Association of Angiotensin-Converting Enzyme ACE Gene Polymorphism with ACE Activity and Susceptibility to Vitiligo in Egyptian Population

Abstract

Aim: The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene is associated with vitiligo in the Indians and Koreans, but not in those of English or Turkish background. We investigated the ACE (I/D) polymorphism in vitiligo patients for the first time in Egypt and compared serum ACE levels between vitiligo patients and controls. The present study was carried out in 100 vitiligo patients (40 males and 60 females) and in 100 healthy controls of an Egyptian population using the polymerase chain reaction genotyping method. Results: The ACE genotype and allele frequency was significantly different between vitiligo patients and controls. Our results revealed a significant increase in the frequency of the ACE I allele (p=0.002; odds ratio: 1.99; 95% confidence intervals: 1.207-3.284) with an overrepresentation of I/D genotype in the vitiligo patient group. Furthermore, there was a significant difference between the segmental, nonsegmental, and focal vitiligo in ACE gene genotype distribution. Serum ACE levels were significantly increased in vitiligo patients compared to controls (p=0.034). Conclusion: This study suggests that, for the first time, ACE gene polymorphism confers susceptibility to vitiligo in the Egyptian population.

Introduction

Vitiligo is a commonly acquired pigmentary disease of unknown etiology and great social importance (Ortonne and Bose, 1993; Passeron and Ortonne, 2005). It affects around 1-2% of the population irrespective of gender and race with a history of familial incidence (Sun et al., 2005; Taïeb and Picardo, 2009). Depigmentation of the skin, with destruction of melanocytes, characterizes this disease with a symmetrical distribution as the common presentation of the disease (Nordlund and Ortonne, 1998; Ezzedine et al., 2015); however, segmental distribution is sometimes seen (Thappa, 2002).

The etiology of vitiligo is multifactorial involving autoimmune background, genetic predisposition, neurogenic factors, and environmental triggers (Dell'Anna and Picardo, 2006; Le Poole and Luiten, 2008; Czajkowski and Męcińska-Jundziłł, 2014). Genetic marker researches have reported several genes to be vitiligo-susceptible genes with diverse results according to different ethnic groups (Kim et al., 2007; Li et al., 2009; Spritz, 2011).

The angiotensin-converting enzyme (ACE) gene was chosen as a candidate gene in this study due to its vasoconstrictor function in the renin-angiotensin system, which regulates the vasculature and inflammation of various diseases in addition to its relationship with various autoimmune diseases (Smallridge et al., 1986; Noveral et al., 1987; Hooper, 1991; Scholzen et al., 2003; Ha, 2014). The ACE gene contains an insertion (I)/deletion (D) polymorphism within intron 16 giving two codominant alleles (Rigat et al., 1990); the D allele of I/D polymorphism of ACE gene in vitiligo patients has been reported in many previous studies. However, in several ethnic populations, the genetic association of ACE gene I/D polymorphism with vitiligo had varied results. Moreover, a predisposition to vitiligo due to the ACE gene D variant has not been identified (Tippisetty et al., 2011).

To the best of our knowledge, this is the first work to address ACE gene I/D polymorphism and its association with vitiligo in Egypt. In this study, the distribution of ACE gene I/D genotypes was investigated in a population of Egyptian patients with vitiligo and age-, sex-, and ethnically matched controls. The role of ACE gene I/D polymorphism as a risk factor and/or activity marker for vitiligo was searched in relation to the ACE level.

Materials and Methods

One hundred vitiligo patients (40 males and 60 females) (age ranged from 20 to 60 years) who attended the Dermatology outpatient clinic at Suez Canal University Hospitals, Ismailia, Egypt, from April 2013 to January 2014, were included in this study. All patients were documented to have vitiligo and were selected randomly from patients attending the clinic and who met the inclusion criteria for vitiligo mentioned before (Thappa, 2002).

A total of 100 healthy age- and sex-matched controls (32 males and 68 females) with no history of vitiligo or other autoimmune diseases were recruited in the study. This study was approved by the Ethics Committee of Suez Canal University, and all subjects provided written informed consent.

ACE gene I/D polymorphism analysis

Blood sampling

Five milliliters of venous blood was drawn from each subject under sterile conditions and divided as follows: 2.5 mL in vacutainer tubes containing anticoagulant (one part of 5% EDTA for each nine parts of blood) and mixed by gentle shaking and 2.5 mL in plain tubes. The former tubes were used for DNA extraction for subsequent polymerase chain reaction (PCR) amplification and the latter ones were centrifuged after clotting formation, stored in aliquots at −20°C, and were not thawed until the assessment of serum level of ACE by enzyme-linked immunosorbent assay (ELISA).

Extraction of genomic DNA

Genomic DNA was extracted from the peripheral blood leucocytes of ethylenediaminetetraacetic acid (EDTA) anticoagulant blood, according to Miller et al. (1988), using the QIAamp DNA Blood Mini Kit (Cat. No. 51106; Qiagen). DNA samples were subjected to DNA quantitation using the NanoDrop^® (ND)-1000 spectrophotometer (NanoDrop Technologies, Inc.).

PCR amplification

Genomic DNA was subjected to PCR amplification using oligonucleotide primers 5′-CTGGAGACCACTCCCATCCTTTCT-3′ (forward) and 5′-GATGTGGCCATCACATTCGTCAGAT-3′ (reverse) (Metabion international AG) to amplify the ACE gene I/D polymorphism.

PCR amplifications were performed on 50-100 ng of genomic DNA in 25 μL reactions containing the following: 12.5 μL of 2×EmeraldAmp GT PCR master mix (Taq DNA polymerase, deoxynucleotide bases [dATP, dCTP, dGTP, and dTTP] and optimized reaction buffer) (Takara Bio, Inc.), 0.5 μL of each primer (10 pmol of each primer), and purified water. The PCR amplifications were performed in an Eppendorf Master cycler gradient thermocycler (Eppendorf North America, Inc.) using the following conditions: initial denaturation at 95°C for 5 min; 10 cycles of amplification at 95°C for 1 min, 57°C for 2 min, and 72°C for 1 min; 22 cycles of amplification at 95°C for 1 min, 57°C for 2 min, and 72°C for 1 min. One final cycle of extension was performed at 72°C for 5 min. Ten microliters of the PCR amplification products was loaded in 1.5% (w/v) agarose gels stained with ethidium bromide using the GeneRuler™ 1 kb Plus DNA Ladder (Ready-To-Use). After electrophoresis, gels were visualized under UV light using the gel documentation system and genotyped according to the following pattern: the presence of a single PCR product of 480-bp represented an insertion homozygous individual (I/I), the presence of a single PCR product of 193-bp represented a deletion homozygous individual (D/D), while a heterozygous individual (I/D) was detected by the presence of both PCR products.

Assessment of serum ACE level by ELISA

The Human ACE Quantikine ELISA Kit for measuring the quantitative determination of human angiotensin I-converting enzyme (ACE) concentrations in cell culture supernates, serum, plasma, and saliva was purchased from R&D Systems, Inc. ACE levels in vitiligo patients' and control subjects' sera were assayed according to the manufacturer's protocol. ACE levels were reported as ng/mL. Intra- and interassay coefficients of variation were <10%.

Statistical analyses

All the statistical analyses were carried out using SPSS software version 19.0 for Microsoft Windows^®. The genotype and allele frequencies were determined by direct counting. The Hardy-Weinberg's equilibrium was evaluated using a chi-square test. Statistical comparisons between healthy controls and vitiligo patients were performed using the chi-square test, Fisher's exact test, and two-way Student's t-test. Associations of genotypes with the ACE level were evaluated by the ANOVA test. A value of p<0.05 was considered significant statistically. All data are presented as mean±SD.

Results

Genotype distribution of ACE polymorphisms between cases and controls

A total of 100 vitiligo patients and 100 healthy controls were included in the study. The baseline demographic characteristics of the study population are given in Table 1. The distribution of ACE I/D genotypes among cases and controls is shown in Table 2. The distribution of all genotypes among control subjects and cases is in agreement with the Hardy-Weinberg equilibrium. The allele and genotype frequencies for the ACE I/D polymorphism in control and patient populations are shown in Table 2. A significant increase in the frequency of the I allele was noted in vitiligo patients compared with controls: of 100 vitiligo alleles, 80 (40%) had the I allele compared to 62/100 (31%) control alleles (p=0.002; odds ratio: 1.99; 95% confidence intervals: 1.207-3.284) (Table 2). The results also indicated that the distribution of the DD, DI and II genotypes was significantly different between the controls and vitiligo patient groups (p=0.0055), with an overrepresentation of the D/I genotype in the vitiligo patient group 56% compared to 32% and 12% for DD and II, respectively, as shown in Table 2.

Table 1.

Descriptive of Case and Control

	Value
Characteristic	Case	Control	p-Value
Gender
Female, n (%)	60 (60%)	68 (68%)	0.567
Male, n (%)	40 (60%)	32 (32%)
Age (years, mean±SD)	28.46±23.28	32.16±26.43	0.71
Disease duration (years, mean±SD)	8.22±6.01	—	—
Family history
Yes, n (%)	60 (60%)	—
No, n (%)	40 (60%)
Other autoimmune diseases
Yes, n (%)	12 (12%)	2 (2%)	0.045^*
No, n (%)	88 (88%)	98 (98%)
Vitiligo classification		—	—
Focal, n (%)	26 (26%)
Nonsegmental, n (%)	64 (64%)
Segmental, n (%)	10 (10%)
DNA	21.39±9.09	49.488±47.08	0.012^*
ACE ELISA/ng/mL	17.966±7.4630	5.956±2.2467	0.034^*

p<0.05 is statistically significant.

ACE, angiotensin-converting enzyme; ELISA, enzyme-linked immunosorbent assay.

Table 2.

The Allele and Genotype Frequencies for the ACE I/D Polymorphism in Control and Patients

	Control		Vitiligo
ACE gene	n (%)		n (%)		p	OR	95% CI
D allele	138	69	120	60	0.002^*	1.99	1.207-3.284
I allele	62	31	80	40
DD	52	52	32	32
ID	34	34	56	56	0.0055^*
II	14	14	12	12

Comparisons were performed by chi square (χ²) and Fisher's exact tests; CI and OR where appropriate, for 2×2 or 2×3 contingency tables for vitiligo patient group versus control group.

p<0.05 is statistically significant.

CI, confidence interval; OR, odds ratio.

The genotype and allele distributions between vitiligo patient subgroups (clinical subtype) showed a significant association between ACE I allele and nonsegmental vitiligo (38) in comparison to (28 and 14 in focal and segmental vitiligo, respectively) an overrepresentation of I/D genotype in nonsegmental vitiligo as well (Table 3).

Table 3.

Comparison of the Genotypes and Allele Distribution Between Vitiligo Patient Clinical Subgroups

	Focal	Nonsegmental	Segmental
ACE gene	n	n	n	p	OR	95% CI
D allele	24	86	10	0.0022^*	2.104	1.169-3.788
I allele	28	38	14
DD	2	28	2	0.0006^*
ID	20	30	6
II	4	4	4

Comparisons were performed by chi-square (χ²) and Fisher's exact tests; CI and OR where appropriate, for 2×3 or 3×3 contingency tables for vitiligo patient subgroups.

p<0.05 is statistically significant.

Serum ACE levels in vitiligo patients and control subjects

Serum ACE levels were determined by ELISA as detailed in the Materials and Methods section. The results indicated that serum ACE levels were significantly elevated in vitiligo patients (n=100) compared to controls (n=100) (17.966±7.4630 vs. 5.956±2.2467 ng/mL, respectively; p=0.034), as shown in Table 1. In addition, significant differences among the three genotypes were found for both the vitiligo patients and healthy controls (p-values were=0.000), with the I/D genotype correlating with the highest levels of serum ACE according to the order I/D>D/D>I/I (p-values were <0.05) in vitiligo patients (Table 4). Furthermore, the serum ACE levels in the vitiligo cases were significantly increased compared with those of the healthy control subjects of the same genotype (all p-values were <0.05) (Table 4).

Table 4.

Serum ACE Levels

ACE	Control	Vitiligo
genotype	ACE ELISA/ng/mL	ACE ELISA/ng/mL	p-Value
DD	5.51±2.12	17.75±6.55	0.000^*
ID	6.05±2.42	18.16±8.34	0.000^*
II	7.40±1.86	15.09±4.92	0.011^*

p<0.05 is statistically significant.

Discussion

Various studies on different populations have verified that ACE gene I/D polymorphism is associated with the development of vitiligo (Shajil et al., 2006). Furthermore, ACE and its related metabolites are known to have multiple roles in the immune system as well as in autoimmune diseases (Kozlowski et al., 1992).

To the best of our knowledge, this is the first study to investigate the association of ACE gene I/D polymorphism with vitiligo in Egyptian populations. In our study, the frequency of D allele in the Egyptian control population is 0.69, which is approximately the same as found by Salem and Batzer (2009) who reported the frequency of the D allele of the ACE gene among Egyptians from Ismailia (0.68), Sinai (0.66), Syrians (0.60), and Jordanians (0.66) and which was also in accordance with that in other Arabian populations, such as Sudanese (0.64), Tunisians (0.76), Algerians (0.73), Moroccans (0.70), Somalis (0.73), Omanis (0.71), and Emiratis (0.61-0.66) (Frossard et al., 1997; Comas et al., 2000; Bayoumi et al., 2006). However, the frequency of D allele is moderate among the western population although the predominance of the D allele over the I one was evident in studies done on the French (0.54), Dutch (0.54), American (0.56), Danish (0.55), and English (0.55) populations (Cambien et al., 1992; Schunkert et al., 1994; Lindpaintner et al., 1995; Tarnow et al., 1995; Chowdhury et al., 1996).

On the other hand, our results are different from that reported by the Chinese (0.29), Japanese (0.33-0.35), Korean, Indian (0.46), and Caucasian (0.46-0.51) populations, indicating a similarity regarding the high frequency and overrepresentation of D allele in the normal Egyptian population with other Arabian populations and dissimilarities with the Asian ones (Barley et al., 1994; Saha et al., 1996).

Such variability with different populations could be explained on the basis of ethnic variation as Barley et al. (1994) suggested that the ethnic origin should be carefully considered in the increasing number of studies on the association between I/D ACE genotype and disease etiology.

In addition, a significant increase in the frequency of I allele was noted in Egyptian vitiligo patients 40% (n=80) compared with controls 31% (n=62) noting that the current study suggests that the polymorphic variant I allele is associated with vitiligo susceptibility, in contrast to those found in the Indian and Korean populations, which indicated that the D allele was significantly overrepresented in vitiligo patients compared with controls (Jin et al., 2004; Deeba et al., 2010).

On the other hand, our results are dissimilar from that done on the English, Turkish, and Gujarat populations, which revealed that the ACE polymorphism did not appear to be associated with such risk. Moreover, Tippisetty et al. (2001) highlighted the protective role of I/I genotype and the significant association of I/D genotype with slow progression of the disease.

Our study also indicated that the distribution of the DD, ID, and II genotypes was significantly different between the control and vitiligo patient groups with an overrepresentation of the I/D genotype in the vitiligo patient group, (56%) compared to 32% and 12% for DD and II, respectively. The results of the genotype distribution rate (I/D>I/I>D/D) in the vitiligo patient population are in accordance with the previously reported distribution rate of I/D for the Chinese (Lee, 1994), Japanese (Ishigami et al., 1995), and Korean populations (Jin et al., 2004). The Indian study done by Deeba et al. (2010) showed a different distribution rate of I/D>D/D>I/I despite the predominance of ID genotype.

Furthermore, there was a significant difference between the segmental, nonsegmental, and focal vitiligo in ACE gene genotype distribution noting that a significant association was found between ACE I allele and nonsegmental vitiligo, while Deeba et al. (2010) clarified that there was no significant difference in the ACE genotype or allele frequencies between the two groups, segmental and nonsegmental, of vitiligo south Indian patients. In the same line, Akhtar et al. (2005) found that the polymorphic variant D allele was not associated with the generalized form of the disease or any of its clinical subtypes in English vitiligo patients.

This diversity in results could be referred to the complexity of the pathogenesis of vitiligo (Zhang et al., 2005) as well as to the racial and ethnic variations with respect to the contributory factors that participate in vitiligo development (Spritz, 2008).

Previous studies have reported that the ACE D/D genotype is associated with significantly higher levels of serum ACE in patients with vitiligo and autoimmune (Patwardhan et al., 2013) disease, which were similarly evident in the current study; significant differences among the three genotypes were found for both the vitiligo patients and the controls, with the I/D genotype correlating with the highest levels of serum ACE according to the order I/D>D/D>I/I (p-values were <0.05) in vitiligo patients. Zhu et al. (2001) reported that although I/D polymorphism has a well-built association with ACE gene regulatory elements that are responsible for the difference in enzyme levels, this did not rule out the changes in mRNA stability or mRNA precursor splicing due to the insertion of an extra base pair. Furthermore, the serum ACE levels in the vitiligo cases were significantly increased compared with healthy controls of the same genotype. Although we have no clear explanation for this result, earlier studies reported similar results in patients with autoimmune disease, but again with no definitive explanation for its rule in the disease (Rigat et al., 1990). Elevated levels of ACE can lead to oxidative stress and consequent tissue damage, which can reinforce the vitiligo underlying etiological mechanisms (Molnár et al., 2004). It has been noted previously (Tiret et al., 1992) that although a significant ACE activity can be found in the circulation, the origins and functional significance of this ACE have not been defined as yet. However, the amount of circulating ACE appears to be related to the I/D genetic polymorphism and the overlap in ACE levels between different ACE genotypes suggests the presence of other, not yet defined, determinants of circulating ACE (Um et al., 2001). Generally, further investigations are required to determine the exact role of ACE in vitiligo pathogenesis.

Conclusion

The statistically significant variation in the frequency of I/D polymorphism in the ACE gene in both populations strongly indicates that ACE gene polymorphism confers susceptibility to vitiligo in the Egyptian population. Our results also suggest that the I allele might be an important risk factor for vitiligo development, thus supporting its autoimmune etiology.

Footnotes

Acknowledgments

We wish to thank the participants who provided us with samples. We also thank Dr. Mona Ghaly (Faculty of Medicine, Suez Canal University, Egypt) for helping with statistical analyses.

Authors' Contributions

All authors participated in the design of the study. H.N. provided the vitiligo patients and reviewed the manuscript. D.B. and R.H. designed the study, carried out the DNA isolation, performed the genotyping, wrote and edited the manuscript. All authors read and approved the final manuscript.

Author Disclosure Statement

No competing financial interests exist.

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