Introduction

The angiotensin-converting enzyme (ACE) is a key enzyme of the renin–angiotensin system (RAS) that has long been recognized as crucial in the regulation of systemic blood pressure and renal electrolyte homeostasis [1, 2]. This enzyme acts by converting the inactive Angiotensin I to the vasoconstrictor Angiotensin II, and inactivates the vasodilator proinflammatory bradykinin and substance P peptides [3]. The ACE gene (encoding enzyme type kinase II, EC 3.4.15.1) is located on chromosome 17 and the gene polymorphisms have been widely investigated. The insertion/deletion (I/D) polymorphism of ACE has been discovered and described by Rigat et al. [4]. The polymorphism is characterized by either the presence/insertion (I) or absence/deletion (D) of a 287-base pair Alu1 element inside intron 16 of the ACE gene, producing three genotypes (DD, II homozygote, and ID heterozygote) [4, 5].

Several investigations have been shown that the D allele of an insertion/deletion (I/D) polymorphism of the gene encoding ACE is associated with higher plasma ACE concentrations [4, 5] and suggested an association of the ACE I/D polymorphism with incidence, pathogenesis and progression of several diseases including diabetes mellitus and its long-term macro- and microvascular complications [6–8], including diabetic nephropathy [9–11]. Moreover, other studies have also provide a positive association of the ACE I/D polymorphism and cardiomyopathy [12, 13] and hypertension [14]. Since the ACE I/D polymorphism is also associated with the overall plasma ACE concentration [15], it was suggested that ACE might be a good candidate gene for the metabolic syndrome, which is clustered together with hypertension, DM and dyslipidemia [16]. However, conflicting results have been reported regarding the association between ACE I/D polymorphism and diseases [17–24]. Moreover, various published reports have suggested inter-ethnic variations in the frequency of allelic forms of the ACE genes [25–27].

The aim of our study was to test the distribution of allele and genotype frequencies of ACE (I/D) polymorphism among Bahrainis and to compare the results with those of other ethnic groups. The Bahraini population is an Arabic peninsula population with a high prevalence of T2DM occurring in 20–30% of adult population [28] and hypertension [29]. The characteristics of Arabic population make them ideal for the study of complex, polygenic, multifactorial disorders such as diabetes. Arabic populations are characterized by genetic homogeneity due to tribal structure of their society, large family size and extensive consanguinity, as a result of these factors, the statistical power of susceptibility and target gene discovery studies is greatly increased if homogeneous and consanguineous populations like the Arabic populations are used.

Materials and methods

Study population

This epidemiological study comprised a total of randomly selected 560 unrelated adults Bahrainis attending the two major hospitals in Bahrain: Salmaniya Medical Center (SMC) and Bahrain Defense Force (BDF) with or without problems. Each subject received a questionnaire which included an informed consent form to sign agreeing to participate in the study. The project also had the relevant ethical clearance from the Ethics Committee from the Arabian Gulf University to undertake the study.

DNA extraction

Venous blood was collected by venipuncture from each individual in vacutainer tubes with EDTA as anticoagulant. Peripheral blood mononuclear cells were used as a source of genomic DNA. Extraction of DNA was performed according to the protocol of the QIAamp DNA Blood mini-spin column (QiagenGmbH, D-40724 Hilden, Germany).

ACE I/D polymorphism genotyping

Genotyping was performed for the insertion/deletion (I/D) polymorphisms in intron 16 of ACE gene by PCR-gel electrophoresis on the extracted DNA using a pair of oligonucleotide primers 5′-CTGGAGAC CACTCCCAT CCTTTCT-3′ and 5′-GATGTG GCCATCACATTCG TCAGAT-3′ (Biobasic, Markham, Canada). The reactions were run with 5 pmol of each primer in combination with Taq PCR Master Mix (Qiagen, Valencia, CA). DNA was amplified using cycling conditions described previously [15]. PCR products were separated on 2% agarose gels after staining with Vistra Green TM. Two DNA fragments were detected; 190 bp indicating the deletion allele (D) and 490 bp indicating the insertion (I) allele. Under some conditions, the ACE D allele amplifies more effectively than the longer I allele, resulting in mistyping of the ID as the DD genotype. To eliminate the possibility, each sample that had the DD genotype was re-amplified with an insertion-specific primer pair, which recognized an insertion-specific sequence DD F 5′-TGG GAC CAC AGC GCC CGC CAC TAC-3′ DD R 5′-TCG CCA GCC CTC CCA TGC CCA TAA-3′ [30]. The interpretation of PCR results was based on the detection of a PCR product of a 335 bp band of insertion (I alleles) to determine whether mistype of the DNA, was present, or whether no PCR product appeared on the gel [31].

Data analysis

Statistical analysis was performed using SPSS version 14.0 statistical package for Windows. Allele and genotype frequencies were determined by allele counting and the χ2 (Chi-squared) were used to compare observed versus expected outcomes. A value of P < 0.05 was taken as significant.

Results

Characteristics of the randomly selected Bahraini population

In this study, the Bahraini subjects (n = 560) comprised of 295 (52.6%) males and 265 (47.2%) females with a mean age (±SD) of 49.5 ± 15 years and a mean body mass index (BMI) of 26.34 ± 6.5 kg/m2 (Table 1). In this population, 65 (11.6%) were diagnosed as T2DM based on 1998 World Health Organization diagnostic and classification criteria [32]. In addition, there were 6.2% with Nephropathy, 2.3% with Retinopathy, 1.8% with Neuropathy, 30.3% with Hypertension, 25.3% with Hypercholesterolemia, and 7.3% with Cardiovascular Diseases (Table 1).

Table 1 Characteristics of 560 Bahrainis
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Genotypes and allele frequencies of I/D polymorphism of the ACE gene in the randomly selected Bahraini population

Allele distribution of ACE I/D polymorphism among the Bahraini subjects was calculated by allele counting using the Hardy–Weinberg (H–W) equation. The results are summarized in Table 2. The frequency of the I allele was 0.38 while that of the D allele was 0.62. Among the 560 Bahraini individuals, 3 of total 228 of DD genotypes were confirmed to be ID and the frequencies of the II, ID, and DD genotypes of the ACE gene in the studied subjects were 15.5, 44.3, and 40.2%, respectively (Table 2). The gene frequency distribution did not deviate from the H–W equilibrium (p 2 = 0.1444; 2pq = 0.4712; q 2 = 0.3844) (Table 2).

Table 2 Frequency of genotypes and allele distribution of ACE I/D polymorphism in Bahraini subjects
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Discussion

The present study investigated for the first time, the frequency of the ACE gene I/D polymorphism among randomly selected unrelated Bahraini subjects and the results obtained were compared with other geographic groups (Table 3). Among the randomly selected unrelated Bahraini subjects, the frequency of the D allele of the ACE I/D gene polymorphism was found to be (0.62), which is consistent with those obtained from the Gulf Region with similar identity to the Omanis (0.71) [33, 34], the Emiratis (0.66) [34, 35], and similar to other Arabs, including such as the Tunisians (0.76) [36], Algerians (0.73) [36], Somalis (0.73) [33, 34], Egyptians from Ismailia (0.68) and from Sinai (0.66) [37], Jordanians (0.66), Sudanese (0.64) [33, 34], and Syrians (0.60) [37].

Table 3 Prevalence of I/D polymorphisms of the ACE gene among different ethnic group
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This high frequency of the D allele among Bahrainis is similar to that observed previously not only among Arabs but also in Africans (0.70–0.60) [25, 38]. This is close to the frequency found in Caucasians (0.46–0.58) [5, 39, 40] but much higher than that found in Japanese (0.33–0.35) [41–43] and Chinese (0.29) [44]. On the other hand, the Yanomami Indians and Samoans have the lowest frequencies, 0.15 and 0.09, respectively [25] (Table 3).

The high level of D allele among Bahrainis might have an impact on the high prevalence of diabetes and its complications in addition to hypertension in Bahraini population, which is one of the highest populations in the world regarding rate of diabetes. The positive association of DD genotype with the development of hypertension was previously reported [7, 14, 45, 46] and with the development of cardiovascular disease [5, 6, 12, 13, 47].

Conclusion

The results obtained for the D allele of the ACE I/D gene polymorphism among Bahrainis is consistent with those obtained from previous studies in Arabs, Africans, and Caucasians. This study adds to the data indicating the wide variations in the distribution of the ACE alleles in different populations and highlights that great care needs to be taken when interpreting clinical data on the association of the ACE alleles with different diseases. Further studies need to be done on diabetic and hypertensive patients among Bahraini population compared with control to study the association of ACE polymorphisms with both diseases.