Pharmacogenetic Study of the Impact of ABCB1 Single Nucleotide Polymorphisms on the Response to Cyclosporine in Psoriasis Patients

Psoriasis is a chronic, T cell-mediated skin disease affecting 2–3% of the Caucasian population. Cyclosporine A is a calcineurin inhibitor that acts selectively on T cells. The cyclosporine A treatment response has been suggested to be modulated by single-nucleotide polymorphisms (SNPs) in the ABCB1 gene. The aim of this research was to evaluate the effect of ABCB1 genetic variants that could affect the response to a cyclosporine treatment in Russian psoriasis patients with the ABCB1 genotype status. The ABCB1 T-129C, G1199A, C1236T, G2677T/A and C3435T SNPs in the 168 patients with psoriasis were genotyped by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) and TaqMan SNP genotyping assays. The ABCB1 C1236T, G2677T/A and C3435T SNPs were significantly associated with a negative response to cyclosporine therapy. A very strong association was evident for the C3435T SNP in the ABCB1 gene in the allele, dominant and recessive models (OR = 2.58, OR = 4.01, OR = 2.50, respectively). ABCB1 C1236T and G2677T/A polymorphisms were significantly associated with a negative response to the cyclosporine therapy in the codominant, dominant and recessive models (p ˂ 0.05). Additionally, the haplotype analysis identified that the TGC haplotype is significantly associated with a negative response to cyclosporine therapy in psoriasis patients (p ˂ 0.05). The current study to the best of our knowledge is the first of its kind to be performed in the Russian population. In conclusion, the present results suggest an association between the ABCB1 genetic variants and unresponsiveness to cyclosporine in the Russian population. Further, larger studies are necessary to confirm our findings and replicate them in various ethnic populations before its implementation in the clinical practice.


Introduction
Psoriasis is a chronic T cell-mediated skin disease affecting 2-3% of the Caucasian population. Cyclosporine A (CsA) is a calcineurin inhibitor that acts selectively on T cells, thereby affecting the production of multiple inflammatory cytokines. CsA is used for the treatment of severe psoriasis, showing significant variability in its efficacy, which is associated with varying degrees of toxicity [1]. In particular, CsA is associated with several side effects including neurotoxicity, hypertension, hyperlipidemia and chronic nephrotoxicity. The absorption of CsA is highly variable in psoriasis patients which can be explained by the function and expression of the multidrug efflux transporter P-gp be explained by the function and expression of the multidrug efflux transporter P-gp Pglycoprotein (MDR1 Multidrug Resistance Protein 1, ABCB1 ATP Binding Cassette Subfamily B Member 1) (Figure 1). Figure 1. PGP (P-gp P-glycoprotein, ABCB1 (ATP-binding cassette sub-family B member 1), MDR1 (multidrug resistance protein 1), and CD243 expression in organs with excretory roles (e.g., liver, kidney and small intestine) and at blood-tissue barriers (e.g., blood-brain, blood-testis and bloodplacenta), plasma membrane, Golgi apparatus, endoplasmic reticulum, lysosomes and endosomes and cyclosporine transport. P-gp performs the function of the ATP-dependent efflux transporter pump for the substrates from the cytoplasm to the extracellular space [2]. ABCB1 plays a key role in the occurrence of adverse events and the efficacy of the treatment with CsA, methotrexate, etanercept, topical steroids, biologic agents and other [3]. At the tissue level, ABCB1 is expressed in the brain, skin, liver, pancreas, kidney, intestine, testis, ovary, endometrium, placenta and adrenal glands as well as in the blood-brain and blood-placenta barriers ( Figure 1) [4]. At the cellular level, the expression of ABCB1 was established in the plasma membrane, Golgi apparatus, endoplasmic reticulum, lysosomes and endosomes [5,6]. The expression of ABCB1 in psoriasis lesional skin was detected to be significantly decreased [7]. ABCB1 expression was inhibited by CsA in the peripheral blood monocytes of rheumatoid arthritis patients [8]. Lown   . PGP (P-gp P-glycoprotein, ABCB1 (ATP-binding cassette sub-family B member 1), MDR1 (multidrug resistance protein 1), and CD243 expression in organs with excretory roles (e.g., liver, kidney and small intestine) and at blood-tissue barriers (e.g., blood-brain, blood-testis and bloodplacenta), plasma membrane, Golgi apparatus, endoplasmic reticulum, lysosomes and endosomes and cyclosporine transport. P-gp performs the function of the ATP-dependent efflux transporter pump for the substrates from the cytoplasm to the extracellular space [2]. ABCB1 plays a key role in the occurrence of adverse events and the efficacy of the treatment with CsA, methotrexate, etanercept, topical steroids, biologic agents and other [3]. At the tissue level, ABCB1 is expressed in the brain, skin, liver, pancreas, kidney, intestine, testis, ovary, endometrium, placenta and adrenal glands as well as in the blood-brain and blood-placenta barriers ( Figure 1) [4]. At the cellular level, the expression of ABCB1 was established in the plasma membrane, Golgi apparatus, endoplasmic reticulum, lysosomes and endosomes [5,6]. The expression of ABCB1 in psoriasis lesional skin was detected to be significantly decreased [7]. ABCB1 expression was inhibited by CsA in the peripheral blood monocytes of rheumatoid arthritis patients [8]. Lown K.S. et al. revealed the significant role of intestinal P-glycoprotein in the first-pass elimination of CsA [9]. In mdr1a (murine MDR1 homologue) knockout mice, which were compared with wild-type mice, the CsA levels in the brain were significantly higher after the intravenous cyclosporine administration [10].
The multidrug resistance 1 (MDR1 or ABCB1) gene, which encodes a 170-kDa P-glycoprotein, is found on chromosome 7 and contains 29 exons. ABCB1 is a highly polymorphic gene. More than 990 ABCB1 SNPs have been identified, the allelic frequencies of which varies widely among different populations [11]. In general, it has been demonstrated that ABCB1 SNPs are detected in all of the studied populations [11,12]. Among the ABCB1 SNPs, three polymorphic variants, C3435T in exon 26, C1236T in exon 12 and G2677T/A in exon 21, were extensively investigated and were identified functionally important. African American people (10%) have the lowest frequencies of polymorphic alleles when they were compared with Caucasian people (42-46%) and Asian people (45%). Tang K. et al. examined the haplotypes and linkage disequilibrium architecture of the ABCB1 gene in Caucasian, African American, Chinese, Malaysian and Indian populations [13]. This study reported that the major haplotype 1236T-2677T-3435T is highly represented in the non-African populations, while the haplotype 1236C-2677G-3435C in the African American population indicating a varied haplotype diversity between the different populations. Kimchi-Sarfaty et al. performed C1236T, G2677T/A and C3435T SNPs analyses, and they determined their haplotypes in the Ashkenazi Jewish population [12]. The Ashkenazi Jewish population is similar to the Caucasian population in the allele and genotype frequencies, and the frequency of the common 1236T-2677T-3435T haplotype is 23.6% [12]. In another study, the frequency of the 3435C allele was lower in near Eastern Jewish population (0.445) and the Mediterranean group (0.58), while the frequencies of the C allele were quite comparable among the Ashkenazi (0.65), Yemenite (0.645), and North-African (0.615) Jewish populations [14].
Though the genetic polymorphism of ABCB1 may affect the disposition to the drug, produce variable drug effects, and may contribute to the disease risk susceptibility, there has been no pharmacogenetic study which analyzed the relationship between ABCB1 and the effectiveness of a cyclosporine treatment in the Russian population. The present study aimed to analyze the effect of ABCB1 polymorphisms on the response to cyclosporine therapies in Russian psoriasis patients who have an ABCB1 genotype status.

Patients
A total of 168 Russian psoriasis patients of European descent were recruited for genotyping ( Table 1). The age of the psoriasis patients ranged from 28 to 93 (mean ± standard deviation (SD), 66.3 ± 15.0; 65 females and 103 males). The patients were treated in the City dispensary of Dermatology and Venereology of Saint Petersburg, Russia. Each patient was evaluated according to the standard protocol, including a complete history and physical examination. Descriptive characteristics on psoriasis patient's demographics, comorbidities, and treatment history were collected. All of the patients had the classical pattern of skin lesions (chronic plaque lesions and psoriasis vulgaris) which were confirmed by a dermatologist. The Psoriasis Area and Severity Index (PASI) was applied to assess the disease activity and the effectiveness of the treatment [45]. All of the patients were treated with cyclosporine Neoral ® (NEO, Novartis, Basel, Switzerland) oral solution containing 100 mg of cyclosporine/mL twice daily in a dose of 3 mg/kg/day. The patients had not been administered any other drugs during the period of three months. The patients were classified as responders (PASI score > 75%) and non-responders (PASI ≤ 50%) after three months of treatment with cyclosporine. This study was conducted in accordance with the Declaration of Helsinki. Each individual provided written informed consent before being entered into study. The study was approved by the local hospital ethics committee.

DNA Isolation and Genotyping
Peripheral blood samples of all of the subjects were collected in ethylenediamine tetraacetic acid (EDTA) tube and stored at −70 • C. The genomic DNA was extracted from peripheral blood samples using a standard phenol-chloroform extraction method [46]. The DNA samples were quantified twice using the NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA, USA). The samples of the genomic DNA were stored at −80 • C until they were used. One hundred and sixty-eight patients treated with cyclosporine were genotyped for ABCB1 polymorphisms using polymerase chain reaction-restriction fragment length polymorphism analysis PCR-RFLP and verified by TaqMan SNP genotyping assays. The ABCB1 T-129C, G1199A, C1236T, G2677T/A and C3435T SNPs were selected from a previous study [29] and the National Center for Biotechnology Information (NCBI) website (http://www.ensembl.org; www.ncbi.nlm.nih.gov/SNP (accessed on 1 January 2021)). We followed the genotyping protocol described by Vasilopoulos et al. in 2014 [29]. Primer sequences and annealing temperatures used for the analysis of each polymorphism are summarized in Table 2. We analyzed the T-129C, G1199A, C1236T, G2677T/A and C3435T SNPs in the ABCB1 gene (Table 2).
We followed the genotyping protocol described by Vasilopoulos et al. in 2014 [45]. The primer sequences and annealing temperatures used for the analysis of each polymorphism are summarized in Table 3. Moreover, the SNPs of the ABCB1 gene were reanalyzed by a Real-Time PCR using TaqMan ® probes-C_27487486_10 was used for T-129C, C_15951365_20 was used for G1199A, C_7586662_10 was used for C1236T, C_11711720C_30 and C_11711720D_40 were used for G2677T/A, and C_7586657_20 for C3435T (Applied Biosystems Inc., Waltham, MA, USA) with TaqMan ® Genotyping Master Mix, respectively.  Table 3. List of SNPs, primer sequences, type of enzymes used for PCR-RFLP analysis in this study.

Restriction
Enzyme, Incubation Conditions

Statistical Analysis
The allele, genotype, and genetic models frequencies and Hardy-Weinberg equilibriums were analyzed using the PLINK version 1.90. The differences in the Hardy-Weinberg equilibrium among all of the individuals in the allele and genetic model frequencies between the studied groups were assessed by Chi square (χ 2 ) test. The statistical significance threshold was set to 0.05 for all of the tests. The odds ratios (ORs) and 95% confidence intervals (CI) were calculated where a statistically significant difference in the allele and genetic model frequencies was found. The linkage disequilibrium (LD) block between the SNP pairs in the genomic region of the ABCB1 locus and haplotypes were estimated using Haploview version 4.1 (Daly Lab, Cambridge, MA, USA) [47,48]. Table 1 shows the clinical characteristics of the psoriasis patients who participated in the current study. The patients were classified as responders (PASI score > 75%) and non-responders (PASI ≤ 50%) after three months of treatment with cyclosporine.

Allele and Genetic Model Association Analysis with Response to Cyclosporine Treatment
All of the SNPs were in the Hardy-Weinberg equilibrium. The results of the ABCB1 T-129C, G1199A, C1236T, G2677T/A and C3435T SNP association analysis with response to the cyclosporine treatment are summarized in Table 3. The ABCB1 C1236T, G2677T/A and C3435T SNPs polymorphisms were significantly associated with a negative response to the cyclosporine therapy. The most significant results were found with C3435T SNP and were associated with an unresponsiveness to the cyclosporine therapy in the allele, dominant and recessive models (Table 4). The allele 3435T in the ABCB1 gene demonstrated an association with a negative response to the cyclosporine therapy (OR = 2.58 95%, CI = 1.64 -4.06) in the Russian population. Namely, the frequency of allele T of the C3435T SNP in the ABCB1 gene was significantly higher in the nonresponders compared to that in the responders (0.57% vs. 0.34%, respectively; Table 4). The risk of no response to the cyclosporine treatment was 1.6-fold higher in the carriers of the dominant model TT + CT vs. CC compared with the recessive TT vs. CC + CT model carriers (OR = 4.01, OR = 2.50, respectively). Concerning the G2677T/A SNP, because only one psoriasis patient had an A allele, the carriers of that allele were excluded from the analyses. Moreover, the ABCB1 C1236T and G2677T/A polymorphisms were significantly associated with a negative response to the cyclosporine therapy in the codominant, dominant and recessive models (p < 0.005). In addition, the association analysis revealed no statistically significant difference in the allele and genotype frequencies between the responders and nonresponders for the T-129C and G1199A SNPs.

Haplotype Association Analysis with Response to Cyclosporine Treatment
The haplotype specific analyses of the ABCB1 polymorphisms are presented in Table 5. The haplotypes with a frequency below 1% were excluded from the analyses, thereby improving the statistical power. The linkage disequilibrium (LD) analysis indicated the existence of one haplotype block (formed by the C3435T, G2677T/A and C1236T SNPs) in the chromosome 7 region among the Russian group. Additionally, the haplotype analysis provided one haplotype that was significantly associated with a negative response to the cyclosporine therapy in the Russian population. Namely, the block 1 haplotype TGC frequency was significantly higher in the nonresponders compared to the responders (0.27% vs. 0.11%, respectively; Table 4). In addition, the association analysis revealed no statistically significant difference in the haplotype frequencies between the responders and nonresponders for the haplotypes CTC, CGC, CTT and TTC.

Discussion
The development of drug resistance decreases the effectiveness of drug treatment and increases the cost of drug development. In addition, the treatment of psoriasis is associated with a considerable economic burden, with the average annual costs per patient being EUR 11928 in Sweden, EUR 8372 in Italy, and EUR 2866-6707 in Germany based on the treatment type [49]. In searching for the discovery in the multidrug resistance phenotype, many studies have been focused on the MDR1 gene. Taking into account that cyclosporine is frequently used in the treatment of various autoimmune diseases, such as psoriasis, rheumatoid arthritis, myasthenia gravis, systemic lupus erythematosus and diabetes mellitus, the identification of MDR1 polymorphisms may help us to find the cause of the ineffectiveness of their therapy.
In this study, we identified the SNPs in the ABCB1 gene as genetic variants with clinically relevant effects on the psoriasis response of cyclosporine. The ABCB1 C3435T, G2677T/A, and C1236T polymorphisms were significantly associated with a negative response to the cyclosporine therapy in the allele, codominant, dominant and recessive models. The most significant result was obtained with ABCB1 C3435T SNP that was associated with a negative response to the cyclosporine therapy. Additionally, the haplotype analysis presented that the TGC haplotype was significantly associated with a negative response to the cyclosporine therapy in the Russian population. Our study had some limitations. Firstly, we did not have a replication cohort in the present study, which would have validated our results, and secondly, our study had a limited sample size. Further studies of different populations are required in order to explore the influence of these variants of the ABCB1 gene on the effectiveness of the cyclosporine treatment in psoriasis patients.
The 3435T allele frequency is known to vary amongst populations with a high prevalence in the Caucasian population [50]. The synonymous C3435T SNP seems to be of the highest biological importance in protein functioning by changing the mRNA stability, performing alternative splicing or by the modification of the translation efficiency [35]. The homozygous TT genotype is associated with more than two-fold lower duodenal ABCB1 protein expression when it was compared with the CC samples [15].
Adult cardiac transplant studies have demonstrated inconsistent results [61,62]. In study of 170 heart transplant recipients, the ABCB1 3435CC genotype was associated with an increased risk of rejection [61]. Taegtmeyer et al. did not find the ABCB1 3435CC genotype to be risk factors for the development of acute rejection [62].
Kwan et al. investigated impact of the ABCB1 C3435T SNP in drug resistance in 746 Han Chinese people, and they showed a significantly higher the TT genotype frequency in the patients with drug-resistant epilepsy [63]. However, the effect the C3435T SNP was not confirmed in some studies that were conducted on Caucasian populations [64,65]. Nevertheless, a meta-analysis in 3,912 drug-resistant epileptic patients and 4,419 epileptic patients established the association C3435T polymorphism with drug resistance in epilepsy in a Caucasian population [66]. A subsequent meta-analysis detected the association of the ABCB1 3435TT genotype with the risk of antiepileptic drugs resistance [67].
Numerous associations' studies and meta-analyses have established the impact of the ABCB1 rs1045642 genetic variant in cancer treatment outcomes concerning chemotoxicity, overall survival, and therapeutic responses. The impact of the C3435T SNP on the imatinib response in chronic myeloid leukemia (CML) patients have been widely estimated, however, the results of the studies were contradictory. A meta-analysis by combining data from a total of 12 studies including 1826 patients indicated that the 3435T allele predicted a worse response to imatinib in CML patients. Loscocco et al. found that the ABCB1 3435TT genotype correlated with a higher probability to achieve an MR3 molecular response in a shorter time in 90 CML patients that were treated with nilotinib [68]. Gregers et al. observed higher liver toxicity values after high-dose methotrexate was administered to acute lymphoblastic leukemia patients with the 3435CC variant versus the 3435CT/TT one [69]. In a meta-analysis involving nine (770 patients), five (566 patients) and three studies (367 patients), no significant association of C3435T in a dominant genetic model with a response to chemotherapy in patients with breast cancer was seen [70]. However, the results did not change after the stratification by ethnicity, cancer type and response criteria [70]. Schaich et al. examined the association of the genetic variants of the ABCB1 gene with the survival of glioblastoma patients who were treated with temozolomide in [71]. The ABCB1 3435CC genotype is associated with a 37% survival rate in glioblastoma patients after a temozolomide treatment [71]. The carriers of the homozygous 3435CC genotype had a higher survival rate when they were compared with the heterozygous genotype carriers in an Indian population [72]. Malmström et al. found a significant correlation with survival for the ABCB1 G1199A SNP in a Swedish population, with the median OS for the homozygous GG patients being 18.2 months versus 11.5 months for the heterozygous AG (p = 0.012) [73].
In total, there are inconsistent results in the studies regarding the effects of ABCB1 polymorphisms on the treatment response in patients with different diseases, and these can be due to them having small sample sizes, inter-ethical variability, differences in concomitant medication and using single time-point sampling [74].

Conclusions
In conclusion, we demonstrated a strong association between the cyclosporine therapy in psoriasis and the ABCB1 C1236T, G2677T/A and C3435T SNPs. Further studies with a larger sample size are needed to verify this issue. The detection of pharmacogenetic markers of a treatment response may be useful in predicting the clinical response to psoriasis therapies, and these would help in the improvement of personalized therapy. In future, combination OMICs technologies with known clinical and immunological data will allow us to identify the potential pharmacogenetic markers and therapeutic targets [3].
Author Contributions: A.C. and E.G. had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Concept and design: E.G. Acquisition, analysis, or interpretation of data: all authors. Drafting of the manuscript: E.G. and T.S. Critical revision of the manuscript for important intellectual content: All authors. Statistical analysis: D.K. and E.G. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement:
This study was conducted in accordance with the Declaration of Helsinki. The study was approved by the local hospital ethics committee (No 37818).

Informed Consent Statement:
Each individual provided written informed consent before being entered into study.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.