Ragip Çam

Dihydrofolate reductase (DHRF) 19-bp intron-1 deletion and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms in breast cancer

The causes of breast cancer (BC) are not clearly known, although different genetic and environmental factors play role in its development. Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate-mediated onecarbon metabolism, regulating the DNA methylation and synthesis. Reduced MTHFR activity is associated with genomic DNA hypomethylation. MTHFR is suspected to play a role in the etiology of cancer, via its effects on DNA methylation and nucleotide synthesis [1]. The C to T substitution at nucleotide 677 of MTHFR gene converts an alanine to a valine is associated with reduced enzyme activity and causes increased plasma homocysteine levels. Associations of MTHFR C677T polymorphism with BC have been investigated [1–4]. Dihydrofolate reductase (DHFR) is also important folate metabolizing enzyme that is essential for DNA synthesis and homocysteine remethylation. Recently, Johnson et al. described a 19-bp deletion polymorphism in intron-1 of DHFR that might affect gene expression [5]. More recently, Gellekink et al. reported that 19-bp del/del genotype was associated with a lower plasma homocysteine level and suggested that 19-bp deletion might increase DHFR expression thereby facilitating homocysteine remethylation [6]. The aim of the present study was to evaluate the association of BC risk with MTHFR C677T and DHFR 19-bp deletion polymorphisms. The study consisted of 110 women with BC. Ninety-five healthy women (in matched control) were used as control. Genomic DNA isolation was performed from peripheral venous blood by standard phenol–chloroform extraction, and polymerase chain reaction of 19-bp deletion polymorphism in intron-1 of DHFR was performed according to previously described method [5], using the following primers: F 50-CCACGGTCGGGGTACCTGGG-30, F 50ACGGTCGGGGTGGCCGACTC-30 and R 50-AAAAGGG GAATCCAGTCGG-30. Amplification was made as follows: initial denaturation of one min at 94 C; followed by 35 cycles of 94 C/55 s, 64 C/55 s, 72 C/55 s, and final extension of 12 min at 72 C (Biometra, Germany). The resulting PCR products were 96-bp and 113-bp. Amplified products were subjected to 4% agarose gel. Genotype data were obtained from 179 subjects. MTHFR C677T polymorphism was determined by using commercially available LightCycler kit (Roche Diagnostic, Roche Molecular Biochemicals, Mannheim, Germany). Genotype data were obtained from 205 cases. The groups were compared using v test and P-value \ 0.05 was considered significant. No differences were observed in the distribution of MTHFR C677T genotype or allele frequencies in the BC cases versus control subjects (P [ 0.05; Table 1). A slightly increased frequency of TT genotype in cancer patients was found but this did not reach statistical significance. We did not find a significant association of DHFR 19-bp deletion polymorphism with BC risk. The frequency of 19-bp deletion allele was lower in BC patients than in control group. Various groups have evaluated the association between MTHFR C677T polymorphism and BC risk, though the findings were conflicting. Some authors observed an increased risk for the carriers of TT genotype. Recent R. Çam A. Eroglu (&) Department of General Surgery, Ankara University Medical School, Cebeci Kampus, Ankara 06590, Turkey e-mail: aydaneroglu@hotmail.com

The 19-bp deletion of dihydrofolate reductase (DHFR), methylenetetrahydrofolate reductase (MTHFR) C677T, Factor V Leiden, prothrombin G20210A polymorphisms in cancer patients with and without thrombosis

Venous thromboembolism (VTE) is a common complication in cancer patients. Several genetic risk factors related to thrombophilia are known; however, their contributions to thrombotic tendency in cancer patients have conflicting results. In the present study, we have focused on the prevalence of methylenetetrahydrofolate reductase (MTHFR) C677T, dihydrofolate reductase (DHFR) 19-bp deletion within intron 1, factor V Leiden (FVL), and prothrombin (PT) G20210A polymorphisms in cancer patients with and without VTE. The study consisted of 63 cancer patients with VTE (group 1) and 124 cancer patients who had no evidence of VTE (group 2). Four gene polymorphisms were determined by the method of polymerase-chain-reaction-based DNA analysis. The prevalence of DHFR 19-bp deletion and MTHFR C677T polymorphisms was similar in two groups (p > 0.05). The frequency of FVL was significantly higher in group1 compared with group 2 (31.7% vs. 1.6%, p < 0.0001), but PT G20210A polymorphism was not associated with VTE. Cancer patients with thrombosis should be evaluated for FVL, but routine screening for PT G20210A, MTHFR C677T and DHFR 19-bp deletion polymorphisms is not suggested.

Factor V Leiden and PT G20210A mutations in cancer patients with and without venous thrombosis

*Department of General Surgery and Surgical Oncology, Numune State Hospital, Selc¸uklu, Konya, Turkey; and Departments of RadiationOncology, Pediatric Molecular Genetic and §General Surgery, Ankara University Medical School, Cebeci, Ankara, TurkeyTo cite this article: Eroglu A, Kurtman C, Ulu A, C¸ am R, Akar N. Factor V Leiden and PT G20210A mutations in cancer patients with,and withoutvenous thrombosis. J Thromb Haemost 2005; 3: 1323–4.

Factor V Leiden and prothrombin G20210A polymorphisms are not associated with disease-free survival in breast cancer.

It is known that cancer and its treatment can induce a hypercoagulable state. On the other hand, coagulation and fibrinolysis can play a significant role in tumor growth, invasion, dissemination and metastasis [1].The significant presence of factor V Leiden (FVL) and/or prothrombin (PT) G20210A polymorphisms which are the two most common defects in Caucasians are responsible for coagulation abnormality in thrombophilia. A few previous reports investigated the relationship between the cancer development and FVL and PT G20210A polymorphisms and found no association of FVL and some types of cancer [2–6]. On the contrary, Pihusch and coworkers demonstrated that the prevalence of PT G20210A polymorphism was significantly increased in patients with gastrointestinal carcinoma as compared to normal subjects [7]. However the role of FVL and PT G20210A polymorphisms in the recurrence risk and prognosis of breast cancer has not been investigated yet. We have read with great interest in the recent publications about experimental metastasis model in FVLdeficient mice [8, 9]. Bruggeman et al. showed that mice carrying FVL mutation were more susceptible to cancer cell metastasis after the injection of melanoma cells into the tail vein [8]. But, other study by Klerk et al. indicated that FVL mutation had no effect on the metastasis of colon cancer to the livers of mice [9]. Encouraged by the reports, we have retrieved data in 123 operable breast cancer and performed an analysis to investigate the role of FVL and PT G20210A polymorphisms in the recurrence (loco-regional and/or distant recurrence) of breast cancer. Genomic DNA isolation was performed from peripheral venous blood by standard phenol-chlorophorm extraction and FVL and PT G20210A polymorphisms were determined by using commercially available LightCycler kits (Roche Diagnostic, Roche Molecular Biochemicals, Mannheim, Germany) [10]. Data regarding patient’s age, tumor type, tumor size, lymph node status, tumor stage, grade, estrogen and progesterone receptor, c-erb B2 expression, FVL and PT G20210A polymorphisms were examined by univariate and multivariate analyses. The Kaplan Meier method was applied to examine the effect of individual variables on disease free survival (DFS). The significance of the observed difference between groups was calculated by the logrank test. The combined and independent effects of the factors on DFS were examined using the Cox proportional hazards model. Statistical analysis was done using SPSS 13.0 for Windows. For all test, a P value of less than 0.05 was considered to be significant. The follow-up period ranged from 4 to 216 months (mean, 57 months). Recurrent disease occurred in 22 patients within a mean time of 26 months (range: 6– 72 months). The univariate analysis factors having significant effects on DFS were tumor size, lymph node status, tumor stage, and c-erb B2 expression (P \ 0.05). FVL was detected in 10 breast cancer patients and the recurrent disease was developed in three of them. As shown in Fig. 1, the 5-year DFS rate in patients with FVL mutation was lower than those without FVL (68% vs. 82%), however this difference did not reach statistically significant A. Eroglu (&) R. Cam Department of General Surgery, Ankara University Medical School, Ankara, Turkey e-mail: aydaneroglu@hotmail.com