GENETIC TESTING AND MOLECULAR BIOMARKERS Volume 19, Number 6, 2015 ª Mary Ann Liebert, Inc. Pp. 1–6 DOI: 10.1089/gtmb.2014.0334
ORIGINAL ARTICLES
Association of the ACE I/D Gene Polymorphisms with JAK2V617F-Positive Polycythemia Vera and Essential Thrombocythemia ¨ zlem Gorukmez,2 Mehmet Ture,2 Ali Topak,2 Orhan Gorukmez,1 S xebnem Ozemri Sag,2 O 2 3 Serdar Sahinturk, Gu¨ven Ozkaya, Tuna Gulten,2 Rıdvan Ali,4 and Tahsin Yakut 2
The renin–angiotensin system contributes to cell growth, proliferation, and differentiation in the bone marrow. We investigated the role of the ACE I/D gene polymorphism in 108 polycythemia vera (PV) and essential thrombocytosis (ET) patients who were positive for the JAK2V617F mutation, with a thrombosis group (TG) of 95 patients who had a history of vascular events, but did not have a history of myeloproliferative neoplasms and compared these to a healthy control group (CG) of 72 subjects. In the patients, II genotype and I allele frequency ( p = 0.009, odds ratio [OR] = 9.716, 95% confidence interval [CI] = 1.242–76.00, p = 0.004, OR = 2.019, 95% CI = 1.243–3.280, respectively) were found to be higher than those in the controls. The DD genotype ( p = 0.021, OR = 0.491, 95% CI = 0.268–0.899) and D allele ( p = 0.004, OR = 0.495, 95% CI = 0.305– 0.805) were found to be correlated with a decreased risk of a myeloproliferative neoplasm. These findings support the hypothesis that the ACE II genotype and I allele may be related to increased risk of ET and PV. Conversely, the DD genotype and D allele may be related to decreased risk of ET and PV. The results also indicated that the ACE I/D gene polymorphism was independent of thrombosis formation.
through the type I receptor of angiotensin (AT) II. Haznedaroglu et al. (1996) proposed that the intrinsic local RAS exists in bone marrow cells and regulates both the physiological and pathological production of blood cells. In addition, the RAS affects hemostasis through various mechanisms (Fatini et al., 2003). Angiotensin converting enzyme (ACE) is the key enzyme in RAS and converts angiotensin 1 to its physiologically active peptide AT 2. Membrane-bound and circulating forms of ACE have been described. The membrane-bound form is available in various cells, including in epithelial cells, neural cells, and macrophages. The circulating form is available in biological fluids, such as plasma and amniotic and seminal fluids. Rigat et al. (1990) reported an indel DNA sequence of 287 bp in intron 16 of the ACE gene that was associated with circulating ACE levels. According to the I/D polymorphism, 3 genotypes have been described: II, ID, and DD. Although the DD genotype is related to highest levels of serum ACE, ID corresponds to medium serum ACE levels and II corresponds to low serum ACE levels (Tiret et al., 1992). Suehiro et al. (2004) reported that the D allele is related to increased ACE mRNA levels. The ACE plays a role in various biological events, including fibrinolysis, thrombocyte aggregation, and blood
Introduction
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hronic myeloproliferative neoplasms (CMN) are a group of diseases characterized by excessive production of mature/functional blood cells arising from hematopoietic stem cell transformation (Barcelos and Santos-Silva, 2011). Polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis are the most common BCR-ABL fusion gene-negative myeloproliferative neoplasms ( Jones et al., 2005). A Janus kinase 2 mutation (JAK2-V617F) was detected in BCR-ABL-negative cases in 2005 (Baxter et al., 2005). It has been reported that this mutation is present at a rate of 96% in PV, 55% in ET, and 65% in PMF (Tefferi and Vainchenker, 2011). Thrombotic complications are the primary problems in patients with CMN. Twelve to thirty-nine percent of ET and PV patients initially present with thrombosis, with even a higher percentage of patients experiencing another thrombotic complication after diagnosis (Elliott and Tefferi, 2005). The renin–angiotensin system (RAS) contributes to cell growth, proliferation, and differentiation in bone marrow through its autocrine and paracrine effects and modulates the reproduction of normal cells and neoplastic hematopoietic cells
1 S xevket Yılmaz Training and Research Hospital, Medical Genetics Unit, Bursa, Turkey. Departments of 2Medical Genetics and 3Biostatistics, School of Medicine, Uludag University, Bursa, Turkey. 4 Division of Hematology, Department of Internal Medicine, School of Medicine, Uludag University, Bursa.
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clotting activation (Fatini et al., 2003). The ACE is expressed at different levels in various carcinomas and may be effective in tumor cell proliferation, migration, angiogenesis, and metastatic behavior (Rocken et al., 2005). In recent studies, an increase in the frequency of the ACE I allele has been reported in lung, breast, prostate, gastric, and oral cancers, as well as in hematologic malignancies (Zhang et al., 2011). In the present study, we aimed to investigate whether the ACE I/ D gene polymorphism is an efficient etiology of myeloproliferative neoplasms and to investigate the relationship between this polymorphism and vascular incidents. Patients and Methods
In this case–control study, we enrolled 52 PV and 56 ET patients who met the WHO criteria (Wadleigh and Tefferi, 2010) for clinical diagnosis of myeloproliferative neoplasms and who referred to the Uludag University Faculty of Medicine, Medical Genetics Department for JAK2V617F mutation analysis with positive results. A thrombosis group (TG) consisted of 95 patients who had at least one vascular event history, had normal complete blood count values, and did not have a history of myeloproliferative neoplasm or cancer. The control group (CG) included 72 subjects with no history of cancer or vascular incident, who were enrolled between the years 2009 and 2013. Clinical and laboratory data [leukocyte and thrombocyte counts, hemoglobin levels, existence of splenomegaly, vascular incident history, and comorbid diseases (dyslipidemia, diabetes mellitus, and hypertension)] were noted for patients with myeloproliferative neoplasms and TG. Vascular events were defined as arterial or venous thrombosis such as transient ischemic attack, ischemic stroke, acute myocardial infarction, peripheral arterial thrombosis, deep vein thrombosis, and pulmonary embolism. Local ethics committee approval was obtained. All of the patients and controls provided written informed consent. Genotyping ACE I/D gene polymorphism
Peripheral blood samples of patients and subjects from all groups were collected in EDTA tubes isolated using Dr. Zeydanlı (DZ) DNA isolation kits and preserved at - 20C. A polymerase chain reaction (PCR) method was used to detect ACE I/D gene polymorphism. The primers used to detect ACE I/D gene polymorphism were F: 5¢-CTG GAG ACC ACT CCCATC CTT TCT 3¢ and R: 5¢ GAT GTG GCC ATC ACATTC GTC AGA T-3¢ and D/D genotype insertion areaspecific primers F: 5¢-TGG GAC CAC AGC GCCCGC CCG CCA CTA C-3¢ and R: 5¢-TCG CCA GCCCTC CCA TGC CCA TAA-3¢. To prevent incorrect D/D genotyping in samples with the ACE D/D genotype, the results were confirmed with a second PCR analysis. The PCR conditions were as follows: after an initial denaturation for 5 min at 94C, 35 cycles of denaturation step for 1 min at 94C, annealing step for 1 min at 57C (for verification of D/D genotype, at 63C), and extension step for 1 min at 72C were run, and ended with a final extension step for 10 min at 72C (Yildiz et al., 2010). After PCR, the products were analyzed on a 2% agarose gel dyed with ethidium bromide, and product bands were observed under ultraviolet light. A 190 bp band was observed in cases with the DD genotype, 490 and 190 bp bands were observed in cases with the ID genotype, and a 490 bp band was found in
GORUKMEZ ET AL.
cases with the II genotype. A second PCR analysis was performed for DD confirmation, with a 335 bp amplification band detected in samples with an insertion band. Statistical analyses
Statistical analyses were performed using the SPSS 20.0 pocket program. Normality of distributions of the data was investigated using the Shapiro–Wilk test. The data showed a skewed distribution and were thus presented as median (minimum–maximum) values. The Mann–Whitney U test was used for two group comparisons, and the Kruskal–Wallis test was used for comparisons of more than two groups. The Pearson chi-square test, Fisher’s exact test, and Fisher– Freeman–Halton test were applied for categorical data. Significance value was set as p < 0.05. Results
The patients in the myeloproliferative group (ET + PV) were between 20 and 84 years old, and the median age was 59.5. Fifty-seven (52.8%) of the patients were female and 51 (47.2%) of them were male. The TG patients were between 19 and 86 years old, with a median age of 58; 56 (58.9%) of them were female and 39 (41.1%) were male. In the CG of subjects without a history of vascular incident neoplasm, the ages were distributed between 49 and 79 years with a median age of 55. Forty-three (59.7%) of the control subjects were female and 29 (40.3%) were male. Between the patient and CGs, no differences were observed in terms of age ( p = 0.065) or gender ( p = 0.565). The clinical features of ET and PV patients are presented in Table 1. The genotype distribution for the ACE I/D gene polymorphism and frequency of the I/D allele are presented comparatively in Table 2 for the ET, PV, TG, and CG groups. The ACE II genotype and the I allele frequency were found to be significantly higher in the ET and PV groups than in the CG ( p = 0.009, odds ratio [OR] = 9.716, 95% confidence interval [CI] = 1.242–76.00; p = 0.004, OR = 2.019, 95% CI = 1.243–3.280, respectively), and the DD genotype ( p = 0.021, OR = 0,491, 95% CI = 0.268–0.899) and the D allele ( p = 0.004, OR = 0.495, 95% CI = 0.305–0.805) frequency were found to be correlated with a decreased risk of ET and PV ( p = 0.004, OR = 0.495, 95% CI = 0.305–0.805). The II genotype and the I allele frequency were found to be
Table 1. Features of ET and PV Patients ET (n = 56)
PV (n = 52)
pValue
Age (years) 63.5 (20–84) 57.5 (28–84) 0.48 Gender 42.9/57.1 51.9/48.1 0.35 (male/female%) Hb (g/dL) 13.8 (10–16.7) 17.95 (10.6–21.3) 0.000 784.5 (288–1560) 490 (173–1221) 0.000 PLT ( · 109/L) a 9 12.5 (5–32.1) 13.2 (5.2–40.5) 0.36 WBC ( · 10 /L) 37 (68.5) 33 (68.8) 0.98 WBCa ( > 10 · 109/L) n (%) WBCa ( £ 10 · 109/L) 17 (31.5) 15 (31.2) n (%) Splenomegaly n (%) 14 (25.5) 13 (27.1) 0.85 a WBC was calculated in 54 ET patients and 48 PV patients. ET, essential thrombocytosis; Hb, hemoglobin; PLT, platelets; PV, polycythemia vera; WBC, white blood cells.
0.432 (0.044–4.241) 0.4 1.328 (0.704–2.505) 0.5 0.817 (0.436–1.529) 0.7 1.097 (0.644–1.870) (21.5)a 38 (20)a (78.5)a 152 (80)a
60 (63.2)a (58.3)a
(40.3)a
32 (33.7)a
0.6 3 (3.2)a (1.4)a
4.196 1 (1.158–15.210) 0.051 1.118 29 (0.997–3.112) 0.001 0.401 42 (0.228–0.707) < 0.001 2.216 31 (1.411–3.480) 113 42 (58.3)a (40.7)a
(35.6)a 31 (21.5)a (64.4)a 113 (78.5)a
29 (40.3)a (47.2)a
39 (34.8)a 38 (36.5)a 73 (65.2)a 66 (63.5)a I D
21 (40.4)a DD 23 (4.1)a
27 (48.2)a 24 (46.2)a ID
a n (%). TG, thrombosis group; CG, control group; CI, confidence interval; OR, odds ratio.
(35.6)a 38 (20)a (6.4)a 152(80)a
(40.7)a 60 (63.2)a
(47.2)a 32 (33.7)a
3 (3.2)a 0.009 7 (13.5)a 0.8
6 (10.7)a II
0.771 13 (0.241–2.467) 0.8 1.086 51 (0.510–2.314) 0.9 1.029 44 (0.477–2.218) 0.8 0.928 77 (0.532–1.620) 139
(12)a
1 (1.4)a
9.716 13 (1.242–76.00) 0.4 1.327 51 (0.725–2.426) 0.021 0.491 44 (0.268–0.899) 0.004 2.019 77 (1.243–3.280) 139
(12)a
0.019
OR (%95 CI) p TG (n = 95) CG (n = 72) OR (%95CI) P TG (n = 95) ET + PV (n = 108) OR (%95 CI) p CG (n = 72) ET + PV (n = 108) OR (%95 CI) p PV (n = 52) ET (n = 56)
Table 2. Comparative Results of Genotype Distribution and Allele Frequency of ACE I/D Gene Polymorphism in ET, PV, TG, and CG Groups
ACE I/D GENE POLYMORPHISMS IN MYELOPROLIFERATIVE DISEASES
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significantly higher in the ET and PV groups than in the TG ( p = 0.019, OR = 4.196, 95% CI = 1.158–15.210; p < 0.001, OR = 2.216, 95% CI = 1.411–3.480, respectively). However, no significant differences were observed between the TG and CG groups in terms of genotype distribution and allele frequency ( p > 0.05). The I and D allele frequencies were 35.6% and 64.4%, respectively, in the myeloproliferative patient group, 20% and 80% in the TG, and 21.5% and 78.5% in the CG. The genotype distribution of the ACE I/D gene polymorphism and the I/D allele frequency were similar between the ET and PV patients. The I and D allele frequencies were 34.8% and 65.2%, respectively, for the ET group and 36.5% and 63.5%, respectively, for the PV group. The clinical and laboratory features of the ET and PV groups with or without a history of vascular events are shown in Table 3. Dyslipidemia was found to be significantly higher in the ET and PV patients with a history of vascular incident ( p = 0.019). The genotype distribution and allele frequencies were similar between the myeloproliferative patients with a history of vascular event and those without such a history (Table 4). The I allele frequency was significantly higher in the myeloproliferative neoplasm group with a history of vascular incident than in the TG ( p = 0.005, OR = 2.40, 95% CI = 1.293–4.454) and the CG ( p = 0.016, OR = 2.187, 95% CI = 1.149–4.162). The I and D allele frequencies were 37.5% and 62.5%, respectively, in the myeloproliferative patients with a history of vascular incident and 20% and 80%, respectively, in the TG (Table 4). The genotype distribution for the ACE I/D gene polymorphism and the I/D allele frequency were similar between the myeloproliferative patients with hypertension (12.5% II, 46.9% ID, 40.6% DD genotype and 35.9% I, 64.1% D allele frequency) and those without hypertension (11.8% II, 47.4% ID, 40.6% DD genotype and 35.5% I, 64.5% D allele frequency). Discussion
In the present study, the distribution of the II genotype and the I allele frequency of the ACE I/D gene polymorphism were found to be significantly higher in the myeloproliferative
Table 3. Clinical Features of Myeloproliferative Patient Group With and Without Vascular Events VEP (n = 32)
VEN (n = 76)
Age (years) 58 (33–86)** 56 (19–84)** Age < 60 16 (50) 38 (50) Age ‡ 60 16 (50) 38 (50) 12.9 (5–40.5) 12.25 (6.3–26.6) WBCa ( · 109/L) a 9 50 (71.4)* WBC ( > 10 · 10 /L 20 (62.5)* 12 (37.5)* 20 (28.6)* WBCa ( £ 10 · 109/L)) Hb (g/dL) 15.2 (10–19.6)* 15.35 (10.4–21.3)* PLT ( · 109/L) 775 (185–1254)* 676.5 (173–1560)* DM 2 (6.2)* 6 (7.9)* DL 6 (18.8)* 3 (3.9)* HT 11 (34.4)* 21 (27.6)* Chronic disease 15 (46.9)* 24 (31.6)*
p value 0.536 1.000 0.511 0.367 0.395 0.407 1 0.019 0.483 0.139
*n (%); **median age. a WBC was calculated in 70 patients for VEN group. DL, dyslipidemia; DM, diabetes mellitus; HT, hypertension; VEP, vascular event positive; VEN, vascular event negative.
0.03 10.143 (1.086–94.759) 0.4 1.483 (0.641–3.427) 0.05 0.429 (0.182–1.008) 0.016 2.187 (1.149–4.162) (1.4)a (40.3)a (58.3)a (21.5)a (78.5)a 0.07 4.381 (0.925–20.756) 4 (12.5)a 1 0.09 1.969 (0.873–4.440) 16 (50)a 29 0.01 0.350 (0.153–0.801) 12 (37.5)a 42 0.005 2.40 (1.293–4.454) 24 (37.5)a 31 40 (62.5)a 113 (3.2)a (33.7)a (63.2)a (20)a (80)a (1.5)a 3 (50)a 32 (37.5)a 60 (37.5)a 38 (62.5)a 152 (11.8)a 1 1.063 (0.302–3.741) 4 (46.1)a 0.7 1.171 (0.512–2.678) 16 (42.1)a 0.7 0.825 (0.353–1.927) 12 (34.9)a 0.7 1.121 (0.611–2.055) 24 (65.1)a 40 9 35 32 53 99 a
n (%).
(12.5)a (50)a (37.5)a (37.5)a (62.5)a 4 16 12 24 40 II ID DD I D
VEP (n = 32)
VEN (n = 76)
p
OR (%95 CI)
VEP (n = 32)
TG (N = 95)
p
OR (%95 CI)
VEP (n = 32)
CG (n = 72)
p
OR (%95 CI)
GORUKMEZ ET AL.
Table 4. Comparative Results of Genotype Distribution and Allele Frequency of ACE I/D Gene Polymorphism in Myeloproliferative Patient Group, With and Without Vascular events, TG and CG Groups
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patient group of ET and PV. The DD genotype and D allele frequency were found to be correlated with decreased ET and PV risk. No relationship was detected between this polymorphism and the development of thrombosis. In recent studies, it has been shown that local RAS exists in many tissues (Abali et al., 2002). In 1996, it was shown for the first time that the local bone marrow RAS affects physiological and neoplastic blood cell production (Haznedaroglu et al., 1996). ACE is the key enzyme in the RAS and plays a role in many biological events such as fibrinolysis, thrombocyte aggregation, blood clotting activation, and blood pressure regulation by converting angiotensin 1 to angiotensin 2. In recent years, there have been many studies showing that this enzyme may be related to cancer etiopathogenesis and that it affects the proliferation, migration, angiogenesis, and metastatic actions of tumoral cells (Fatini et al., 2003; Zhang et al., 2011). The effect of local RAS in bone marrow was investigated in myeloproliferative diseases, and it was shown that angiotensin is an autocrine growth factor. Aksu et al. (2006) showed that the ACE surface antigen (CD 143) is overexpressed in leukemic cells in acute myeloid leukemia (AML). In a study on 83 chronic myeloid leukemia (CML) patients, Sayitoglu et al. (2009) found that the ACE, angiotensinogen, and renin mRNA levels were high at the time of diagnosis and that mRNA levels decreased during imatinib mesylate treatment in the 3rd, 6th, and 12th month in 35 of the CML patients. Aksu et al. (2005) reported that mRNA levels and synthesis of major RAS components (ACE, renin, and angiotensinogen) in the bone marrow were elevated in PV patients. Akalin et al. (2011) found that 80.4% of patients represented the ID/II genotype in hematologic malignancies, such as acute and chronic leukemia, myelodysplastic syndrome, and multiple myeloma, whereas the ID/II genotype was present in 55.9% of the CG. They also found that a 3.2-fold increase in disease risk can be attributed to the existence of the insertion allele (ID/II). In the present study, we found that the II genotype and the I allele frequency were significantly and similarly higher in the ET and PV groups. In previous studies, the DD genotype was found to be associated with high serum ACE levels (Rigat et al., 1990; Tiret et al., 1992). We found a higher frequency of the I allele in myeloproliferative diseases than in the D allele, possibly due to local RAS activity, which acts excessively through a local mechanism independent of RAS in systemic circulation. Abali et al. (2002) detected higher levels of ACE in bone marrow specimens of leukemia patients than in peripheral blood specimens. Beyazıt et al. (2007) reported significantly higher mRNA expression rates of major RAS components (ACE, renin, and angiotensinogen) in bone marrow aspirates of 10 AML patients than in those of 8 patients followed for nonmalignant hematologic diseases. Koca et al. (2007) showed elevated expressions of ACE, renin, and angiotensinogen in K562 leukemic blast cells, which are multipotential hematopoietic malignant cells. The effect of ACE gene polymorphism on other malignant tumors was investigated, and the D allele was found to be a poor prognostic factor for various cancers such as breast cancer, intestinal-type gastric cancer, and prostate cancer (Ro¨cken et al., 2007; Yaren et al., 2007; Yigit et al., 2007). However, some studies have shown that the I allele is associated with poor prognosis in oral and endometrial cancers (Freitas-Silva et al., 2004; Vairaktaris et al., 2007). As a consequence, it may be
ACE I/D GENE POLYMORPHISMS IN MYELOPROLIFERATIVE DISEASES
comprehended that different genotypes of ACE I/D gene polymorphisms may act differently in different tissues of various malignant diseases. Our study and in agreement to the study by Akalin et al. (2011) suggested that the I allele may be a potential risk factor for disease development, especially for hematological malignancies. It should be noted that insertion alleles may enhance local ACE mRNA expression, leading to potential proliferative growth effects of angiotensin 2 in leukemic bone marrow cells. Thrombotic events for both arterial and venous systems are observed in 30–50% of PV and ET patients and account for 35–45% of deaths in this population (Besses et al., 1999; Marchioli et al., 2005; Mehtap et al., 2012). In our study, nearly one-third of the patients with ET and PV had a history of vascular event. Several studies have reported age, history of a previous vascular event, hypertension, hyperlipidemia, presence of JAK2V617F mutation and leukocytosis to be risk factors for thrombosis in ET and PV patients (Barbui et al., 2013). Moreover, thrombotic events in PV and ET were attributed to increased oxidative stress (Durmus et al., 2013, 2014). Mehtap et al. (2012) reported that older age and leukocytosis were risk factors for vascular incidents in ET and PV patients. In our study, we did not detect any differences aside from dyslipidemia in clinical and laboratory features of the ET and PV patients with or without a history of vascular incident. Coexisting dyslipidemia was found to be a risk factor for vascular events in the ET and PV patients in our study. Hsiao and Hsu used a meta-analysis comprising 14 studies to evaluate the relationship between ACE I/D gene polymorphism and venous thromboembolism; the DD genotype was found to have protective effect in 3 studies, whereas 5 studies showed that the DD genotype is a risk factor for venous thromboembolism. In another 6 studies, no relationships were observed between the DD genotype and venous thromboembolism. It was reported that the diversity between the studies may be derived from ethnic heterogeneity, study design differences, patient features, and gene–gene and gene–environment interactions (Hsiao and Hsu, 2011). Poorgholi et al. (2013) did not detect any association between ACE I/D gene polymorphisms and coronary artery disease. Zhang et al. (2012) found that a D allele of ACE I/D polymorphism is a low-penetrance susceptibility marker of ischemic stroke in a meta-analysis of 50 case–control studies. Mehtap et al. (2012) did not report significant differences between the ACE I/D genotype and the development of vascular incidents in 64 ET and PV patients. In our study, we found similar results of the ACE II genotype and I allele frequency in groups of patients having myeloproliferative neoplasm with or without a history of vascular incident. Thus, we could not detect any relationship between this polymorphism and vascular incident risk in myeloproliferative diseases. In addition, a significantly higher frequency of the I allele was observed in the patients in the myeloproliferative disease group with a history of vascular incident than in the TG and CG, whereas similar results of the genotype and the allele frequency between the TG and the CG support that the I allele would be a risk factor for ET and PV, but would not be a risk for the development of thrombosis. In conclusion, the II genotype of the ACE I/D polymorphism and the I allele frequency were found to be signifi-
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cantly higher in the myeloproliferative neoplasia group consisting of ET and PV independent from vascular incident history. It was found that the II genotype and the I allele frequency are risk factors for ET and PV and that the DD genotype and the D allele frequency are associated with decreased risk of ET and PV. In addition, the ACE I/D gene polymorphism did not contribute to risk for thrombotic events. Future studies with larger patient groups will provide more accurate information for establishing a connection between ACE I/D gene polymorphism and myeloproliferative diseases and vascular events. Author Disclosure Statement
The authors declare no conflicts of interest. References
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Haznedaroglu IC, Tuncer S, Gursoy M (1996) A local reninangiotensin system in the bone marrow. Med Hypotheses 46:507–510. Hsiao FC, Hsu LA (2011) Meta-analysis of association between insertion/deletion polymorphism of the Angiotensin I-converting enzyme gene and venous thromboembolism. Clin Appl Thromb Hemost 17:51–57. Jones AV, Kreil S, Zoi K, et al. (2005) Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood 106:2162–2168. Koca E, Haznedaroglu IC, Acar K, et al. (2007) Renin-angiotensin system expression in the K562 human erythroleukaemic cell line. J Renin Angiotensin Aldosterone Syst 8:145–147. Marchioli R, Finazzi G, Landolfi R, et al. (2005) Vascular and neoplastic risk in a large cohort of patients with polycythemia vera. J Clin Oncol 23:2224–2232. Mehtap O, Birtas-Atesoglu E, Tarkun P, et al. (2012) The Association Between Gene Polymorphisms and Leukocytosis with Thrombotic Complications in Patients with Essential Thrombocythemia and Polycythemia. Vera Turk J Hematol 29:162–169. Poorgholi L, Saffar H, Fathollahi MS, et al. (2013) Angiotensin- converting enzyme insertion/deletion polymorphism and its association with coronary artery disease in an Iranian population. J Tehran Heart Cent 8:89–94. Rigat B, Hubert C, Alhenc-Gelas F, 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. Rocken C, Lendeckel U, Dierkes J, et al. (2005) The number of lymph node metastases in gastric cancer correlates with the angiotensin I-converting enzyme gene insertion/deletion polymorphism. Clin Cancer Res 11:2526–2530. Ro¨cken C, Rohl FW, Diebler E, et al. (2007) The angiotensin II/ angiotensin II receptor system correlates with nodal spread in intestinal type gastric cancer. Cancer Epidemiol Biomarkers Prev 16:1206–1212. Sayitoglu M, Haznedaroglu IC, Hatirnaz O, et al. (2009) Effects of imatinib mesylate on renin-angiotensin system (RAS) activity during the clinical course of chronic myeloid leukaemia. J Int Med Res 37:1018–1028. Suehiro T, Morita T, Inoue M, et al. (2004) Increased amount of the angiotensin-converting enzyme (ACE) mRNA originating from the ACE allele with deletion. Hum Genet 115:91–96.
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Tefferi A, Vainchenker W (2011) Myeloproliferative neoplasms: molecular pathophysiology, essential clinical understanding, and treatment strategies. J Clin Oncol 29:573–582. Tiret L, Rigat B, Visvikis S, et al. (1992) Evidence, from combined segregation and linkage analysis, that a variant of the angiotensin I-converting enzyme (ACE) gene controls plasma ACE levels. Am J Hum Genet 51:197–205. Vairaktaris E, Yapijakis C, Tsigris C, et al. (2007) Association of angiotensin-converting enzyme gene insertion/deletion polymorphism with increased risk for oral cancer. Acta Oncol 46:1097–1102. Wadleigh M, Tefferi A (2010) Classification and diagnosis of myeloproliferative neoplasms according to the 2008 World Health Organization criteria. Int J Hematol 91:174–179. Yaren A, Turgut S, Kursunluoglu R, et al. (2007) Insertion/ deletion polymorphism of the angiotensin I-converting enzyme gene in patients with breast cancer and effects on prognostic factors. J Investig Med 55:255–261. Yigit B, Bozkurt N, Narter F, et al. (2007) Effects of ACE I/D polymorphism on prostate cancer risk, tumor grade and metastatis. Anticancer Res 27:933–936. Yildiz M, Karkucak M, Yakut T, et al. (2010) Lack of association of genetic polymorphisms of angiotensin-converting enzyme gene I/D and glutathione-S-transferase enzyme T1 and M1 with retinopathy of prematures. Genet Mol Res 9:2131–2139. Zhang Y, He J, Deng Y, et al. (2011) The insertion/deletion (I/ D) polymorphism in the Angiotensin-converting enzyme gene and cancer risk: a meta-analysis. BMC Med Genet 12:159. Zhang Z, Xu G, Liu D, et al. (2012) Angiotensin-converting enzyme insertion/deletion polymorphism contributes to ischemic stroke risk: a meta-analysis of 50 case-control studies. PLoS One 7:e46495.
Address correspondence to: Tahsin Yakut, MD, PhD Department of Medical Genetics School of Medicine Uludag University Go¨ru¨kle Bursa 16059 Turkey E-mail: [email protected]
ORIGINAL ARTICLES
Association of the ACE I/D Gene Polymorphisms with JAK2V617F-Positive Polycythemia Vera and Essential Thrombocythemia ¨ zlem Gorukmez,2 Mehmet Ture,2 Ali Topak,2 Orhan Gorukmez,1 S xebnem Ozemri Sag,2 O 2 3 Serdar Sahinturk, Gu¨ven Ozkaya, Tuna Gulten,2 Rıdvan Ali,4 and Tahsin Yakut 2
The renin–angiotensin system contributes to cell growth, proliferation, and differentiation in the bone marrow. We investigated the role of the ACE I/D gene polymorphism in 108 polycythemia vera (PV) and essential thrombocytosis (ET) patients who were positive for the JAK2V617F mutation, with a thrombosis group (TG) of 95 patients who had a history of vascular events, but did not have a history of myeloproliferative neoplasms and compared these to a healthy control group (CG) of 72 subjects. In the patients, II genotype and I allele frequency ( p = 0.009, odds ratio [OR] = 9.716, 95% confidence interval [CI] = 1.242–76.00, p = 0.004, OR = 2.019, 95% CI = 1.243–3.280, respectively) were found to be higher than those in the controls. The DD genotype ( p = 0.021, OR = 0.491, 95% CI = 0.268–0.899) and D allele ( p = 0.004, OR = 0.495, 95% CI = 0.305– 0.805) were found to be correlated with a decreased risk of a myeloproliferative neoplasm. These findings support the hypothesis that the ACE II genotype and I allele may be related to increased risk of ET and PV. Conversely, the DD genotype and D allele may be related to decreased risk of ET and PV. The results also indicated that the ACE I/D gene polymorphism was independent of thrombosis formation.
through the type I receptor of angiotensin (AT) II. Haznedaroglu et al. (1996) proposed that the intrinsic local RAS exists in bone marrow cells and regulates both the physiological and pathological production of blood cells. In addition, the RAS affects hemostasis through various mechanisms (Fatini et al., 2003). Angiotensin converting enzyme (ACE) is the key enzyme in RAS and converts angiotensin 1 to its physiologically active peptide AT 2. Membrane-bound and circulating forms of ACE have been described. The membrane-bound form is available in various cells, including in epithelial cells, neural cells, and macrophages. The circulating form is available in biological fluids, such as plasma and amniotic and seminal fluids. Rigat et al. (1990) reported an indel DNA sequence of 287 bp in intron 16 of the ACE gene that was associated with circulating ACE levels. According to the I/D polymorphism, 3 genotypes have been described: II, ID, and DD. Although the DD genotype is related to highest levels of serum ACE, ID corresponds to medium serum ACE levels and II corresponds to low serum ACE levels (Tiret et al., 1992). Suehiro et al. (2004) reported that the D allele is related to increased ACE mRNA levels. The ACE plays a role in various biological events, including fibrinolysis, thrombocyte aggregation, and blood
Introduction
C
hronic myeloproliferative neoplasms (CMN) are a group of diseases characterized by excessive production of mature/functional blood cells arising from hematopoietic stem cell transformation (Barcelos and Santos-Silva, 2011). Polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis are the most common BCR-ABL fusion gene-negative myeloproliferative neoplasms ( Jones et al., 2005). A Janus kinase 2 mutation (JAK2-V617F) was detected in BCR-ABL-negative cases in 2005 (Baxter et al., 2005). It has been reported that this mutation is present at a rate of 96% in PV, 55% in ET, and 65% in PMF (Tefferi and Vainchenker, 2011). Thrombotic complications are the primary problems in patients with CMN. Twelve to thirty-nine percent of ET and PV patients initially present with thrombosis, with even a higher percentage of patients experiencing another thrombotic complication after diagnosis (Elliott and Tefferi, 2005). The renin–angiotensin system (RAS) contributes to cell growth, proliferation, and differentiation in bone marrow through its autocrine and paracrine effects and modulates the reproduction of normal cells and neoplastic hematopoietic cells
1 S xevket Yılmaz Training and Research Hospital, Medical Genetics Unit, Bursa, Turkey. Departments of 2Medical Genetics and 3Biostatistics, School of Medicine, Uludag University, Bursa, Turkey. 4 Division of Hematology, Department of Internal Medicine, School of Medicine, Uludag University, Bursa.
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clotting activation (Fatini et al., 2003). The ACE is expressed at different levels in various carcinomas and may be effective in tumor cell proliferation, migration, angiogenesis, and metastatic behavior (Rocken et al., 2005). In recent studies, an increase in the frequency of the ACE I allele has been reported in lung, breast, prostate, gastric, and oral cancers, as well as in hematologic malignancies (Zhang et al., 2011). In the present study, we aimed to investigate whether the ACE I/ D gene polymorphism is an efficient etiology of myeloproliferative neoplasms and to investigate the relationship between this polymorphism and vascular incidents. Patients and Methods
In this case–control study, we enrolled 52 PV and 56 ET patients who met the WHO criteria (Wadleigh and Tefferi, 2010) for clinical diagnosis of myeloproliferative neoplasms and who referred to the Uludag University Faculty of Medicine, Medical Genetics Department for JAK2V617F mutation analysis with positive results. A thrombosis group (TG) consisted of 95 patients who had at least one vascular event history, had normal complete blood count values, and did not have a history of myeloproliferative neoplasm or cancer. The control group (CG) included 72 subjects with no history of cancer or vascular incident, who were enrolled between the years 2009 and 2013. Clinical and laboratory data [leukocyte and thrombocyte counts, hemoglobin levels, existence of splenomegaly, vascular incident history, and comorbid diseases (dyslipidemia, diabetes mellitus, and hypertension)] were noted for patients with myeloproliferative neoplasms and TG. Vascular events were defined as arterial or venous thrombosis such as transient ischemic attack, ischemic stroke, acute myocardial infarction, peripheral arterial thrombosis, deep vein thrombosis, and pulmonary embolism. Local ethics committee approval was obtained. All of the patients and controls provided written informed consent. Genotyping ACE I/D gene polymorphism
Peripheral blood samples of patients and subjects from all groups were collected in EDTA tubes isolated using Dr. Zeydanlı (DZ) DNA isolation kits and preserved at - 20C. A polymerase chain reaction (PCR) method was used to detect ACE I/D gene polymorphism. The primers used to detect ACE I/D gene polymorphism were F: 5¢-CTG GAG ACC ACT CCCATC CTT TCT 3¢ and R: 5¢ GAT GTG GCC ATC ACATTC GTC AGA T-3¢ and D/D genotype insertion areaspecific primers F: 5¢-TGG GAC CAC AGC GCCCGC CCG CCA CTA C-3¢ and R: 5¢-TCG CCA GCCCTC CCA TGC CCA TAA-3¢. To prevent incorrect D/D genotyping in samples with the ACE D/D genotype, the results were confirmed with a second PCR analysis. The PCR conditions were as follows: after an initial denaturation for 5 min at 94C, 35 cycles of denaturation step for 1 min at 94C, annealing step for 1 min at 57C (for verification of D/D genotype, at 63C), and extension step for 1 min at 72C were run, and ended with a final extension step for 10 min at 72C (Yildiz et al., 2010). After PCR, the products were analyzed on a 2% agarose gel dyed with ethidium bromide, and product bands were observed under ultraviolet light. A 190 bp band was observed in cases with the DD genotype, 490 and 190 bp bands were observed in cases with the ID genotype, and a 490 bp band was found in
GORUKMEZ ET AL.
cases with the II genotype. A second PCR analysis was performed for DD confirmation, with a 335 bp amplification band detected in samples with an insertion band. Statistical analyses
Statistical analyses were performed using the SPSS 20.0 pocket program. Normality of distributions of the data was investigated using the Shapiro–Wilk test. The data showed a skewed distribution and were thus presented as median (minimum–maximum) values. The Mann–Whitney U test was used for two group comparisons, and the Kruskal–Wallis test was used for comparisons of more than two groups. The Pearson chi-square test, Fisher’s exact test, and Fisher– Freeman–Halton test were applied for categorical data. Significance value was set as p < 0.05. Results
The patients in the myeloproliferative group (ET + PV) were between 20 and 84 years old, and the median age was 59.5. Fifty-seven (52.8%) of the patients were female and 51 (47.2%) of them were male. The TG patients were between 19 and 86 years old, with a median age of 58; 56 (58.9%) of them were female and 39 (41.1%) were male. In the CG of subjects without a history of vascular incident neoplasm, the ages were distributed between 49 and 79 years with a median age of 55. Forty-three (59.7%) of the control subjects were female and 29 (40.3%) were male. Between the patient and CGs, no differences were observed in terms of age ( p = 0.065) or gender ( p = 0.565). The clinical features of ET and PV patients are presented in Table 1. The genotype distribution for the ACE I/D gene polymorphism and frequency of the I/D allele are presented comparatively in Table 2 for the ET, PV, TG, and CG groups. The ACE II genotype and the I allele frequency were found to be significantly higher in the ET and PV groups than in the CG ( p = 0.009, odds ratio [OR] = 9.716, 95% confidence interval [CI] = 1.242–76.00; p = 0.004, OR = 2.019, 95% CI = 1.243–3.280, respectively), and the DD genotype ( p = 0.021, OR = 0,491, 95% CI = 0.268–0.899) and the D allele ( p = 0.004, OR = 0.495, 95% CI = 0.305–0.805) frequency were found to be correlated with a decreased risk of ET and PV ( p = 0.004, OR = 0.495, 95% CI = 0.305–0.805). The II genotype and the I allele frequency were found to be
Table 1. Features of ET and PV Patients ET (n = 56)
PV (n = 52)
pValue
Age (years) 63.5 (20–84) 57.5 (28–84) 0.48 Gender 42.9/57.1 51.9/48.1 0.35 (male/female%) Hb (g/dL) 13.8 (10–16.7) 17.95 (10.6–21.3) 0.000 784.5 (288–1560) 490 (173–1221) 0.000 PLT ( · 109/L) a 9 12.5 (5–32.1) 13.2 (5.2–40.5) 0.36 WBC ( · 10 /L) 37 (68.5) 33 (68.8) 0.98 WBCa ( > 10 · 109/L) n (%) WBCa ( £ 10 · 109/L) 17 (31.5) 15 (31.2) n (%) Splenomegaly n (%) 14 (25.5) 13 (27.1) 0.85 a WBC was calculated in 54 ET patients and 48 PV patients. ET, essential thrombocytosis; Hb, hemoglobin; PLT, platelets; PV, polycythemia vera; WBC, white blood cells.
0.432 (0.044–4.241) 0.4 1.328 (0.704–2.505) 0.5 0.817 (0.436–1.529) 0.7 1.097 (0.644–1.870) (21.5)a 38 (20)a (78.5)a 152 (80)a
60 (63.2)a (58.3)a
(40.3)a
32 (33.7)a
0.6 3 (3.2)a (1.4)a
4.196 1 (1.158–15.210) 0.051 1.118 29 (0.997–3.112) 0.001 0.401 42 (0.228–0.707) < 0.001 2.216 31 (1.411–3.480) 113 42 (58.3)a (40.7)a
(35.6)a 31 (21.5)a (64.4)a 113 (78.5)a
29 (40.3)a (47.2)a
39 (34.8)a 38 (36.5)a 73 (65.2)a 66 (63.5)a I D
21 (40.4)a DD 23 (4.1)a
27 (48.2)a 24 (46.2)a ID
a n (%). TG, thrombosis group; CG, control group; CI, confidence interval; OR, odds ratio.
(35.6)a 38 (20)a (6.4)a 152(80)a
(40.7)a 60 (63.2)a
(47.2)a 32 (33.7)a
3 (3.2)a 0.009 7 (13.5)a 0.8
6 (10.7)a II
0.771 13 (0.241–2.467) 0.8 1.086 51 (0.510–2.314) 0.9 1.029 44 (0.477–2.218) 0.8 0.928 77 (0.532–1.620) 139
(12)a
1 (1.4)a
9.716 13 (1.242–76.00) 0.4 1.327 51 (0.725–2.426) 0.021 0.491 44 (0.268–0.899) 0.004 2.019 77 (1.243–3.280) 139
(12)a
0.019
OR (%95 CI) p TG (n = 95) CG (n = 72) OR (%95CI) P TG (n = 95) ET + PV (n = 108) OR (%95 CI) p CG (n = 72) ET + PV (n = 108) OR (%95 CI) p PV (n = 52) ET (n = 56)
Table 2. Comparative Results of Genotype Distribution and Allele Frequency of ACE I/D Gene Polymorphism in ET, PV, TG, and CG Groups
ACE I/D GENE POLYMORPHISMS IN MYELOPROLIFERATIVE DISEASES
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significantly higher in the ET and PV groups than in the TG ( p = 0.019, OR = 4.196, 95% CI = 1.158–15.210; p < 0.001, OR = 2.216, 95% CI = 1.411–3.480, respectively). However, no significant differences were observed between the TG and CG groups in terms of genotype distribution and allele frequency ( p > 0.05). The I and D allele frequencies were 35.6% and 64.4%, respectively, in the myeloproliferative patient group, 20% and 80% in the TG, and 21.5% and 78.5% in the CG. The genotype distribution of the ACE I/D gene polymorphism and the I/D allele frequency were similar between the ET and PV patients. The I and D allele frequencies were 34.8% and 65.2%, respectively, for the ET group and 36.5% and 63.5%, respectively, for the PV group. The clinical and laboratory features of the ET and PV groups with or without a history of vascular events are shown in Table 3. Dyslipidemia was found to be significantly higher in the ET and PV patients with a history of vascular incident ( p = 0.019). The genotype distribution and allele frequencies were similar between the myeloproliferative patients with a history of vascular event and those without such a history (Table 4). The I allele frequency was significantly higher in the myeloproliferative neoplasm group with a history of vascular incident than in the TG ( p = 0.005, OR = 2.40, 95% CI = 1.293–4.454) and the CG ( p = 0.016, OR = 2.187, 95% CI = 1.149–4.162). The I and D allele frequencies were 37.5% and 62.5%, respectively, in the myeloproliferative patients with a history of vascular incident and 20% and 80%, respectively, in the TG (Table 4). The genotype distribution for the ACE I/D gene polymorphism and the I/D allele frequency were similar between the myeloproliferative patients with hypertension (12.5% II, 46.9% ID, 40.6% DD genotype and 35.9% I, 64.1% D allele frequency) and those without hypertension (11.8% II, 47.4% ID, 40.6% DD genotype and 35.5% I, 64.5% D allele frequency). Discussion
In the present study, the distribution of the II genotype and the I allele frequency of the ACE I/D gene polymorphism were found to be significantly higher in the myeloproliferative
Table 3. Clinical Features of Myeloproliferative Patient Group With and Without Vascular Events VEP (n = 32)
VEN (n = 76)
Age (years) 58 (33–86)** 56 (19–84)** Age < 60 16 (50) 38 (50) Age ‡ 60 16 (50) 38 (50) 12.9 (5–40.5) 12.25 (6.3–26.6) WBCa ( · 109/L) a 9 50 (71.4)* WBC ( > 10 · 10 /L 20 (62.5)* 12 (37.5)* 20 (28.6)* WBCa ( £ 10 · 109/L)) Hb (g/dL) 15.2 (10–19.6)* 15.35 (10.4–21.3)* PLT ( · 109/L) 775 (185–1254)* 676.5 (173–1560)* DM 2 (6.2)* 6 (7.9)* DL 6 (18.8)* 3 (3.9)* HT 11 (34.4)* 21 (27.6)* Chronic disease 15 (46.9)* 24 (31.6)*
p value 0.536 1.000 0.511 0.367 0.395 0.407 1 0.019 0.483 0.139
*n (%); **median age. a WBC was calculated in 70 patients for VEN group. DL, dyslipidemia; DM, diabetes mellitus; HT, hypertension; VEP, vascular event positive; VEN, vascular event negative.
0.03 10.143 (1.086–94.759) 0.4 1.483 (0.641–3.427) 0.05 0.429 (0.182–1.008) 0.016 2.187 (1.149–4.162) (1.4)a (40.3)a (58.3)a (21.5)a (78.5)a 0.07 4.381 (0.925–20.756) 4 (12.5)a 1 0.09 1.969 (0.873–4.440) 16 (50)a 29 0.01 0.350 (0.153–0.801) 12 (37.5)a 42 0.005 2.40 (1.293–4.454) 24 (37.5)a 31 40 (62.5)a 113 (3.2)a (33.7)a (63.2)a (20)a (80)a (1.5)a 3 (50)a 32 (37.5)a 60 (37.5)a 38 (62.5)a 152 (11.8)a 1 1.063 (0.302–3.741) 4 (46.1)a 0.7 1.171 (0.512–2.678) 16 (42.1)a 0.7 0.825 (0.353–1.927) 12 (34.9)a 0.7 1.121 (0.611–2.055) 24 (65.1)a 40 9 35 32 53 99 a
n (%).
(12.5)a (50)a (37.5)a (37.5)a (62.5)a 4 16 12 24 40 II ID DD I D
VEP (n = 32)
VEN (n = 76)
p
OR (%95 CI)
VEP (n = 32)
TG (N = 95)
p
OR (%95 CI)
VEP (n = 32)
CG (n = 72)
p
OR (%95 CI)
GORUKMEZ ET AL.
Table 4. Comparative Results of Genotype Distribution and Allele Frequency of ACE I/D Gene Polymorphism in Myeloproliferative Patient Group, With and Without Vascular events, TG and CG Groups
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patient group of ET and PV. The DD genotype and D allele frequency were found to be correlated with decreased ET and PV risk. No relationship was detected between this polymorphism and the development of thrombosis. In recent studies, it has been shown that local RAS exists in many tissues (Abali et al., 2002). In 1996, it was shown for the first time that the local bone marrow RAS affects physiological and neoplastic blood cell production (Haznedaroglu et al., 1996). ACE is the key enzyme in the RAS and plays a role in many biological events such as fibrinolysis, thrombocyte aggregation, blood clotting activation, and blood pressure regulation by converting angiotensin 1 to angiotensin 2. In recent years, there have been many studies showing that this enzyme may be related to cancer etiopathogenesis and that it affects the proliferation, migration, angiogenesis, and metastatic actions of tumoral cells (Fatini et al., 2003; Zhang et al., 2011). The effect of local RAS in bone marrow was investigated in myeloproliferative diseases, and it was shown that angiotensin is an autocrine growth factor. Aksu et al. (2006) showed that the ACE surface antigen (CD 143) is overexpressed in leukemic cells in acute myeloid leukemia (AML). In a study on 83 chronic myeloid leukemia (CML) patients, Sayitoglu et al. (2009) found that the ACE, angiotensinogen, and renin mRNA levels were high at the time of diagnosis and that mRNA levels decreased during imatinib mesylate treatment in the 3rd, 6th, and 12th month in 35 of the CML patients. Aksu et al. (2005) reported that mRNA levels and synthesis of major RAS components (ACE, renin, and angiotensinogen) in the bone marrow were elevated in PV patients. Akalin et al. (2011) found that 80.4% of patients represented the ID/II genotype in hematologic malignancies, such as acute and chronic leukemia, myelodysplastic syndrome, and multiple myeloma, whereas the ID/II genotype was present in 55.9% of the CG. They also found that a 3.2-fold increase in disease risk can be attributed to the existence of the insertion allele (ID/II). In the present study, we found that the II genotype and the I allele frequency were significantly and similarly higher in the ET and PV groups. In previous studies, the DD genotype was found to be associated with high serum ACE levels (Rigat et al., 1990; Tiret et al., 1992). We found a higher frequency of the I allele in myeloproliferative diseases than in the D allele, possibly due to local RAS activity, which acts excessively through a local mechanism independent of RAS in systemic circulation. Abali et al. (2002) detected higher levels of ACE in bone marrow specimens of leukemia patients than in peripheral blood specimens. Beyazıt et al. (2007) reported significantly higher mRNA expression rates of major RAS components (ACE, renin, and angiotensinogen) in bone marrow aspirates of 10 AML patients than in those of 8 patients followed for nonmalignant hematologic diseases. Koca et al. (2007) showed elevated expressions of ACE, renin, and angiotensinogen in K562 leukemic blast cells, which are multipotential hematopoietic malignant cells. The effect of ACE gene polymorphism on other malignant tumors was investigated, and the D allele was found to be a poor prognostic factor for various cancers such as breast cancer, intestinal-type gastric cancer, and prostate cancer (Ro¨cken et al., 2007; Yaren et al., 2007; Yigit et al., 2007). However, some studies have shown that the I allele is associated with poor prognosis in oral and endometrial cancers (Freitas-Silva et al., 2004; Vairaktaris et al., 2007). As a consequence, it may be
ACE I/D GENE POLYMORPHISMS IN MYELOPROLIFERATIVE DISEASES
comprehended that different genotypes of ACE I/D gene polymorphisms may act differently in different tissues of various malignant diseases. Our study and in agreement to the study by Akalin et al. (2011) suggested that the I allele may be a potential risk factor for disease development, especially for hematological malignancies. It should be noted that insertion alleles may enhance local ACE mRNA expression, leading to potential proliferative growth effects of angiotensin 2 in leukemic bone marrow cells. Thrombotic events for both arterial and venous systems are observed in 30–50% of PV and ET patients and account for 35–45% of deaths in this population (Besses et al., 1999; Marchioli et al., 2005; Mehtap et al., 2012). In our study, nearly one-third of the patients with ET and PV had a history of vascular event. Several studies have reported age, history of a previous vascular event, hypertension, hyperlipidemia, presence of JAK2V617F mutation and leukocytosis to be risk factors for thrombosis in ET and PV patients (Barbui et al., 2013). Moreover, thrombotic events in PV and ET were attributed to increased oxidative stress (Durmus et al., 2013, 2014). Mehtap et al. (2012) reported that older age and leukocytosis were risk factors for vascular incidents in ET and PV patients. In our study, we did not detect any differences aside from dyslipidemia in clinical and laboratory features of the ET and PV patients with or without a history of vascular incident. Coexisting dyslipidemia was found to be a risk factor for vascular events in the ET and PV patients in our study. Hsiao and Hsu used a meta-analysis comprising 14 studies to evaluate the relationship between ACE I/D gene polymorphism and venous thromboembolism; the DD genotype was found to have protective effect in 3 studies, whereas 5 studies showed that the DD genotype is a risk factor for venous thromboembolism. In another 6 studies, no relationships were observed between the DD genotype and venous thromboembolism. It was reported that the diversity between the studies may be derived from ethnic heterogeneity, study design differences, patient features, and gene–gene and gene–environment interactions (Hsiao and Hsu, 2011). Poorgholi et al. (2013) did not detect any association between ACE I/D gene polymorphisms and coronary artery disease. Zhang et al. (2012) found that a D allele of ACE I/D polymorphism is a low-penetrance susceptibility marker of ischemic stroke in a meta-analysis of 50 case–control studies. Mehtap et al. (2012) did not report significant differences between the ACE I/D genotype and the development of vascular incidents in 64 ET and PV patients. In our study, we found similar results of the ACE II genotype and I allele frequency in groups of patients having myeloproliferative neoplasm with or without a history of vascular incident. Thus, we could not detect any relationship between this polymorphism and vascular incident risk in myeloproliferative diseases. In addition, a significantly higher frequency of the I allele was observed in the patients in the myeloproliferative disease group with a history of vascular incident than in the TG and CG, whereas similar results of the genotype and the allele frequency between the TG and the CG support that the I allele would be a risk factor for ET and PV, but would not be a risk for the development of thrombosis. In conclusion, the II genotype of the ACE I/D polymorphism and the I allele frequency were found to be signifi-
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cantly higher in the myeloproliferative neoplasia group consisting of ET and PV independent from vascular incident history. It was found that the II genotype and the I allele frequency are risk factors for ET and PV and that the DD genotype and the D allele frequency are associated with decreased risk of ET and PV. In addition, the ACE I/D gene polymorphism did not contribute to risk for thrombotic events. Future studies with larger patient groups will provide more accurate information for establishing a connection between ACE I/D gene polymorphism and myeloproliferative diseases and vascular events. Author Disclosure Statement
The authors declare no conflicts of interest. References
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Address correspondence to: Tahsin Yakut, MD, PhD Department of Medical Genetics School of Medicine Uludag University Go¨ru¨kle Bursa 16059 Turkey E-mail: [email protected]
