Objective: Methotrexate may be used as a prophylactic agent against graft-versus-host disease in hematopoietic cell transplant. The drug exerts its effect on folate metabolism; 5,10-methylenetetrahydrofolate reductase is a critical enzyme involved in this cycle and is related to the toxicity of methotrexate.
Methods: We examined the association of a single nucleotide polymorphism at position 677 in the 5,10-methylenetetrahydrofolate reductase gene and the clinical outcomes of patients treated with allogeneic hematopoietic cell transplant. Genotyping of 5,10-methylenetetrahydrofolate reductase was performed by polymerase chain reaction-restriction fragment length polymorphism on 30 patients receiving hematopoietic cell transplant and their HLA-matched related donors. Patients were given a short course of methotrexate as prophylaxis to prevent graft-versus-host disease.
Results: Donors and recipients who carried a 677T allele showed mildly higher total bilirubin, aspartic transaminase, and alanine transaminase levels, but these increases above the normal values were not statistically significant (P > .05). The platelet recovery to 20 000/µL and granulocyte recovery to 500/µL were slower for patients who carried a 677T allele, but these correlations also were not statistically significant. The 5,10-methylenetetrahydrofolate reductase genotypes of neither the donors nor the recipients had any effect on the incidence of acute graft-versus-host disease.
Conclusions: No association was observed between the C677T polymorphism and the outcome parameters for any of the different genotypes studied here. Additional studies with larger samples are necessary to further elucidate the influence of 5,10-methylenetetrahydrofolate reductase genotyping on clinical outcomes of patients treated with hematopoietic cell transplant who receive methotrexate.
Key words : Graft-versus-host disease, Transplantation, Methotrexate, Polymorphism, Prophylaxis
Methotrexate, usually in combination with other immunosuppressive drugs, is commonly used in recipients of allogeneic hematopoietic cell transplants as prophylaxis to prevent graft-versus-host disease (1). Methotrexate inhibits graft-versus-host disease by preventing activated, mature donor T lymphocytes from expanding; it induces apoptosis and modulates cytokine responses at several levels (2,3,4). Methotrexate competes with intracellular folate for binding to the enzyme dihydrofolate reductase, which results in depletion of folate and interferes with synthesis of pyrimidines and purines, nucleotides required for DNA and RNA synthesis and for DNA methylation substrates (5). Methotrexate exerts a powerful effect on donor T-cell proliferation, thereby inhibiting graft-versus-host disease (6).
Methylenetetrahydrofolate reductase directs intracellular folate toward homocysteine metabolism and away from nucleotide synthesis. The common methylenetetrahydrofolate reductase polymorphism, C677T, is associated with reduced enzyme activity (5,6). The toxicities of methotrexate include myelosuppression, hepatic dysfunction, and mucosal injury (7,8). To evaluate whether the C677T polymorphism of methylenetetrahydrofolate reductase is related to the toxicity of methotrexate used in allogeneic stem cell transplant, we analyzed the associations between this genetic polymorphism and the clinical outcomes of patients treated by human leukocyte antigen (HLA)-matched sibling hematopoietic cell transplant.
Materials and Methods
Subjects in this retrospective cohort study were 30 patients (20 males, 10 females; median age, 29 years; range, 14-48 years) receiving an allogeneic hematopoietic cell transplant at the Bone Marrow Transplant Center of the Shiraz University of Medical Sciences in Shiraz, Iran, between 2006 and 2007. All patients received methotrexate and cyclosporine for prophylaxis to prevent graft-versus-host disease. Written informed consent was obtained from all patients, donors, and controls, allowing analyses of the clinical data and testing for all mutations and polymorphisms mentioned in this article. The study was conducted according to the guidelines of the 1975 Declaration of Helsinki, and the study protocol was approved prior to its beginning by the ethics committee of Shiraz University. Clinical data were obtained from medical, laboratory, and radiologic records before methylenetetrahydrofolate reductase genotyping. The data collected were liver and renal function values including total bilirubin, aspartic transaminase, alanine transaminase, and creatinine levels; platelet and granulocyte counts, as well as complications of transplant such as acute graft-versus-host disease. We evaluated liver toxicity based on peak total bilirubin, aspartic transaminase, and alanine transaminase levels during the first 30 days after allogeneic hematopoietic cell transplant. Granulocyte engraftment was defined as the time to the first 3 consecutive days of counts exceeding 500/µL, and platelet engraftment was defined as the time to the first 7 consecutive days of counts exceeding 20 000/µL without transfusion. Acute graft-versus-host disease was graded from 0 to IV using conventional criteria (9,10). Patient characteristics, including underlying disease and conditioning regimen, are shown in Table 1. Acute leukemia was the most common underlying diagnosis (10 acute leukemia, 7 non-Hodgkin's lymphoma, 4 metastatic carcinoma, and 9 major thalassemia).
The median follow-up of survivors was 100 days (range, 90-100 days). The commonly used conditioning regimen consisted of busulfan, cyclophosphamide, and antithymocyte globulin (n=21; 61%). Conditioning regimens for thalassemic patients were composed of cyclophosphamide and busulfan (n=9; 39%). Twenty-three patients (77%) received bone marrow as a stem cell source, and 7 patients (23%) received peripheral blood stem cells.
Genomic DNA was extracted from a peripheral blood sample by means of a commercial extraction kit (DNG plus DNA Extraction Kit, Sinagene Company, Tehran, Iran) and stored at –20°C until the analysis was performed. DNA fragments containing the C677T methylenetetrahydrofolate reductase variant were amplified by polymerase chain reaction using the oligonucleotides published by Kowa and associates (11). The sense primer sequence was 5'-TGA AGG AGA AGG TGT CTG CGG GA-3', and the antisense sequence was 5'-AGG ACG GTG CGG TGA GAG TG-3'. The thermal cycle parameters included 1 cycle at 95°C for 3 minutes for an initial denaturation, followed by 35 cycles of denaturation for 1 minute at 94°C, primer annealing for 1 minute at 65°C, primer extension for 2 minutes at 72°C, and a final extension for 10 minutes at 72°C. This amplification reaction resulted in the synthesis of a 198-bp fragment. The C to T substitution at nucleotide 677 in the methylenetetrahydrofolate reductase gene introduces a restriction site for the HinfI enzyme (New England Biolabs, Herts, United Kingdom). This enzyme was used to distinguish the alleles by cleaving the mutant fragment into 175-bp and 23-bp fragments. Samples were fractionated by using a 3% agarose gel, stained with ethidium bromide (Sinagene Company, Tehran, Iran), and visualized under ultraviolet light. To confirm the results, 10 separate samples were also genotyped using an ABI PRISM 310 genetic analyzer (Global Medical Instrumentation, Inc., Ramsey, MN), which confirmed the results of the polymerase chain reaction-restriction fragment length polymorphism.
Analyses were performed with SPSS software (Statistical Package for the Social Sciences, version 13, SSPS Inc, Chicago, IL, USA). Statistical analyses to evaluate differences between the clinical outcomes of the methylenetetrahydrofolate reductase genotype groups were performed using the t test for discrete data. Mean peak levels of total bilirubin, aspartic transaminase, alanine transaminase, and creatinine were calculated for individual genotypes. Engraftment rates and the incidences of acute graft-versus-host disease were compared between genotypes.
The frequencies of the methylenetetrahydrofolate reductase C677T genotype were 70% for 677CC, 10% for 677CT, and 20% for 677TT in recipients and 67%, 23%, and 10% in donors, respectively (Table 2). Because the 677TT genotype was so infrequent, those genotypes were modeled as a dichotomous variable (677CC vs 677TT and 677CT taken together) in the statistical analyses. Analyses were performed in recipients and donors separately for methylenetetrahydrofolate reductase C677T (Tables 3 and 4). In terms of the C677T polymorphism in donors and recipients, no associations with total bilirubin, aspartic transaminase, alanine transaminase, or creatinine levels were found (P > .05). Regarding the C677T polymorphisms, there was no statistically significant difference between recipients and donors regarding peak levels of serum creatinine during the first 30 days (P > .05) (Table 3). No statistically significant differences were found regarding platelet recovery to 20 000/µL or granulocyte recovery among the C677T polymorphisms in either the donors or the recipients (P > .05) (Table 3).
Nine patients (30%) developed grade II-IV acute graft-versus-host disease. None of the C677T polymorphisms were found to be risk factors for acute graft-versus-host disease in recipients or donors (P = 1.00 and P = .63 respectively). There was no evidence that inheritance of the C allele had a protective effect in patients (P = 1.00) (Table 4).
In this study, we investigated the effects of the C677T polymorphisms in donors and recipients on the toxicity of methotrexate used as a prophylaxis for graft-versus-host disease in allogeneic hematopoietic cell transplant. Methotrexate inhibits dihydrofolate reductase, which results in the depletion of tetrahydrofolates. In addition, methotrexate metabolites are known to inhibit methylenetetrahydrofolate reductase (3). Homocysteine levels have been reported as being increased in patients with the methylenetetrahydrofolate reductase polymorphism and associated with lower enzyme activity (T allele at the C677T), which could cause endothelial damage and hepatic fibrosis (12,13). In a previous study (14) that investigated the role of methylenetetrahydrofolate reductase polymorphisms on patients with rheumatoid arthritis treated with methotrexate, the T allele of the C677T polymorphism was found to render patients sensitive to methotrexate toxicity. The C677T polymorphism also was associated with hyperhomocysteinemia related to chronic allograft nephropathy in renal transplant recipients (15), although another report indicated no statistically significant differences in terms of methotrexate toxicity, possibly reflecting ethnic differences between whites and nonwhites (16). The homozygous 677TT genotype, which occurs in approximately 10% to 15% of white and Asian populations (3), has been shown to have 30% of the methylenetetrahydrofolate reductase wild-type enzyme activity in vitro, and the heterozygous (CT) genotype has approximately 60% of the wild-type enzyme activity (6).
In patients receiving hematopoietic cell transplants, methotrexate is administered at a low dosage and for a short duration as prophylaxis to prevent graft-versus-host disease. Patients treated with hematopoietic stem cell transplant are at high risk of folate deficiency owing to decreased dietary intake (as a result of nausea, anorexia, and mucositis), increased folate requirements during recovery from the hematopoietic cell transplant, and exposure to several drugs (ie, methotrexate and trimethoprim-sulfamethoxazole). Patients with the 677TT genotype undergoing hematopoietic cell transplant, regardless of methotrexate use, show more disturbance of the folate pathway than do patients with other genotypes. This greater disturbance results in a greater disruption of DNA methylation, altered transcriptional regulation, and increased homocysteine levels, all of which affect nucleotide pools (5). Such a reduced DNA repair capacity in persons with the 677TT genotype might result in delayed healing after hematopoietic cell transplant. Previous studies in bone marrow transplant individuals have shown that patients with the 677TT genotype are at high risk of developing hepatic toxicity and have a poor likelihood of survival (17). In a study by Murphy and associates, the combined donor 677 CT and TT genotypes were associated with a decreased incidence of graft-versus-host disease in recipients undergoing hematopoietic stem cell transplants (18). Robien showed that those with the variant 677T allele had a decreased risk of detectable acute graft-versus-host disease (19) as well as a higher risk of relapse with the 677CC genotype (20). Pihusch and associates (21) performed a study consisting of an equal number of related and unrelated donor hematopoietic cell transplants that examined methylenetetrahydrofolate reductase polymorphisms in recipients and donors and reported no effect of the recipient methylenetetrahydrofolate reductase genotype on the incidence of graft-versus-host disease. No analyses of the donor genotypes appear to have been performed (21). However, in our study, no association between clinical outcomes, incidence of graft-versus-host disease, and the 677TT methylenetetrahydrofolate reductase polymorphisms in either donors or recipients was observed.
Our study has several limitations. The analysis was based on a small sample size. Therefore, our results must be confirmed in a larger prospective cohort. The methotrexate level in blood during transplant and other methotrexate toxicities such as oral and intestinal mucositis were not investigated. The study is also limited by the short follow-up (100 days), which precludes any conclusion about longer term toxicities. Further examination of polymorphisms in other key folate cycle enzymes such as the reduced folate carriers, folyl-γ polyl glutamate synthetase, thymidylate synthase, and dihydrofolate reductase are required to establish whether polymorphisms in these genes also contribute to an increased risk of graft-versus-host disease.
Volume : 5
Issue : 2
Pages : 693 - 697
From the Organ Transplant Research Center, Hematology Transplant Research Center,
Shiraz University of Medical Sciences, Shiraz, Iran
Address reprint requests to: Negar Azarpira MD, Organ Transplant Research Center, Nemazi Hospital, Shiraz University of Medical Sciences, Iran
Phone: 00 98 711 627 6211
Fax: 00 98 711 627 6211
Table 1. Patient characteristics
Table 2. Frequencies of the MTHFR 677 genotype in controls, patients, and donor populations
Table 3. Comparisons of laboratory values according to MTHER genotypes
Table 4. MTHFR C677T genotype/haplotype frequencies in donors and recipients graded as absent or mild (grade 0-I) and clinically significant (grade II-IV) acute GVHD