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Keywords:

  • ACE;
  • ATr1;
  • gender;
  • genetic polymorphism;
  • panic disorder

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Aims:  Angiotensins were shown to have some role in the development of panic disorder (PD). In this study, we aimed to determine the frequency of polymorphisms in two angiotensin-related genes, angiotensin I-converting enzyme (ACE) and angiotensin II type I receptor (ATr1), in a sample of Turkish patients with PD and to evaluate their association with PD development.

Methods:  Polymerase chain reaction and restriction fragment length polymorphism was used to analyze ATr1 A1166C polymorphism, and only polymerase chain reaction was used to analyze functional ACE insertion/deletion polymorphism in 123 patients with PD and in 169 similarly aged disease-free controls.

Results:  There was no significant difference in the genotype distribution between PD patients and controls for each polymorphism (P > 0.05). Allele frequency of ACE insertion/deletion was borderline statistically significant between the groups (P = 0.055; odds ratio: 1.39; 95% confidence interval: 0.99–1.95), and allele frequency of ATr1 A1166C was not significantly different between the groups (P = 0.32; odds ratio: 0.81; 95% confidence interval: 0.53–1.22).

Conclusion:  This study suggests that polymorphisms of ACE I/D and ATr1 A1166C are not associated with risk of PD in Turkish patients. However, in ACE insertion/deletion polymorphism, the insertion allele was found to be more frequent in the male subgroup of patients (χ2 = 4.61, P = 0.032) than in controls, suggesting a potential male-specific role of the less active ACE insertion allele in the pathogenesis of PD.

PANIC DISORDER (PD) is an anxiety disorder characterized by sudden attacks of intense fear and often accompanied with agoraphobia and loss of control. PD has a life-time prevalence of 1–3%.1 Family and twin studies suggest that, there is a strong genetic contribution to the pathogenesis of PD, with an estimated heritability of 50%.2–4 Women are affected 2:1 relative to men.5 There is no simple pattern of inheritance in segregation studies,6 so PD can be considered a polygenic, multifactorial genetically complex disorder.

Although sensitivity to anxiety is the major risk factor for PD,7 other concomitant factors play important roles in the development of PD, including family history, age, gender, personality type and stressful life events. Some factors affecting the respiratory system are the characteristic features of panic attacks including shortness of breath, a feeling of being smothered and hyperventilation, suggesting the involvement of central or peripheral regulation of respiration in the pathophysiology of PD.8 Other accompanying physical symptoms may also be observed during panic attacks such as chest pain, heart palpitations, dizziness and abdominal distress.9 The existence of a significant correlation between respiratory symptoms and increased responsiveness to CO2 in PD patients has been reported.10

Recently, some studies have hypothesized that polymorphisms in angiotensin-related genes affect anxiety disorders such as PD.7,8,11,12 Although the exact pathogenetic mechanism of PD has not been fully clarified, the possible involvement of polymorphisms on angiotensin-related genes in PD pathogenesis may indicate the role of enzymes of the renin-angiotensin system in psychiatric disorders. Consistent with this hypothesis, Olsson et al. and Bandelow et al. stated that polymorphisms involved in the angiotensin I-converting enzyme (ACE) gene may be one of the genetic factors for predisposition to an increased risk of PD.8,11

Angiotensins are known to act on cardiovascular, renal, endocrine and peripheral autocrine nervous systems. In addition, angiotensins are known to have a central effect within the brain. ACE catalyzes the conversion of angiotensin I to the active octapeptide angiotensin II. Angiotensin II controls blood pressure and the fluid-electrolyte balance.13,14 Some previous studies have indicated that angiotensin II is a potential risk factor in the pathogenesis of panic attacks in animal models.15,16

ACE has been shown to degrade a neurotransmitter called substance P (SP),17 which belongs to the tachykinin neuropeptide family. SP interacts selectively with the SP receptor and is degraded by ACE.18 It has been reported that an increase in SP concentration produces anxiogenic-like responses in rats.19,20

Polymorphisms of the ACE gene may play important roles in anxiety disorders. Data concerning the role of the functional insertion (I)/deletion (D) polymorphism in PD risk in different populations are conflicting.7,8,11 The functional I/D polymorphism is characterized by the presence (allele I) or absence (allele D) of a 287-bp Alu repeat within intron 16 of the ACE gene located on chromosome 17q23.21ACE I/D polymorphism is associated with serum and tissue ACE levels whereas the DD genotype shows the highest levels.21–24

The human angiotensin II type I receptor (ATr1) gene encodes ATr1, which is activated by angiotensin II in the brain. ATr1 mediates most of the effects of angiotensin II.25 It has also been reported that many of the classical and hypothetical functions of brain angiotensin II are mediated by stimulation of ATr1.26 An A/C substitution at position 1166 of the ATr1 gene has been identified in the 3′-untranslated region.27,28 The single nucleotide polymorphism (SNP) A1166C in the ATr1 gene was hypothesized to be involved in PD pathogenesis.8

Polymorphisms in the ACE and ATr1 genes may have some phenotypic significance in the development of PD. Screening for the possible relationship between polymorphisms of angiotensin-related genes and PD may contribute to an understanding of the pathogenesis of PD and may be useful in the prevention of this disease. There have been few studies exploring the relationship between angiotensin-related gene polymorphisms and PD susceptibility.7,8,11 However, there have been no studies examining the relationship between angiotensin-related gene polymorphisms and PD in a sample of Turkish patients. As the polymorphisms in ACE I/D and ATr1 A1166C are common in the population and have functional significance, we determined the frequency of the polymorphisms in a sample of Turkish patients with PD and evaluated their association with PD development.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Subject recruitment and psychiatric assessment

This case-control study included a total of 123 patients with PD and 168 disease-free controls. The PD patients and controls were patients at Istanbul University Cerrahpasa Medical Faculty (Istanbul, Turkey); the PD patients were in the Department of Psychiatry. All patients provided informed consent before they took part in the study. The study was approved by the Local Ethics Committee of the Cerrahpasa Medical Faculty.

Each subject underwent a psychiatric examination. Diagnosis of PD was made by four clinicians according to the DSM-IV-TR criteria. The severity of the symptoms was determined based on the Panic and Agoraphobia Scale (PAS). Each PAS item is scored on a 5-point scale (score range, 0–4), and the total score range was 0–52.

Any other psychiatric disorders were assessed by the Structured Clinical Interview for DSM-IV, and patients with comorbid psychiatric diagnosis were excluded. Also, patients and controls with chronic illnesses such as diabetes or hypertension were excluded from the study. The mean age of the PD group was 36.72 ± 11.3 years (range, 15–70 years), and 81 (66%) of the 123 patients were women.

Control subjects presented with the absence of neurological and psychiatric disorders to our outpatient department. Subjects were excluded if they had any self-reported personal or familial psychiatric history, or psychotropic medication history. They were age- and sex-matched, healthy volunteers without any psychiatric disorders. The mean age of the control group was 37.81 ± 16.26 years (range, 14–84 years), and 104 (62%) of the 168 patients were women.

Blood samples and DNA isolation

Venous blood samples were obtained from the patient and control groups and collected into ethylenediaminetetraacetic acid tubes. Immediately after collection, whole blood was stored in aliquots at −20°C until use. Genomic DNA was extracted from whole blood using a High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions.

Genotyping of ACE gene I/D polymorphism

ACE genotypes were determined only by polymerase chain reaction (PCR). I/D polymorphism in intron 16 of the ACE gene was amplified to form a fragment of 190 bp in the absence of the insertion and 490 bp in the presence of the insertion with the primers 5′-CTG GAG ACC ACT CCC ATC CTT TCT-3′(forward) and 5′-GAT GTG GCC ATC ACA TTC GTC AGA T-3′ (reverse).20 Insertion/insertion individuals had only a 490-bp fragment, I/D individuals had 490- and 190-bp fragments, and deletion/deletion individuals had only a 190-bp fragment. The ACE deletion (D) allele amplifies more effectively than the longer I allele. And this results in mistyping of the ID as the DD genotype.29 All DD samples were re-amplified with an insertion-specific primer pair as in the previously described protocol.30

Genotyping of ATr1 gene A1166C polymorphism

ATr1 gene A1166C polymorphism was determined by PCR-restriction fragment length polymorphism. An A/C substitution at position 1166 in the 3′-untranslated region of the ATr1 gene was amplified to form an undigested 350-bp fragment with the primers 5′-CAA GAA GCC TGC ACC ATG TTT-3′ (forward) and 5′-AGG ACA AAA GCA GGC TAG GGA-3′ (reverse).8 PCR products were digested with DdeI (Roche Diagnostics) at 37°C overnight. DdeI digestion resulted in one 350-bp fragment for wild-type homozygotes (AA), two fragments of 230 and 120 bp for the variant homozygotes (CC) and three fragments of 350, 230 and 120 bp for the heterozygotes (AC).

Statistical analysis

The ages of the patient and control groups were compared with Student's t-test. χ2 analyses were used to compare the gender distribution, to compare the association between the genotypes and alleles in relation to the cases and controls, and to test for deviation of genotype distribution from Hardy-Weinberg equilibrium (HWE). P < 0.05 was considered statistically significant. The odds ratio (OR) and their 95% confidence intervals (CI) were calculated to determine the strength of the association between polymorphism genotype alleles and the cases and controls. Additionally, power analyses were performed with G*Power software (University of Kiel, Kiel, Germany).31 The statistical power ranged from 0.21–0.99.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Demographic data and PAS scores of patients

The study included 123 cases with PD and 168 healthy controls. The groups were not statistically significant with respect to age (P = 0.49) or gender (P = 0.48). As shown in Table 1, the patients were divided into two groups based on the existence of agoraphobia. A significant difference was observed between the mean age of patients with and without agoraphobia (P = 0.033). Also, patients with agoraphobia had lower age onset relative to patients without agoraphobia (P = 0.008). A highly significant difference was observed between patients with agoraphobia having PAS scores less than 11 and greater than or equal to 11 (P = 0.01). There was an extremely significant difference between the mean PAS scores of patients with agoraphobia and without agoraphobia (P < 0.001). A significant difference was observed between male and female patients having PAS scores greater than 11 (P = 0.021) (data not shown).

Table 1.  Demographic data of panic disorder patients with and without agoraphobia
 Agoraphobia (+)Agoraphobia (−)P-value
  1. PAS, Panic Agoraphobia Scale.

Total (n = 123)4182
Women (n (%))30 (37%)51 (63%)
Men (n (%))11 (26.2%)31 (73.8%)0.23
Age (years (mean ± SD))33.66 ± 12.8938.24 ± 10.150.033
Age onset (years (mean ± SD))26.41 ± 7.6130.27 ± 7.380.008
PAS < 11 (n (%))1 (2.4%)16 (19.5%)
PAS ≥ 11 (n (%))40 (97.6%)66 (80.5%)0.01
PAS average value (mean ± SD)30.39 ± 9.9520.68 ± 10.20<0.001

HWE for functional ACE I/D, and ATr1 A1166C SNP

The genotype distributions of ACE I/D and ATr1 A1166C polymorphisms are presented in Table 2. The distributions of the ACE I/D and ATr1 A1166C genotypes were in accordance with the HWE among the patients (P = 0.53 and P = 0.06, respectively) and controls (P = 0.08 and P = 0.75, respectively). The frequencies of each genotype were consistent with the HWE in the whole sample (P > 0.05).

Table 2.  The distribution of ACE I/D and ATr1 A1166C genotype and allele frequencies among panic disorder patients and healthy controls
ACE geneI/II/DD/DP-valueI allele frequencyD allele frequencyP-value
Panic disorderTotal (n (%))25 (20.3)57 (46.3)41 (33.3)0.300.430.570.055*
Women (n (%))12 (14.8)41 (50.6)28 (34.6)0.4510.400.600.82**
Men (n (%))13 (31)16 (38.1)13 (31)0.1310.500.500.032***
ControlsTotal (n (%))29 (17.3)68 (40.5)71 (42.3)0.360.64
Women (n (%))19 (18.3)43 (41.3)42 (40.4)0.390.61
Men (n (%))10 (15.6)25 (39.1)29 (45.3)0.350.65
ATr1 geneAAACCCP-valueA allele frequencyC allele frequencyP-value
  • *

    χ2 test, total panic disorder patients versus total controls. **χ2 test, female panic disorder patients versus female controls. ***χ2 test, male panic disorder patients versus male controls.

  • Inadequate number to analyze.

  • ACE, angiotensin converting enzyme; ATr1, angiotensin II type I receptor; D, deletion; I, insertion.

Panic disorderTotal (n (%))80 (65.0)34 (27.6)9 (7.3)0.4950.790.210.32*
Women (n (%))47 (58)26 (32.1)8 (9.9)0.4560.740.260.19**
Men (n (%))33 (78.6)8 (19)1 (2.4)0.880.120.65***
ControlsTotal (n (%))115 (68.5)46 (27.4)7 (4.2)0.820.18
Women (n (%))68 (65.4)30 (28.8)6 (5.8)0.800.20
Men (n (%))47 (73.4)16 (25)1 (1.6)0.860.14

Functional ACE I/D and ATr1 A1166C polymorphisms

No statistically significant differences were observed in the genotype frequencies of functional ACE I/D and ATr1 A1166C gene polymorphisms between the PD patients and control group (Table 2). Borderline significance was observed in the allele frequency of ACE I/D polymorphism (P = 0.055).

Interactions between angiotensin-related genes and PD subtypes

We also explored the possible selective effect of polymorphisms on the patients' subgroups by analyzing the effect of the polymorphisms depending on PAS scores (< 11 vs ≥11) and having agoraphobia. Statistical analyses revealed no association between the alleles or the genotype frequencies of the ACE I/D and ATr1 A1166C gene polymorphisms and agoraphobia and PAS scores of patient subtypes (P > 0.05) (data not shown). However, with respect to the ACE I/D polymorphism, the I allele was found to be more frequent in the male subgroup of PD patients than in controls (χ2 = 4.61, P = 0.032). In PD patients, the genotype distributions were also compared between male and female subgroups for SNP ACE I/D (χ2 = 4.588, P = 0.10, power = 0.55) and for SNP ATr1 A1166C (χ2 = 5.623, P = 0.06, power = 0.99; data not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

In addition to its endocrine functions, the renin-angiotensin system (RAS) plays an important role within individual tissues such as the brain. The brain RAS is thought to control blood pressure and may regulate metabolism through mechanisms that remain undefined.32 RAS has been studied in numerous psychiatric diseases including schizophrenia, bipolar disorders, major depression, migraine development,33–37 and PD.7,8,11,12

Many anxiety disorders, including PD, have genetic components, and an association between angiotensin-related genes and PD has been shown in previous findings based on the defects in respiratory and cardiovascular control in PD patients. Busjahn et al. reported lower heart rate variability in II and ID genotype carriers of the ACE gene compared to DD variants.38 As such, the existence of a significant correlation between regulation of respiration and pathophysiology of PD can be suggested. However, future studies of PD patients must divide patients into subgroups with and without any aberrations in respiration or heart rate variability.

Olsson et al. reported that the II or ID variants of the ACE gene were more common in PD patients compared to controls, which is consistent with our results.8 However, our results were not statistically significant. No statistically significant association was found between PD and ACE I/D, and ATr1 A1166C polymorphisms in this case-control study. In this study, D allele frequency of ACE gene in Turkish Caucasians (D allele 64 %) was confirmed as described previously.36 The C allele frequency of ATr1 gene in Turkish population (18%) was also confirmed as reported previously in a Turkish hypertension study.39

In the general population, PD occurs more frequently in women than in men.40 From this gender difference, candidate genes may emerge in PD pathogenesis. Thus, some studies expressing gonadal effects on ACE activity reported that women may be more influenced by the ACE gene than mens.41 Another study on ACE I/D polymorphism reported that the less-active I allele was found to be associated with PD in the male subgroup of patients, suggesting a potential gender-specific effect, which is in line with another study's report of a Caucasian sample.8,11 Furthermore, Erhardt et al. have identified an association of two SNP (rs4311 and rs4333), other than I/D polymorphism (rs4340), in the ACE gene with syndromal panic attacks.12 No statistically significant association was found between genotype distributions of genders and PD in this case-control study. This may be ascribed to ethnic, genetic and environmental differences in allele frequency for the investigated polymorphisms, which might affect the results in genetic studies. However, a statistically significant difference was found in the less-active I allele frequency in the male PD subgroup, which is consistent with the findings of Olsson et al. and Bandelow et al.8,11 A previous negative association was found in a Japanese sample of patients with PD.7 This negative finding can be attributed to allelic differences in the ACE I/D polymorphism between ethnic Japanese and Caucasian populations.42

Previous studies have shown that ACE catalyzes the degradation of SP by hydrolysation.43 The less-active I allele can lead to decreased degradation of SP, and thus, increased SP levels have been reported to induce anxiogenic-like responses.19,20 SP receptor (neurokinin 1 receptor) antagonists have been suggested as anxiolytic compounds that avail themselves to a specific therapy based on the ACE I/D genotype, where homozygous carriers of the less-active I allele might profit most.11

It has been suggested that angiotensin II affects central serotonin and dopamine turnover.44 RAS also has a complex influence on brain dopaminergic transmission. It has been shown that ACE inhibitors reduce the formation of angiotensin II and enhance dopamine release and turnover.45,46 Angiotensin II has been reported to facilitate the formation of dopaminergic nerve cells.47 Few studies have explored the interaction between angiotensin and serotonin. However, it has been shown that angiotensin II reduces central serotonin release in rats,48,49 and the ATr1 antagonist enhances serotonin formation.45 Thus, angiotensin-related gene polymorphisms may affect the serotonergic and dopaminergic system in PD patients. Thus, the role of the polymorphisms affecting the serotonergic and dopaminergic system may shed light on the pathogenesis of PD.

The results of this investigation suggest that the I/D polymorphism of the ACE gene and the A1166C polymorphism of the ATr1 gene may not be associated with PD pathogenesis. However, the allele frequency of the ACE gene has a borderline significance in PD patients. Furthermore, with respect to the ACE I/D polymorphism, the I allele was found to be more frequent in the male subgroup of PD patients than in controls, suggesting a potential gender-specific role for the less-active ACE I allele in the pathogenesis of PD. In contrast, no evidence for an association between ATr1 gene allele frequency and PD was found.

In conclusion, although the sample sizes of the groups of PD patients and healthy controls were not large enough to detect the significance between the groups, this is the first study to evaluate the possible association between angiotensin-related genes and PD development in a sample of Turkish patients. The results of our investigation suggest that the two well-known angiotensin-related genes, ACE and ATr1 polymorphisms, are not significantly associated with PD development in the study population. However, D allele frequency was borderline significant in patients with PD, and the less-active I allele frequency of the male subgroup suggests a potential gender-specific effect in the pathogenesis of PD.

The genes involved in the susceptibility and development of PD are still unknown. Given the findings, genome-wide association studies may enable the exploration of a large number of genes together. Furthermore, expression of non-coding RNA may lead to new genomic mechanisms of PD in future studies.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

This work was supported by Scientific Research Projects Coordination Unit (project #3155) of Istanbul University (Istanbul, Turkey).

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES