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Relationship between MTHFR gene polymorphisms (C677T and A1298C) and chronic lymphocytic leukemia in the Turkish population
Meta Gene 17 (2018) 232–236
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Relationship between MTHFR gene polymorphisms (C677T and A1298C) and chronic lymphocytic leukemia in the Turkish population Ahmet Umaya,
⁎,1
T
, Ramazan Bilginb, Ersin Akgöllüc,1, Emel Gürkand, Cem Kisd
a
Osmaniye Korkut Ata University, Department of Environmental Protection and Control Technology, Osmaniye, Turkey Department of Chemistry, Arts and Science Faculty, Çukurova University, Adana, Turkey c Çukurova University, Faculty of Medicine, Department of Gastroenterology, Adana, Turkey d Çukurova University, Faculty of Medicine, Department of Haematology, Adana, Turkey b
A R T I C LE I N FO
A B S T R A C T
Keywords: MTHFR Polymorphisms C677T A1298C CLL
Objective: Chronic lymphocytic leukemia (CLL) is a neoplastic disease that influences B cell lymphocytes. CLL is the most common kind of leukemia accounting for approximately 30% of all hematological malignancies. The functions of MTHFR enzyme are a critical juncture in the DNA synthesis, and in methylation of metabolic reactions. Two common genetic polymorphisms, C677T and A1298C, are associated with reduced enzyme activity. We evaluated the association between MTHFR gene polymorphisms and the CLL risk. Materials and methods: In the present study, MTHFR gene polymorphisms were investigated in a case-control study of 91 patients with CLL and 101 healthy control subjects using real time polymerase chain reaction (RTPCR) assay in the Turkish population. Results & conclusion: The patients carrying 1298C allele or 1298CC genotype had a higher risk of CLL (P = .04, P = .005, respectively). Moreover, the patients carrying 677TT genotype had a lower risk of CLL than the other patients carrying 677CC and 677CT genotypes in recessive model (P = .03). Additionally, the CA haplotype block showed a protective effect against the risk of CLL (the C677/A1298, P = .0013). In conclusion, our results demonstrate for the first time MTHFR gene C677T and A1298C polymorphisms, especially A1298C, have a major effect on the risk of CLL in the Turkish patients. We suggest that considering the first contradictory results in Caucasians, further additional studies are necessary to elucidate the relationship of these polymorphisms with the risk of CLL disease in Caucasian populations.
1. Introduction Chronic lymphocytic leukemia (CLL) is a neoplastic disease that influences B cell lymphocytes. B cell lymphocytes grow by an uncontrolled proliferation, and accumulate in the blood and bone marrow. Therefore, B cells dominate healthy blood cells result in failed homeostasis (Goldin et al., 2004). CLL is the most common kind of leukemia accounting for approximately 30% of the whole cases (Rudd et al., 2004). The United States Surveillance, Epidemiology, and End Results (SEER) Registry reported that the CLL incidence in the time 1996–2000 to be 3.7 per 100,000 with a median age 72 years in U.S. (Goldin et al., 2004). Even though older age, male gender, the white race, the history of hematologic malignancy of family have recognized as risk factors, the etiology of CLL is not yet known (Sgambati et al., 2001). Several studies reported that genetic predisposition to CLL has significantly recognized 3- to 7-fold highly risk in primary relatives of CLL cases
⁎
1
(Houlston et al., 2003). Folate metabolism plays a major role in carcinogenesis because of its association in DNA methylation and nucleotide synthesis. DNA methylation is extremely important for gene regulation and cellular differentiation (Choi and Mason, 2002). Abnormal genomic DNA methylation, especially hypomethylation, is related with the genesis of several cancers (Jones, 1986; Ehrlich, 2002). The methylenetetrahydrofolate reductase (MTHFR) is the most important enzyme for cellular DNA methylation. MTHFR catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Reduced MTHFR activity has an inhibitory influence on the 5-methyltetrahydrofolate pathway, and it causes increased 5,10-methylenetetrahydrofolate which if not reduced results in the transfer of a methyl group converting dUMP to dTMP during DNA synthesis. In contrast, if uracil synthesis is increased, uracil may be misincorporated attached to DNA, and leads to DNA double-strand breaks and chromosomal detriment (Blount et al.,
Corresponding author at: Osmaniye Korkut Ata University, Department of Environmental Protection and Control Technology, 80010 Osmaniye, Turkey. E-mail address: [email protected] (A. Umay). These authors contributed equally to this work.
https://doi.org/10.1016/j.mgene.2018.07.001 Received 2 March 2018; Received in revised form 14 June 2018; Accepted 2 July 2018 Available online 04 July 2018 2214-5400/ © 2018 Elsevier B.V. All rights reserved.
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heterozygote samples produce two melting peaks (For the C677T polymorphism, C allele at 63.0 °C and T allele at 54.5 °C; for the A1298C polymorphism, A allele at 64.5 °C and C allele at 59.0 °C).
1997; Skibola et al., 1999; Dianov et al., 1991). The some of the studies reported that folate deficient diets cause insufficient DNA repair capacity (Choi and Mason, 2002), elevated dUMP/dTMP ratios and DNA damage in humans (Blount et al., 1997), DNA hypomethylation (Jacob et al., 1998), and premutagenic apyrimidinic lesions (Pogribny et al., 1997). Two common genetic polymorphisms, C677T (rs1801133) and A1298C (rs1801131), have been described for MTHFR enzyme. The functions of MTHFR enzyme are a critical juncture in the DNA synthesis, and in methylation of metabolic reactions. Two variants of the MTHFR gene at the chromosome 1p36.3, C677T in exon 4 (Ala222Val) and A1298C in exon 7 (Glu429Ala), are associated with reduced enzyme activity (Van der Put et al., 1998; Lucock, 2000; Frosst et al., 1995). The aim of the current study, for the first time, was to analyze correlation between the MTHFR gene (A1298C and C677T) polymorphisms and the risk of chronic lymphocytic leukemia (CLL), folateadequate condition, in the Turkish population. To test the hypothesis that MTHFR gene polymorphisms were investigated in a case–control study of 91 patients with CLL and 101 healthy control subjects using real time polymerase chain reaction (RT-PCR) assay in the Turkish population.
2.3. Statistical analysis Data analysis was performed using the computer software IBM Statistical Package for Social Sciences (SPSS; SPSS, Inc., Chicago, IL, USA) for Windows (version 20). Continuous variables are presented as the mean ± SD (standard deviation) for normal distributions or median (min-max) for non-normal distributions and categorical variables are presented as (%) frequencies. Comparisons in the distributions of demographical characteristics between the patients with chronic lymphocytic leukemia and healthy control subjects were evaluated using the Student's t-test or Mann-Whitney U test for continuous variables depending on their Gaussian distribution, and chi-square test for categorical variables. The observed genotype frequencies were compared with expected values calculated from Hardy–Weinberg equilibrium theory. Significant variables after univariate regression analysis were entered into a stepwise logistic regression analysis to identify factors of CLL risk. Statistical analysis of genotypes, haplotype estimation and linkage disequilibrium (LD) were analyzed using the website for SNP Statistics: http://bioinfo.iconcologia.net/snpstats/start.htm. Logistic regression analysis with adjustment for age and sex was used to analysis the association of genotypes in inheritance models (co-dominant, dominant, recessive) in the case and control groups. Results are expressed as odds ratios with 95% confidence interval (CI). All tests were two-sided and P value < .05 was considered significant.
2. Materials and methods 2.1. Patients and methods This study was approved by the Committee for Ethics of Medical Experiment on Human Subjects, Çukurova University Faculty of Medicine in Adana in Turkey. CLL and control groups were recruited at Çukurova University Balcalı Hospital. Voluntariness of the individuals to the study was conditioned by an obtained written informed consent form regarding the use of their blood samples for research studies. In this study, Peripheral blood samples of 91 individuals with CLL and 101 healthy individuals are used. Peripheral blood samples were taken from patients and controls in the department of haematology between May 2013 to February 2015. Blood specimens were stored at +4 °C until analysis. Hemoglobin (HGB), hematocrit (HCT), white blood cell (WBC) and platelet (PLT) levels were evaluated with statistical analysis between two groups. Lactate dehydrogenase (LDH), vitamin B12, folate levels of CLL patients were evaluated with statistical analysis.
3. Results The clinical and demographic data of the case and control groups were shown in Table 1. All of individuals were Turkish and Caucasian in the present study. The most of the individuals were male (62.6%) and were 65 (median) years old for case group. On the other hand, most of healthy control subjects were female (55.4%) and were 50 (median) years old. As expected, there were significant differences between the characteristics of the patient and the control group including age, gender, and the levels of blood parameters WBC, HBG, HCT, PLT, ferritin and folate (Table 1). Additionally, the clinical symptom (lymphadenomegaly, hepatomegaly, and splenomegaly) rates of cases were shown in Table 1.
2.2. MTHFR gene C677T and A1298C genotype detection Genomic DNA was purified from peripheral whole blood using High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the producer protocol. Genotyping for C677T and A1298C at the MTHFR gene were performed by RT-PCR and high-resolution melting curve analysis on the Roche LightCycler 480 System according to the LightCycler 480 Genotyping Master protocol (Roche Diagnostics, Switzerland). For detection of the C677T (rs1801133) and A1298C (rs1801131) polymorphisms' gene loci were amplified by using primers; 5′- TGA CTG TCAT CCC TAT TGGC3′(forward) and 5′- CTT GCC CCA TCG TCC CCG GG-3′(reverse); 5′GCC TTT GGG GAG CTG AAG GA-3′(forward) and 5′- CCC CTG GCC TGA CCC CAG CT-3′(reverse), respectively. These primers were designed using the NCBI Primer-Blast Tool (http://www.ncbi.nlm.nih. gov/tools/primer-blast/). The genotyping technology is based on SimpleProbe design (TIB MOLBIOL, Berlin, Germany). Shortly, 5 μl of the DNA extract was mixed with 10 μl of LightCycler 480 Probe Master, 4 μl of ddH2O and 1 μl of SimpleProbe reagent for a 20 μl single reaction according to the TIBMOLBIOL protocol. The following cycling conditions were: denaturation at 95 °C (10 min), and then denaturation at 95 °C (5 s), annealing at 60 °C (10 s), and extension at 72 °C (15 s) for 45 cycles with final melting conditions at 95 °C (30 s) and 40 °C (2 min). Each allele has a melting peak at a specific temperature thereby
Table 1 The Clinical and demographic data of CLL patient group and control group.
Age⁎ Sex, (%) Female Male HGB⁎⁎ (g/dL) HCT⁎⁎ (%) WBC⁎ (103μL) PLT⁎ (103μL) Folate⁎ (ng/mL) Vit B12⁎ (pg/mL) Ferritin⁎ (ng/mL) LDH⁎ (U/L) Hepatomegaly, n (%) Splenomegaly, n (%) Lymphadenomegaly, n (%) a
CLL, n = 91
Control, n = 101
P-value
65 (42–91)
50 (18–78)
0.001a 0.012b
34 (% 37.4) 57 (% 62.6) 12.50 ± 1.95 37.6 ± 5.71 40.5 (8–391) 207 (23–782) 7.85 (1.50–19.0) 254 (90–1500) 56.30(6.80–1444) 178.5 (30–511) 18 (19,8) 27 (29.7) 28 (30.8)
56 (% 55.4) 45 (% 44.6) 13.43 ± 1.70 40.40 ± 4.97 8 (4.8–14.5) 249.50 (140–462) 8.26 (3.5–17.3) 286 (47–426) 32 (3.10–200.40) 185 (130–270) – – –
P value were calculated by Mann-Whitney test. P value were calculated by chi-square test. c P value were calculated by student t-test. ⁎ Data were shown as median (min-max). ⁎⁎ Data were shown as mean ± SD. b
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0.001c 0.001c 0.001a 0.001a 0.036a 0.89a 0.024a 0,33a
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Table 2 Alleles and genotypes frequency distribution of MTHFR polymorphisms among cases and controls as well as the association with the risk of chronic lymphocytic leukemia.a CLL n = 91, (%) For C677T, Allele frequency C T Co-dominant model CC CT TT Dominant model CC CT + TT Recessive model CC + CT TT For A1298C, Allele frequency A C Co-dominant model AA AC CC Dominant model AA AC + CC Recessive model AA + AC CC
a b
Controls n = 101, (%)
136 (% 74.7) 46 (% 25.3)
140 (% 69,3)
48 (% 52.7) 40 (% 44) 3 (% 3.3)
P-value
Genotypes
OR (95% CI) A1298C AA AC CC P-value C677T CC CT TT P-value
1.00 (Reference) 0.24a
0.76 (0.48–1.19)
51 (% 50.5) 38 (% 37.6) 12 (% 11.9)
0.35b 0.07b
1.00 (Reference) 1.42 (0.68–2.96) 0.27 (0.06–1.18)
48 (% 52.8) 43 (% 47.2)
51 (% 50.5) 50 (% 49.5)
0.77b
1.00 (Reference) 1.11 (0.55–2.21)
88 (% 96.7) 3 (% 3.3)
89 (% 88.1) 12 (% 11.9)
0.03
92 (% 50.5) 90 (% 49.5)
123 (% 60.9) 79 (% 39.1)
0.04a
23 (% 25.3) 46 (% 50.5) 22 (% 24.2)
35 (% 34.6) 53 (% 52.5) 13 (% 12.9)
0.09b 0.005b
23 (% 25.3) 68 (% 74.7)
35 (% 34.6) 66 (% 65.3)
0.01b
69 (% 75.8) 22 (% 24.2)
88 (% 87.1) 13 (% 12.9)
0.005b
62 (% 30.7)
Table 3 The relationship between MTHFR A1298 and C677T genotypes with blood parameters in patients with CLL.
b
LDH, (U/L) Median (min-max)
Vit B12 (pg/mL) Median (min-max)
Folate (ng/mL) Median (min-max)
168 (30–506) 179 (108–511) 178 (117–304) 0.54
229 (115–603) 245 (90–1500) 258 (176–985) 0.25
8.10 (2.20–13) 7.96 (1.50–13.30) 6.62 (3.30–19) 0.91
169 (117–439) 187 (30–511) 203 (143–330) 0.97
231 (164–1500) 206 (126–432) 297 (195–399) 0.90
8,12 (3.30–19) 7.41 (1,50–13) 5.62 (2.20–9) 0.37
P values were calculated by Kruskal Wallis test.
model (OR = 0.23; 95% 0.05–0.98, P = .03) (Table 2). Additionally, there was no a significant difference between the levels of folate, LDH, vitamin B12 of CLL patients and MTHFR polymorphisms (Table 3).
1.00 (Reference) 0.23 (0.05–0.98)
1.00 (Reference) 1.52 (1.02–2.28)
3.3. Linkage disequilibrium and haplotype analysis of MTHFR gene C677T and A1298C polymorphisms in case and control groups
1.00 (Reference) 1.95 (0.88–4.32) 6.16 (1.94–19.57)
Linkage disequilibrium (LD) was strong for C677T and A1298C polymorphisms (D′ = 0.94, P < .0001). When the haplotype analysis was performed, there were no significant differences between the case and control groups in terms of haplotype blocks, except for the CA haplotype block showed a protective effect against the risk of CLL (for the C677/ A1298, OR = 0.34; 95% 0.17–0.65, P = .0013) (Table 4).
1.00 (Reference) 2.54 (1.19–5.41) 1.00 (Reference) 4.09 (1.45–11.53)
4. Discussion
P value were calculated by chi-square test. Data were calculated by logistic regression analysis. Adjusted for age and
In the current study we investigated, for the first time, the effect of the MTHFR gene polymorphisms (C677T and A1298C) on the risk of CLL using control group with healthy persons in Turkish population who were Caucasians. Several important studies independently showed that the MTHFR gene polymorphisms were found to be associated with CLL, and decreased the activity of MTHFR enzyme in the different diseases including the cancers of cervical, gastric, esophageal, breast, and CML, AML, MM (Semenza et al., 2003; Campbell et al., 2002; Esteller et al., 1997; Moon et al., 2007). Rudd et al. (2004) reported that these polymorphisms of MTHFR gene do not significantly contribute to genetic susceptibility to CLL. While the patients carrying 677TT or 677CT genotypes, respectively, showed to have 30% and 60% of MTHFR enzyme activity, the patients carrying 1298CC had a lower MTHFR enzyme activity about 60% when compared with those carrying 1298AA genotype (Van der Put et al., 1998; Robien and Ulrich, 2003). Nonetheless, few studies reported that the association between these polymorphisms and CLL is controversial (Rudd et al., 2004; Hussain et al., 2012). Therefore, the MTHFR gene polymorphisms were selected as the candidate polymorphisms due to their crucial role in folate metabolism and DNA synthesis. In the current study, the allele frequencies of MTHFR
sex.
3.1. The allele and genotype frequencies of MTHFR gene C677T and A1298C polymorphisms The overall allelic frequencies of MTHFR gene C677T polymorphism were 71.9% and 28.1% for C and T, respectively. On the other hand, the overall allelic frequencies for A1298C polymorphism were 56% and 44% for A and C, respectively. There was a significant relationship between the control and the case groups for A1298C allele frequencies (P = .04), but it was not significant for C677T (Table 2). Additionally, the genotype frequencies of A1298C and C677T polymorphisms were not significantly different between case and control groups (P = .09 and P = .08, respectively). Similarly, the genotype frequencies were not significant for these polymorphisms according to gender (P = .98, data not shown). Additionally, the patients' and controls' frequencies were also in Hardy–Weinberg equilibrium and had no selection bias (P = 1.0 and P = .4, respectively). 3.2. The relationship between MTHFR genotypes and the risk of CLL
Table 4 Frequency distribution of haplotypes of MTHFR gene C677T and A1298C polymorphisms in cases and controls.
To identify whether there was a significant difference increased risk of CLL in terms of the MTHFR gene C677T and A1298C genotypes, we performed logistic regression analysis with adjustment for age and sex between the groups (Table 2). For the A1298C polymorphism, the patients carrying C allele or CC genotype had a higher risk of CLL when compared with those carrying A allele or AA genotype, and it was significant (P = .04 and P = .005, respectively) (Table 2). For the C677T polymorphism, there was no a significant difference for the allele frequencies. The patients carrying TT genotype had a lower risk of CLL than the other patients carrying CC and CT genotypes in recessive
A1298C
C A A C
234
C677T
C C T T
Frequency CLL
Control
0.4945 0.2527 0.2527 0
0.3769 0.3161 0.2928 0.0141
OR (95% CI)
P-value
1 0.34 (0.17– - 0.65) 0.53 (0.28– - 1.0) 0.87 (0.0– - 9.418)
–— 0.0013 0.051 0.98
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also adequate, 677TT genotype decreases the risk of CLL in recessive genetic model (P = .03), and our finding is the same as the results of Ma et al. (1997). Additionally, when the folate status inadequate, 677TT genotype is associated with increased risk of colorectal cancer (Ulrich et al., 2000). Furthermore, the having 677TT genotype was significantly associated with a lower folate levels in the neural-tube defects (Van der Put et al., 1998). In contrast to these reports, Friso et al. (2002) observed higher red blood cell folate levels in individuals carrying the mutant 677TT genotype. MTHFR enzyme plays a crucial role in folate metabolism, DNA synthesis, DNA methylation, and cancer susceptibility (Deligezer et al., 2006). Therefore, the investigation of MTHFR gene polymorphisms, which effect MTHFR enzyme activity, in different cancer types is an important subject. Additionally, the association between the folate status and MTHFR gene polymorphisms of individuals should be explored for cancer susceptibility. Folate inadequate status decreases deoxythymidylate synthesis from deoxyuridylate result in nucleotide imbalance, and increasing uracil misincorporation into DNA (Choi and Mason, 2002). Thereby, it can cause DNA strand-breaks, enhance cellular transformation as well as increase the risk of cancer. There are various limitations in the current study: Firstly, the individuals in the current study were selected at Çukurova University Balcalı Hospital in Adana from South Turkey, the study may, hence, not be representative of all CLL patients in the overall population. Secondly, this study is limited by the small number of patients, but it is noteworthy to mention that the CLL incidence in the time 1996–2000 to be 3.7 per 100,000 U.S. There is no study about the incidence of CLL in Turkey. We therefore think that the sample size may be sufficient. Thirdly, the current study focused on two polymorphisms on single gene without taking into consideration the gene–gene interactions, gene–environment and any interactions between different locuses on the same gene, which may affect the MTHFR enzyme activity.
polymorphisms in control group were found 30.7% and 39.1% for 677 T and 1298C alleles, respectively. According to the previous results, the frequencies of 677 T and 1298C alleles among the populations were distributed as 29% and 40% in British, 31% and 37% in German, 37% and 30% in Canadian, 45% and 30% in Italian, 35% and 31% in Slovakian, 40% and 23% in Korean, 44% and 23% in Brazilian, respectively (Skibola et al., 1999; Schnakenberg et al., 2005; Krajinovic et al., 2004; Gemmati et al., 2004; Petra et al., 2007; Hur et al., 2006; Franco et al., 2001). While the allele frequencies reported in some of studies in the Caucasian populations are almost in agreement with our results (Skibola et al., 1999; Schnakenberg et al., 2005), others studies' results were not consistent when compared with our results (Krajinovic et al., 2004; Gemmati et al., 2004; Petra et al., 2007). Although acute lymphoblastic leukemia (ALL) was associated with the C677T polymorphism in a few of these studies (Skibola et al., 1999; Krajinovic et al., 2004; Gemmati et al., 2004; Petra et al., 2007; Franco et al., 2001), A1298C polymorphism was not found to be relationship with ALL(Schnakenberg et al., 2005; Gemmati et al., 2004; Petra et al., 2007; Franco et al., 2001). In these studies, the T allele of C677T polymorphism was a predominantly associated with decreased risk of ALL. Additionally, there are a few studies concerned with these polymorphisms in chronic myelogenous leukemia (CML), and these studies' results are controversial (Moon et al., 2007; Hussain et al., 2012; Kim et al., 2009). It is noteworthy to mention that while this manuscript is being prepared, just three studies have examined both the MTHFR gene C677T and MTHFR gene A1298C variants in relation to CLL in the Caucasian populations, so far (Rudd et al., 2004; Nückel et al., 2004; Gra et al., 2008). None of these studies found to be a significant association for these polymorphisms in any genetic model. These studies' results were not consistent with our results. According to our results, the C allele and CC genotype of A1298C polymorphism significantly increased the risk of CLL (OR = 1.52, P = .04; OR = 6.16, P = .005, respectively), but C677T allele and genotypes, except for recessive model, were not significant for the risk of CLL. Moreover, A1298C polymorphism was found to be associated with CLL in dominant and recessive genetic models (P = .01, P = .005). In the other study reported by Hussain et al. (2012). 677TT genotype increased the risk of CLL in the Indian population. This observation reported was not same trend with our results. As per our finding, 677TT genotype decreases the risk of CLL in recessive genetic model (P = .03). Moreover, we also performed haplotype analysis for association between the CLL risks with these polymorphisms. In the current study, the wild-type CA haplotype block showed a significant protective effect against the risk of CLL (For C677/A1298, OR = 0.34; 95% 0.17–0.65, P = .0013). In contrast to our data, Rudd et al. (2004) reported no association was found in the distribution of haplotypes between case and control groups. In addition to this result, Nückel et al. (2004) reported that MTHFR haplotype (677CC plus 1298CC or A/C) was the best independent prognostic factor for progression-free survival, although allele and genotype distributions for both polymorphisms were not significantly different. This result is nearly similar to result presented in our study. Different results observed in these studies might be owing to ethnic and geographic diversity. There is no another study reported the association of these polymorphisms with CLL to compare with our results. Furthermore, we performed analysis the association of these polymorphisms with folate status in CLL patients, but not significant. There is no study showed the relationship folate status with both polymorphisms in CLL. However, there are some data reported by different studies. Castro et al. (2004) reported that the mutant genotype 1298CC, independently of folate level, and individuals carrying the mutant 677TT genotype with accompanying folate insufficient levels, might be a risk factor for DNA hypomethylation status causing different diseases. Moreover, Ma et al. (1997) reported that the persons having the 677TT genotype with adequate folate status is related to a lower risk of developing colorectal cancer. Since folate levels of our study patients are
5. Conclusion In conclusion, our results demonstrate for the first time MTHFR gene C677T and A1298C polymorphisms, especially A1298C, have a major effect on the risk of CLL with folate-adequate in the Turkish patients. According to our current knowledge, considering the first contradictory results in Caucasians, further additional studies are necessary to elucidate the relationship of these polymorphisms with the risk of CLL disease in Caucasian populations. Competing interests The authors declare that no conflict of interest. Authors' contributions The contributions of all the authors on the paper are equal. All authors are in agreement with the contents of the article. Funding This study was supported by ÇUKUROVA UNIVERSITY SCIENTIFIC RESEARCH PROJECTS UNIT (FEF2013D17). Acknowledgements The authors are grateful to all patients who contributed to this study. References Blount, B.C., Mack, M.M., Wehr, C.M., MacGregor, J.T., Hiatt, R.A., Wang, G., 1997. Wickramasinghe SN, Everson RB, Ames BN. Folate deficiency causes uracil
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Relationship between MTHFR gene polymorphisms (C677T and A1298C) and chronic lymphocytic leukemia in the Turkish population Ahmet Umaya,
⁎,1
T
, Ramazan Bilginb, Ersin Akgöllüc,1, Emel Gürkand, Cem Kisd
a
Osmaniye Korkut Ata University, Department of Environmental Protection and Control Technology, Osmaniye, Turkey Department of Chemistry, Arts and Science Faculty, Çukurova University, Adana, Turkey c Çukurova University, Faculty of Medicine, Department of Gastroenterology, Adana, Turkey d Çukurova University, Faculty of Medicine, Department of Haematology, Adana, Turkey b
A R T I C LE I N FO
A B S T R A C T
Keywords: MTHFR Polymorphisms C677T A1298C CLL
Objective: Chronic lymphocytic leukemia (CLL) is a neoplastic disease that influences B cell lymphocytes. CLL is the most common kind of leukemia accounting for approximately 30% of all hematological malignancies. The functions of MTHFR enzyme are a critical juncture in the DNA synthesis, and in methylation of metabolic reactions. Two common genetic polymorphisms, C677T and A1298C, are associated with reduced enzyme activity. We evaluated the association between MTHFR gene polymorphisms and the CLL risk. Materials and methods: In the present study, MTHFR gene polymorphisms were investigated in a case-control study of 91 patients with CLL and 101 healthy control subjects using real time polymerase chain reaction (RTPCR) assay in the Turkish population. Results & conclusion: The patients carrying 1298C allele or 1298CC genotype had a higher risk of CLL (P = .04, P = .005, respectively). Moreover, the patients carrying 677TT genotype had a lower risk of CLL than the other patients carrying 677CC and 677CT genotypes in recessive model (P = .03). Additionally, the CA haplotype block showed a protective effect against the risk of CLL (the C677/A1298, P = .0013). In conclusion, our results demonstrate for the first time MTHFR gene C677T and A1298C polymorphisms, especially A1298C, have a major effect on the risk of CLL in the Turkish patients. We suggest that considering the first contradictory results in Caucasians, further additional studies are necessary to elucidate the relationship of these polymorphisms with the risk of CLL disease in Caucasian populations.
1. Introduction Chronic lymphocytic leukemia (CLL) is a neoplastic disease that influences B cell lymphocytes. B cell lymphocytes grow by an uncontrolled proliferation, and accumulate in the blood and bone marrow. Therefore, B cells dominate healthy blood cells result in failed homeostasis (Goldin et al., 2004). CLL is the most common kind of leukemia accounting for approximately 30% of the whole cases (Rudd et al., 2004). The United States Surveillance, Epidemiology, and End Results (SEER) Registry reported that the CLL incidence in the time 1996–2000 to be 3.7 per 100,000 with a median age 72 years in U.S. (Goldin et al., 2004). Even though older age, male gender, the white race, the history of hematologic malignancy of family have recognized as risk factors, the etiology of CLL is not yet known (Sgambati et al., 2001). Several studies reported that genetic predisposition to CLL has significantly recognized 3- to 7-fold highly risk in primary relatives of CLL cases
⁎
1
(Houlston et al., 2003). Folate metabolism plays a major role in carcinogenesis because of its association in DNA methylation and nucleotide synthesis. DNA methylation is extremely important for gene regulation and cellular differentiation (Choi and Mason, 2002). Abnormal genomic DNA methylation, especially hypomethylation, is related with the genesis of several cancers (Jones, 1986; Ehrlich, 2002). The methylenetetrahydrofolate reductase (MTHFR) is the most important enzyme for cellular DNA methylation. MTHFR catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Reduced MTHFR activity has an inhibitory influence on the 5-methyltetrahydrofolate pathway, and it causes increased 5,10-methylenetetrahydrofolate which if not reduced results in the transfer of a methyl group converting dUMP to dTMP during DNA synthesis. In contrast, if uracil synthesis is increased, uracil may be misincorporated attached to DNA, and leads to DNA double-strand breaks and chromosomal detriment (Blount et al.,
Corresponding author at: Osmaniye Korkut Ata University, Department of Environmental Protection and Control Technology, 80010 Osmaniye, Turkey. E-mail address: [email protected] (A. Umay). These authors contributed equally to this work.
https://doi.org/10.1016/j.mgene.2018.07.001 Received 2 March 2018; Received in revised form 14 June 2018; Accepted 2 July 2018 Available online 04 July 2018 2214-5400/ © 2018 Elsevier B.V. All rights reserved.
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heterozygote samples produce two melting peaks (For the C677T polymorphism, C allele at 63.0 °C and T allele at 54.5 °C; for the A1298C polymorphism, A allele at 64.5 °C and C allele at 59.0 °C).
1997; Skibola et al., 1999; Dianov et al., 1991). The some of the studies reported that folate deficient diets cause insufficient DNA repair capacity (Choi and Mason, 2002), elevated dUMP/dTMP ratios and DNA damage in humans (Blount et al., 1997), DNA hypomethylation (Jacob et al., 1998), and premutagenic apyrimidinic lesions (Pogribny et al., 1997). Two common genetic polymorphisms, C677T (rs1801133) and A1298C (rs1801131), have been described for MTHFR enzyme. The functions of MTHFR enzyme are a critical juncture in the DNA synthesis, and in methylation of metabolic reactions. Two variants of the MTHFR gene at the chromosome 1p36.3, C677T in exon 4 (Ala222Val) and A1298C in exon 7 (Glu429Ala), are associated with reduced enzyme activity (Van der Put et al., 1998; Lucock, 2000; Frosst et al., 1995). The aim of the current study, for the first time, was to analyze correlation between the MTHFR gene (A1298C and C677T) polymorphisms and the risk of chronic lymphocytic leukemia (CLL), folateadequate condition, in the Turkish population. To test the hypothesis that MTHFR gene polymorphisms were investigated in a case–control study of 91 patients with CLL and 101 healthy control subjects using real time polymerase chain reaction (RT-PCR) assay in the Turkish population.
2.3. Statistical analysis Data analysis was performed using the computer software IBM Statistical Package for Social Sciences (SPSS; SPSS, Inc., Chicago, IL, USA) for Windows (version 20). Continuous variables are presented as the mean ± SD (standard deviation) for normal distributions or median (min-max) for non-normal distributions and categorical variables are presented as (%) frequencies. Comparisons in the distributions of demographical characteristics between the patients with chronic lymphocytic leukemia and healthy control subjects were evaluated using the Student's t-test or Mann-Whitney U test for continuous variables depending on their Gaussian distribution, and chi-square test for categorical variables. The observed genotype frequencies were compared with expected values calculated from Hardy–Weinberg equilibrium theory. Significant variables after univariate regression analysis were entered into a stepwise logistic regression analysis to identify factors of CLL risk. Statistical analysis of genotypes, haplotype estimation and linkage disequilibrium (LD) were analyzed using the website for SNP Statistics: http://bioinfo.iconcologia.net/snpstats/start.htm. Logistic regression analysis with adjustment for age and sex was used to analysis the association of genotypes in inheritance models (co-dominant, dominant, recessive) in the case and control groups. Results are expressed as odds ratios with 95% confidence interval (CI). All tests were two-sided and P value < .05 was considered significant.
2. Materials and methods 2.1. Patients and methods This study was approved by the Committee for Ethics of Medical Experiment on Human Subjects, Çukurova University Faculty of Medicine in Adana in Turkey. CLL and control groups were recruited at Çukurova University Balcalı Hospital. Voluntariness of the individuals to the study was conditioned by an obtained written informed consent form regarding the use of their blood samples for research studies. In this study, Peripheral blood samples of 91 individuals with CLL and 101 healthy individuals are used. Peripheral blood samples were taken from patients and controls in the department of haematology between May 2013 to February 2015. Blood specimens were stored at +4 °C until analysis. Hemoglobin (HGB), hematocrit (HCT), white blood cell (WBC) and platelet (PLT) levels were evaluated with statistical analysis between two groups. Lactate dehydrogenase (LDH), vitamin B12, folate levels of CLL patients were evaluated with statistical analysis.
3. Results The clinical and demographic data of the case and control groups were shown in Table 1. All of individuals were Turkish and Caucasian in the present study. The most of the individuals were male (62.6%) and were 65 (median) years old for case group. On the other hand, most of healthy control subjects were female (55.4%) and were 50 (median) years old. As expected, there were significant differences between the characteristics of the patient and the control group including age, gender, and the levels of blood parameters WBC, HBG, HCT, PLT, ferritin and folate (Table 1). Additionally, the clinical symptom (lymphadenomegaly, hepatomegaly, and splenomegaly) rates of cases were shown in Table 1.
2.2. MTHFR gene C677T and A1298C genotype detection Genomic DNA was purified from peripheral whole blood using High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the producer protocol. Genotyping for C677T and A1298C at the MTHFR gene were performed by RT-PCR and high-resolution melting curve analysis on the Roche LightCycler 480 System according to the LightCycler 480 Genotyping Master protocol (Roche Diagnostics, Switzerland). For detection of the C677T (rs1801133) and A1298C (rs1801131) polymorphisms' gene loci were amplified by using primers; 5′- TGA CTG TCAT CCC TAT TGGC3′(forward) and 5′- CTT GCC CCA TCG TCC CCG GG-3′(reverse); 5′GCC TTT GGG GAG CTG AAG GA-3′(forward) and 5′- CCC CTG GCC TGA CCC CAG CT-3′(reverse), respectively. These primers were designed using the NCBI Primer-Blast Tool (http://www.ncbi.nlm.nih. gov/tools/primer-blast/). The genotyping technology is based on SimpleProbe design (TIB MOLBIOL, Berlin, Germany). Shortly, 5 μl of the DNA extract was mixed with 10 μl of LightCycler 480 Probe Master, 4 μl of ddH2O and 1 μl of SimpleProbe reagent for a 20 μl single reaction according to the TIBMOLBIOL protocol. The following cycling conditions were: denaturation at 95 °C (10 min), and then denaturation at 95 °C (5 s), annealing at 60 °C (10 s), and extension at 72 °C (15 s) for 45 cycles with final melting conditions at 95 °C (30 s) and 40 °C (2 min). Each allele has a melting peak at a specific temperature thereby
Table 1 The Clinical and demographic data of CLL patient group and control group.
Age⁎ Sex, (%) Female Male HGB⁎⁎ (g/dL) HCT⁎⁎ (%) WBC⁎ (103μL) PLT⁎ (103μL) Folate⁎ (ng/mL) Vit B12⁎ (pg/mL) Ferritin⁎ (ng/mL) LDH⁎ (U/L) Hepatomegaly, n (%) Splenomegaly, n (%) Lymphadenomegaly, n (%) a
CLL, n = 91
Control, n = 101
P-value
65 (42–91)
50 (18–78)
0.001a 0.012b
34 (% 37.4) 57 (% 62.6) 12.50 ± 1.95 37.6 ± 5.71 40.5 (8–391) 207 (23–782) 7.85 (1.50–19.0) 254 (90–1500) 56.30(6.80–1444) 178.5 (30–511) 18 (19,8) 27 (29.7) 28 (30.8)
56 (% 55.4) 45 (% 44.6) 13.43 ± 1.70 40.40 ± 4.97 8 (4.8–14.5) 249.50 (140–462) 8.26 (3.5–17.3) 286 (47–426) 32 (3.10–200.40) 185 (130–270) – – –
P value were calculated by Mann-Whitney test. P value were calculated by chi-square test. c P value were calculated by student t-test. ⁎ Data were shown as median (min-max). ⁎⁎ Data were shown as mean ± SD. b
233
0.001c 0.001c 0.001a 0.001a 0.036a 0.89a 0.024a 0,33a
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Table 2 Alleles and genotypes frequency distribution of MTHFR polymorphisms among cases and controls as well as the association with the risk of chronic lymphocytic leukemia.a CLL n = 91, (%) For C677T, Allele frequency C T Co-dominant model CC CT TT Dominant model CC CT + TT Recessive model CC + CT TT For A1298C, Allele frequency A C Co-dominant model AA AC CC Dominant model AA AC + CC Recessive model AA + AC CC
a b
Controls n = 101, (%)
136 (% 74.7) 46 (% 25.3)
140 (% 69,3)
48 (% 52.7) 40 (% 44) 3 (% 3.3)
P-value
Genotypes
OR (95% CI) A1298C AA AC CC P-value C677T CC CT TT P-value
1.00 (Reference) 0.24a
0.76 (0.48–1.19)
51 (% 50.5) 38 (% 37.6) 12 (% 11.9)
0.35b 0.07b
1.00 (Reference) 1.42 (0.68–2.96) 0.27 (0.06–1.18)
48 (% 52.8) 43 (% 47.2)
51 (% 50.5) 50 (% 49.5)
0.77b
1.00 (Reference) 1.11 (0.55–2.21)
88 (% 96.7) 3 (% 3.3)
89 (% 88.1) 12 (% 11.9)
0.03
92 (% 50.5) 90 (% 49.5)
123 (% 60.9) 79 (% 39.1)
0.04a
23 (% 25.3) 46 (% 50.5) 22 (% 24.2)
35 (% 34.6) 53 (% 52.5) 13 (% 12.9)
0.09b 0.005b
23 (% 25.3) 68 (% 74.7)
35 (% 34.6) 66 (% 65.3)
0.01b
69 (% 75.8) 22 (% 24.2)
88 (% 87.1) 13 (% 12.9)
0.005b
62 (% 30.7)
Table 3 The relationship between MTHFR A1298 and C677T genotypes with blood parameters in patients with CLL.
b
LDH, (U/L) Median (min-max)
Vit B12 (pg/mL) Median (min-max)
Folate (ng/mL) Median (min-max)
168 (30–506) 179 (108–511) 178 (117–304) 0.54
229 (115–603) 245 (90–1500) 258 (176–985) 0.25
8.10 (2.20–13) 7.96 (1.50–13.30) 6.62 (3.30–19) 0.91
169 (117–439) 187 (30–511) 203 (143–330) 0.97
231 (164–1500) 206 (126–432) 297 (195–399) 0.90
8,12 (3.30–19) 7.41 (1,50–13) 5.62 (2.20–9) 0.37
P values were calculated by Kruskal Wallis test.
model (OR = 0.23; 95% 0.05–0.98, P = .03) (Table 2). Additionally, there was no a significant difference between the levels of folate, LDH, vitamin B12 of CLL patients and MTHFR polymorphisms (Table 3).
1.00 (Reference) 0.23 (0.05–0.98)
1.00 (Reference) 1.52 (1.02–2.28)
3.3. Linkage disequilibrium and haplotype analysis of MTHFR gene C677T and A1298C polymorphisms in case and control groups
1.00 (Reference) 1.95 (0.88–4.32) 6.16 (1.94–19.57)
Linkage disequilibrium (LD) was strong for C677T and A1298C polymorphisms (D′ = 0.94, P < .0001). When the haplotype analysis was performed, there were no significant differences between the case and control groups in terms of haplotype blocks, except for the CA haplotype block showed a protective effect against the risk of CLL (for the C677/ A1298, OR = 0.34; 95% 0.17–0.65, P = .0013) (Table 4).
1.00 (Reference) 2.54 (1.19–5.41) 1.00 (Reference) 4.09 (1.45–11.53)
4. Discussion
P value were calculated by chi-square test. Data were calculated by logistic regression analysis. Adjusted for age and
In the current study we investigated, for the first time, the effect of the MTHFR gene polymorphisms (C677T and A1298C) on the risk of CLL using control group with healthy persons in Turkish population who were Caucasians. Several important studies independently showed that the MTHFR gene polymorphisms were found to be associated with CLL, and decreased the activity of MTHFR enzyme in the different diseases including the cancers of cervical, gastric, esophageal, breast, and CML, AML, MM (Semenza et al., 2003; Campbell et al., 2002; Esteller et al., 1997; Moon et al., 2007). Rudd et al. (2004) reported that these polymorphisms of MTHFR gene do not significantly contribute to genetic susceptibility to CLL. While the patients carrying 677TT or 677CT genotypes, respectively, showed to have 30% and 60% of MTHFR enzyme activity, the patients carrying 1298CC had a lower MTHFR enzyme activity about 60% when compared with those carrying 1298AA genotype (Van der Put et al., 1998; Robien and Ulrich, 2003). Nonetheless, few studies reported that the association between these polymorphisms and CLL is controversial (Rudd et al., 2004; Hussain et al., 2012). Therefore, the MTHFR gene polymorphisms were selected as the candidate polymorphisms due to their crucial role in folate metabolism and DNA synthesis. In the current study, the allele frequencies of MTHFR
sex.
3.1. The allele and genotype frequencies of MTHFR gene C677T and A1298C polymorphisms The overall allelic frequencies of MTHFR gene C677T polymorphism were 71.9% and 28.1% for C and T, respectively. On the other hand, the overall allelic frequencies for A1298C polymorphism were 56% and 44% for A and C, respectively. There was a significant relationship between the control and the case groups for A1298C allele frequencies (P = .04), but it was not significant for C677T (Table 2). Additionally, the genotype frequencies of A1298C and C677T polymorphisms were not significantly different between case and control groups (P = .09 and P = .08, respectively). Similarly, the genotype frequencies were not significant for these polymorphisms according to gender (P = .98, data not shown). Additionally, the patients' and controls' frequencies were also in Hardy–Weinberg equilibrium and had no selection bias (P = 1.0 and P = .4, respectively). 3.2. The relationship between MTHFR genotypes and the risk of CLL
Table 4 Frequency distribution of haplotypes of MTHFR gene C677T and A1298C polymorphisms in cases and controls.
To identify whether there was a significant difference increased risk of CLL in terms of the MTHFR gene C677T and A1298C genotypes, we performed logistic regression analysis with adjustment for age and sex between the groups (Table 2). For the A1298C polymorphism, the patients carrying C allele or CC genotype had a higher risk of CLL when compared with those carrying A allele or AA genotype, and it was significant (P = .04 and P = .005, respectively) (Table 2). For the C677T polymorphism, there was no a significant difference for the allele frequencies. The patients carrying TT genotype had a lower risk of CLL than the other patients carrying CC and CT genotypes in recessive
A1298C
C A A C
234
C677T
C C T T
Frequency CLL
Control
0.4945 0.2527 0.2527 0
0.3769 0.3161 0.2928 0.0141
OR (95% CI)
P-value
1 0.34 (0.17– - 0.65) 0.53 (0.28– - 1.0) 0.87 (0.0– - 9.418)
–— 0.0013 0.051 0.98
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also adequate, 677TT genotype decreases the risk of CLL in recessive genetic model (P = .03), and our finding is the same as the results of Ma et al. (1997). Additionally, when the folate status inadequate, 677TT genotype is associated with increased risk of colorectal cancer (Ulrich et al., 2000). Furthermore, the having 677TT genotype was significantly associated with a lower folate levels in the neural-tube defects (Van der Put et al., 1998). In contrast to these reports, Friso et al. (2002) observed higher red blood cell folate levels in individuals carrying the mutant 677TT genotype. MTHFR enzyme plays a crucial role in folate metabolism, DNA synthesis, DNA methylation, and cancer susceptibility (Deligezer et al., 2006). Therefore, the investigation of MTHFR gene polymorphisms, which effect MTHFR enzyme activity, in different cancer types is an important subject. Additionally, the association between the folate status and MTHFR gene polymorphisms of individuals should be explored for cancer susceptibility. Folate inadequate status decreases deoxythymidylate synthesis from deoxyuridylate result in nucleotide imbalance, and increasing uracil misincorporation into DNA (Choi and Mason, 2002). Thereby, it can cause DNA strand-breaks, enhance cellular transformation as well as increase the risk of cancer. There are various limitations in the current study: Firstly, the individuals in the current study were selected at Çukurova University Balcalı Hospital in Adana from South Turkey, the study may, hence, not be representative of all CLL patients in the overall population. Secondly, this study is limited by the small number of patients, but it is noteworthy to mention that the CLL incidence in the time 1996–2000 to be 3.7 per 100,000 U.S. There is no study about the incidence of CLL in Turkey. We therefore think that the sample size may be sufficient. Thirdly, the current study focused on two polymorphisms on single gene without taking into consideration the gene–gene interactions, gene–environment and any interactions between different locuses on the same gene, which may affect the MTHFR enzyme activity.
polymorphisms in control group were found 30.7% and 39.1% for 677 T and 1298C alleles, respectively. According to the previous results, the frequencies of 677 T and 1298C alleles among the populations were distributed as 29% and 40% in British, 31% and 37% in German, 37% and 30% in Canadian, 45% and 30% in Italian, 35% and 31% in Slovakian, 40% and 23% in Korean, 44% and 23% in Brazilian, respectively (Skibola et al., 1999; Schnakenberg et al., 2005; Krajinovic et al., 2004; Gemmati et al., 2004; Petra et al., 2007; Hur et al., 2006; Franco et al., 2001). While the allele frequencies reported in some of studies in the Caucasian populations are almost in agreement with our results (Skibola et al., 1999; Schnakenberg et al., 2005), others studies' results were not consistent when compared with our results (Krajinovic et al., 2004; Gemmati et al., 2004; Petra et al., 2007). Although acute lymphoblastic leukemia (ALL) was associated with the C677T polymorphism in a few of these studies (Skibola et al., 1999; Krajinovic et al., 2004; Gemmati et al., 2004; Petra et al., 2007; Franco et al., 2001), A1298C polymorphism was not found to be relationship with ALL(Schnakenberg et al., 2005; Gemmati et al., 2004; Petra et al., 2007; Franco et al., 2001). In these studies, the T allele of C677T polymorphism was a predominantly associated with decreased risk of ALL. Additionally, there are a few studies concerned with these polymorphisms in chronic myelogenous leukemia (CML), and these studies' results are controversial (Moon et al., 2007; Hussain et al., 2012; Kim et al., 2009). It is noteworthy to mention that while this manuscript is being prepared, just three studies have examined both the MTHFR gene C677T and MTHFR gene A1298C variants in relation to CLL in the Caucasian populations, so far (Rudd et al., 2004; Nückel et al., 2004; Gra et al., 2008). None of these studies found to be a significant association for these polymorphisms in any genetic model. These studies' results were not consistent with our results. According to our results, the C allele and CC genotype of A1298C polymorphism significantly increased the risk of CLL (OR = 1.52, P = .04; OR = 6.16, P = .005, respectively), but C677T allele and genotypes, except for recessive model, were not significant for the risk of CLL. Moreover, A1298C polymorphism was found to be associated with CLL in dominant and recessive genetic models (P = .01, P = .005). In the other study reported by Hussain et al. (2012). 677TT genotype increased the risk of CLL in the Indian population. This observation reported was not same trend with our results. As per our finding, 677TT genotype decreases the risk of CLL in recessive genetic model (P = .03). Moreover, we also performed haplotype analysis for association between the CLL risks with these polymorphisms. In the current study, the wild-type CA haplotype block showed a significant protective effect against the risk of CLL (For C677/A1298, OR = 0.34; 95% 0.17–0.65, P = .0013). In contrast to our data, Rudd et al. (2004) reported no association was found in the distribution of haplotypes between case and control groups. In addition to this result, Nückel et al. (2004) reported that MTHFR haplotype (677CC plus 1298CC or A/C) was the best independent prognostic factor for progression-free survival, although allele and genotype distributions for both polymorphisms were not significantly different. This result is nearly similar to result presented in our study. Different results observed in these studies might be owing to ethnic and geographic diversity. There is no another study reported the association of these polymorphisms with CLL to compare with our results. Furthermore, we performed analysis the association of these polymorphisms with folate status in CLL patients, but not significant. There is no study showed the relationship folate status with both polymorphisms in CLL. However, there are some data reported by different studies. Castro et al. (2004) reported that the mutant genotype 1298CC, independently of folate level, and individuals carrying the mutant 677TT genotype with accompanying folate insufficient levels, might be a risk factor for DNA hypomethylation status causing different diseases. Moreover, Ma et al. (1997) reported that the persons having the 677TT genotype with adequate folate status is related to a lower risk of developing colorectal cancer. Since folate levels of our study patients are
5. Conclusion In conclusion, our results demonstrate for the first time MTHFR gene C677T and A1298C polymorphisms, especially A1298C, have a major effect on the risk of CLL with folate-adequate in the Turkish patients. According to our current knowledge, considering the first contradictory results in Caucasians, further additional studies are necessary to elucidate the relationship of these polymorphisms with the risk of CLL disease in Caucasian populations. Competing interests The authors declare that no conflict of interest. Authors' contributions The contributions of all the authors on the paper are equal. All authors are in agreement with the contents of the article. Funding This study was supported by ÇUKUROVA UNIVERSITY SCIENTIFIC RESEARCH PROJECTS UNIT (FEF2013D17). Acknowledgements The authors are grateful to all patients who contributed to this study. References Blount, B.C., Mack, M.M., Wehr, C.M., MacGregor, J.T., Hiatt, R.A., Wang, G., 1997. Wickramasinghe SN, Everson RB, Ames BN. Folate deficiency causes uracil
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