A pharmacogenetic study of ABCB1 polymorphisms and cyclosporine treatment response in patients with psoriasis in the Greek population

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Abstract

Psoriasis affects 2–3% of the population, causing significant morbidity and financial burden. Immunosuppressive drugs such as cyclosporine are first line systemic therapies for moderate-to-severe forms. However, patients exhibit heterogeneity in their response to therapy, possibly due to genetic factors. The aim of the present study was to assess the ABCB1 T-129C, G1199A, C1236T, G2677T and C3435T single-nucleotide polymorphisms (SNPs) as candidate predictive markers of response to cyclosporine treatment in 84 psoriasis patients. 62% of the patients were defined as responders and 38% as nonresponders. All SNPs complied with Hardy–Weinberg equilibrium. SNP and haplotype analyses were performed to access responsiveness to treatment. Association analysis revealed statistically significant association of SNP 3435 T with negative response (P=0.0075), a result that was further validated in haplotype analysis. This study is the first in the field of the pharmacogenetics of cyclosporine in psoriasis whose results merit further exploitation in larger independent cohorts.

Introduction

Psoriasis is a chronic inflammatory skin disease affecting 2–4% of the Caucasian population.1 The level of severity may depend on patient’s age as well as the strong interplay between genetic and immunological factors.2 In patients with moderate-to-severe psoriasis or with a high percentage of affected body surface, systemic therapies like cyclosporine (Neoral) are administered as first line of defense against the progression of the disease.2 However, their efficacy varies from 50–70% that couples with the development of severe side effects including nephrotoxicity and gastrointestinal symptoms, which creates the need for more careful selection of patients that will be treated by these regimens.3 This substantial heterogeneous response to treatment may reflect complex genetic heterogeneity. Indeed recent pharmacogenetic studies, mainly in biological agents for psoriasis, have indicated a role for single-nucleotide polymorphisms (SNPs) in response to the anti-tumor necrosis factor drugs used to treat psoriasis.4, 5 Therefore polymorphisms that may affect the function of genes regulating the transport, activity and/or effectiveness of cyclosporine could be good candidates to test for association with prediction to therapy.

Such a gene is the efflux transporter ABCB1 (MDR1) encoding for the P-glycoprotein.6 Today the important role of P-glycoprotein in drug disposition is well established, acting either alone or synergistically with the major drug metabolizing enzyme CYP3A4 thus significantly influencing the influx–efflux of active drug into target cells.7 ABCB1 appears to be a functionally highly polymorphic gene with more than 100 SNPs identified in the coding regions of the gene.6, 8 In the case of cyclosporine, there is no pharmacogenetic study found in literature in the field of psoriasis, but only in pediatric renal transplantation candidates.9

Therefore the aim of the current study, which was based on a Greek collaboration, was to perform the first pharmacogenetic analysis on cyclosporine, by selecting functional polymorphisms covering most of the coding regions of the gene locus that could influence the absorption and disposition of P-glycoprotein substrate drugs like cyclosporine in psoriasis patients to assess prediction of response to drug therapy.

Materials and methods

Patients’ profile and clinical data

Eighty-four patients (39 male, 45 female) with psoriasis who underwent treatment with cyclosporine for at least 3 months at the outpatient clinics of the Department of Dermatology at the University General Hospital Larissa and the Second Department of Dermatology at the Aristotle University of Thessaloniki at Papageorgiou Hospital in Greece, were included in this study. All the patients were treated with cyclosporine (Neoral) under standard doses described in its product special sheet (3 mg kg−1 daily). The patients received no other drug or emollient during the period of the three months. The mean age of the patients was 37.9 years and the mean duration of the disease was 4.3 years. No other special selection criteria were applied for entry in the study. This study adheres to the Declaration of Helsinki guidelines. Written informed consent to participation was obtained from each individual and the research protocol was approved by the local ethics committee.

Clinical and laboratory assessments

The Psoriasis Area and Severity Index (PASI) was applied to assess disease activity, whereas response to treatment after three months was evaluated using the modified PASI index, as before.4 Responders were defined as the ones that had a change in PASI >75%, whereas nonresponders were defined as the ones that had a change in PASI 50%, after three months of therapy with cyclosporine. All psoriasis patients were examined for the presence of comorbidities, most of which included hypertension, hypothyroidism and hyperlipidemia, and a small percentage (<5%) also had arthritis. Blood samples were obtained from consenting patients when they required a blood test as part of routine care.

DNA isolation and genotyping

Genomic DNA was isolated using a standard phenol/chloroform extraction method. Genotyping for the selected ABCB1 SNPs was performed using polymerase chain reaction-restriction fragment length polymorphism analysis or sequencing. Briefly the following primers and annealing temperatures were used for PCR amplification: T-129C (rs3213619)—forward 5′IndexTermATTGGCTGGGCAGGAACA3′, reverse 5′IndexTermTTTGGAAGATACTCCGAC3′, 58 oC; G1199A (rs2229109)—forward 5′IndexTermCAGCTATTCGAAGAGTGGGC3′, reverse 5′IndexTermCCGTGAGAAAAAAACTTCAAGG3′, 57 oC; C1236T (rs1128503)—forward 5′IndexTermTCTTTGTCACTTTATCCAGC3′, reverse 5′IndexTermTCTCACCATCCCCTCTGT3′, 58 oC; G2677T (rs2032582)—5′IndexTermTGCAGGCTATAGGTTCCAGG3′, reverse 5′IndexTermTTTAGTTTGACTCACCTTCCCG3′, 58 oC and C3435T (rs1045642)—forward 5′IndexTermTAGGCCAGAGAGGCTGCC3′, reverse 5′IndexTermAGTGGCTCCGAGCACACC3′ 58 oC. The following PCR conditions were used at a final volume of 50 μl: Kapa (Kapa Biosystems, Wilmington, MA, USA) polymerase buffer (1X), 2 mM MgCl2, 1 μl dNTPs (deoxynucleotide triphosphates, 10 mM), 0.25 mM of each dNTPs 1U Kapa Taq polymerase, 50 pmol of each primer (forward–reverse), 100 ng μl−1 DNA template. The following cycler conditions were used: 95 oC for 4 min, 95 oC for 40 s, 57–58 oC for 45 s, 72 oC for 40 s (35 cycles) and 72 oC for 10 min.

Statistical analysis

For SNP analysis, responders and nonresponders were compared nonparametrically by means of using 2 × 2 tables by grouping the heterozygous and homozygous for the rare allele and comparing the risk for the individuals grouped for the common allele, in each case. The χ2 test was used to test for Hardy–Weinberg equilibrium. Test for association of each SNP with disease status was performed using the Cochran–Armitage trend test, as well as allelic and genotypic tests and tests of dominant or recessive gene action. In addition to analyses performed for each individual SNP, haplotypes were constructed using the HapStat software platform.10 The threshold haplotype frequency value for inclusion in the analysis was set at 5%. Fisher’s exact test was used to compare genotype groups. All the analyses were performed using SPSS 14 (Chicago, IL, USA). A value of P<0.05 was used to identify a significant result. All significant P-values were corrected for multiple testing after applying Bonferroni correction. The power of the study was assessed with the G*Power software applying the post hoc regression omnibus F-test.11

Results

The major clinical characteristics of the psoriasis patients who participated in this study are summarized in Table 1. On the basis of the ΔPASI change, overall 52 patients (62%) were defined as responders to cyclosporine treatment by achieving a reduction of >75% in their PASI after 3 months of treatment, a finding that falls within the range of the drug’s efficiency, despite variations that may exist due to ethnic and population characteristics of the cohort under study, compared with the ones used in clinical trials. Furthermore comorbidities were found in 46% of the psoriasis patients, including mainly hypertension and hyperlipidemia that are components of the metabolic syndrome, a term given to characterize a cluster of clinical parameters with its prevalence in concordance with psoriasis being continuously rising (Table 1).12

Table 1 Clinical characteristics of patient population (n=84) used in the present study

All SNPs studied complied with Hardy–Weinberg equilibrium distribution. Results of ABCB1 SNP association analysis with response to cyclosporine treatment are shown in Table 2. This analysis showed no statistically significant difference in allele and genotype frequencies between responders and nonresponders for SNPs T-129C, G1199A, C1236T and G2677T. Interestingly, ABCB1 C3435T was found to be associated with response to cyclosporine treatment, even after Bonferroni correction (PC=0.0075). In detail, the frequency of the rare allele 3435T was found to be significantly increased in nonresponders (0.58) compared with responders (0.35) (odds ratio: 2.995 (1.448, 6.226); PC=0.0075). Further haplotype analysis strengthened this association with allele 3435T being the responsible risk marker (Table 3; P<0.001).

Table 2 Results of association tests between psoriasis patients’ ABCB1 genotype and response to cyclosporine therapy at 3 months
Table 3 Results of haplotype association tests with response to cyclosporine therapy

Discussion

The use of cyclosporine in the therapeutic portfolio against psoriasis some decades ago has improved patients’ quality of life.2 However, the long-term usage of such therapeutic regimens showed variability in efficiency and unpredictable risk of serious side effects, including nephrotoxicity and gastrointestinal symptoms, without a significant clinical benefit in many cases. From such findings stemmed the need of identifying good noninvasive biomarkers to efficiently predict response to treatment. To address the latter, we performed the first multicenter pharmacogenetic study in the field of psoriasis and cyclosporine.

Our results revealed an association of ABCB1 C3435T polymorphism with negative response to cyclosporine therapy in psoriatic patients, a finding that was confirmed by both SNP and haplotype analysis. This polymorphism is a synonymous one and it could affect P-glycoprotein levels by an effect either in mRNA stability, alternative splicing or translation efficiency.13, 14 Interestingly a previous study has shown that subjects homozygous for the allele 3435T exhibit half the activity of P-glycoprotein, compared with the ones that carry the C3435 allele.15 Lower levels of P-glycoprotein, in principle, could suggest impaired drug transport and thus reduced efficiency of therapy. In addition, as depicted from the frequencies of our haplotypes analysis, the associated marker C3435T would be informative for approximately 20% of the psoriatic patients suggesting that other genes and/or pathways could also be involved in the prediction of response to therapy, something that systems biology could reveal as the next target for future pharmacogenetic studies in psoriasis and cyclosporine therapy.16

Our study had sufficient power for the reported analyses. Specifically, our analysis had power over 0.85 to detect an effect of the ABCB1 genotype, explaining at least 50% of the variance in ΔPASI in patients treated with cyclosporine. No further analysis of patients for smaller ΔPASI percentiles was performed because these strata were underpowered. However it should be noted that the results in the present article should be taken cautiously as our study had some limitations, mainly being the medium-size sample size. Replicating our findings in larger independent cohorts could overcome this limitation and provide sufficient power to test for genetic predisposition that affects patient response to cyclosporine therapy, thus increasing its efficiency while reducing cost and unwanted toxicity.

In conclusion to the best of our knowledge this is the first study in the field of cyclosporine pharmacogenetics in psoriasis, demonstrating a significant association of ABCB1 C3435T polymorphism in predicting response to cyclosporine treatment in psoriasis. Notwithstanding the importance of our findings both from a clinical and economical perspective, our results merit further exploitation and validation in larger independent cohorts constructed by multicenter as well as multinational collaborations to identify additional efficient biomarkers in the prognosis of psoriasis patients’ response to cyclosporine therapy.

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Acknowledgements

We are very grateful to all the participants of the study. This work was supported by the postgraduate programs ‘Biotechnology-Quality Assessment in Nutrition and the Environment’ and ‘Molecular Biology and Genetics Applications—Diagnostic Markers’ of the Department of Biochemistry and Biotechnology, University of Thessaly, Greece.

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Correspondence to Y Vasilopoulos.

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Vasilopoulos, Y., Sarri, C., Zafiriou, E. et al. A pharmacogenetic study of ABCB1 polymorphisms and cyclosporine treatment response in patients with psoriasis in the Greek population. Pharmacogenomics J 14, 523–525 (2014) doi:10.1038/tpj.2014.23

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