II-63Short invited lectures – abstracts
GENETIC POLYMORPHISMS IN FOLATE METABOLIC PATHWAY
INFLUENCE THE RISK FOR DEVELOPING ADVERSE EFFECTS
DURING METHOTREXATE TREATMENT IN RHEUMATOID
ARTHRITIS
Petra Bohanec-Grabar1, Dušan Logar2, Boris Lestan2, Vita Dolžan1
1 Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana,
Slovenia
2 Department of Rheumatology, University Medical Centre, Vodnikova 62, Ljubljana, Slovenia;
petra.bohanec@mf.uni-lj.si
Introduction Methotrexate (MTX) is the most widely prescribed disease-modifying antirheumatic drug
(DMARD) for the treatment of rheumatoid arthritis (RA). MTX enters the cells through
reduce folate carrier (RFC1) and directly inhibits several enzymes of the folate metabolic
pathway. Intracellular folate metabolism is complex and involves enzymes such as 5,10-
methylenetetrahydrofolate reductase (MTHFR), thymidylate synthase (TS), methionine
synthase (MS) and methionine synthase reductase (MTRR) that channel the methyl group
of tetrahydrofolate (THF) into the direction of the DNA synthesis or methylation. MTHFR is
the central enzyme of the folate metabolic pathway that defines the ratio between the two
forms of THF. Two common polymorphisms C677T and A1298C are known in the MTHFR
gene resulting in a lower enzyme activity. The promoter enhancer region of the TS gene
contains a double (2R) or a triple (3R) 28-base pairs (bp) tandem repeat polymorphism.
3R is associated with a higher TS gene expression and therefore higher conversion rate of
dUMP to dTMP. MS and MTRR are enzymes crucial for the efficient methionine synthesis
and methylation reactions. A2756G polymorphism in the MS gene results in a lower en-
zyme activity, while the function of MTRR A66G polymorphism is not yet understood. RFC1
gene also contains a genetic polymorphism (A80G) that may have an impact on affinity of
the transporter for the folate. Common genetic polymorphisms in RFC1 and in enzymes in
the folate metabolic pathway alter enzymatic activity and thus may modify MTX uptake or
influence MTX metabolic effect.
Aim The aim of our study was to analyse if genetic polymorphisms in RFC1 and the folate
metabolic pathway influence the efficacy and/or toxicity of the RA treatment.
Methods Study population consisted of the group of 214 consecutive, unrelated RA patients, 26 male
(12.1 %) and 188 female (87.9 %), median age 60.5 (range 51–69) years. At the entry into
the study 157 out of 214 patients were treated with methotrexate, while the remaining 57
patients were treated with other disease modifying antirheumatic drugs. A Genotyping
approach using both restriction fragment length polymorphism (RFLP) and/or TaqMan
genotyping assays (Applied Biosystems) was used to determine RFC1 A80G, MTHFR C677T/
A1298C, MS A2756G, MTRR A66G and TS 2R/3R polymorphisms in 214 RA patients. Bina-
ry logistic regression analysis with the addition of independent variables, such as sex, dis-
ease and treatment duration, RF seropositivity and the amount of the folate in the erythro-
cytes was utilized to determine the risk for inefficacy of the treatment and/or developing
adverse effects. The risk was expressed as odds ratio (OR) with 95 % confidence intervals
(CI).
Results MTX treatment was well tolerated by 157 patients (73.3 %), 58 of them (27.1 %) were co-
treated with additional disease-modifying anti-rheumatic drug (DMARD). Efficient MTX
treatment was achieved in 136 patients (63.6 %), 36 patients (16.8 %) had intermediate
MTX treatment efficacy, while 17 (7.9 %) and 25 (11.7 %) patients experienced poor and
inefficient MTX treatment, respectively. Adverse effects were present in 148 patients (61.8
%), among them 38 (17.7 %) developed severe adverse effects and were discontinued from
the MTX treatment. No significant association between genetic polymorphisms in the folate
metabolic pathway and the efficacy of the RA treatment was observed however, specific
genetic polymorphisms influenced the risk for developing adverse effects. Carriers of RFC1
II-64 Zdrav Vestn 2007; 76: SUPPL II
80GG and TS 3R/3R genotype had a 2.65-fold and a 2.46-fold higher risk for developing
MTX adverse effects than carriers of at least one RFC1 80A allele and TS 2R allele, respec-
tively (P = 0.021 and P = 0.025, respectively; 95 % CI = 1.160–6.033 and 95 % CI = 1.120–
5.396, respectively). Furthermore, patients with RFC1 80GG genotype had an 11.17-fold
higher risk for developing infections than patients with RFC1 80AA/AG genotype (P = 0.006,
95 % CI = 2.030–61.486). A combination of at least one MTHFR 677T or 1298C polymor-
phic allele and MS 2756AG/GG and MTRR 66AG/GG genotypes, which may lead to lower
methylation capacity reduced the risk for developing adverse effects 3.175-fold (P = 0.032,
OR = 0.315, 95 % CI = 0.109–0.907).
Conclusion Our results suggest that genetic polymorphisms in RFC1 and the folate metabolic pathway
do not modify the MTX efficacy of RA treatment but influence the risk for developing MTX
adverse effects.