Methotrexate effect on biocheMical indices of psoriasis patients depends on MTHFR gene polyMorphisM

Methotrexate (MTX) is the immunosuppressive anti-inflammatory drug and the antagonist of the enzyme dihydrofolate reductase. Pharmacogenomic studies and clinical evidences suggest that altered response to MTX in patients with different diseases is associated with polymorphisms of genes that regulate folate metabolism. The purpose of the article was to analyze the methotrexate effect on the biochemical indices of psoriasis patients depending on methylenetetrahydrofolate reductase gene (MTHFR) polymorphisms. Effects of two single-nucleotide polymorphisms, C677T and A1298C, were studied. An increase of alanine aminotransferase and aspartate aminotransferase activity above the normal level in the patients with both MTHFR gene polymorphisms after methotrexate intake was observed. In patients with CC, TT, CT genotypes for C677T polymorphism and AA genotype for A1298C polymorphism of MTHFR gene, significant differen ces in alphaamylase activity before and after treatment with methotrexate were detected. Analysis of the biochemical indices of patients with arthropathic and vulgaris psoriasis showed that the positive effect of MTX treatment could be associated with wild-type alleles in both polymorphisms of MTHFR gene, while the ineffectiveness of methotrexate was associated with the dihеterozygous genotype. The largest number of smokers was found within the ctaa genotype group (37.5%), while no smokers were observed within ttaa patients and most of CCAA patients. The data obtained testify the utility of the individual approach to the psoriasis patients therapy taking into account genetic background.

Methotrexate (MTX) is the immunosuppressive anti-inflammatory drug and the antagonist of the enzyme dihydrofolate reductase. Pharmacogenomic studies and clinical evidences suggest that altered response to MTX in patients with different diseases is associated with polymorphisms of genes that regulate folate metabolism. The purpose of the article was to analyze the methotrexate effect on the biochemical indices of psoriasis patients depending on methylenetetrahydrofolate reductase gene (MTHFR) polymorphisms. Effects of two single-nucleotide polymorphisms, C677T and A1298C, were studied. An increase of alanine aminotransferase and aspartate aminotransferase activity above the normal level in the patients with both MTHFR gene polymorphisms after methotrexate intake was observed. In patients with CC, TT, CT genotypes for C677T polymorphism and AA genotype for A1298C polymorphism of MTHFR gene, significant differen ces in alphaamylase activity before and after treatment with methotrexate were detected. Analysis of the biochemical indices of patients with arthropathic and vulgaris psoriasis showed that the positive effect of MTX treatment could be associated with wild-type alleles in both polymorphisms of MTHFR gene, while the ineffectiveness of methotrexate was associated with the dihеterozygous genotype. The largest number of smokers was found within the ctaa genotype group (37.5%), while no smokers were observed within ttaa patients and most of CCAA patients. The data obtained testify the utility of the individual approach to the psoriasis patients therapy taking into account genetic background. I ndividualization of pharmacotherapy based on the patients' genetic background allows to in crease the effectiveness of treatment and reduce costs [1,2]. Psoriasis is a heterogeneous chronic painful, disfiguring disease. Joints deformations and disability are the most prominent extracutane ous manifestations of this condition [3]. Currently, psoriasis is considered as a systemic disease affect ing multiple organs. Cardiovascular, neurological and mental disorders could be observed in psoriasis patients [4]. The use of drugs of different pharmaco logical groups could be accompanied by a various response of patients to therapy, including serious complications depending on the symptoms, organ function, gender, age, diet, genotype, and others [5,6].
Methylenetetrahydrofolate reductase (MTHFR ) is one of the main regulatory enzymes in the folate and homocysteine metabolic pathways. Poly morphisms of MthFR (OMIM 607093) gene lead to decreased activity of enzyme, lower effi ciency of the homocysteinemethionine cycle and hyperhomocystei nemia. MthFR gene polymor phisms effects are not only detected in patients with cardiovascular, nervous, mental, dermatological, reproductive, oncological, chromosomal and other diseases but are also shown to depend on smoking, coffee and alcohol consumption, diet features, physi cal activity [7,8].
Therefore, multifactorial pathology could be considered in terms of psoriasis, reflecting the interac tion between genetic and environmental factors.
One of the main objectives of psoriasis phar macotherapy is to find treatment options that quickly and firmly suppress and prevent further progression of the disease. Methotrexate (MTX) is the drug with antiinflammatory and immunosuppressive action and an antagonist of the enzyme dihydrofolate re ductase, that blocks the synthesis of tetrahydrofolate and therefore prevents the synthesis of purines and pyrimidines [9,10]. Pharmacogenomic studies and clinical evidences suggest that altered response to MTX in patients with different diseases is associated with polymorphisms of folate metabolism genes [11].
Methylenetetrahydrofolate reductase is known as an enzyme that mediates MTX pharmacokine tics [12]. The interconnection between MthFR gene polymorphisms (OMIM 607093) responses to MTX treatment and adverse reactions to MTX was discove red in patients with rheumatoid arthritis [13]. MTX was effective without any adverse effects in psoriasis patients with positive presumed 677C 1298A SNP haplotypes. At the same time no asso ciation between SNPs in FPGS, GGh and MthFR genes and either MTX efficacy or toxicity in patients with psoriasis in the UK were shown. The assump tion about the high risk of adverse effects develop ment after MTX administration in carriers of the 677T allele of the MthFR gene was not confirmed in patients with psoriasis arthritis [14]. According to the results of researches in Algeria, association between the 677T allele, methotrexateinduced toxi city and adverse effects was detected, while the C allele, in contrast, had a protective effect against MTX toxicity in patients with rheumatoid arthri tis. A response to the treatment (good or moderate) was observed more often in A1298 allele carriers than in carriers of 1298C allele associated with a reduced response [15]. Generally, research results evaluating the role of individual SNPs in response to MTX were ambiguous. This could be attributed to different study designs, insufficient statistical power and folate supplementation, MTX dose, duration and route of administration and concurrent therapies [15]. Moreover, it is known that alleles and geno types frequencies distribution, of folate metabolism genes showed ethnic and geographical patterns, so studies of onecarbon metabolism genes are actua lly carried out in each population. Pharmacogenetic studies on methotrexate therapy in the Ukrainian population enable the development of an effective and targeted therapeutic strategy.
The purpose of the article was to analyze methotrexate effect on the biochemical parameters of psoriasis patients depending on methylenetetrahy drofolate reductase gene (MthFR) polymorphisms and to evaluate the response to therapy.

Materials and Methods
The study included a retrospective analysis of the medical history of 41 patients with psoriasis. Patients age ranged from 19 to 77 years, mean age was 46.27±1.78 years. All participants had signed the informed consent before the study. Screening in cluded standardized questionnaires for personal data and clinical measurements such as age, sex, medical and family history of psoriasis. The liver function tests were carried out in all patients at the beginning and at the end of methotrexate treatment. Genoty ping of patients according to the C677T and A1298C polymorphisms was carried out by PCRRFLP and allelespecific PCR [16,17]. The data were ac cumulated on clinical observations of adverse and favorable reactions in patients with psoriasis who were on methotrexate treatment [18]. The data dis tribution was tested for normality before further statistical analyses by the KolmogorovSmirnov and ShapiroWilk tests. Wilcoxon test was used for determination of difference between the means of two groups before and after methotrexate treatment. KruskalWallis test value was calculated in order to establish significant differences between continu ous dependent variable by a categorical independent variable. Biochemical parameters were presented as mean ± standard error of mean (M±SEM). All statis tical tests were twotailed and a probability (P) value of 5% or less was considered statistically significant.

results and discussion
We evaluated the relation of MthFR gene poly morphisms with the main biochemical meta bolic parameters before/after taking methotrexate.
Stratification of psoriatic patients by genotype according to C677T of MthFR gene showed the prevalence of heterozygotes CT (25 persons) and homozygotes CC (12 persons) compared to TT ho mozygotes (4 persons Among arthropathic psoriasis patients with SNP C677T, those with СT genotype had the lowest alanine aminotransferase (ALT) activity compared to CC and TT genotypes. The level of activity be fore and after methotrexate treatment by patients with CT genotype was within the normal range, as opposed to persons with CC and TT genotypes. The same trend was noted for aspartate aminotransferase (AST) activity (Table 1). A significant change in the parameters of αamylase activity and urea level was noted for all three genotypes, and there were differen ces between the genotypes. The deviations from the normal range were noted for creatinine in TT genotype, total protein in CC genotype, and total cholesterol in CT genotype (Table 1).
Positive effect of C677T and A1298C in MthFR genotypes on the biochemical parameters of arthropathic psoriasis patients followed the pat tern CTAC > CTAA > CCAC > CCAA > TTAA (Table 3). A similar trend was observed for the bio chemical parameters in psoriasis vulgaris patients, genotyped by two SNPs in MthFR gene: CTAC > CCAC > CTAA > CCAA (Table 4). Our results correspond to the data obtained by other authors, who studied the reaction of patients with rheuma toid arthritis on MTX T allele was associated with nonresponsiveness to the drug [19]. According to the litera ture data, the response of psoriasis patients to drug therapy could be determined by MthFR polymorphisms, with MthFR 677CC serving as a predictor of high drug sensitivity, and the MthFR 677 (CT, TT) and MthFR 677T1298A haplotypes serving as possible predictors of MTX adverse ef fects [13].
Considering the dynamics of transaminases activity, the protective effect of several SNPs in MthFR gene against liver toxicity was assumed in psoriasis patients. In the studies the increased ALT and AST activity by more than 1.5 times was con sidered as significant hallmark of hepatotoxicity. Along with structural and functional liver damage, the serious complication of methotrexate treatment is nephrotoxicity [20], that could be noted by the dy namics of creatinine levels for some genotypes in our research (Table 14).
In the presented study, αamylase activity in psoriasis patients after methotrexate treatment in most cases was within the normal range or lower. Kidney function is a crucial modifier that affects the clearance of circulating amylase in the blood [21]. Certain pharmacotherapeutics were reported to in crease serum amylase level [22]. It is known that T allele leads to a decreased rate of 5.10MTHF con version to 5MTHF, which results in a low rate of homocysteine remyelation, thereby increasing the homocysteine level and decreasing folic acid level in blood plasma [23]. It is also known that low levels of folate contribute to ALT elevation in serum [24]. It is proved that smoking increases plasma homocystei ne level [25]. Therefore, polymorphisms C677T and A1298C and smoker status could affect the level of ALT and AST in serum.
Our analysis showed that among the examined group of patients, 81.3% of persons were smokers or had a long history of smoking in the past. The smoker experience ranged from four to seve ral decades. The distribution of genotypes could be represented as CTAA (37.5%) > CTAC (25.0%) > CCAC (12.5%) > CCAA (6.25%) > TTAA (0%), the largest number of smokers were found within CTAA genotype group. TTAA patients and most of CCAA patients did not smoke. The distribu tion of genotypes and ALT parameters in smokers before/after taking methotrexate was: CTAA (0.63±0.06/0.83±0.10) > CTAC (0.57±0.05/0.60±0.02) > CCAC (0.53±0.02/0.65±0.03). Patients with geno types TTAA and CCAA showed the highest rates and dynamics of ALT -0.98±0.25/1.42±0.06 and 0.65±0.08/0.85±0.10, which was probably one of the smoking prevention factors in these persons.
The differences in the gene pool indicate both specific risks and a unique system of genetic markers for each ethnic group, which could be considered while forming risk groups and choosing a treatment strategy.