Investigation of Association Between Angiotensin-Converting Enzyme Gene Insertion/Deletion Polymorphism Frequency in Turkish Patients with Schizophrenia

Abstract

Aim: The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene is of increasing interest in etiology and treatment of various neuropsychiatric disorders. The present study aimed at detecting the incidence of ACE gene I/D polymorphism in patients with schizophrenia living in the Eskisehir region (Turkey) and also at determining whether this illness could be associated to ACE gene I/D polymorphism and serum ACE concentrations. Methods: In our study, genomic DNA was studied in a total of 237 individuals, 132 of them having been diagnosed as patients with schizophrenia and 105 of them being used as control subjects. In addition, sera from 31 patients with schizophrenia and 26 healthy subjects were used to compare serum ACE concentrations. By using polymerase chain reaction, we determined the frequency of ACE gene I/D polymorphism and measured the serum ACE concentrations by ELISA. Results and Conclusion: Distribution of ACE gene I/D polymorphism and allele frequencies between the control group genotype proportions (II 19%, ID 44%, DD 37%) and the patient group (II 19%, ID 45%, DD 36%) were not significantly different. Serum ACE concentrations were 293.15 ± 23.29 ng/mL in the control group and 362.61 ± 19.96 ng/mL in the patients. It was observed that serum ACE concentrations significantly increased in patients with schizophrenia compared with those of the control group (p < 0.05). However, no significant difference could be observed according to genotypes in serum ACE concentration.

Introduction

Schizophrenia is a complex neuropsychiatric syndrome influencing about 0.5%-1% of the world population. Since the etiology and pathology of schizophrenia have not been entirely solved, the relation between schizophrenia and genes has drawn the attention of neuroscientists and genetic scientists (Chen et al., 1997; Sobell et al., 2002; Akyol, 2004; Kaplan et al., 2004).

Schizophrenia is a chronic relapsing psychotic disorder with positive (paranoia, psychotic, and hallucination) negative (alogia, avolition) and disorganized symptoms. The risk of being a patient with schizophrenia is 10 times higher among the first-degree relatives compared with that of the rest of the population. The exact biological and inheritance mechanisms of this illness are not clearly known (Chen et al., 1997; Gelernter et al., 1998; American Psychiatric Association (DSM-IV-TR), 2000; Ohara et al., 1998; Stober et al., 1998; Prasad et al., 1999; Midtsuyasu et al., 2001; Sobell et al., 2002) However, it has been argued that schizophrenia inheritance might be polygenic (Chen et al., 1997; Wahlbeck et al., 1998; Prasad et al., 1999).

Angiotensin-converting enzyme (ACE) is an ectoenzyme, and its primary function is to convert angiotensin I (Ang I) to angiotensin II (Ang II) (Kumar et al., 1989; Hubert et al., 1991; Holla et al., 1999). Ang II is a multifunction neuropeptide in the brain (Ganong, 2001; Mckinley et al., 2003).

The ACE gene, known to be associated with cardiovascular disorders, which, in turn, are accompanied with an increased susceptibility to depression, is, therefore, a promising candidate gene for affective disorders (Baghai et al., 2006). In some reports, brain, renin-angiotensin system, and ACE play crucial roles in the growth and differentiation of nerve cells (Wahlbeck et al., 2000). For example, increased ACE activity has been reported in the substantia nigra of a suicide group compared with nonsuicide controls (Arregui et al., 1980).

It has been recently shown that there is a relation between the insertion-deletion polymorphism of the ACE gene present in chromosome 17q23 and ACE level (Mattei et al., 1989; Tsutaya et al., 1997; Niimi et al., 1998; Holla et al., 1999). The genotype is classified into three types: insertion homozygotes (II); deletion homozygotes (DD), and heterozygotes (ID) (Arbustini et al., 1996; Sohn et al., 1997; Tomita et al., 1997; Gelernter et al., 1998). When the relationship between ACE insertion/deletion (I/D) polymorphism and serum ACE level is examined, it has been determined that the ACE activity is higher in DD type than that of II type (Ouyang et al., 2001). The tendency of early schizophrenia is higher in individuals carrying DD genotype (Illi et al., 2003). Rigat et al. (1990) have shown that DD polymorphism is closely related to the increase of ACE concentration in the circulation.

The aim of the present study was to detect the incidence of ACE gene I/D polymorphism in patients with schizophrenia living in the Eskisehir region (Turkey), and also we wanted to determine whether this illness could be related to ACE gene I/D polymorphism and serum ACE concentrations.

Methods

In this study, we used DNA samples collected, extracted, and stored from patients with schizophrenia in our previously studied research project between 2002 and 2004 (grant no. 200011027) that was approved by the Ethics Committee of Eskisehir Osmangazi University, Medical Faculty, Eskisehir, Turkey (grant no. 02/104).

The patients with schizophrenia were recruited in the Psychiatric Service of the Hospital Eskisehir Osmangazi University on the basis of Diagnostic and Statistical Manual of Mental Disorders criteria (DSM-IV) (APA, 1994). In total, 132 subjects were diagnosed with schizophrenia and used as the patient group, and 105 subjects were used as the control group. By using polymerase chain reaction (PCR), we determined the frequency of ACE gene (GenBank accession no. NM 000789.2) I/D polymorphism and measured the serum ACE concentrations by ELISA. Also, to compare the serum ACE concentrations, sera of 31 schizophrenia patients and 26 control subjects were used. Patient and control group subjects were selected from individuals without any accompanying organic disease (dementia, organic brain syndrome, mental retardation, etc.) or mental illness (substance and drug addiction) in the study. Patients' relatives were informed about the study, and written consent was obtained. Treatment protocols of patients was planned by psychiatrists.

Determination of ACE genotypes

Genomic DNA was isolated from peripheral blood leucocytes. Genomic DNA was extracted from 10 mL of venous blood, anticoagulated with 1.6 mg/mL EDTA, by a salt method. The D and I alleles were identified on the basis of PCR amplification of the respective fragments from intron 16 of the ACE gene and size fractionation and visualization with standard techniques. PCR was performed according to the method described by Urhan et al. (2004). The sense and antisense primers (ACE primers) were 5′-CTG CAG ACC ACT CCC ATC CTT TCT-3′ and 5′-GAT GTG GCC ATC ACA TTC GTC AGA T-3′, respectively. The PCR mixture (50 μL) contained genomic DNA, 10 pmol of each primer, 0.2 mM of each deoxynucleotide triphosphates, 1.5 mM of MgCl₂, 50 mM of KCl, 10 mM of Tris-HCl (pH 8.0), and 2 U of taq polymerase). Amplification was performed in a thermal cycler (Eppendorf Mastercycle) for 30 cycles with denaturation at 94°C for 40 s, annealing at 56°C for 40 s, extension at 72°C for 40 s, and followed by a final extension at 72°C for 10 min. PCR products were separated by electrophoresis on 2% agarose-gel and identified with ethidium bromide staining. The polymorphism detected by PCR was characterized by an ∼490 bp fragment in the presence of the insertion (I) allele and as an ∼190 bp fragment in the absence of the deletion (D) allele. To increase the specifity of DD genotyping, PCR amplifications were performed with an insertion-specific primer pair (ACE X primers) 5′-TGG GAC CAC AGC GCC CGC CAC TAC-3′ and 5′-TCG CCA GCC CTC CCA TGC CCA TAA-3′ (17), with 50 μL reactions (genomic DNA; 10 pmol of each primer [ACEX]; 0.2 mM of each deoxynucleotide triphosphates, 1.5 mM of MgCl₂, 50 mM of KCl, 10 mM of Tris-HCl (pH 8.0), and 2 U of taq polymerase). Amplification was performed in a thermal cycler for 30 cycles with denaturation at 94°C for 40 s, annealing at 63°C for 40 s, extension at 72°C for 40 s, and followed by a final extension at 72°C for 10 min. Only the I allele produces a 335 bp amplicon. The 335 bp fragment was separated on a 2% agarose gel and identified with ethidium bromide staining. The reaction yielded no products in the samples of DD genotype.

Enzyme assay

Serum ACE concentrations were determined by using an ELISA kit (Chemicon International, Inc., Temecula, CA).

Statistical analysis

The allele ratio and genotype distribution of patients with schizophrenia and control subjects were statistically analyzed by using the chi-square test. Analysis of serum ACE level was performed using one-way analysis of variance (ANOVA). Analysis of serum ACE level was performed using the Student t-test for comparison of the two groups and the analysis of variance for three genotypes. A p-value < 0.05 was considered as significant. Values were expressed as mean ±standard error.

Results

The genotype and allele frequencies of the ACE gene I/D polymorphism are given in Table 1. As can be followed from Table 1, valid for the individuals of the control subject (n = 105); number of individuals having II genotype 20 (19%), number of individuals having ID genotype 46 (44%), and number of individuals having DD genotype is 39 (37%). Frequencies of I and D alleles were determined as 41% and 59%, respectively.

Table 1.

Genotypic and Allelic Frequencies of Insertion/Deletion Polymorphism of the Angiotensin-Converting Enzyme Gene in the Control Subjects and Patients with Schizophrenia

		Genotypes		Alleles
Groups	n	II n (%)	ID n (%)	DD n (%)	I n (%)	D n (%)
Control	105	20 (19)	46 (44)	39 (37)	86 (41)	124 (59)
Patients with schizophrenia	132	25 (19)	60 (45)	47 (36)	110 (42)	154 (58)

χ² = 0.74; SD = 2; p > 0.05.

For the patients with schizophrenia (n = 132), the number of individuals having II genotype was 25 (19%), the number of individuals having ID genotype was 60 (45%), and the number of individuals having DD genotype was 47 (36%). Frequencies of I and D alleles were determined as 42% and 58%, respectively.

When the control subject and patients with schizophrenia were compared, a statistical treatment revealed that there is no significant difference between ACE gene I/D polymorphism genotype distribution and allele frequency (p > 0.05).

As seen from Table 2, the ACE serum concentrations for the control subjects and patients with schizophrenia were (n = 26) 293.15 ± 23.29 ng/mL and (n = 31) 362.60 ± 19.96 ng/mL, respectively.

Table 2.

Activity of Angiotensin-Converting Enzyme in the Control Subjects and Patients with Schizophrenia

Groups	n	ACE activity (ng/mL)
Control	26	293.15 ± 23.29
Patients with schizophrenia	31	362.60 ± 19.96^a

Student's t test, t = (test) value, t = 2.277.

p < 0.05.

ACE, angiotensin-converting enzyme.

ACE serum concentrations for the genotype distributions of the control subject and patients with schizophrenia are given in Table 3. Table 3 shows that ACE concentration levels of the control subjects were 273.77 ± 28.42 ng/mL for the II genotype, 292.62 ± 43.62 ng/mL for the ID genotype, and 316.12 ± 46.68 ng/mL for the DD genotype. Also, for the patients with schizophrenia, ACE concentration levels were 360.48 ± 34.38 ng/mL for the II genotype, 319.89 ± 29.81 ng/mL for the ID, and 403.36 ± 36.62 ng/mL for the DD.

Table 3.

Association Between the Serum Angiotensin-Converting Enzyme Levels and Genotypes in the Control Subjects and Patients with Schizophrenia

Groups	Control		Patients with Schizophrenia
Genotypes	n	ACE (ng/mL)	n	ACE (ng/mL)
II	8	273.76 ± 28.42	10	360.48 ± 34.38
ID	11	292.62 ± 43.62	10	319.89 ± 29.81
DD	7	316.12 ± 46.68	11	403.36 ± 36.62

p > 0.05.

ACE serum levels of the patients with schizophrenia have been statistically determined as highly significant (p < 0.05). However, when evaluated from the point of view of genotypes, no significant difference has been found (p > 0.05).

Discussion

Being a key enzyme in the renin angiotensin system, ACE plays an important role in the catalyzation of secretion of Ang I to Ang II (Ouyang et al., 2001). Ang II controls both the liquid electrolyte stability and the systemic blood pressure. In addition to its effects on the cardiovascular, renal, otocrine, and peripheral systems, it has a central effect on the brain (Phillips and Sumners, 1998; Shimizu et al., 2004). It has been reported that schizophrenia causes an increase in the dopaminergic activity of the limbic structure of brain, contributing to the regulation of the dopamine activity of ACE (Ouyang et al., 2001; Vuoristo et al., 2002).

As a result of our study, when the control subject and patients with schizophrenia were compared, statistically no important difference was found between their ACE gene I/D polymorphism genotype distribution and allele frequency. Such a finding is in line with the findings of Arinami et al. (1996). In studies conducted by Ouyang et al. (2001) and Illi et al. (2003), despite the differences in their II, ID, and DD genotype distributions compared with those of our study, no important difference has been found between ACE gene I/D polymorphism genotype distribution and allele frequencies of the control subject and patients with schizophrenia.

When the serum ACE concentrations of the control subjects and patients with schizophrenia were compared, in our study, it was determined that ACE concentrations of the patients with schizophrenia were considerably higher than those of the control subjects. However, a study conducted by Wahlbeck et al. (1998) does not report any significant difference between the serum ACE concentrations of the control subjects and patients with schizophrenia.

In our study, a statistical comparison of serum ACE concentration values according to their genotypes revealed that serum concentration levels did not significantly differ. However, it has been determined that the ACE concentration of the DD genotype was higher than that of the II and ID type for both the control subjects and patients with schizophrenia. Similar findings were obtained in a study conducted by Illi et al. (2003) and Rigat et al. (1990), who have concluded that there is a significant relation between ACE I/D polymorphism genotypes and serum ACE levels.

It has been reported that renin angiotensin system and ACE play an important role in regulating the growing and differentiation of neuron cells (Wahlbeck et al., 1993; Wahlbeck et al., 2000). In schizophrenia, particularly in CSF, increased ACE levels have been reported (Wahlbeck et al., 1993, 1998, 2000). In contrast, Wahlbeck et al. (2000) observed no relation between schizophrenia and ACE levels. The results of some other studies suggest that ACE levels in serum and CSF are not related to age, sex, the starting age of the illness, tallness, and blood pressure (Wahlbeck et al., 1993, 1998, 2000).

As a result, it is possible to suggest that the findings obtained in this study can partly provide information about ACE gene I/D polymorphism genotype and allele frequency. We strongly stress that further studies are necessary to determine the roles of ACE gene I/D polymorphism genotype, allele frequency, and serum ACE concentrations in schizophrenia.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

Akyol

. 2004. Şizofrenide Oksidatif Stres. Kocatepe Tip Dergisi, 5:15-25.

American Psychiatric Association2000. Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) American Psychiatric Association: Washington, DC.

[APA] American Psychiatry Association. 1994. Diagnostic and Statistical Manual of Mental Disorders. Fourth(DSM-IV)Washington, DC.

Arbustini

, Grasso

, Leo

et al. 1996. Polymorphism of angiotensin-converting enzyme gene in sarcoidosis. Am J Respir Crit Care Med, 153:851-854.

Arinami

, Li

, Mitsushio

et al. 1996. An insertion/deletion polymorphism in the angiotensin converting enzyme gene is asssociated with both brain substance P contents and affective disorders. Biol Psychiatry, 40:1122-1127.

Arregui

, Mackoy

, Spokes

, Inversen

. 1980. Reduced activity of angiotensin-converting enzyme in basal ganglia in early onset schizophrenia. Psychol Med, 10:307-313.

Baghai

, Binder

, Schule

et al. 2006. Polymorphisms in the angiotensin-converting enzyme gene are associated with unipolar depression, ACE activity and hypercortisolism ACE SNP rs4291 is associated with depression. Mol Psychiatry, 11:1003-1015.

Chen

C-H

, Liu

M-Y

, Wei

F-C

et al. 1997. Further evidence of no association between ser9gly polymorphism of dopamine D3 receptor gene schizophrenia. Am J Med Genet (Neuropsychiatr Genet), 74:40-43.

Ganong

. 2001. Review of Medical Physiology, Twentieth. Lange Medical Books/Mcgraw-Hill Book Co.: New York, 439-443.

10.

Gelernter

, Kranzler

, Cubells

et al. 1998. Drd2 allele frequencies and linkage disequilibria, including the −141 Ins/Del promoter polymorphism, in European-American, African-American, and Japanese Subjects. Genomics, 51:21-26.

11.

Holla

, Vasku

, Znojil

et al. 1999. Association of 3 gene polymorphisms with atopic diseases. J Allergy Clin Immunol, 103:702-708.

12.

Hubert

, Houot

, Corvol

et al. 1991. Structure of the angiotensin I-converting enzyme gene. J Biol Chem, 266:15377-15383.

13.

Illi

, Kampman

, Anttila

et al. 2003. Interaction between angiotensin-converting enzyme and catechol-O-methyltransferase genotypes in schizophrenics with poor response to conventional neuroleptics. Eur Neuropsychopharmacol, 13:147-151.

14.

Kaplan

, Sadock

. Abay

. Calıyurt

, Tuglu

. 2004. Klinik Psikiyatri. Nobel Tıp Kitabevi, İstanbul, 121-138. translated from Pocket Handbook of Clinical Psychiatry 2nd. Williams & Wilkins, 1996.

15.

Kumar

, Kusari

, Roy

et al. 1989. Structure of testicular angiotensin-converting enzyme. J Biol Chem, 264:16754-16758.

16.

Mattei

, Hubert

, Alhenc-Gelas

et al. 1989. Angiotensin I converting enzyme gene is on chromosome 17. Cytogenet Cell Genet, 51:1041-1045.

17.

Mckinley

, Albiston

, Allen

et al. 2003. The brain renin-angiotensin system: location and physiological roles. Int J Biochem Cell Biol, 35:901-918.

18.

Midtsuyasu

, Hirata

et al. 2001. Association analysis of polymorphisms in the upstream region of the human dopamine D4 receptor gene (Drd4) with schizophrenia and personality traits. J Hum Genet, 46:26-31.

19.

Niimi

, Tomita

, Sato

et al. 1998. Bronchial responsiveness and angiotensin-converting enzyme gene polymorphism in sarcoidosis patients. Chest, 114:495-499.

20.

Ohara

, Nagai

, Tani

et al. 1998. Functional polymorphism of −141c Ins/Del in the dopamine D2 receptor gene promoter and schizophrenia. Psychiatry Res, 81:117-123.

21.

Ouyang

W-C

, Wang

Y-C

, Hong

C-J

et al. 2001. Association study of angiotensin-converting enzyme gene polymorphism with schizophrenia and polydipsia. Neuropsychobiology, 44:31-35.

22.

Phillips

, Sumners

. 1998. Angiotensin II in central nervous system physiology. Regul Pept, 78:1-11.

23.

Prasad

, Deshpande

, Bhatia

et al. 1999. Association study of schizophrenia among Indian families. Am J Med Genet (Neuropsychiatr Genet), 88:298-300.

24.

Rigat

, Hubert

, Gelas

et al. 1990. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest, 86:1343-1346.

25.

Shimizu

, Hashimoto

, Kobayashi

et al. 2004. Lack of association between angiotensin I-converting enzyme insertion/deletion gene functional polymorphism and panic disorder in humans. Neurosci Lett, 363:81-83.

26.

Sobell

, Mikesell

, Mcmurray

. 2002. Genetics and etiopathophysiology of schizophrenia. Mayo Clin Proc, 77:1068-1082.

27.

Sohn

, Kwon

, Kim

. 1997. The genetic polymorphism of angiotensin I-converting enzyme in a Korean population. Pharmogenetics, 7:425-427.

28.

Stober

, Jatzke

, Heils

et al. 1998. Insertion/deletion variant (-141 C Ins/Del) in the 5′ regulatory region of the dopamine D2 receptor gene: lack of association with schizophrenia and bipolar affective disorder. J Neural Transm, 105:101-109.

29.

Tomita

, Ina

, Sugiura

et al. 1997. Polymorphism in the angiotensin-converting enzyme (ACE) gene and sarcoidosis. Am J Respir Crit Care Med, 156:255-259.

30.

Tsutaya

, Kitaya

, Saito

et al. 1997. Angiotensin-converting enzyme gene polymorphism and its enzyme activity in serum in young Japanese females. Tohoku J Exp Med, 182:151-155.

31.

Urhan

, Degirmenci

, Harmanci

et al. 2004. High frequency of DD polymorphism of the angiotensin-converting enzyme gene in Turkish asthmatic patients. Allergy Asthma Proc, 25:243-247.

32.

Vuoristo

. 2002. Human GNAL, C18orf2, and MPPEI Genes, Genomic Organization of the Human GNAL Gene and Characterization of Two Novel Genes, C18orf2 and Mppe1, on Chromosome 18p11.2, a Susceptibility Region for Schizophrenia and Bipolar Disorder. Oulu University Press: Finland.

33.

Wahlbeck

, Ahokas

, Miettinen

et al. 1998. Higher cerebrospinal fluid angiotensin converting enzyme levels in neuroleptic treated than in drug free patients with schizophrenia. Schizphr Bull, 24:391-397.

34.

Wahlbeck

, Ahokas

, Nikkilä

et al. 2000. Cerebrospinal fluid angiotensin-converting enzyme (ACE) correlates with length of illness in schizophrenia. Schizophr Res, 41:335-340.

35.

Wahlbeck

, Rimón

. 1998. CSF ACE correlates with duration of illness in schizophrenia. Schizophr Res, 29:103.

36.

Wahlbeck

, Rimon

, Fyhrquist

. 1993. Elevated angiotensin-converting enzyme (kininase II) in the cerebrospinal fluid of neuroleptic-treated schizophrenic patients. Schizophr Res, 9:77-82.