ACE gene I/D polymorphism and risk of sarcoidosis development in Turkish patients
Dilber YILMAZ1, Mutlu KARKUCAK2, Funda COŞKUN3, Tahsin YAKUT2, Esra KUNT UZASLAN3
1Çorum Göğüs Hastalıkları Hastanesi, Göğüs Hastalıkları Kliniği, Çorum,
2Uludağ Üniversitesi Tıp Fakültesi, Tıbbi Genetik Anabilim Dalı, Bursa,
3Uludağ Üniversitesi Tıp Fakültesi, Göğüs Hastalıkları Anabilim Dalı, Bursa.
ÖZET
Türk sarkoidoz hastalarında ACE gen polimorfizmi ve sarkoidoz gelişme riski
Giriş: Sarkoidozun etyolojisi bilinmemektedir fakat farklı etnik gruplar ve tek yumurta ikizlerindeki prevalansı, ailevi özel- liklerine bakıldığında genetik predispozisyon olası bir faktör olabilir. Sarkoidozun patofizyolojisinde anjiyotensin dönüştürücü enzim (ACE) gösterilmiştir. Bu araştırmanın amacı; ACE I/D (Insertion/Deletion) polimorfizminin sarkoidoz gelişimine olan etkisini araştırmaktır.
Hastalar ve Metod: Çalışmamız 70 Türk, histopatolojik olarak tanı konmuş sarkoidoz hastası ve 69 sağlıklı, cinsiyet ve yaş açısından eşlenmiş kontrol içeriyordu. ACE I/D polimorfizmini analiz etmek için polimeraz zincir reaksiyonu kullanıldı.
Agaroz jel elektroforezinde oluşan bantlara göre genotip tayini yapıldı. İstatistiksel analiz için ki-kare testi kullanıldı ve an- lamlılık değeri p< 0.05 olarak kabul edildi.
Bulgular: Sarkoidoz grubunda D allelinin sıklığı daha fazla olmasına rağmen, D allelinin sıklığı sırasıyla sarkoidoz ve kont- rol grubunda %67 ve %54 idi. Sarkoidoz ve kontrol grubu arasında I/I, I/D, D/D polimorfizmlerinin sıklığı açısından anlam- lı farklılık saptanmadı (p> 0.05). Benzer olarak I/I, I/D, D/D polimorfizmlerinin sıklığı açısından akciğer dışı organ tutulu- mu olan sarkoidoz hastalarıyla akciğer dışı organ tutulumu olmayan hastalar arasında farklılık yoktu (p> 0.05).
Sonuç: Bizim bulgularımıza göre ACE gen polimorfizminin Türk sarkoidoz hastalarında hastalığın gelişimine katkısı sağ- lıklı kontrollerden farklı değildi.
Anahtar Kelimeler: Sarkoidoz, polimorfizm, ACE geni.
SUMMARY
ACE gene I/D polymorphism and risk of sarcoidosis development in Turkish patients
Dilber YILMAZ1, Mutlu KARKUCAK2, Funda COŞKUN3, Tahsin YAKUT2, Esra KUNT UZASLAN3
Yazışma Adresi (Address for Correspondence):
Dr. Dilber YILMAZ, Bağlarbaşı Mahallesi, Bozcaada Sokak No: 17 Osmangazi, BURSA - TURKEY
e-mail: drdilberyilmaz@gmail.com
INTRODUCTION
Sarcoidosis is a chronic systemic granulomatous disease of unknown etiology characterized by accumulation of activated T-lymphocytes and mononuclear phagocytes forming non-caseating epitheloid granuloma in the affec- ted organs (1,2). The recognition of race as an important risk factor clearly suggests a genetic predisposition to de- velop sarcoidosis. The underlying pathophysiology of the disorder could be explained as an antigen-driven process, which in genetically susceptible people leads to an exag- gerated Th1 type immunological response (3).
One of the possible mechanisms is functional polymorp- hism of the angiotensin converting enzyme (ACE) gene.
The ACE gene, located on chromosome 17q23, contains many polymorphisms. The insertion/deletion (I/D) poly- morphism in intron 16 is the most studied polymorphism (4). The genotype is classified into three types: deletion homozygotes (D/D), insertion homozygotes (I/I) and he- terozygotes (I/D). Serum ACE levels corresponding to ACE I/D genotypes in the order I/I < I/D < D/D encoura- ged investigating the influence of this variation on sarco- idosis susceptibility (5,6). Some studies have not found association between ACE I/D polymorphism and the in- cidence of sarcoidosis (4,7-9). By contrast D/D genoty- pe conferred a threefold increased in risk for sarcoidosis in African-Americans though lack of association between ACE gene polymorphism and sarcoidosis in Caucasians in the same study (10). Also D allele is a possible risk factor for development of sarcoidosis in Japanese women
(11). Additionally a striking over-representation of the D/D genotype in sarcoidosis patients and in unaffected relatives has established in Germans (12).
The aim of our study was to examine the distribution of the ACE I/D genotypes in Turkish patients with sarco- idosis and healthy controls to identify a whether it is ge- netic risk factor for sarcoidosis.
PATIENTS and METHODS Study Subjects
Between September 2010 and December 2010, 70 sar- coidosis patients were enrolled from the outpatient clinic of Chest Diseases Department, Uludag University Medi- cal Faculty. Diagnosis of sarcoidosis was based on histo- logical evidence. Skin and lymph node involvement was attributed to sarcoidosis only if it was proven histologi- cally. Spleen involvement was based on radiological fin- dings. All patients had received the appropriate treatment for care of the disease. As a control group we studied 69 healthy age and sex matched subjects. All patients and control subjects were Turkish and unrelated to each other.
All participated (patients and control subjects) in the study after they had given their full informed and written consent. The study was approved by the Ethics Commit- tee of the Uludag University Medical School.
DNA Extraction and Genotyping
Blood samples were obtained in EDTA tubes both from the patients and controls. DNA was isolated according
1Clinic of Chest Diseases, Corum Chest Diseases Hospital, Corum, Turkey,
2Department of Medical Genetic, Faculty of Medicine, Uludag University, Bursa, Turkey,
3Department of Chest Diseases, Faculty of Medicine, Uludag University, Bursa, Turkey.
Introduction: Etiology of sarcoidosis is unknown but the prevalence of disease in different ethnic groups and identical twins, family characteristics indicate that genetic predisposition is a possible factor. The angiotensin-converting enzyme (ACE) has been implicated in the pahophysiology of sarcoidosis. The aim of this study is to investigate the influence of a polymorphism in I/D (Insertion/Deletion) of the ACE gene on the susceptibility to sarcoidosis.
Patients and Methods: Our study included 70 Turkish patients who had histopathological diagnosis of sarcoidosis and 69 healthy age and sex matched control subjects. Polymerase chain reaction was used for analysing an I/D polymorphism in the gene coding for ACE. Genotyping was done according to bands that were formed on the agarose gel electrophoresis.
Chi-square test was used for statistical analysis and p< 0.05 was accepted as significance.
Results: Although the D allele was more frequent in the sarcoidosis patients group, the frequency of the D allele was 67%
and 54% respectively in the sarcoidosis and the control group. No significant difference in allele frequencies of I/I, I/D, D/D polymorphisms was observed between the sarcoidosis and control group (p> 0.05). Similarly allele frequencies of I/I, I/D, D/D polymorphisms was not different between sarcoidosis patients with extrapulmonary involvement and sarcoidosis patients without extrapulmonary involvement (p> 0.05).
Conclusion: Our findings have showed that contribution of ACE gene polymorphisms to susceptibility of disease develop- ment in Turkish sarcoidosis patients is not different from the healthy control subjects.
Key Words: Sarcoidosis, polymorphism, ACE gene.
to the Dr. Zeydanlı (DZ) DNA isolation kit procedure and then stored at -20°C. The ACE gene I/D polymorp- hism was determined using the polymerase chain reac- tion (PCR) method. The primers used to determine ACE I/D polymorphism were F: 5’-CTG GAG ACC ACT CCC ATC CTT TCT 3’ and R: 5’ GAT GTG GCC ATC ACA TTC GTC AGA T-3’, and the D/D genotype insertion area specific primer: F: 5’-TGG GAC CAC AGC GCC CGC CCG CCA CTA C-3’ and R: 5’-TCG CCA GCC CTC CCA TGC CCA TAA-3’. To prevent incorrect D/D genotyping in samples of ACE D/D genotype, the re- sults were confirmed with a second PCR analysis. PCR conditions were as follows: after the first denaturation for 5 min at 94°C, denaturation continued for 1 min at 94°C, which continued with 35 cycles of annealing for 1 min at 57°C (for verification of D/D genotype, at 63°C), and extension for 1 min at 72°C, which ended with final extension for 10 min at 72°C (13).
After PCR, the samples were separated by 2% agarose gel electrophoresis, stained with ethidium bromide and photographed for the amplification study. As a result of the amplification study, the agarose gel showed an amp- lification band of 190 bp in samples with the DD genoty- pe, bands of 490 and 190 bp in samples with the ID ge- notype and a band of 490 bp in samples with the II ge- notype (Figure 1). In the second PCR analysis conduc- ted for DD confirmation, an amplification band of 335 bp was observed with samples that had the insertion band.
Statistical Analysis
The SPSS 13.0 program was used for the analysis. All data were reported in terms of mean ± standard devi- ation (SD). A chi-squared test was used to compare genotypes and also genotypes/extrapulmonary invol-
vement. P value less than 0.05 was accepted as statis- tical significance.
RESULTS
The mean age was 46.64 ± 10.5 years and 44.86 ± 10.83 for the patient group (15 males and 55 females) and the control group (24 males and 45 females) res- pectively. There was no significant difference in terms of age and gender between the two groups (Table 1).
Nine patients had skin involvement, one patient had lymph node involvement and one patient had spleen involvement.
Characteristics and genotype distributions of ACE gene I/D polymorphism for both groups are tabulated (Table 1). No significant difference in allele frequencies for all polymorphisms was observed between sarcoidosis and control groups, although the D allele was more frequent in the sarcoidosis group (p> 0.05) (Table 1, Figure 2).
The D allele frequencies of ACE gene I/D polymorp- hism in sarcoidosis in published studies before are sum- marized (Table 2). Out of nine patients with skin invol- vement, three patients had D/D, four patients had I/D and two patients had I/I allele. One patient with spleen involvement had I/D allele and one patient with lymph node involvement had I/D allele. Similarly there was not significant difference between sarcoidosis patients with extrapulmonary involvement and sarcoidosis patients without extrapulmonary involvement in terms of D/D, I/D and I/I allele frequencies (p> 0.05). Further analysis showed that also ACE genotype was not associated with skin involvement. However due to low numbers of sarcoidosis patients with extrapulmonary involvement, we can not exclude type II error.
DISCUSSION
The discovery that an I/D polymorphism in intron 16 of the ACE gene contributes to the ACE activity in a he- althy population was soon followed by studies of the ro- le for this polymorphism in sarcoidosis patients (14).
The D allele is reported to be a predisposing factor to high serum ACE levels both in normal subjects and in patients with sarcoidosis (15).
We have reported that there is no association between ACE gene I/D gene polymorphism and risk of sarco- idosis in Turkish people based on our prospective study. Although frequency of D allele was higher in the sarcoidosis patients group, significant difference was not observed between the D allele frequencies of two groups.
Many studies have investigated the relationship betwe- en ACE gene I/D polymorphism and sarcoidosis to da- Figure 1. Detection ACE gene I/D polymorphism by polyme-
rase chain reaction. Line MARKER is 100 bp DNA Ladder.
Lines 1 and 4 are D/D genotype (190 bp/190 bp), line 2 I/I genotype (490 bp/490 bp) and line 3 is I/D genotype (490 bp/190 bp).
200 bp
100 bp 500 bp
1
MARKER 2 3 4
te. As a result these studies have showed that frequ- ency of genotypes can be different according to the po- pulation (4,7-12,14-24). While Biller et al. studied ACE gene I/D polymorphism in Caucasian sarcoidosis pati- ents from Northern Germany, Tomita et al. studied pa- tients from Japan (7,16). In both reports, distribution of ACE genotypes of patients with sarcoidosis and he- althy controls were not different. Similar results were found among United Kingdom, Czech, Scandinavian and Caucasian sarcoidosis patients in Sweden (9,17,18). Arbustini et al. analyzed ACE genotyping of Italian Caucasian population and Garrib et al. analyzed the genotyping of Caucasian patients in United King-
dom (4,19). In the both studies, D/D was the most fre- quent genotype observed but this difference did not at- tain statistical significance. Similarly, Pietinalho et al.
analyzed ACE gene I/D polymorphism in Finnish popu- lation (8). Although the frequency of the D allele was higher in control subjects and sarcoidosis patients, the difference was not statistically significant. However, Furuya et al. found an over-represantation of the allele D in Japanese sarcoidosis patients as compared with Japanese control (11). While this phenomenon was significant in female, there was no clear difference in male patients. Maliarik et al. studied African Americans and Caucasians (10). There was not significant diffe- Table 1. Characteristics features and genotype distribution of ACE gene I/D polymorphism in sarcoidosis patient and controls groups.
Patients group Control group
Variables (n= 70) (n= 697) p
Age (years) (mean ± SD) 46.64 ± 10.5 44.86 ± 10.83 0.08
Gender
Female 55 45 0.333
Male 15 24
Genotypes
I/I 8 13 0.073
I/D 30 37
D/D 32 19
Frequency of allele
I (%) 33 46 0.060
D (%) 67 54
SD: Standard deviation, I: Insertion, D: Deletion, ACE: Angiotensin converting enzyme.
100
90
80
70
60
50
40
30
20
10
0
67
Patient group
54
46
D allele frequency (%) I allele frequency (%)
Control group 33
Figure 2. Distribution of all allel frequencies of ACE gene I/D polymorphism in the sarcoidosis patients and control groups.
rence in the distribution of ACE I/D genotypes in Ca- ucasian cases and controls. But they found a three fold increase in sarcoidosis risk in African Americans with the D/D genotype. Additionally in the former populati- on D/D genotype showed a marked increase in risk for cases with a positive family history compared with tho- se who had a negative family history. Schürmann et al.
studied German families with more than one member suffering from sarcoidosis (12). There was a striking over-representation of the D/D genotype in patients with sarcoidosis and their families compared to cont- rols. Also Salobir et al. evaluated Slovenian sarcoidosis patients (20). In the sarcoidosis patient group a higher frequency of the D allele was detected, while in the control group the D allele frequency was lower. So ACE gene I/D polymorphism in Slovenian sarcoidosis pati-
ents, an increased risk of the disease susceptibility was demonstrated in homozygous carriers of the D allele.
Hatemi et al. studied the ACE I/D gene polymorphism in 48 Turkish sarcoidosis patients (21). As a result fre- quency distribution was not different in patients and healthy control subjects. Frequency of D allele in our study was higher compared to Hatemi et al. (67% and 58%, respectively) (21). Additionally the number of sarcoidosis patients was much higher (70 and 48 res- pectively).
Data about extrathoracic involvement of sarcoidosis and ACE gene polymorphism is very limited in litera- ture. Maliarik et al. showed that ACE genotype was not associated with extrathoracic involvement in sarcoido- sis patients (10). Similarly we did not find any associ- ation between ACE gene polymorphism and extratho- Table 2. D allele frequencies of ACE gene I/D polymorphism of sarcoidosis patient group and control group in published studies to date.
D allele frequency D allele
Number of of sarcoidosis Number of frequency of Study sarcoidosis patients patients (%) control subjects control group (%)
Arbustini et al. (4) 61 64 80 60
Tomita et al. (7) 207 38 314 34
Pietinalho et al. (8) 59 58 70 51
McGrath et al. (9)
(United Kingdom) 180 52 386 54
McGrath et al. (9)
(Czech) 56 47 179 47
Maliarik et al. (10)
(African Americans) 183 57 111 44
Maliarik et al. (10)
(Caucasian) 60 52 48 58
Furuya et al. (11) 103 39 341 33
Schürmann et al. (12) 140 44 100 62
Kruit et al. (14) 99 50 327 51
Takemoto et al. (15) 100 37 96 39
Biller et al. (16) 95 55 264 54
Papadopoulos et al. (17) 62 52 107 52
Planck et al. (18) 73 49 65 46
Garrib et al. (19) 54 63 100 53
Salobir et al. (20) 105 57 80 47
Hatemi et al. (21) 48 58 106 59
Császár et al. (22) 31 60 29 65
Tahir et al. (23) 72 40 199 65
Alia et al. (24) 177 61 104 57
Our study 70 67 69 54
ACE: Angiotensin converting enzyme, I: Insertion, D: Deletion.
racic involvement. But number of patients with extra- pulmonary involvement so we could not exclude type II error.
In conclusion, while our study did not find a significant correlation between the ACE gene I/D polymorphism and sarcoidosis in Turkish population, we believe that a larger sample size and further elucidation of other single nucleotide polymorphisms are required to reach a definitive conclusion.
CONFLICT of INTEREST None declared.
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