Оценка влияния полиморфизмов генов-транспортеров (RFC1, MDR1) и GGH на эффективность метотрексата при ревматоидном артрите

The efficacy of methotrexate (MT) in patients with rheumatoid arthritis (RA) may be determined by genetic factors.Objective: to evaluate the isolated and combined effects of single nucleotide polymorphisms (SNPs) of membrane transporter proteins (RFC1 80G>A and MDR1 3435C>T) and the GGH -401C>T gamma-glutamyl hydrolase enzyme genes on the efficacy of MT in patients with RA.Material and methods. The study group consisted of 85 patients with a confirmed diagnosis of RA, who received therapy with MT starting at 10 mg/week and increasing in dose to a maximum of 25 mg/week. Efficacy was assessed after six months of treatment using the dynamics of the DAS28 index, identifying patients who responded and those who did not respond to MT therapy.Genotyping of RFC1, MDR1 and GGH gene polymorphisms was performed by real-time polymerase chain reaction. Three different approaches were used to analyze the results: 1) analysis for each of the genes; 2) logistic regression; and 3) multifactor dimensionality reduction (MDR).Results and discussion. Single gene analysis was used to determine the most likely predictors of non-response to therapy: 1) for GGH-401C>T, TT genotype (odds ratio, OR 5.09; 95% confidence interval, C11.11—23.3); 2) forMDR13435C>T, the TT genotype (OR 2.38; 95% CI0.89-6.37); 3) for RFC180G>A, not - AA genotype (OR 1.87; 95% CI 0.93-3.76).The logistic regression model showed a significant effect of homozygous genotype GGH -401TT on the efficacy of MT with low sensitivity of the method. The multifactorial dimensionality reduction results show a significant synergistic effect of the MT transport genes (MDR1, RFC1) and the GGH enzyme encoding the conversion of MT to the elimination form.Conclusion. Using various statistical methods, the following results were obtained: Single gene analysis revealed the most likely predictors of nonresponse to MT therapy: GGH -401C>T - TT genotype, MDR1 3435C>T - TT genotype, RFC1 80G>A - not-AA genotype; the method of multiple logistic regression allowed to determine the significant effect of GGH -401ТТ genotype on the effect of the drug with a low sensitivity of the method; the isolated effect of polymorphisms is probably less pronounced than their combined effect on the effectiveness of MT. SNP synergism is a major contributor to the development of treatment resistance. MDR is a promising method that can be used in the future to assess the impact of SNPs.Эффективность метотрексата (МТ) у больных ревматоидным артритом (РА) может определяться генетическими факторами.Цель исследования — оценить изолированное и совместное влияние однонуклеотидных полиморфизмов (SNP) генов мембранных белков-транспортеров (RFC1 80G>A и MDR1 3435C>T) и гена фермента гамма-глутамилгидролазы GGH -401C>T на эффективность МТ у больных РА.Материал и методы. Исследуемую группу составили 85 больных с достоверным диагнозом РА, которым проводилась терапия МТ начиная с 10 мг/нед с повышением дозы максимально до 25 мг/нед. Эффективность оценивалась после 6 мес лечения по динамике индекса DAS28, были определены пациенты, отвечающие и не отвечающие на терапию МТ.Генотипирование полиморфизмов генов RFC1, MDR1 и GGH выполнено методом полимеразной цепной реакции в режиме реального времени. Для анализа результатов использовались три различных подхода: 1) анализ по каждому из генов; 2) логистическая регрессия и 3) многофакторное снижение размерности (Multifactor Dimensionality Reduction, MDR).Результаты и обсуждение. С помощью анализа по отдельным генам идентифицированы наиболее вероятные предикторы отсутствия ответа на терапию: 1) для GGH-401C>T — генотип TT(отношение шансов, ОШ5,09; 95% Доверительный интервал, ДИ 1,11—23,3); 2) для MDR13435C>T-генотип TT(ОШ2,38; 95% ДИ0,89-6,37); 3) Для RFC180G>A - генотип не АА (ОШ 1,87; 95% ДИ 0,93-3,76).Модель логистической регрессии свидетельствует о значимом влиянии на эффективность МТ носительства гомозиготного генотипа GGH -401TT при низкой чувствительности метода. Результаты многофакторного снижения размерности демонстрируют значимое синергичное влияние генов транспорта МТ (MDR1, RFC1) и фермента GGH, кодирующего конверсию МТ в форму Для элиминации.Заключение. С помощью разных статистических методов получены следующие результаты: анализ по отдельным генам выявил наиболее вероятные предикторы отсутствия ответа на терапию МТ: GGH - 401C>T - генотип TT, MDR1 3435C>T - генотип TT, RFC1 80G>A - генотип не AA; метод множественной логистической регрессии позволил определить значимое влияние генотипа GGH - 401ТТ на эффект препарата при низкой чувствительности метода; изолированное влияние полиморфизмов, вероятно, менее выражено, чем их совместное воздействие на эффективность МТ. Синергизм SNP вносит большой вклад в формирование резистентности к терапии. MDR - перспективный метод, который может быть использован в дальнейшем для оценки влияния SNP

Allelic polymorphisms of thymidylate synthase gene and their haplotypes as predictors of the therapeutic response to methotrexate in patients with rheumatoid arthritis

Methotrexate (MT) is recognized as the main component of the treat to target strategy in patients with rheumatoid arthritis (RA). Unfortunately, MT therapy is not equally effective in all patients with RA, some of them are resistant to such treatment, and have steady progression of destructive changes of the joints, reducing quality of life and leading to disability.Objective: examination of genetic predictors of therapeutic efficacy and resistance to MT among single-nucleotide polymorphisms (SNP) of folate cycle genes, in particular polymorphisms of the thymidylate synthase gene TSER 2R/3R and TS 6bp del/ins and their haplotypes.Subjects and methods. The study included 85 patients with RA, who were prescribed MT at a dose of 10 to 17.5 mg/week as a the «first line» disease-modifying antirheumatic drug. The therapeutic response was evaluated with DAS28 after 6 months of continuous treatment. According to the results of this assessment groups of «respondents» and «non-respondents» to MT were identified. Genetic typing of SNP TSER 2R/3R and TS 6bp del/ins was performed by real-time polymerase chain reaction.Results and discussion. In RA patients with different efficacy of MT, statistically significant differences in the frequency of alleles and genotypes of polymorphic variants of the gene thymidylate synthase were revealed. Thus, in patients resistant to MT homozygous genotype TS 6bp ins/ins frequency was significantly increased (OR 4,3; 95% CI of 1.58 to 11.7; p=0.003), whereas response to the treatment was associated with occurrence of TS 6bp allele del (HR OF 0.48; 95% CI of 0.23 to 1.0; p=0.049), of TSER 2R/3R (OR 0,32; 95% CI 0,12–0,88; p=0.042) and TS 6bp del/ins (OR 0.23; 95% CI 0.08–0.65; p=0.008) genotypes. We have not established statistically significant differences in the frequency of occurrence of haplotypes of the thymidylate synthase gene in RA patients with different therapeutic efficacy of MT, but we found a tendency to increase of the haplotype TS 3R-6bp del (OR 0.39; 95% CI 0.24–1.09; p=0.081) frequency in the «respondents» to the treatment.Conclusion. Our results may indicate the relationship of the thymidylate synthase gene single nucleotide polymorphisms with therapeutic response to MT in patients with RA

MOLECULAR BIOLOGY ANALYSIS OF IL -1Β, IL -4, AND IL -1Β RA GENE POLYMORPHISMS IN ETHNIC BASHKIR PATIENTS WITH RHEUMATOID ARTHRITIS, LIVING IN C HELYABINSK REGION

Abstract. Common polymorphisms of interleukin-1β, receptor antagonist interleukin-1, and interleukin-4 genes have been studied among the patients with rheumatoid arthritis of Bashkir origin, living in Chelyabinsk Region. As a result of study, an increased frequency of interleukin-1β homozygosity for a high-producer allele was found. An association has been revealed between the homozygosity for 2-repeat allele of interleukin-4, and the age of disease onset