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The effect of ESR1 and ESR2 gene polymorphisms on the outcome of rheumatoid arthritis treatment with leflunomide Aim: Leflunomide is the drug used in the therapy for rheumatoid arthritis (RA). Previous studies indicated that the efficacy of the therapy with antirheumatic drugs is more effective in men than in women. Moreover, estrogens can decrease the anti-inflammatory action of leflunomide. Estrogens act through the estrogen receptors ESR1 and ESR2. In ESR1 and ESR2 genes, several polymorphisms have been detected. The aim of the present study was to examine the association between polymorphisms in the ESR1 and ESR2 genes and the response to treatment of RA patients with leflunomide. Materials & methods: The study was carried out on 115 women, mean age 54.1 ± 11.0 years, diagnosed with RA and treated with leflunomide (20 mg daily). Results: Our results indicated a better response to treatment in patients with ESR1 rs9340799 AA and rs2234693 TT genotypes after 12 months of therapy. In these patients, the improvement of erythrocyte sedimentation rate, patient’s global assessment of disease activity on a 100 mm visual analog scale and disease activity score values was greater than in patients with other genotypes. The ESR1 rs9340799–rs2234693 A–T haplotype was associated with a better response to treatment, the G–C haplotype with a worse response and the A–C haplotype was neutral. There were no statistically significant associations of response to treatment with ESR2 gene rs4986938 and rs1256049 polymorphisms. Conclusion: The results of the present study suggest that ESR1 gene polymorphisms in females with RA may be associated with the response to treatment with leflunomide. KEYWORDS: estrogen receptors n genetic polymorphism n leflunomide n rheumatoid arthritis
Rheumatoid arthritis (R A) is a systemic inf lammatory disease which is characterized by destructive changes in the bone and cartilage of affected joints. Its treatment is mainly based on disease-modifying antirheumatic drugs such as: arechin, sulfasalazine, methotrexate and leflunomide. Leflunomide is an isoxazole derivative that is structurally and functionally unrelated to other known immunomodulatory drugs [1] . In the intestinal mucosa and plasma, almost 100% of the compound is nonenzymatically converted into the active open-ring malononitrile metabolite A77 1726. The main molecular target of A77 1726 is dihydroorotate dehydrogenase, a key enzyme of de novo pyrimidine synthesis [2] . Consequently, the main effect of leflunomide in immune-mediated diseases has been attributed to the inhibition of B- and T-cells proliferation and the synthesis of proinflammatory cytokines [3,4] . Moreover, polymorphism in the DHODH gene may be associated with the response to treatment and toxicity of leflunomide [5,6] . Previous studies indicated that women have a poorer response to treatment than men [7,8] . Moreover it has been indicated that estrogens may modulate the action of
leflunomide in cell cultures [9,10] . Sex hormones, together with other factors, play an important role in the regulation of the immune response in R A [11] . Two estrogen receptors (ERs) have been identified and designated as ESRa (ESR1) and ESRb (ESR2). They are members of the superfamily of nuclear receptors, which can transduce extracellular signals into transcriptional responses. ERs act as ligand-activated transcription factors that reside in the cytosol and translocate into the nucleus upon ligand binding. Subsequently, ERs form dimers that interact with estrogen response elements in the promoter region of target genes. In addition, estrogens bind to plasma membrane-associated subpopulations of ERa and ERb, thereby activating a variety of rapid intracellular signaling cascades [12] . The human ESR1 gene is a large genetic unit that spans approximately 300 kb (including the 140 kb sequence containing the eight proteincoding exons), and is located on chromosome 6q25.1 [13] . The coding region has a length of 1785 nucleotides and it is translated into a protein of 595 amino acids and 66 kDa. The ESR1 gene is alternatively transcribed from at least seven promoters into multiple transcripts
10.2217/PGS.10.164 © 2011 Future Medicine Ltd
Pharmacogenomics (2011) 12(1), 41–47
Violetta Dziedziejko1, Mateusz Kurzawski2, Krzysztof Safranow1, Dariusz Chlubek1 & Andrzej Pawlik†3 Department of Biochemistry & Medical Chemistry, Pomeranian Medical University, Szczecin, Poland 2 Department of Experimental & Clinical Pharmacology, Pomeranian Medical University, Szczecin, Poland 3 Department of Pharmacokinetics & Therapeutic Drug Monitoring, Pomeranian Medical University, Szczecin, Poland † Author for correspondence: Tel.: +48 914 661 589 Fax: +48 914 661 600
[email protected] 1
ISSN 1462-2416
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Dziedziejko, Kurzawski, Safranow, Chlubek & Pawlik
that all vary in their 5’-UTRs. The primary transcripts are then spliced to a common splice acceptor site located at position +163 bp relative to the transcription start site generated by the most proximal promoter [14] . The ESR2 gene is located on chromosome 14q23.2 and comprises of nine exons spanning approximately 62 kb. ESR2 has multiple transcriptional isoforms, each representing a full-length transcript despite truncations at the 3´-end [15] . Two of the polymorphisms identified in the ESR1 gene: rs9340799:A>G (previously identified as XbaI restriction site) and rs2234693:T>C (previously identified as PvuII restriction site) are most widely investigated. They are located in the first intron of ESR1 gene, 397- and 351-bp upstream of exon 2, respectively [16,17] . In the ESR2 gene, two common polymorphisms have been investigated: synonymous rs1256049:G>A (Val328Val) in exon 6 and rs4986938:G>A located in 3´-UTR region [17,18] . The aim of the present study was to examine the association between polymorphisms in the ESR1 and ESR 2 genes and the response to treatment of female R A patients with leflunomide.
Material & methods Patients The study was carried out on 115 women, mean age 54.1 ± 11.0 years old, diagnosed with RA and treated with leflunomide (20 mg daily). During therapy with leflunomide, the patients were not administered any other antirheumatic drugs. R A was diagnosed according to the criteria of the American College of Rheumatology (ACR). All patients underwent a monthly evaluation for 1 year from the start of leflunomide treatment, applying the ACR definition of improvement in RA [19] . We analyzed the ACR core set of variables, including the number of swollen joints, the number of tender joints, patient’s global assessment of disease activity on a 10 cm visual analog scale (VAS), erythrocyte sedimentation rate (SR), C-reactive protein and disease activity score for 28 joints (DAS28). DAS28 was calculated according to 28 swollen joint and tender joint counts [20] , from the formula: DAS28 = (0.56 square root [tender joint count] + 0.28 square root [swollen joint] + 0.70 natural logarithm [erythrocyte SR]) + 0.014 patient’s global VAS [21] . The study was approved by the local ethics committee and written informed consent was obtained from all subjects. 42
Pharmacogenomics (2011) 12(1)
Genotyping Common SNPs in ESR1 (rs9340799:A>G, rs2234693:T>C) and ESR2 (rs4986938:G>A, rs1256049:G>A) genes were selected for the purpose of the current study. Genomic DNA was extracted from 200 µl of whole blood samples using GeneMATRIX Quick Blood DNA Purif ication Kit (EUR x, Poland). Prevalidated allelic discrimination TaqMan® (Applied Biosystems, CA, USA) real-time PCR assays (assay IDs: C___3163591_10, C _ _ _ 316359 0 _10, C _ _114 62726 _10, C___7573265_1) were used for detection of the respective SNPs in ESR1 and ESR2 genes. Fluorescence data were captured using an ABI PRISM 7500 FAST Real-Time PCR System (Applied Biosystems), after 40 cycles of PCR. Statistical analysis Distributions of the disease activity parameters were significantly different from normal (p C) and ESR2 (rs4986938:G>A and rs1256049:G>A) genes were examined using real-time PCR analysis. A significantly greater improvement of erythrocyte sedimentation rate, patient’s global assessment of disease activity on visual analog scale and disease activity score for 28 joints (DAS28) values were observed in patients with the AA rs9340799 genotype compared to patients with AG and GG genotypes. With regard to the ESR1 rs2234693 polymorphism, a significantly greater improvement of erythrocyte sedimentation rate, visual analog scale and DAS28 values were observed in patients with the TT genotype than in patients with CC genotype. There were no statistically significant associations of the improvement of disease activity parameters after 12 months of therapy with ESR2 genotypes and haplotypes. Conclusion The results of the present study suggest that ESR1 gene polymorphisms in females with RA may be associated with the response to treatment with leflunomide.
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