Syed Kashif Nawaz, Shahida Hasnain*. Pleiotropic effects of ACE polymorphism. Biochemia Medica 2009;19(1):36-49. http://dx.doi.org/10.11613/BM.2009.004
Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan
Corresponding author*: genetic [at] brain [dot] net [dot] pk
Angiotensin converting enzyme (ACE) has vital role in normal functioning of the human body due to its direct involvement in the renin-angiotensin-aldosterone system (RAAS), kinin-kallikrein system, in vitro degradation of amyloid beta-peptide, GPIase (glycosylphosphatidylinositol, GPI) activity and in signal transduction. As ACE activity level is strongly influenced by ACE insertion/deletion (I/D) polymorphism, a huge body of data has been generated to elucidate the association of I/D polymorphism with cardiovascular and non cardiovascular diseases like diabetes, diabetic nephropathy, diabetic retinopathy, atherosclerosis, coronary heart diseases and stroke, hypertension, Alzheimer’s disease, cancer and Parkinson’s disease. This review will be limited to the effect of ACE I/D polymorphism on longevity considering the pathophysiology of several diseases.
Key words: ACE polymorphism; longevity; early mortality; cardiovascular diseases
Angiotensin converting enzyme
Angiotensin converting enzyme (ACE) is a chloride and zinc dependent carboxypeptidase enzyme present on the surface of epithelial and endothelial cells. In humans, two isoforms exist. One is larger protein, composed of 1300 amino acids (150–180 kDa) and is called somatic ACE (sACE), due to its presence in somatic tissues. sACE can be anchored in plasma membrane through transmembrane domain, or be present in plasma in the soluble form (1). The other isoform is a smaller protein composed of 730 amino acids (100–110 kDa), present only in testicles and called germinal form or testicular form (tACE). Its function is not clear, but it appears to be involved in male fertility (2). These isoforms differ in active sites: sACE has two active sites, whereas tACE has only one active site (3).
Major functions of ACE are as follows:
- conversion of decapeptide angiotensin I (inactive) to octapeptide angiotensin II (active compound). Angiotensin II causes vasoconstriction, release of aldosterone, mediation of cell growth, and proliferation and induction of endothelial dysfunction (4-7);
- inactivation of bradykinin (a vasodilating neurokinin) allowing for vasoconstriction (3);
- signal transduction pathway (8);
- in vitro degradation of amyloid beta peptide (9); and
- GPI-ase activity which allows for the release of membrane glycosylphosphatidylinositol (GPI) – anchored protein (10).
Genetics of angiotensin converting enzyme
A single gene is responsible for the expression of both sACE and tACE. It is located at locus 17q23. ACE gene is 21 kb long and composed of 26 exons and 25 introns. Alternative promoters are responsible for the expression of each isoform. The promoter for sACE lies in the 5’ flanking region of the first exon and transcribes exon 1-12 and 14-26, whereas the promoter for tACE lies within intron 12 and transcribes exon 13-26 (11,12). The initiation sites for the transcription of two mRNAs encoding these isoforms are 5.7 kb apart and polyadenylation sites are 628 bp apart (13).
Insertion/deletion (I/D) polymorphism in ACE gene
In 1990, Rigat et al. observed a polymorphism involving insertion of 287 bp sequence (NCBI ref. SNP ID: rs1799752) resulting in insertion (I) allele, whereas deletion (D) allele is present in the absence of insertion (14). This polymorphism is responsible for the ACE activity level, which increases 2-fold in homozygous deletion carriers (D/D), as compared to homozygous insertion carriers (I/I). I/D carriers show intermediate ACE activity. This codominance was observed both, in plasma and tissue ACE levels (15).
Detection of I/D polymorphism
Rigat et al. (1990)used a set of primers flanking the insertion region for the detection of I/D polymorphism through polymerase chain reaction (PCR) (14). In this method, there was a problem of 5%-10% of mistyping, causing preferred amplification of the D allele depicting I/D heterozygotes as D/D carriers (16). For accurate genotyping, this mistyping had to be eliminated. Different strategies were adopted by different scientists. Shanguman et al. (1993) used 5% dimethyl sulfoxide (DMSO) and sense primer from the 5’ end of the insertion sequence, along with the standard antisense primer (17). Later on, step down PCR modification in this method improved the accuracy of the method (18).
Multiplex PCR method and use of real time PCR were also devised for the detection of I/D polymorphism,but both methods failed to replace Shangumans method, due to problems related to post PCR handling, such as agarose diagonal gel electrophoresis in case of multiplex PCR and expensive nature of real time PCR (19,20). Recently, Koyama et al. (2008) have described a quick and easy technique involving DHPLC (denaturing high performance liquid chromatography) in non denaturing conditions to analyze the PCR product for screening for I/D polymorphisms in genetic epidemiological studies (21). This method removes the chances of mistyping and yields 100% accuracy in I/D heterozygotes, but expenses of chromatography do not prove it to be cost effective.
Effects of I/D polymorphism on human health
The influence of I/D polymorphism on pathophysiological conditions mediated through ACE activity has generated a lot of data showing its association with several diseases (Table 1). In the present review, its role in deciding on the health status is discussed in the light of previous studies on several health indicators.
Table 1. Diseases in association with ACE polymorphism
Growth and ACE polymorphism
Intrauterine environment have obvious effects on the gene expression of ACE, which ultimately influences the period of gestation and birth weight of the newborn. Kajantie et al. (2004) noticed the association of I/I genotype with shorter gestation duration and higher birth weight, which indirectly means less chances for development of coronary heart diseases, type 2 diabetes, insulin resistance and metabolic syndrome in adults (22-24). So, a concept was developed on I allele to be responsible for higher birth weight. This concept remained acceptable until Hindmarsh et al. (2007) have claimed that there is no association of ACE genotype of the newborn or his/her parents with birth weight (25). However, I/I genotype exhibited advantageous role in the early growth of babies, showing more gain in weight, body mass index and midarm circumference in one fiscal year as compared to the babies with D/D genotype, the majority of which showed no change or catch-down. I/D genotype was distributed equally across all the categories. These effects were more prominent in males. So, I/I genotype has positive effects on the early growth after birth.
Diabetes and ACE polymorphism
There are many association studies showing influence of ACE I/D polymorphism on the onset of diabetic mellitus (26-29).However, recent findings do not support this statement. For example, a large follow up of 10.2 years in 24,309 Caucasian women free from diabetes at baseline failed to show any association of ACE genotype with diabetes (30). This result was replicated in many other studies in different ethnic groups, both in patients with and without nephropathy (31-33).So, ACE polymorphism cannot be supposed as an independent factor responsible for diabetes. Anyhow, further research can investigate other effective genetic factors and environmental factors to find out the possible role of ACE in the onset of diabetes.
Diabetic nephropathy and ACE polymorphism
Diabetic nephropathy is a major cause of mortality in chronic diabetic patients. The clinical course of the disease is variable among patients. A meta-analysis by Ng et al. (2005) comprising 14,727 subjects showed a significantly higher risk of diabetic nephropathy in the carriers of D allele as compared with the I/I genotype group (OR = 1.28; 95% CI = 1.14-1.45) (34). Findings of Movva et al. (2007) also demonstrated D allele carriers with type 2 diabetes to be more vulnerable to the development of diabetic nephropathy (35).
Diabetic retinopathy and ACE polymorphism
Diabetic retinopathy is blindness due to retinal damage as a complication of diabetes mellitus. The ACE gene has been the main probable candidate gene predisposing the development of diabetic retinopathy. Findings reported by Globocnik-Petrovic et al. (2003) show no association of ACE genotype with diabetic retinopathy, non-proliferative, proliferative or severe proliferative type (36). However, Matsumoto et al. (2000) and Feghhi et al. (2008) observed frequent occurrence of D/D allele in patients with proliferative diabetic retinopathy in Japanese and Iranian population, respectively (37,38). Contradictory results reported by Wiwanitkit (2008) also reject involvement of ACE polymorphism in the development of diabetic retinopathy (39). So, because of contradictory findings, it is difficult to define the role of ACE polymorphism in the expression of diabetic retinopathy.
Atherosclerosis and ACE polymorphism
Atherosclerosis can be diagnosed by measuring intima media thickness (IMT), on autopsy and by determining coronary calcification. Sayed-Tabatabaei et al. (2003) conducted a meta-analysis of 23 articles published until October 2002, including 9,833 subjects to study the association of ACE polymorphism with atherosclerosis based on IMT measurement (40). Data suggested strong relation between D allele and common carotid IMT. This association was more prominent in subjects with cerebrovascular disease, diabetes or hypertension. Similar results have been reported by Pawel et al. (2008) and Kretowski et al. (2007) (41,42). It is noteworthy that experiments involving autopsy measurements and coronary calcification for the detection of atherosclerosis showed no association of ACE polymorphism with atherosclerosis (43,44). Discordant findings due to different diagnostic methods pose the need of more reliable diagnostic methods to confirm the presence of disease, which will help identify the role of ACE polymorphism in atherosclerosis.
Hypertension and ACE polymorphism
D/D genotype is predicted to be associated with hypertension, because of the increased level of ACE activity. Different ACE variants showed different chances to develop hypertension, due to aging and abnormality in nocturnal blood pressure (45,46).The first meta-analysis based on 23 studies consisting of 28 case-control groups with 6,923 subjects showed a 10% increased, but statistically non-significant risk, of hypertension in D/D versus I/I genotype. Anyhow, there was a significant correlation between D genotype and hypertension in women and in Asians (47). Another meta-analysis restricted to Caucasians, also failed to show any association between ACE genotype and hypertension (48). Findings by Miyama et al. (2007) and Glavnik and Petrovic (2007) also showed no influence of ACE genotype on hypertension (49,50). These contradictory results indicate the complex interaction of ACE polymorphism with the environmental and other genetic factors for the expression of hypertension. Recently, Bautista et al. (2008) have reported on D/D genotype as an independent factor for developing hypertension among Hispanics. Similar findings have been reported for the Chinese population and male population of Bangladesh, Japan and Argentina (51-55).However, Napoles et al.(2007) found no association of ACE genotype and hypertension in Cuban population (56). It is also noteworthy that the effects of ACE polymorphism in different ethnic groups are different. It predicts the effects of different lifestyles and environmental factors causing hypertension in different ethnic groups.
Coronary heart disease and ACE polymorphism
The role of D/D genotype in myocardial infarction observed by Cambien et al. (1992) generated huge scientific interest, but contradictory results make its role controversial (57). A meta-analysis involving 1,918 white subjects (1,196 cases and 722 controls) showed no difference in ACE genotype (P > 0.05) or allele frequency (P > 0.05) between cases and controls. The overall OR for D allele as an independent risk factor in ischemic stroke was 1.31 under a recessive model, and 1.14 under a dominant model. This result indicates that D allele, acting recessively, is a modest but independent risk factor for ischemic stroke onset (58). These results, however, were not confirmed by a large meta-analysis of 46 studies including a total of 32,715 white individuals (48). Results showed an association of ACE polymorphism with plasma activity level, but not with cardiovascular diseases. Meta-analysis studies in Asian population also showed the absence of any role of ACE polymorphism in disease occurrence (59). Later on, similar results were replicated in the next years (60,61). It can be concluded that ACE genotyping has no prominent association with myocardial infarction.
Alzheimer’s disease and ACE polymorphism
In vitro degradation of amyloid beta-peptide by ACE predicted its possible protective role against Alzheimer’s disease (AD). First of all, Kehoe et al. (1999) observed positive association between I allele and AD (62). Later on, they found that SNPrs 4343 was more associated with AD in spite of SNPrs 4291, which was previously thought to be a strong genetic variation responsible for AD (63). Effects of ACE polymorphism on AD were further verified by a meta-analysis by Lehmann et al. (2005) (64). The meta-analysis included 39 studies comprising 6,037 AD cases and 12,099 controls. Findings suggested that D/D carriers were at a reduced risk (OR = 0.81; 95% CI = 0.72-0.90; P < 0.001); I/I homozygotes exhibited no association with AD, while heterozygotes were more vulnerable to AD. Similar results were seen among North Europeans, South Caucasians, and East Asians. Anyhow, in North Europeans, both association and Hardy-Weinberg analysis indicated partial heterogeneity, due to unknown reason. These results were also replicated in other studies (65,66). Some discordant results have also been reported, thus opening way to further investigations to confirm the role of ACE in disease manifestation (67-69). The greater body of data confirming association as compared to those denying association appears to suggest the influence of several factors on deciding about ACE polymorphism as a candidate gene variation responsible for AD.
Little work has been done to find out any relation of ACE polymorphism with Parkinson’s disease (PD). Lin et al. (2003) conducted a case-control study comprising of 127 sporadic PD patients and 198 healthy controls, and observed the presence of homozygote D/D genotype to be more frequent in patients with PD than in controls (P = 0.048), although there was no significant difference in the allelic frequency (P = 0.133) (70). A stepwise logistic regression analysis verified the independent role of D/D genotype as a risk factor for PD (OR = 1.32; 95% CI = 1.12-2.16).
Lin et al. (2007) also observed that ACE I/D polymorphism was primary predictor for the occurrence of psychosis in L-dopa patients (71). So, ACE genotyping is recommended in PD patients for identification of subjects at risk and for minimizing the chances of L-dopa induced psychosis. These studies are not sufficient for conclusive role of ACE polymorphism but seek confirmation through a cohort followed until the late phase of PD.
Cancer and ACE polymorphism
ACE polymorphism involvement in the occurrence of several malignancies, tumor cell proliferation, tumor cell migration, angiogenesis and metastatic behavior is mediated by angiotensin I/I which has been proven to be an angiogenic and growth factor (72). This information has become a basis for many association studies related to different types of cancer. We shall discuss the outcomes of some of them.
In 2003, Koh et al. analyzed 189 incident breast cancer cases and 671 female cohort control subjects for sorting out any impact of ACE polymorphism on breast cancer in Singapore (73). It was observed that I allele carriers were at a low risk as compared to D allele carriers, suggesting that the renin-angiotensin system may serve as a therapeutic target for breast cancer treatment and prevention. This finding was also supported by many other studies (74-76).
Vairaktaris et al. (2007) recorded a three-fold risk in I/I homozygotes for developing oral cancer, regardless of smoking habit or alcohol consumption, early or advanced stage of cancer, and presence or absence of a family history of cancer or thrombophilia (77). For confirmation of the above mentioned results, additional analyses with a larger sample size are required.
No relation of ACE polymorphism with gastric cancer was observed by Röcken et al. (2005) but in the same year, discordant results were obtained by Goto et al. (72,78). Their results were based on a study including 454 Japanese subjects undergoing health checkup and 202 gastric cancer patients. There was no effect of the polymorphism on Helicobacter pylori seropositivity or gastric atrophy. However, I/D carriers were at an increased risk of gastric cancer (OR = 1.59; 95% CI = 1.02-2.48).
Röcken et al. (2007) conducted experiments to assess local expression of ACE by using quantitative reverse transcription-polymerase chain reaction and by immunohistochemistry in colorectal carcinomas and adenomas (79). Results showed greater production of ACE protein in adenomas (17 Š81%Ć) and cancer epithelial cells (22 Š100%Ć) than in the corresponding non-neoplastic crypt and surface epithelium (2 Š10%Ć and 2 Š9%Ć, respectively). Moreover, I/D polymorphism was found to be associated with gender specific differences in primary tumor size and patient survival. Female colorectal carcinoma patients were found to more frequently have I/D genotype and less frequently I/I and D/D genotypes as compared to male patients. Tumors of I/D and D/D male carriers were larger than those of I/I genotype. In the same year, the findings reported by Nikiteas et al. negated the above results suggesting controversial predisposition of ACE polymorphism for colorectal cancers (80).
Muscle performance and ACE polymorphism
Endothelium-dependent vasodilation can be increased with aerobic exercise in healthy individuals due to the increase in nitric oxide (NO) production and decreased NO inactivation, leading to an increase in NO bioavailability. Improvement of vasodilation by regular isotonic exercise varies with different alleles of ACE carriers. ACE I/I carriers can better improve this vasodilation as compared to I/D and D/D carriers (81). So, I/I genotype promotes the chances for improvement of vasodilation during aerobic exercise.
Frequency distribution of ACE genotype indicates that I/I and I/D genotypes are frequent in endurance athletes, long distance runners, rowers and mountaineers, whereas D/D genotype is found mostly among top level professional French cyclists (82,83).Moran et al. (2006) observed a relation of I allele with phenotypes related more to strength than to endurance in 1,027 teenage Greeks (84). It suggests a more complicated role for the ACE gene in human physical performance than previously described. This finding apparently opposes the results of previous experiments. However, those studies are not comparable due to high selection and relatively small population. So, a modest influence of ACE gene on physical performance is clear in general population. Now, the only challenge is to trace out the mechanism of ACE influence on performance related phenotypes.
Immunity and ACE polymorphism
Effects of ACE genotype on immunity and immune disorders have also been a topic of interest during the previous decade. We shall precisely discuss a few of them pointing out the importance of ACE polymorphism.
The role of ACE I/D polymorphism in defense against sepsis in children was studied in detail by Cogulu et al. (2008) (85). They noted that I allele carriers (I/I or I/D genotype) were at an increased risk as compared to D/D carriers. This finding speculates the protective role of D/D genotype against sepsis. As this finding is based on the experiment with a small number of subjects (N = 287), it is needed to repeat the experiment in a large population to make a doubt free conclusion.
Asthma and allergic rhinitis
Development of symptoms of asthma in some patients and of allergic rhinitis in other patients conjectures the involvement of genetic factors. Lue et al. (2006) tried to find out the genetic reason for the two different phenotypes and speculated the ACE polymorphism for it (86). They performed genotyping for ACE in 106 children with allergic rhinitis but no asthma, 105 age- and gender-matched children with allergic rhinitis and asthma, and 102 healthy children. Serum level of total immunoglobulin E (IgE), allergen-specific IgE sensitivity, and eosinophil count were also measured for each sample. A more frequent occurrence of D/D genotype in children with both allergic rhinitis and asthma than in children with allergic rhinitis but no asthma showed the protective role of D/D genotype in the development of asthma phenotype in children with allergic rhinitis.
Systemic lupus erythematosus
Systemic lupus erythematosus (SLE) manifestation due to ACE polymorphism was confirmed by Lee et al. (2006) in a meta-analysis study of 13 comparison studies including 1,411 patients with SLE and 1,551 controls (87). No effect of ACE I/D polymorphism was observed on SLE either in total sample or according to ethnic groups. A trend for association of D/D genotype (OR = 1.212; 95% CI = 0.966-1.520; P = 0.097) and D allele with SLE was observed in Caucasian patients (OR = 1.157; 95% CI = 0.991-1.349; P = 0.064); however, it was not statistically significant. So, it is obvious that there is no relation of ACE polymorphism with SLE.
Bones and ACE polymorphism
Osteoporosis, a bone disease, is a multifactorial problem reported in elderly subjects of both sexes. Scarce data are available showing the role of ACE I/D polymorphism in the manifestation of osteoporosis and its treatment. There is evidence showing an association of ACE polymorphism with osteoporosis (88). Findings suggest that low ACE activity associated with ACE I/I genotype has a beneficial role in its treatment. In a cross-sectional study of 3,887 Chinese men (N = 1958) and women (N = 1,929), Lynn et al. (2006) observed that I/I carriers showed better results with ACE inhibitor therapy as compared to I/D or D/D carriers (89,90). Similar advantageous effects of ACE I/I genotype have been reported by Woods et al. (2001) for hormone replacement therapy (91). So, ACE I/D polymorphism is also important in relation to bones.
Longevity and ACE polymorphism
The status of several diseases in association with ACE polymrophism (Table 1) predicts it as a marker of longevity. For confirmation of this hypothesis, Schachter et al. (1994) genotyped 338 centenarians and 164 control individuals aged 20 to 70 years, from a cohort ascertained by the Centre d’Etude du Polymorphisme Humain (CEPH) in Paris, France (92). Their findings were unexpected showing frequent occurrence of D/D genotype in the centenarian group compared with the control group (40% vs. 26%, P = 0.01). In 2000, the experiment was repeated with 560 additional French centenarians, each paired with a younger individual of the same sex and geographic origin. However, the results now failed to reveal a difference between the centenarian and control populations (93). A similar conclusion has been reported by Nacmias et al. (2007) (67).
In a population based study, Arias-Vasquez et al. (2003) genotyped 6,968 elderly individuals and found no relation of ACE polymorphism with longevity but with early mortality, which was common in smokers with D/D genotype, whereas frequency distribution was not significantly different in non smokers (94). So, ACE polymorphism has significant relation with mortality at early age due to cardiovascular and non-cardiovascular diseases in the presence of other physical and environmental factors.
Aging is due to a complex interaction of genetic, epigenetic, and environmental factors, but a strong genetic component appears to have an impact on survival to extreme ages. ACE polymorphism was thought to be responsible for longevity because of its role in the expression of many diseases. In the beginning, I/I genotype was thought to be associated with high birth weight, which is a sign of healthy future in old ages. But later on, it was found that there is no relation between birth weight of the newborn but that early growth after birth is influenced by I/I or I/D genotype. The protective effects of I/I allele against cardiovascular diseases and diabetic complications are not obvious and are still controversial. These facts show that ACE polymorphism has a low-level role in determining longevity.
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