A STUDY ON ANGIOTENSIN CONVERTING ENZYME ......Ain Shams University 3Department of Psychiatry,...

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The Egyptian Journal of Biochemistry & Molecular Biology VOL 30 (N.2)203- 216 Dec. 2012

A STUDY ON ANGIOTENSIN CONVERTING ENZYME GENE

INSERTION/DELETION POLYMORPHISM AND SERUM

CORTISOL AMONG A SAMPLE OF EGYPTIAN PATIENTS

WITH MAJOR DEPRESSION DISORDER

Mohga S. Abdullah1, Adel G. El-Missiry

2, Hayat M. Sharada

1,

Ahmad A.El-Missiry3, Fatma A. Abu Zahra

4*

1Department of Chemistry, Faculty of Science,

Helwan University, Cairo, Egypt 2Department of Parasitology, Faculty of Medicine,

Ain Shams University 3Department of Psychiatry, Faculty of Medicine,

Ain Shams University 4Medical Research Center, Faculty of Medicine,

Ain Shams University

Received 4/7/2012– Accepted 3/9/2012

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ABSTRACT

Angiotensin-converting enzyme (ACE) is assumed to influence

the activity of the hypothalamic-pituitary-adrenocortical (HPA)

system, which shows hyperactivity in the majority of patients with

major depression. The ACE gene, known to be associated with

cardiovascular disorders, which in turn are accompanied with an

increased susceptibility for depression, is therefore a promising

candidate gene for effective disorders. However, the results are

conflicting, with no reported studies on Egyptian depressed patients.

This study aimed to assess ACE insertion / deletion (I/D) gene

polymorphism among Egyptian depressed patients in order to clarify

HPA system dysregulation, and to determine its possible association

with severity of depression. This case/control study was conducted on

42 adult depressed patients, and 37 healthy controls were screened to

detect genetic associations with unipolar major depression.

Determination of ACE genotypes was performed for all subjects by

real time PCR. Total serum cortisol levels were measured by ELISA.

The frequencies of the DD, ID and II genotypes were 26.2%, 45.2%,

Mohga S. Abdullah et all


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and 28.2%, respectively among the cases, and 17.49%, 25.2%, and

56.41%, respectively among the controls. Significant differences in

ACE genotype distribution were observed between cases and controls

(p = 0.0384). Serum cortisol in patients show the highest value in the

ID polymorphism while II polymorphism shows the lowest value of

a.m. cortisol. Data illustrated a significant association of ID

polymorphism with the more severity of illness. Our findings support

that ACE gene I/D polymorphism and high D allele frequency are

associated with depression, also hypercortisolimia is significantly

higher in individuals with major depression compared to control

among Egyptian adults. ACE gene polymorphism might provide a

common link between developing depressive episode and

dysfunctional HPA-axis.

Keywords: ACE gene –insertion\deletion polymorphism –cortisol –

depression

INTRODUCTION

Depression is undoubtedly a common disabling disorder (Alonso et

al., 2004). The economic burden on society arises not only from the

direct health and social care costs, but also from the indirect costs, due

to the reduced work productivity of patients and premature death due

to suicide (Baghai et al., 2012). Experimental studies have

investigated many different types of stress, and their effects on the

hypothalamic-pituitary- adrenocortical (HPA) axis in many different

circumstances (Sower et al., 2009). Cortisol is released in response to

stress and a low level of blood glucocorticoids acting to restore

homeostasis (Holsboer, 2000). Changes in the activity of the renin-

angiontensin-aldosterone system (RAAS) in depression have recently

been reported (Murck et al., 2006). Angiotensin I is converted to

angiotensin II by angiotensin-converting enzyme (ACE) (Paul et al.,

2006). ACE is assumed to influence the activity of the HPA system,

which shows hyperactivity in the majority of patients with major

depression. The ACE gene, known to be associated with

cardiovascular disorders, which in turn are accompanied with an

increased susceptibility for depression, is therefore a promising

candidate gene for affective disorders (Baghai et al., 2006).

Given the abundance of the renin–angiotensin system (RAS)

components in the brain, their importance in behavior and cognition,

A STUDY ON ANGIOTENSIN CONVERTING ENZYME GENE ……..


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and the data that implicates them in the etiology and treatment of

depression, it is possible that those RAS gene polymorphisms

associated with increased RAS activity may also be associated with

depression (Saab et al., 2007).

This study aimed to examine the ACE/ID polymorphism and

estimation of serum cortisol level as a sign of the extent of HPA-axis

system dysregulation in relation to this polymorphism in a sample of

Egyptian patients with depression.

MATERIALS AND METHODS

1-Study population

The 42 cases of major depression disorder (MDD) patients were

selected from the Institute of Psychiatry, Ain Shams University

Hospitals. The control group consisted of 39 subjects of apparently

healthy recruited from the visitors of Ain Shams University Hospitals

and were selected to match the study group as much as possible.

The Structured Clinical Interview for DSM-IV Axis I Disorders

(SCID-I) has done by a clinician, semistructured interview with

psychiatric patients or with non-patient community subjects who are

undergoing evaluation for psychopathology for diagnosis of MDD.

Both groups have permanently resided in the same region, with sex

and age matched. The local committee for ethics of medical

experiments on human subjects approved the study, and all

participants gave their written informed consent. The Beck Depression

Inventory (BDI) (Beck et al., 1961) was used to measure the

behavioral manifestations of depression. On the other hand, Hamilton

Rating Scale for Depression (Ham-D) (Appendix-VI) was designed to

measure the severity of depressive symptoms in patients with primary

depressive illness (Hamilton, 1960).

The reliability of the scale varies with conditions but is generally

accepted, with internal consistency tends to be 0.92. It tends to be

higher with the structured than unstructured interview (Potts et al.,

1990). The inter rater reliability reached up to 0.9 (Rehm and

O’Hara, 1985).

2-Estimation of serum cortisol (a.m.): Samples of patients and

control had been estimated for the results of serum cortisol for in vitro

diagnostic use with the IMMULITE 1000 Analysis –for quantitative

measurement of cortisol (Siemens Healthcare, Diagnostics Products




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Ltd Llaberis, GwyneddLL55 4EL, United kingdom).

The candidate gene selected in this study was Angiotensin

converting enzyme gene (ACE gene).

3-Genotyping

Gene typing of genomic DNA samples obtained from 42 unrelated

patients with MDD (20 women and 22 men, aged 34.79±9.1 years)

and 39 unrelated healthy controls (20 women and 19 men, aged

35.56±10.8 years) was performed.

The molecular part of this work was carried out at the Medical

Research Center (MRC) Faculty of Medicine, Ain Shams University.

Genomic DNA was isolated from whole blood using the Qiagen-

Kit. PCR (polymerase chain reaction) amplification of the of ACE I/D

polymorphism was carried out using the

primers sequence:

Forward : 5’-GAT GTG GCC ATC ACA TTG GTC AGA T -3’.

Reverse : 5’-CTG GAG ACC ACT CCC ATC ATT TCT -3’.

The insertion (I-allele) resulted in a product of 478 bp, deletion (D-

allele) in 191 bp. We report here the development of a single-tube

real-time PCR assay to determine the ACE gene I/D polymorphism.

This method takes advantages of the SYBR Green I fluorescent dye

for real-time 45 ng of DNA is sufficient to use as a template for PCR.

Amplification with 45 cycles only was optimal for ligation

efficiencies. A mixture containing Taq polymerase and a proofreading

polymerase, Taq must be in excess of a 10:1 ratio to ensure the

presence of 3’A-overhangs on the PCR product.

The PCR in a 20 µl volume containing:

DNA Template 45 ng

10X PCR Buffer 2 µl

50 mM dNTPs 0.5 µl

Primers 1 µM each

Sterile water to a total volume of 19 µl

Taq Polymerase 1 unit

Total Volume 20 µl

Special care to avoid sources of nuclease contamination and long

exposure to UV light to prevent degradation of the single A-

overhangs.

Real-time PCR

Light cycler-based real-time PCR (LC-PCR) assay was used. Real-

time PCR were performed with a Light Cycler 1.5 instrument (Roche




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Molecular Diagnostics, Penzberg, Germany) by using a total

reaction volume of 20µl in PCR capillaries. Optimization reactions

were first performed until the best primer concentrations.

Melting curve analysis reveals only one peak at the characteristic

melting temperature, when PCR results only in the specific amplicon

as in insertion (490 bp) or Deletion (190 bp). Two peak at the

characteristic melting temperature, when PCR results only in the two

specific amplicon (as in heterozygot insertion and Deletion case 490

bp and 190 bp). Primer dimers and other nonspecific products

would cause additional peaks.

According to the presence or absence of the insertion allele, the

genotype of the subjects could be classified as II (homozygote for the

insertion allele), DD (homozygote for the deletion allel), or ID

(heterozygote).

The PCR products were separated on a 2% agarose gel and visualized

by ethidium bromide staining under UV light. The insertion (I-allele)

resulted in a product of 490 bp, the deletion (D-allele) in 191 bp

(Bocchetta et al., 1999).

Statistical analyses: In some occasions Fisher-Exact probability was

used if less than 5 observations are expected in a cell. Student-t- test

was used to detect difference between 2 groups' means and ANOVA if

more than 2 groups. All statistical manipulation and data analyses

were performed using the 16th

version of SPSS (Statistical Package for

Social Sciences).




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RESULTS

Table (1) shows Socio-demographic characteristics of cases compared

to controls which show no significant differences between the two

groups.

Table (1): Socio-demographic characteristics of cases compared to

controls

Cases

n=

Controls

n=

Test of

Significance

df P

value

Education

years

Mean ±

SD

Mean ±

SD t= 1.89

◊ 79

0.062

(NS) Mean (SD) 6.4±5.3 8.8±6.1

Min – Max 0 – 16 0 – 17

Occupation N (%) N (%)

X2=6.80 5 0.236

(NS)

Jobless 4 (9.5) 3 (7.7)

House Wife 14 (33.3) 6 (15.4)

Non-skilled -- 2 (5.1)

Semi-skilled 6 (14.3) 5 (12.8)

Semi-

Professional

12 (28.6) 12 (30.8)

Professional 6 (14.3) 11 (28.2)

Social class N (%) N (%)

X2= 5.95 3 0.114

(NS)

High-Mid -- 5 (12.8)

Low Mid 20 (47.6) 18 (46.2)

Low 18 (42.9) 13 (33.3)

Very Low 4 (9.5) 3 (7.7)

Marital Status N (%) N (%)

X2= 1.161 3 0.762

(NS)

Single 10 (23.8) 7 (17.9)

Married 28 (66.7) 26 (66.7)

Divorced 2 (4.8) 4 (10.3)

Widow 2 (4.8) 2 (5.1)

In this study, II polymorphism was encountered more

frequently in the control group compared to only 28.6% in the cases.

ID polymorphism and DD polymorphism were significantly and

frequently found in the cases group than the controls (45.2% and

26.2% versus 25.6% and 17.94% respectively) (Figure 1).




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Figure (1): ACE Genotype among the studied depression patients

compared to controls

Table (2) shows significant difference between Insertion I and

deletion D alleles frequencies among the studied patients with

depression compared with matched controls by using the Fisher Exact

Probability. Data revealed that the D allele was significantly

prominent in the depressed group compared to controls (48.8% and

30.77% respectively).

Table (2): ACE Allele frequencies among the depression patients vs.

controls

Cases Controls χ2 P value

• I 43(51.19%) 54

(69.23%) 4.754 0.029

• D 41

(48.81%)

24

(30.77%) ¤Fisher Exact probability has been used

Table (3) illustrates a high significant level of serum cortisol in cases

as compared to controls.

0

10

20

30

40

50

60

II DD ID

CasesControls




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Table (3): Serum cortisol level in cases compared with control group

Cases Controls

Mean ±SD Mean ±SD

Plasma

Cortisol

Mean (SD)

Min – Max

17.33±5.2

15.7 – 18.9

10.68±5.3

8.9 – 12.4

t-value 5.69

P value <0.001*

Table (4) shows the distribution of different allele among depressed

patients illustrating higher percentage of ID allele in comparison to II

and DD alleles. We performed genotyping of the I/D polymorphism

using polymerase chain reactions (PCR) amplification of the ACE I/D

polymorphism in cases who were diagnosed according to DSM-VI

criteria to have major depression compared to matched healthy

controls.

Table (4): ACE polymorphism among the studied depression patients

RAS

Polymorphism N (%) 95% CI

• II 12 (28.6) 16.2 – 44.8

• DD 11 (26.2) 14.4 – 42.3

• ID 19 (45.2) 30.2 – 61.2

Table (5): Serum cortisol levels among the studied depression patients

in relation to ACE genotypes

II�� DD�� ID�� f value�� P value

Serum

Cortisol

Mean

(SD)

95% CI

14.9

(2.1)

13.9 –

15.9

17.5

(6.6)

13.1 –

22.0

� 21.0

(5.3)

17.6 –

24.4

6.438 0.004*

�F value (ANOVA test), df= 2 Between groups and within groups 39.




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Figure (2): represents agarose gel electrophoresis illustrating 100 base

pair (bp) loading marker in first and last lanes. In lanes 1 and 6 show

homozygote insertion ACE gene polymorphism at 490 bp, in lanes 2

and 5, heterozygote insertion/deletion ACE gene polymorphism at 490

bp and 190 bp while lanes 3 and 4 represent homozygote deletion

ACE gene polymorphism at 190 bp, and lane 7 represents control

sample (no polymorphism).

DISCUSSION

Depression is a prevalent disorder and one of the leading disease

causing disability and economic burden according to the World Bank

reports (Kato and Serretti, 2010). Since epidemiological data denotes

that depression may affect 10% of population, we except that we have

about 8 million people with depression in Egypt (Ghanem et al.,

2009). That is why this study was designed to be the first step to find one

important parameter for a biological marker which helps for the early

detection of depression and reconfirmation of the diagnosis. This also

may lead to early intervention hoping for recovery from this

devastating disorder.

To fulfill our aim we investigated 42 patients suffering from major

depression according to the DSM IV criteria for diagnosis of

depression and we compared them with 39 matched healthy controls.

Recent studies have shown that genetics variants of angiotensin

converting enzyme (ACE) can contribute to disease susceptibility and




212

inter-individual differences in antidepressants response (Yanfeng et

al., 2011). A genetic polymorphism is characterized by an insertion (I)

or deletion (D). Both alleles have concomitant effects on the level of

ACE. Thus individuals who are Homozygous for the I allele has the

lowest level of the enzyme, while those who are homozygous for the

D allele have the highest; while, heterozygous subjects have an

intermediate level (Zintzaras et al., 2008).

The renin-angiotensim-aldosterone system (RAAS) has been

implicated in mood disorders (Hallberg et al., 2011; Nasr et al.,

2011). The contribution of the RAAS in controlling mood has been

attributed to the angiotensin II that interacts with dopamine (DA) in

specific brain regions and the ACE has an important role in the

modulation of DA turnover. ACE is part of the renin-angiotensin

system (RAS) and is involved in the conversion of angiotensin I to

angiotensin II (Kobori et al., 2007), which is a peptide hormone acts

as a stimulator of proinflammatory cytokines and interferes with

hypothalamic pituitary adrenal (HPA) axis activation in response to

stress (Ising and Jolsboer, 2006; Yanfeng et al., 2011).

There has been a growing interest in the study of ACE/ID

polymorphism as a potential factor for developing depression (Baghai

et al., 2006). However, despite the large number of studies with

different designed, the role of the ACE gene I/D polymorphism on

depression is still controversial. Previous association studies for ACE

and depression conducted by genotyping ACE-I/D polymorphism

have yielded inconsistent results (Heck et al., 2009). On the other

hand, previous data pointed to the possibility that ACE/ID

polymorphism could be a finger point or a biological marker of major

depression and response to treatment (Baghai et al., 2003 and 2006;

Murck et al., 2006).

Wu et al. (2012) provided a strong evidence of a positive association

of the ACE/ID polymorphism as risk factor for depression in

Caucasians but not in Asians. On the other hand, they failed to detect

significant differences between cases and control groups as regards

ACE/ID polymorphism in Chinese population.

In a similar culture to ours, Saab et al. (2007) in Lebanese

population proved that the angiotensin receptor type I was

significantly associated with depression, however he could not prove

that the type of the ACE/ID polymorphism is associated with unipolar

depression.




213

It seems that variation of results across ethnic groups could account

for the differences in race specific genetics effects, sample selection

diagnostic criteria, different methodology and different patient

recruitment methods for patients and controls.

The dysregulation of the activity of the hypothalamic-pituitary-

adrenocorticaI (HPA) system is one of the major neuroendocrine

abnormalities in major depressive disorder. These include elevated

circulating plasma levels of both corticotropin (adrenocorticotropic

hormone, ACTH) and cortisol (Holsboer, 2000). RAAS is closely

related to HPA-axis as ACTH is a common stimulus for both cortisol

and aldosterone, moreover, cortisol release is suppressed by

mineralocorticoide receptor agonists and finaly angiotensim II

releases corticotropin-releasing hormone (CRH) and vasopression

from the hypothalamus (Murck et al., 2003).

In this study, serum cortisol in patients show the highest value in the

ID polymorphism while II polymorphism shows the lowest value of

a.m. cortisol and DD polymorphism shows the intermediate value

(Table 5). The HPA-axis is suggested to be hyperactive in major

depressive disorder (MDD) (Bao et al., 2008) and chronic depression

is proved to be associated with increased level of cortisol (Lindquist

et al., 2008). Not only hypercortisolism but also reduced feedback

inhibition of the hypothalamus-pituitary adrenocortical (HPA) system

has been reported in patients with depression (van den Bos et al.,

2006).

The current research data are in agreement with previous reports

which found that serum cortisol is significantly higher in patients with

depression compared to controls (Murck et al., 2005; Baghi et al.,

2006; Häfner et al., 2012). In conclusion, our findings support that ACE gene I/D polymorphism

and high D allele frequency are associated with depression, also

hypercortisolimia is significantly higher in individuals with major

depression compared to control. ACE gene polymorphism might

provide a common link between developing depressive episode and

dysfunctional HPA-axis.

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A STUDY ON ANGIOTENSIN CONVERTING ENZYME ......Ain Shams University 3Department of Psychiatry,...

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