erba XL-600 (Mannheim, Germany).
Plasma superoxide dismutase (SOD) activity was measured using commercially available kit (Randox Laboratories Ltd, Ireland; #SD125). Catalase activity was determined by the reduction of H2O2 at 240 nm.18
2.3 DNA extraction, ACE, and AT1R genotyping
DNA was extracted from peripheral blood samples using the phenol chloroform extraction method according to the standard protocol.25 The AT1R A1166C polymorphism was determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and ACE I/D was detected by the PCR technique.18, 26
The forward 3′-GCACCATGTTTTGAGTTG-5′ and reverse 5′-CGACTACTGCTTAGCATA-3′ primers were used to identify ACE I/D polymorphism. PCR reaction mixtures containing DNA, primers, and dNTPs were heated at 94°C for 2 minutes, followed by 35 cycles at 94°C for 30 seconds, 58°C for 48 seconds, and 72°C for 1 minute, with a final extension at 72°C for 10 minutes. The size of PCR products was 190 bp for the D allele and 490 bp for the I allele. In PCR, when I allele is placed next to the deletion, duplication of D allele is prevailing. The second PCR was accomplished to avoid missing ID genotype and identify DD genotype. All samples from the first PCR displaying DD genotype came under second PCR. The presence of a fragment with 335 bp indicated the I allele; it means that when I allele was emerged in the given sample DD genotype was converted into ID one.27 Amplification of AT1R A1166C by PCR-RFLP has been described previously.18
2.4 Statistical analyses
The allelic frequencies were calculated by the gene counting method. The χ2 test was used to verify the agreement of the observed genotype frequencies with those expected according to the Hardy-Weinberg equilibrium. Statistical significance was assumed at the P ≤ 0.05. The genotypes and allele frequencies of AT1R A1166C in psoriasis patients were compared with the control group and between patients with various disease activity (mild, moderate, and severe) using χ2 test in three different models: the genotype codominant model, the minor genotype dominant/recessive model, and the minor genotype heterozygous model. Odds ratios (ORs) were calculated as estimates of relative risk for the disease and 95% confidence intervals (CI) obtained by SPSS logistic regression. The correlation values of serum VAP-1, MDA, HDL-C, LDL-C, TC, APOA1, APOB, LP(a), uric acid, and total and direct bilirubin levels, and serum activities of ARE, SOD, and CAT with the AT1R A1166C polymorphism between the two groups were calculated using linear regression and an unpaired t test. A two-tailed Student t test, analysis of variance and nonparametric independent sample Mann-Whitney analysis were used to compare quantitative data. The SPSS statistical software (SPSS for windows 16; SPSS Inc, Chicago, IL) was used for the statistical analysis. Statistical significance was assumed at the P ≤ 0.05.
3 RESULTS
The characteristics and laboratory test results of patients with psoriasis and controls are shown in Table 1. Patients had significantly higher levels of MDA (P < 0.001), VAP-1 (P < 0.001), LDL-C (P = 0.035), and total cholesterol (P = 0.048) compared with control as previously reported.5-7, 18 The OR with 95% confidential interval and the frequencies of ACE I/D genotypes and alleles are demonstrated in Table 2. The frequency of ACE I/I genotype was higher in patients (16%) compared with that of healthy controls (8%, P = 0.02) and it was associated with 3.11-fold increased risk of psoriasis. Also, the frequencies of ACE genotypes according to dominant model (I/D+I/I vs DD), I/D genotype and I allele were higher in patients compared with control subjects. According to these data, the I allele and I/D genotype increased the susceptibility to psoriasis by 1.63 times (P = 0.019) and 1.77-fold (P = 0.068), respectively.
| Parameters | Psoriasis patient N = 100 | Control subjects N = 100 | P values |
|---|---|---|---|
| Age | 35.3 ± 10.9 | 35.7 ± 13.2 | 0.82 |
| Sex (male/female) | 43/57 | 44/56 | 0.88 |
| MDA, µmol/L | 4.01 ± 0.87 | 2.22 ± 0.56 | <0.001 |
| VAP-1, ng/mL | 290 ± 135 | 193 ± 41.7 | <0.001 |
| LDL-C, mg/dL | 112 ± 37 | 102.4 ± 23.3 | 0.035 |
| HDL-C, mg/dL | 43.3 ± 15 | 46.2 ± 13.4 | 0.16 |
| Total cholesterol, mg/dL | 167 ± 42 | 156.7 ± 31.1 | 0.048 |
| Triacylglycerol, mg/dL | 123 ± 67 | 113.3 ± 44.3 | 0.226 |
| APOA1, mg/dL | 139.3 ± 53.8 | 151.5 ± 49.5 | 0.1 |
| APOB, mg/dL | 107.5 ± 41 | 99.8 ± 76.5 | 0.37 |
| LP(a), mg/dL | 19.2 ± 16.5 | 17.1 ± 17 | 0.41 |
| APOB/APOA1 | 0.89 ± 0.48 | 0.73 ± 0.47 | 0.027 |
| Duration of psoriasis, y | 10.2 ± 5.8 | ||
| BMI, kg/m2 | 25.53 ± 3.8 | 24.66 ± 2.95 | 0.052 |
| PASI score | 16.7 ± 5.2 |
- Abbreviations: BMI, body mass index; HDL-C, high-density lipoprotein cholesterol; MDA, malondialdehyde; LDL-C, low-density lipoprotein cholesterol; VAP-1, Vascular adhesion protein-1.
- Compared serum MDA, HDL-C, LDL-C, TC, TG, APOA1, APOB, and levels LP(a) and ratio of APOB/APOA1, BMI and age between patients and controls were used two-tailed Student t test and sex by the χ2 test.
- Scores on the psoriasis area and-severity index (PASI) range from 0 to 72, with higher scores indicating more severe disease.
| Psoriasis patients (n = 100) | Control subjects (n = 100) | |
|---|---|---|
| ACE I/D genotypes | ||
| D/D | 27 (27%) | 42 (42%) |
| I/D | 57 (57%) | 50 (50%) |
| (χ2 = 3.3, df = 2, P = 0.067) | ||
| 1.77 (0.96-3.3, P = 0.068) | ||
| I/I | 16(16%) | 8 (8%) |
| (χ2 = 5.4, df = 2, P = 0.02) | ||
| 3.11 (1.17-8.3, P = 0.023) | ||
| (χ2 = 6.3, df = 2, P = 0.041) | ||
| Dominant model of ACE genotypes | ||
| D/D | 27 (27%) | 42 (42%) |
| I/D+I/I | 73 (73%) | 58 (58%) |
| (χ2 = 5, df = 2, P = 0.026) | ||
| 1.96 (1.08-3.54, P = 0.027) | ||
| ACE(I/D) alleles | ||
| D | 111 (55.5) | 134 (67%) |
| I | 89 (44.5%) | 66 (33%) |
| (χ2 = 5.6, df = 1, P = 0.018) 1.63 (1.09-3.6, P = 0.019) | ||
- OR calculated by 95% confidential interval.
The presence of I/D+I/I genotype of ACE gene in psoriasis patients was strongly associated with higher concentrations of VAP-1 (P = 0.021), MDA (P ≤ 0.001), and APOB/APOA1 (P = 0.051; Table 3). Healthy individuals with I/D+I/I genotype had significantly higher SOD, arylsulfatase, and catalase activities compared with psoriasis patients with the same genotype (P ≤ 0.001; Table 3).
| Dominant model of ACE genotypes (D/D, I/D+I/I) | Psoriasis patients | Control subjects | P values |
|---|---|---|---|
| D/D | N = 27 | N = 42 | |
| VAP-1, ng/mL | 239 (205-371) | 200 (175-213) | 0.021 |
| MDA, µmol/L | 4.05 (3.4-4.4) | 2.15 (1.8-2. 4) | <0.001 |
| LDL-C, mg/dL | 99.4 ± 37 | 105 ± 21 | 0.04 |
| HDL-C, mg/dL | 44.1 ± 17.1 | 42.5 ± 11.8 | 0.64 |
| Cholesterol, mg/dL | 155 ± 42.2 | 160.5 ± 28.3 | 0.49 |
| TG, mg/dL | 106 ± 54.7 | 126 ± 60 | 0.16 |
| APOA1, mg/dL | 150 ± 47.3 | 149 ± 62.7 | 0.96 |
| APOB, mg/dL | 106 (81-128) | 89 (79-102) | 0.051 |
| LP(a), mg/dL | 17.4 ± 11.9 | 17.5 ± 18 | 0.98 |
| APOB/APOA1 | 0.79 ± 0.41 | 0.77 ± 0.57 | 0.88 |
| SOD (KU/mL_1) | 75 (65-87) | 99 (85-109) | <0.001 |
| ARE, U/mL | 137 (92-145) | 160 (137-183) | 0.001 |
| CAT, KU/mL | 44 (35-46) | 54.5 (47.8-61.3) | <0.001 |
| I/D+I/I | |||
| VAP-1, ng/mL | 2247 (185-379) | 203 (164-229) | 0.001< |
| MDA, µmol/L | 3.9 (3.3-4.4) | 2.15 (1.79-2.59) | <0.001 |
| LDL-C, mg/dL | 117 ± 36.2 | 107 ± 32.6 | 0.11 |
| HDL-C, mg/dL | 43.1 ± 17.2 | 47 ± 14.8 | 0.13 |
| Cholesterol, mg/dL | 173 ± 42.2 | 163 ± 41.2 | 0.18 |
| TG, mg/dL | 130 ± 70.5 | 1263 ± 67.4 | 0.73 |
| APOA1, mg/dL | 135 ± 55.7 | 153 ± 47.3 | 0.067 |
| APOB, mg/dL | 100 (80-132) | 95 (70-116) | 0.13 |
| LP(A), mg/dL | 20.8 ± 18.9 | 15.6 ± 16.4 | 0.11 |
| APOB/APOA1 | 0.92 ± 0.51 | 0.69 ± 0.34 | 0.008 |
| SOD (KU/mL) | 77.5 (60-95) | 95.5 (81.6-102) | <0.001 |
| ARE, U/mL | 123 (99-145) | 145 (123-162) | <0.001 |
| CAT (KU/mL) | 43.5 (37-51) | 53 (47-63.8) | <0.001 |
- Abbreviations: BMI, body mass index; HDL-C, high-density lipoprotein cholesterol; MDA, malondialdehyde; LDL-C, low-density lipoprotein cholesterol; SOD, superoxide dismutase; VAP-1, Vascular adhesion protein-1.
The frequency of AT1R A1166C genotypes and alleles in psoriasis patients and control group are shown in Table 4 and were described in details previously.7
| Psoriasis patients (n = 100) | Control subjects (n = 100) | |
|---|---|---|
| AT1R A1166C genotypes | ||
| A/A | 64 (64%) | 82 (82%) |
| A/C | 31 (31%) | 17 (17%) |
| *(χ2 = 5.5, df = 2, P = 0.019) | ||
| C/C | 5 (5%) | 1 (1%) |
| *(χ2 = 8.2, df = 2, P = 0.016) | ||
| AT1R A1166C alleles | ||
| A | 159 (79.5) | 181 (90.5%) |
| C | 41 (20.5%) | 19 (9.5%) |
| *(χ2 = 8.8, df = 1, P = 0.003) | ||
| AT1R genotype | Psoriasis patients reference group OR (95%confidential interval) | Control reference group |
| Codominant | ||
| C/C vs A/A | 2.5 (0.85-7.4, P = 0.096) | (n = 1 vs n = 82) |
| (n = 5 vs n = 64) | ||
| A/C vs A/A | 2.23 (1.13-4.4, P = 0.021) | (n = 17 vs n = 82) |
| (n = 31 vs n = 64) | ||
| Dominant | ||
| C/C+A/C vs A/A | 2.45 (1.3-4.7, P = 0.007) | (n = 18 vs n = 82) |
| (n = 36 vs n = 64) | ||
| Recessive | ||
| C/C vs A/A+A/C | 2.28 (0.77-6.7, P = 0.135) | (n = 1 vs n = 99) |
| (n = 5 vs n = 95) | ||
| Overcodominant | ||
| A/C vs C/C+A/A | 2.1 (1.07-4.1, P = 0.032) | (n = 17 vs n = 83) |
| (n = 31 vs n = 69) | ||
| AT1R alleles | ||
| A | n = 159 | n = 181 |
| C | 2.46 (1.15-6.07, P = 0.004, n = 41) | n = 19 |
To investigate the interaction between ACE I/D and AT1R A1166C alleles in patients with psoriasis, logistic regression analysis was used and results are presented in Table 5. For the first time our results demonstrated that the concurrent presence of C allele of AT1R A1166C and I allele of ACE strongly increased the risk of psoriasis by 6.42-fold (P < 0.001).
| AT1R A1166 C | ACE (I/D) | Psoriasis patients | Control group | OR (95%CI) | |
|---|---|---|---|---|---|
| C | I | ||||
| – | – | n = 20 (20%) | n = 31 (31%) | References | |
| + | – | n = 7 (7%) | n = 11 (11%) | 0.99 (0.3-3, P = 0.98) | (χ2 = 0.01, df = 1, P = 0.98) |
| – | + | n = 44 (44%) | n = 51 (51%) | 1.34 (0.7-2.6, P = 0.41) | (χ2 = 0.7, df = 1, P = 0.4) |
| + | + | n = 29 (29%) | n = 7 (7%) | 6.42 (2.37-17.4, P < 0.001) | (χ2 = 14.7, df = 1, P < 0.001) |
- Overall distribution of ACE(I/D) and AT1R A1166C receptor alleles in psoriasis compared with control group was significantly differences (χ2 = 17.2, df = 3, P = 0.001).
4 DISCUSSION
Numerous studies have demonstrated that genes and environmental factors play a crucial role in the etiology of psoriasis.14, 28 But, to the best of our knowledge, current study is the first to show that ACE I/D and AT1R A1166C polymorphisms and lipid profiles, MDA and VAP-1 are associated with psoriasis susceptibility in Kurdish population from west of Iran. Results of our study showed that the concurrent presence of C allele of AT1R A1166C and I allele of ACE significantly increased the risk of psoriasis in the studied population by 6.42-fold. The frequencies of A/C and C/C genotypes of AT1R A1166C were significantly higher in patients with psoriasis that increased the risk of psoriasis. In addition, the presence of II genotype of ACE I/D polymorphism significantly increased susceptibility to psoriasis. The levels of MDA, LDL-C, total cholesterol, and VAP-1 were considerably higher in psoriasis patients compared with healthy controls. Also, BMI was in direct association with psoriasis. Although the MDA level was significantly higher in psoriasis patients with I/D+I/I genotype of ACE, but the SOD and ARE activities were higher in control subjects having the same genotype. Contrary to our results, Huang et al29 reported that the distribution of DD genotype of ACE I/D polymorphism among cases was significantly higher than controls and D allele was considerably associated with psoriasis susceptibility in Chinese Han population. One case-control study including 312 patients with psoriasis and 312 matched healthy controls indicated a significantly higher frequency of the II genotype in patients compared with control subjects and this genotype increased the risk of the disease by 1.45 time in Chinese Taiwanese population.30 Yang et al3 also demonstrated that the distribution of the II genotype and I allele of ACE I/D polymorphism was significantly higher in patients with psoriasis than in the control group. Results of Ozkur et al31 indicated that I allele had higher frequency in type I psoriasis patients (onset <40 years and positive family history) than in type II patients with psoriasis (onset ≥40 years with no family history). Weger et al32 cited the prevalence of the homozygous ACE II genotype in patients with early-onset psoriasis with an OR of 1.88 for early-onset disease. However, no association has been found between ACE I/D polymorphism and progression or susceptibility to psoriatic arthritis or psoriasis in Europeans.33
ROS may be involved in the onset, pathogenesis, and development of psoriasis.34, 35 It has been shown that the activity of erythrocyte-SOD and catalase are low and serum level of MDA, nitric oxide end product, is high in psoriasis, suggesting a decreased in antioxidant potential in psoriasis.35 Psoriasis is a chronic, multifactorial genetic, and inflammatory disease. So, knowledge of association between these polymorphisms with increased disease susceptibility and the impact of genetic variations on expression of ACE enzyme and AT1 receptor could be important in detection of risk factors of the disease. Moreover, effect of mentioned polymorphisms and inflammatory biomarkers in disease trigger, development and severity has to be considered. Also, genetic differences in the pattern of haplotypes of the AT1R and ACE gene between different ethnic races, the influence of sample size and gene-environmental interactions are the crucial factors leading to controversial results from different populations.
5 CONCLUSION
In this study for the first time, we demonstrated that the simultaneous presence C allele of AT1R A1166C and I allele of ACE strongly increased the risk of psoriasis by 6.42-fold. In addition, II+I/D genotype of ACE I/D significantly increased susceptibility to psoriasis and was associated with lipid peroxidation and antioxidant defense.
ACKNOWLEDGMENTS
This study was performed in partial fulfillment of requirements for an MD degree in Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran (Badieh Falahi). This study was funded by Kermanshah University of Medical Sciences, Kermanshah, Iran; Grant #91323.
CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest.
