ABCC1, ABCG2 and FOXP3: Predictive Biomarkers of Toxicity from Methotrexate Treatment in Patients Diagnosed with Moderate-to-Severe Psoriasis

Citation: Membrive-Jiménez, C.; Vieira-Maroun, S.; Márquez-Pete, N.; Cura, Y.; Pérez-Ramírez, C.; Tercedor-Sánchez, J.; Jiménez-Morales, A.; Ramírez-Tortosa, M.d.C. ABCC1, ABCG2 and FOXP3: Predictive Biomarkers of Toxicity from Methotrexate Treatment in Patients Diagnosed with Moderate-to-Severe Psoriasis. Biomedicines 2023,11, 2567. https://doi.org/10.3390/ biomedicines11092567 Academic Editor: Dae Joon Kim Received: 7 August 2023 Revised: 1 September 2023 Accepted: 15 September 2023 Published: 19 September 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biomedicines Article ABCC1,ABCG2 and FOXP3: Predictive Biomarkers of Toxicity from Methotrexate Treatment in Patients Diagnosed with Moderate-to-Severe Psoriasis Cristina Membrive-Jiménez 1, Sayleth Vieira-Maroun 1, Noelia Márquez-Pete 1, Yasmin Cura 1, Cristina Pérez-Ramírez 1,2,* , Jesús Tercedor-Sánchez 3, Alberto Jiménez-Morales 4,† and María del Carmen Ramírez-Tortosa 2,† 1Pharmacogenetics Unit, Pharmacy Service, University Hospital Virgen de las Nieves, 18014 Granada, Spain; [email protected] (N.M.-P.) 2Department of Biochemistry and Molecular Biology II, Faculty of Pharmacy, University of Granada, 18011 Granada, Spain; [email protected] 3Dermatology Service, University Hospital Virgen de las Nieves, 18014 Granada, Spain 4Hospital Pharmacy Department, University Hospital Virgen de las Nieves, 18014 Granada, Spain *Correspondence: cper[email protected] †These authors contributed equally to this work. Abstract: Background: Methotrexate (MTX) is one of the most extensively used drugs in the treatment of moderate-to-severe psoriasis (PS). However, it frequently must be suspended owing to the toxicity in certain patients. Objective: To evaluate the influence of ABCC1, ABCG2, and FOXP3 in the development of MTX toxicity in PS. Methods: Retrospective cohort study with 101 patients. Five single-nucleotide polymorphisms (SNPs) were genotyped using real-time polymerase chain reaction with TaqMan probes. Results: Patients carrying ABCC1 rs2238476-AG genotype (AG vs. GG: OR = 8.04; 95% CI = 1.48–46.78; p= 0.015); FOXP3 rs376154-GT and GG genotypes (GT vs. TT/GG: OR = 3.86; 95% CI = 1.17–13.92; p= 0.031) and ABCG2 rs13120400-T allele (T vs. CC: OR = 8.33; 95% CI = 1.24–164.79; p= 0.059) showed a higher risk of developing more than one adverse effect. The toxicity analysis by subtypes showed that the ABCC1 rs2238476-AG genotype (AG vs. GG: OR = 8.10; 95% CI = 1.69–46.63; p= 0.011) and FOXP3 rs376154-GT genotype (OR = 4.11; 95% CI = 1.22–15.30; p= 0.027) were associated with the appearance of asthenia. No association of the other ABCC1 polymorphisms (rs35592 and rs246240) with MTX toxicity was found. Conclusion: ABCC1, ABCG2, and FOXP3 polymorphisms can be considered to be risk biomarkers of toxicities in PS patients treated with MTX. Keywords: psoriasis; pharmacogenetics; methotrexate; genetic polymorphisms; toxicity; adverse events; adverse drug reactions; hepatotoxicity 1. Introduction Psoriasis (PS) is a chronic, inflammatory, autoimmune disease affecting 1–3% of the population worldwide [ 1 , 2 ]. The main characteristic is the production of skin lesions, but it is also associated with potentially disabling pathologies, such as erectile dysfunction in 35% of patients and arthropathy in 40% [ 3 – 8 ]. Consequently, psoriasis is considered a systemic entity that severely impacts the quality of life of patients [9]. Etiology is not completely defined; however, it is believed that different factors are involved [ 10 , 11 ]. Regarding genetics, it has been observed that there are multiple chromosomal loci associated with susceptibility to psoriasis (PSORS), the most prominent being PSORS1, responsible for 50% of the heritability of the disease [ 12 ]. In addition, the incidence of psoriasis is different according to ethnicity, and higher among relatives and even more so among monozygotic twins [ 13 ]. The presence of the human leukocyte antigen HLA-Cw6 is Biomedicines 2023,11, 2567. https://doi.org/10.3390/biomedicines11092567 https://www.mdpi.com/journal/biomedicines Biomedicines 2023,11, 2567 2 of 16 particularly associated with phenotypic features, along with early development and course of the disease [14,15]. Genetic and environmental factors such as stress, infections, or unhealthy lifestyle habits predispose to the onset of the pathology, while abnormalities in cutaneous immune responses are responsible for the development and maintenance of psoriatic inflammation [ 10 , 14 ]. When plasmacytoid dendritic cells, keratinocytes, natural killer cells, and macrophages are activated they secrete various cytokines (tumor necrosis factor and Interleukin 1, respectively) that stimulate myeloid dendritic cells [ 16 ]. The activated dendritic cells promote the production of a cytokine cascade that activates keratinocyte proliferation in the epidermis. Consequently, hyperproliferation of keratinocytes in the epidermis and vascular endothelium occurs, resulting in epidermal hyperplasia typical of psoriatic lesions [11]. There are different types of psoriasis, but the most prevalent is “psoriasis vulgaris or plaque psoriasis” characterized by clinical manifestations in the form of erythematous plaques on the scalp, elbows, knees and back covered with whitish scales [ 17 – 19 ]. The severity of the lesions is measured with the indicators of psoriasis area severity index (PASI), body surface area (BSA), and dermatologic quality-of-life index (DLQI). When these indicators are greater than 10, moderate–severe psoriasis is considered [ 20 ]. The treatments employed are aimed at blocking the inflammatory response [ 21 ]. Pharmacological treatment is chosen according to the severity, in mild psoriasis the treatment is mainly based on topical and symptomatic therapy [ 22 , 23 ]. However, in cases of moderate–severe psoriasis, treatment with systemic therapy (methotrexate, cyclosporine, acitretin, apremilast, fumaric acid esters), phototherapy, or photochemotherapy is indicated [ 9 , 24 , 25 ]. In patients diagnosed with moderate–severe psoriasis who do not respond to systemic treatment or who are contraindicated, treatment with biologics is used [ 20 ]. Generally, the drug most frequently used in the treatment of moderate-to-severe PS is methotrexate (MTX) at low doses, because of the benefits it shows (proven effectiveness, low cost, relatively simple administration, and its usefulness in combination with other treatments) [ 26 ]. Furthermore, it can be administered orally or subcutaneously, the latter route being the more advantageous, with better absorption and greater bioavailability [27,28]. Methotrexate is an antimetabolite with antiproliferative, anti-inflammatory, and immunosuppressive activity. Although the mechanism of action at low doses is not clear, it is related to the ability to form intracellular polyglutamates and to increased adenosine formation due to inhibition of the ATIC (5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase) enzyme, which leads to apoptosis in activated lymphocytes or to inhibition of the activation and expression of certain adhesion molecules in them [ 29 – 31 ]. Consequently, the action of MTX may be conditioned at various points on the metabolic pathway [32–34]. This drug has proved effective, reaching PASI75 and PASI90 after 16 and 24 weeks of treatment. However, it is frequently associated with reversible adverse effects (AEs) which entail temporary interruption of treatment or modification of the dose [ 35 ]. Long-term studies show that AEs occur in around 61–95% of patients [ 36 ]. The most common AEs are nausea, anorexia, and asthenia, depending on the dose and usually occurring at the start of therapy [25]. Moreover, prolonged use of MTX is associated with hepatotoxicity and hematological, renal, pulmonary, and cutaneous toxicity, among others [ 37 ]. Specifically, long-term treatment with MTX is associated with progressive and dose-dependent hepatotoxicity. Previous studies have observed that 23–33% of patients show an increase in liver enzyme levels; however, in the absence of excessive alcohol consumption, it rarely causes clinically significant liver damage [ 38 ]. They have also observed that it may cause hematological toxicity through hematopoietic suppression of MTX, specifically pancytopenia, which is reversible with dose reduction or temporary withdrawal of MTX [ 39 , 40 ]. In 3% of the patients studied, renal toxicity occurred in prolonged treatment with MTX [ 37 , 40 ]. Pulmonary toxicity has also been described as a risk associated with MTX treatment; in particular, there are reports of pulmonary fibrosis and pneumonitis [ 26 ]. Severe skin reactions were Biomedicines 2023,11, 2567 3 of 16 observed in patients with long-term MTX; in particular, 4% of the patients studied showed alopecia [ 40 , 41 ]. Finally, it is important to note the association of MTX treatment with a higher incidence of certain malignancies, such as lymphoma [41]. Despite the risks involved in using MTX, it has been observed that AEs limit treatment in only 6.9% of patients treated with MTX for 6 months, and therefore the risk-benefit profile of MTX is acceptable [ 42 ]. However, it is crucial to conduct exhaustive pharmacotherapeutic follow-ups of patients receiving treatment with MTX and to use folic acid supplementation [43]. In conclusion, although low-dose MTX treatment in patients diagnosed with moderateto-severe PS is safe and effective, certain patients do not attain an optimum response or they experience various degrees of toxicity [ 44 ]. This variability in response and toxicity may be due to genetic factors. Interindividual variations in the genes implicated in the disease environment of the condition, pharmacokinetics, pharmacodynamics, metabolism, or in the mechanism of action of MTX may explain the emergence of adverse effects in certain patients treated with the same therapeutic conditions [ 45 ]. Specifically, genetic polymorphisms in the membrane efflux transporters responsible for the intracellular entry of MTX (ABCC1-4, ABCB1, and ABCG2), as well as other genes involved in the physiopathogeny of the disease have proved to play a crucial role in interindividual variability in response to and toxicity from MTX [46]. In particular, the ABCC1 gene is located on chromosome 16 and encodes the ABCC1 protein, also known as multidrug-resistance-associated protein 1 (MRP1). It is found mainly in the basolateral plasma membranes of the enterocytes transporting endogenous substances, as well as xenobiotics and their metabolites [ 46 – 48 ]. Genetic polymorphisms in ABCC1 (rs35592 T>C; rs246240 A>G, rs2238476 G>A) have been associated with response to and toxicity from MTX in psoriasis patients [49]. Similarly, the ABCG2 gene, on chromosome 4, codes for a membrane transporter characterized by being essentially a xenobiotic transporter that plays an important part in resistance to numerous drugs, such as MTX. The ABCG2 protein transports MTX and its forms of polyglutamate, specifically polyglutamate 2 and 3, and therefore plays a key role in MTX’s mechanism of action [ 50 ]. Previous studies have found an association between response to and toxicity from MTX treatment in patients diagnosed with PS and the polymorphism of this gene (ABCG2 rs13120400, C>T) [ 50 ]. Consequently, genetic alterations in ABCC1 and ABCG2 may modify the metabolism of MTX and lead to variability in the toxicity of this drug. On the other hand, the FOXP3 gene, located on the X chromosome, encodes a protein from the fork-winged helix family of transcription factors which plays an essential role in modulating regulatory T cells and can act as a transcriptional repressor or activator depending on its interactions with other transcription factors, histones, acetylases, and deacetylases [ 51 , 52 ]. Alterations in FOXP3 (rs3761548; G>C/T/A) can potentiate the inflammatory cascade in psoriasis and consequently not respond to and/or provoke AEs due to MTX treatment [53,54]. Based on all the foregoing, the object of this study was to evaluate the influence of polymorphisms of the ABCC1, ABCG2, and FOXP3 genes on the development of toxicity from treatment with MTX in moderate-to-severe psoriasis. 2. Materials and Methods 2.1. Study Design Retrospective observational cohort study. 2.2. Study Subjects The study included 101 Caucasian patients, over the age of 18, diagnosed with moderate-to-severe PS (BSA and PASI > 10), under treatment with MTX as monotherapy or in combination with biologic medications, for at least two months, from the Dermatology Department of the HUVN, during the period between January 2019 and November 2020. Biomedicines 2023,11, 2567 4 of 16 The starting dose of oral MTX was 15 mg/week, in combination with folic acid (5 mg/week, administered 24 h after the MTX), a dose approved in clinical practice guidelines [55]. 2.3. Sociodemographic and Clinical Variables The sociodemographic variables collected were sex, age at the start of MTX therapy, family history, and smoking and drinking status. We also collected data on the clinical features of the PS, including the type of psoriasis (plaque, pustular, inverse, guttate, or a combination of different types of psoriasis: plaque and guttate, plaque and pustular, plaque and inverse, plaque, guttate and inverse) and the location of the lesions (scalp and face, nails, palmoplantar, torso, and upper and lower extremities or flexures). In addition, we studied the development of psoriatic arthritis, concomitant diseases, therapy adherence, duration of MTX treatment, route of administration of MTX, concomitant medication, and maximum dose of MTX (mg/week). The adverse reactions collected were classified as gastrointestinal toxicity (nausea, vomiting, diarrhea, and stomatitis), hepatotoxicity (appearance of abnormalities in hepatic parameters [ALT/AST, GGT, total bilirubin, procollagen peptide], exacerbation or reactivation of hepatitis, fibrosis, or cirrhosis), hematological toxicity (anemia, leukopenia, thrombocytopenia, and/or pancytopenia), asthenia, infections, neurological toxicity (dizziness, headache disorders), skin toxicity (hair loss, skin rash), nephrotoxicity (appearance of abnormalities in renal parameters [creatinine, urea, uric acid] or renal disorders). 2.4. Sample Processing and Genotyping 2.4.1. DNA Isolation DNA was obtained from saliva samples with buccal swabs (OCR-100 kit), after the inclusion of the patients and signing of the informed consent. Subsequently, DNA extraction was performed, using the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany), following the manufacturer’s instructions for the purification of DNA from saliva, and stored at − 20 ◦ C. To check DNA concentration and purity values, a NanoDrop 2000 UV spectrophotometer was used with an absorbance ratio of 280/260 and 280/230. 2.4.2. Detection of Gene Polymorphisms The ABCC1 rs35592 (TaqMan assay ID C___1003671_10), ABCC1 rs246240 (assay ID C___1003698_10), ABCC1 rs2238476 (assay ID C___16172578_10), ABCG2 rs13120400 (assay ID C___9510480_10), and FOXP3 rs3761548 (assay ID C___27476877_10) gene polymorphisms were determined by real-time polymerase chain reaction (PCR) using TaqMan ® probes (ABI Applied Biosystems, 7300 Real-Time PCR System, Foster City, CA, USA). 2.5. Toxicity Variables Toxicity was evaluated according to the common terminology criteria for adverse events (CTCAE) using version 5.0 (U.S. Department of Health and Human Services (HHS), Washington DC, USA). The severity of the adverse events was classified as presence (toxicity grade 1–4) or absence (no presence of toxicity). General toxicity was defined as present when there was at least one adverse event (grade 1–4). The occurrence of more than one and more than two adverse events was also analyzed. 2.6. Statistical Analysis Statistical analysis was performed using the freely available software R 3.5.1 (R Foundation for Statistical Computing, Vienna, Austria). Normal quantitative variables were expressed as mean ( ± standard deviation) and as median (p50) and percentiles (25 and 75) for non-normal variables. Normality analysis was performed by applying the Kolmogorov– Smirnov test. Pearson’s chi-square test was used for bivariate analysis between toxicity and polymorphisms. Fisher’s exact test was applied for associations between toxicity and qualitative variables, while Student’s t-test was used for normal quantitative variables, and the Mann–Whitney test for non-normal variables. The association with SNPs was Biomedicines 2023,11, 2567 5 of 16 evaluated in multiple models (genotypic, dominant, and recessive), which were defined as follows: genotypic (DD vs. Dd vs. dd), dominant ((DD, Dd) vs. dd) and recessive (DD vs. (Dd, dd)), where D is the major allele (wild-type) and d the minor allele (variant). The adjusted odds ratio (OR) and 95% confidence interval (95% CI) were obtained by logistic or linear regression from multivariate analysis. The Hosmer–Lemeshow test was applied to determine the goodness-of-fit of each model. In addition, the omnibus test and the Cox-Snell and Nagelkerke r2 coefficients were calculated. We considered a statistically significant probability with a value of 0.05 or less. For the analysis of polymorphisms, haplotype frequencies were calculated, as well as Hardy-Weinberg equilibrium and linkage disequilibrium (Lewontin’s D (D’) and linkage disequilibrium coefficient (r2)). This genetic analysis was performed with the PLINK application for genome-wide association analysis and SNPstats, a web-based tool for association study analysis [56–59]. 3. Results 3.1. Patient Characteristics A total of 101 Caucasian patients diagnosed with moderate-to-severe PS under treatment with MTX were included in this study. As Table 1shows, the median age of the patients at diagnosis was 27.25 (18.42–44.25) years, and the majority were women (52/101; 51.49%), non-smokers (49/101; 48.51%) and non-drinkers (61/101; 60.40%). The information on family history of PS was positive in 51.49% of the patients (52/101). A total of 73.27% of them had PS vulgaris with plaques (74/101), with the lesions located mainly on the torso and upper and lower extremities (93/101; 92.08%). Thirty-one patients developed psoriatic arthritis (31/101; 30.69%). The mean age of starting MTX therapy was 45.60 ± 14.79 years, and the median duration of MTX treatment was 15 [ 5 – 33 ] months. In most of the patients, MTX was orally administered (60/101; 59.41%), as monotherapy (93/101; 92.08%), with a median maximum dose of 12.5 [10.0–15.0] mg/week, and 69.31% of the patients adhered to the treatment (70/101). Table 1. Clinicopathological characteristics of the 101 patients with moderate–severe psoriasis treated with methotrexate. Variable n% Media ±SD Gender Female 52 51.49 - Male 49 48.51 - Age diagnosis PS 101 - 27.25 (18.42–44.25) Family history PS 52 51.49 Smoking Smoker 31 30.69 - Non-smoker 49 48.51 - Former Smoker 21 20.79 - Alcoholic drinking Drinker 38 37.62 - Non-drinker 61 60.40 - Former drinker 2 1.98 - Type of PS Plaque 74 73.27 - Pustular 5 4.95 - Inverse 1 0.99 - Guttate 5 4.95 - Plaque and Guttate 12 11.88 - Plaque and Inverse 2 1.98 - Plaque and pustular 1 0.99 - Plaque, guttate and inverse 1 0.99 - Biomedicines 2023,11, 2567 6 of 16 Table 1. Cont. Variable n% Media ±SD Location of lesions Trunk and lower and upper limbs 93 92.08 - Scalp and face 77 76.24 - Nails 58 57.43 - Palmoplantar 19 18.81 - Flexures 28 27.72 - Psoriatic Arthritis 31 30.69 - Comorbidities 57 56.44 - Age of onset of MTX 101 - 45.60 ±14.79 Duration of MTX treatment (months) 101 - 15 (5–33) Administration type of MTX Oral 47 46.53 - Subcutaneous 30 29.70 - Both 24 23.76 - Type of MTX therapy Monotherapy 93 92.08 - Combination Therapy 8 7.92 - Maximum MTX dose (mg/week) 101 - 12.5 (10–15) Medication Adherence Adherent 70 69.31 - Non-adherent 31 30.69 - Toxicity (Grade 1–4) 69 68.32 - Gastrointestinal toxicity (Grade 1–4) 29 28.71 - Hepatotoxicity (Grade 1–4) 37 36.63 - Hematological toxicity (Grade 1–4) 3 2.97 - Nephrotoxicity (Grade 1–4) 1 0.99 - Asthenia (Grade 1–4) 28 27.72 - Nervous system toxicity (Grade 1–4) 9 8.91 - Skin Toxicity (Grade 1–4) 8 7.92 - Infections (Grade 1–4) 6 5.94 - Occurrence of adverse events - More than 1 (grade 1–4) 36 35.64 - More than 2 (grade 1–4) 15 14.85 - Qualitative variables: frequency (percentage, %). Quantitative variables: Normal distribution: mean ± standard deviation (SD). Non-normal distribution: P50 (P25–P75). The patients showed the following grade 1–4 toxicity values: 36.63% hepatotoxicity (37/101), 28.71% gastrointestinal toxicity (29/101), 27.72% asthenia (28/101), 8.91% neurotoxicity (9/101), 7.92% cutaneous toxicity (8/101), 5.94% infections (6/101), 2.97% hematological toxicity (3/101), and 0.99% nephrotoxicity (1/101). The clinical and sociodemographic characteristics are described in Table 1. 3.2. Influence of Clinical-Pathological Characteristics on Toxicity 3.2.1. Global Toxicity In the bivariate analysis a significant association was found between the presence of toxicity and the development of psoriatic arthritis (OR = 6.60, 95% CI = 2.08–29.44, p= 0.002; Table S1), and also with the MTX administration route (both routes: OR = 4.04, 95% CI = 1.28–15.53; subcutaneous: OR = 2.65, 95% CI = 1.02–7.78; p= 0.028; Table S1). In addition, a tendency was found toward an association between patients in whom PS was in the nails and the risk of toxicity from MTX (OR = 2.24, 95% CI = 0.89–5.81, p= 0.058; Table S1). In the multivariate analysis, no significant association was found between developing toxicity through MTX and the clinical and sociodemographic variables studied. Biomedicines 2023,11, 2567 7 of 16 Furthermore, a significant association was found between the presence of more than one adverse effect and patients who were women (OR = 3.20, 95% CI = 1.37–7.82, p= 0.007; Table S2), ex-smokers (OR = 5.56, 95% CI = 1.67–20.48, p= 0.019; Table S2) and with psoriatic arthritis (OR = 3.26, 95% CI = 1.36–8.01, p= 0.007; Table S2). The MTX administration route was also associated with the risk of developing more than one adverse effect (both: OR = 13.88, 95% CI = 4.45–49.21; subcutaneous: OR = 3.81, 95% CI = 1.32–11.81; p< 0.001; Table S2). Moreover, a tendency was found toward an association between the presence of more than one adverse effect and that of inverse PS (OR = 2.31, 95% CI = 0.94–5.72, p= 0.062; Table S2), as well as with the longer duration of MTX therapy (OR = 1.02, 95% CI = 1.00–1.04, p= 0.063; Table S2). The multivariate analysis showed an association between the emergence of more than one adverse effect, development of psoriatic arthritis (OR = 3.49, 95% CI = 1.29–9.91, p= 0.015), and MTX administration route (both: OR = 14.67, 95% CI = 4.51–54.89, p< 0.001; subcutaneous: OR = 3.32; 95% CI = 1.09–10.68, p= 0.036) (details of these values are shown in Table 2). Table 2. Multivariate regression analysis for the presence of more than adverse events based on clinical characteristics and genetic variables. Occurrence of Adverse Events OR (CI95%)p-Value >1 Adverse Event Development of psoriatic arthritis (yes) 3.49 (1.29–9.91) 0.015 MTX administration Subcutaneous 3.32 (1.09–10.68) 0.036 Both 14.67 (4.50–54.89) <0.001 The presence of more than two adverse effects during MTX therapy, after the bivariate analysis, was associated with female sex (OR = 4.16, 95% CI = 1.35–21.24, p= 0.017; Table S3) and the MTX administration route (both: OR = 6.04, 95% CI = 1.49–30.62; subcutaneous: OR = 2.93, 95% CI = 0.66–15.29; p= 0.038; Table S3). The multivariate analysis did not reveal an association between the presence of more than two adverse effects and the clinical-pathological variables studied. 3.2.2. Toxicity Subtypes In the bivariate analysis, no significant association was found between the emergence of hepatotoxicity and the clinical-pathological variables studied (Table S4). Conversely, the emergence of gastrointestinal toxicity was related to female sex (OR = 2.78, 95% CI = 1.14– 7.20, p= 0.026; Table S5), smoking status (non-smoker: OR = 1.88, 95% CI = 0.62–6.45; ex-smoker: OR = 5.72, 95% CI = 1.66–22.34; p= 0.016; Table S5), alcohol (non-drinker: OR = 3.99, 95% CI = 1.46–12.96; ex-drinker: OR = 6.60, 95% CI = 0.24–186.55; p= 0.013; Table S5), and MTX administration route (subcutaneous: OR = 2.08, 95% CI = 0.66–6.68; both: OR = 8.00, 95% CI = 2.65–26.52; p= 0.001, Table S5). Moreover, a tendency was found toward an association between gastrointestinal toxicity and PS located in the nails (OR = 2.48, 95% CI = 1.00–6.64, p= 0.053; Table S5). Similarly, developing asthenia was related to the MTX administration route (p< 0.001), showing a higher risk in those who used both routes (OR = 9.93; 95% CI = 3.07–36.90) and those who used only the subcutaneous route (OR = 4.20; 95% CI = 1.31–15.04) (details of all these values are given in Table S6). The bivariate analysis showed that the route of administration of MTX (subcutaneous: OR = 7.07, 95% CI = 0.98–142.41; both: OR = 9.19, 95% CI = 1.26–186.15; p= 0.047; Table S7), as well as the presence of inverse psoriasis (OR = 6.36, 95% CI = 1.55–32.17, p= 0.006; Table S7), was associated with a higher risk of showing neurotoxicity in our patients. In addition, the development of cutaneous adverse events was associated with patients suffering from psoriatic arthritis (OR = 8.16, 95% CI = 1.75–58.25, p= 0.009; Table S8). Biomedicines 2023,11, 2567 8 of 16 Similarly, the bivariate analysis revealed that patients with lesions on the scalp and face had a lower risk of developing infections (OR = 7.50, 95% CI = 1.36–56.97, p= 0.027; Table S9). No significant association was found between the emergence of hematological toxicity or nephrotoxicity and the clinical variables analyzed (Tables S10 and S11). 3.3. Influence of Genetic Polymorphisms on Toxicity 3.3.1. Genotype Distribution The distribution of all polymorphisms studied is consistent with that expected according to the Hardy-Weinberg equilibrium model (Supplementary Table S12). The linkage disequilibrium values D’ and r2 are presented in Supplementary Table S13. The minor allele frequencies of all polymorphisms were higher than 1% and therefore, none of them have been excluded from the analysis (Table S14). 3.3.2. Global Toxicity The bivariate analysis showed that patients carrying the ABCG2 rs13120400-TT and CT genotypes showed a higher risk of grade 1–4 toxicity during MTX therapy (TT vs. CC/CT: OR = 13.93, 95% CI = 2.02–279.52; CT vs. CC/TT: OR = 11.15, 95% CI = 1.59–225.34, p= 0.023; Table S15). Moreover, the ABCG2 rs13120400-T allele emerged as the allele for risk of MTX toxicity in our patients (T vs. CC: OR = 12.59, 95% CI = 1.92–247.28, p= 0.012; Table S15). In addition, in the multivariate analysis a tendency was found towards association between patients carrying the ABCG2 rs13120400-T allele (T vs. CC: OR = 8.33; 95% CI = 1.24–164.79; p= 0.059) with psoriatic arthritis (OR = 5.60; 95% CI = 1.74–25.18; p= 0.009) and the risk of developing toxicity during treatment with MTX (all these values are shown in Table 3). Table 3. Multivariate regression analysis for overall toxicity based on clinical characteristics and genetic variables. Overall Toxicity OR (CI95%)p-Value Development of psoriatic arthritis (yes) 5.60 (1.74–25.18) 0.009 ABCG2 rs13120400-T (T vs. CC) 8.33 (1.24–164.79) 0.059 In the study of the emergence of multiple adverse effects, the bivariate analysis showed an association between patients carrying the FOXP3 rs3761548-GT and GG genotypes and a higher risk of having more than one adverse effect (GT vs. TT/GG: OR = 3.24, 95% CI = 1.20–9.15; GG vs. TT/GT: OR = 1.03, 95% CI = 0.36–2.91; p= 0.034; Table S16). The multivariate analysis showed an association between the FOXP3 rs3761548-GT genotype (GT vs. TT/GG: OR = 3.86, 95% CI = 1.17–13.92, p= 0.031) and the risk of having more than one adverse effect, adjusted for MTX administration route (both: OR = 15.92, 95% CI = 4.60–64.78, p< 0.001; subcutaneous: OR = 4.07, 95% CI = 1.26–14.43, p= 0.022) and developing psoriatic arthritis (OR = 4.28, 95% CI = 1.48–13.45, p= 0.009) (all these values are shown in Table 4). Similarly, a significant association was found between the presence of more than two adverse effects and the ABCC1 rs2238476-AG genotype (OR = 4.85, 95% CI = 1.09–19.89, p= 0.039; Table S17) and FOXP3 rs3761548-GT and GG genotypes (GT vs. TT/GG: OR = 5.55, 95% CI = 1.47–27.20; GG vs. TT/GT: OR = 1.28, 95% CI = 0.22–7.34, p= 0.021; Table S17). After the multivariate analysis was performed, it was confirmed that patients carrying the ABCC1 rs2238476-AG genotype (AG vs. GG: OR = 8.04, 95% CI = 1.48–46.78, p= 0.015) and the FOXP3 rs3761548-GT genotype (GT vs. TT/GG: OR = 7.48, 95% CI = 1.68–46.23, p= 0.014) showed a higher risk of suffering more than two adverse effects, adjusted for MTX administration route (both: OR = 7.32, 95% CI = 1.54–47.02, p= 0.018; subcutaneous: OR = 4.89, 95% CI = 0.95–32.46, p= 0.069) (all these values are shown in Table 5). Biomedicines 2023,11, 2567 9 of 16 Table 4. Multivariate regression analysis for the presence of more than one adverse event based on clinical characteristics and genetic variables. More than 1 Adverse Event OR (CI95%)p-Value Development of psoriatic arthritis (yes) 4.28 (1.48–13.45) 0.009 MTX administration Both 15.92 (4.60–64.78) <0.001 Subcutaneous 4.07 (1.26–14.43) 0.022 FOXP3 rs3761548 (GT vs. TT/GG) 3.86 (1.17–13.92) 0.031 Table 5. Multivariate regression analysis for the presence of more than 2 adverse events based on clinical characteristics and genetic variables. More than 2 Adverse Events OR (CI95%)p-Value MTX administration Both 7.35 (1.54–47.02) 0.018 Subcutaneous 4.89 (0.95–32.46) 0.069 ABCC1 rs2238476 (AG vs. GG) 8.04 (1.48–46.78) 0.015 FOXP3 rs3761548 (GT vs. TT/GG) 7.48 (1.68–46.23) 0.014 3.3.3. Toxicity Subtypes The bivariate analysis showed a tendency towards an association between the presence of the ABCG2 rs13120400-TT genotype and the risk of developing hepatotoxicity during MTX treatment (C vs. TT: OR = 0.44, 95% CI = 0.19–1.02, p= 0.058; Table S18). As for the emergence of asthenia, patients carrying the ABCC1 rs2238476-AG genotype showed a higher risk of having this adverse event during therapy with MTX (OR = 4.70, 95% CI = 1.23–19.87, p= 0.026; Table S20). Moreover, a tendency was found toward an association between the FOXP3 rs3761548-GT and GG genotypes and asthenia during MTX therapy (GT vs. TT/GG: OR = 3.25, 95% CI = 1.14–9.79; GG vs. TT/GT: OR = 1.12, 95% CI = 0.35–3.53, p= 0.051; Table S20). The multivariate analysis, adjusted for MTX administration route, showed that the ABCC1 rs2238476-AG genotype (AG vs. GG: OR = 8.10, 95% CI = 1.69–46.63, p= 0.011) and the FOXP3 rs3761548-GT genotype (OR = 4.11, 95% CI = 1.22–15.30, p= 0.027) were associated with a higher risk of having asthenia during MTX therapy (these values are shown in Table 6). Conversely, the bivariate analysis of gastrointestinal toxicity risk and the polymorphisms studied revealed no significant results (Table S19). Table 6. Multivariate regression analysis for asthenia based on clinical characteristics and genetic variables. Asthenia OR (CI95%)p-Value MTX administration Subcutaneous 6.45 (1.78–28.51) 0.007 Both 13.01 (3.49–60.27) <0.001 ABCC1 rs2238476-AG (AG vs. GG) 8.10 (1.69–46.63) 0.011 FOXP3 rs3761548-GT (GT vs. TT/GG) 4.10 (1.22–15.30) 0.027 Biomedicines 2023,11, 2567 16 of 16 68. Attwa, E.M.; Elkot, R.A.; Abdelshafey, A.S.; Hafez, A.R. Subcutaneous methotrexate versus oral form for the treatment and prophylaxis of chronic plaque psoriasis. Dermatol. Ther. 2019,32, e13051. [CrossRef] 69. Fráˇnová, J.; Fingerhutová, Š.; Kobrová, K.; Srp, R.; Nˇemcová, D.; Hoza, J.; Uher, M.; Saifridová, M.; Linková, L.; Doležalová, P.; et al. Methotrexate efficacy, but not its intolerance, is asso-ciated with the dose and route of administration. Pediatric Rheumatol. 2016,14, 1–11. [CrossRef] 70. Amital, H.; Arnson, Y.; Chodick, G.; Shalev, V. Hepatotoxicity rates do not differ in patients with rheumatoid arthritis and psoriasis treated with methotrexate. Rheumatology 2009,48, 1107–1110. [CrossRef] 71. Jansen, G.; de Rotte, M.; de Jonge, R. Smoking and Methotrexate Inefficacy in Rheumatoid Arthritis: What About Underlying Molecular Mechanisms? J. Rheumatol. 2021,48, 1495–1497. [CrossRef] 72. Romero, M.Z.; Martínez, F.; Montoro, M.M.; Ramírez, C.; Hernández, M.C.; Pete, N.; Martín, A.; Morales, A.; Tortosa, M.R. Association of genetic poly-morphisms on methotrexate toxicity in patients with rheumatoid arthritis. Arch. Med. Sci. 2020 ,16, e97360. [CrossRef] 73. Zhu, J.; Wang, Z.; Tao, L.; Han, L.; Huang, Q.; Fang, X.; Yang, K.; Huang, G.; Zheng, Z.; Yawalkar, N.; et al. MTHFR Gene Polymorphism Association with Psoriatic Arthritis Risk and the Efficacy and Hepatotoxicity of Methotrexate in Psoriasis. Front. Med. 2022,9, 869912. [CrossRef] [PubMed] 74. Jeiziner, C.; Allemann, S.S.; E Hersberger, K.; Schwabedissen, H.E.M.z. Is Pharmacogenetic Panel Testing Applicable to Low-Dose Methotrexate in Rheumatoid Arthritis?—A Case Report. Pharmacogenomics Pers. Med. 2022,15, 465–475. [CrossRef] [PubMed] 75. Takano, M.; Yumoto, R.; Murakami, T. Expression and function of efflux drug transporters in the intestine. Pharmacol. Ther. 2006 , 109, 137–161. [CrossRef] [PubMed] 76. Vlaming, M.L.; Pala, Z.; van Esch, A.; Wagenaar, E.; de Waart, D.R.; van de Wetering, K.; van der Kruijssen, C.M.; Oude Elferink, R.P.; van Tellingen, O.; Schinkel, A.H. Functionally overlapping roles of ABCG2 (Bcrp1) and Abcc2 (Mrp2) in the elimination of methotrexate and its main toxic metabolite 7-hydroxymethotrexate in vivo .Clin. Cancer Res. 2009 ,15, 3084–3093. [CrossRef] [PubMed] 77. Vlaming, M.L.; van Esch, A.; van de Steeg, E.; Pala, Z.; Wagenaar, E.; van Tellingen, O.; Schinkel, A.H. Impact of abcc2 [multidrug re-sistance-associated protein (MRP) 2], abcc3 (MRP3), and ABCG2 (breast cancer resistance protein) on the oral pharmacokinetics of methotrexate and its main metabolite 7-hydroxymethotrexate. Drug Metab. Dispos. 2011,39, 1338–1344. [CrossRef] 78. Vlaming, M.L.; van Esch, A.; Pala, Z.; Wagenaar, E.; van de Wetering, K.; van Tellingen, O.; Schinkel, A.H. Abcc2 (Mrp2), Abcc3 (Mrp3), and ABCG2 (Bcrp1) are the main determinants for rapid elimination of methotrexate and its toxic metabolite 7-hydroxymethotrexate in vivo. Mol. Cancer Ther. 2009,8, 3350–3359. [CrossRef] 79. Lui, G.; Treluyer, J.M.; Fresneau, B.; Piperno-Neumann, S.; Gaspar, N.; Corradini, N.; Gentet, J.C.; Marec Berard, P.; Laurence, V.; Schneider, P.; et al. A Pharmacokinetic and Pharmacogenetic Analysis of Osteosarcoma Patients Treated With High-Dose Methotrexate: Data From the OS2006/Sarcoma-09 Trial. J. Clin. Pharmacol. 2018,58, 1541–1549. [CrossRef] 80. Hegyi, M.; Arany, A.; Semsei, A.F.; Csordas, K.; Eipel, O.; Gezsi, A.; Kutszegi, N.; Csoka, M.; Muller, J.; Erdelyi, D.J.; et al. Pharmacogenetic analysis of high-dose methotrexate treatment in children with osteosarcoma. Oncotarget 2017,8, 9388–9398. [CrossRef] 81. Indhumathi, S.; Rajappa, M.; Chandrashekar, L.; Ananthanarayanan, P.H.; Thappa, D.M.; Negi, V.S. Pharmacogenetic markers to predict the clinical response to methotrexate in south Indian Tamil patients with psoriasis. Eur. J. Clin. Pharmacol. 2017 ,73, 965–971. [CrossRef] 82. Wu, Z.; Xu, Q.; Qiu, X.; Jiao, Z.; Zhang, M.; Zhong, M. FOXP3 rs3761548 polymorphism is associated with tacrolimus-induced acute nephrotoxicity in renal transplant patients. Eur. J. Clin. Pharmacol. 2017,73, 39–47. [CrossRef] 83. Saxena, D.; Misra, M.; Parveen, F.; Phadke, S.; Agrawal, S. The transcription factor Forkhead Box P3 gene variants affect idiopathic recurrent pregnancy loss. Placenta 2015,36, 226–231. [CrossRef] [PubMed] 84. Gandolfo, M.T.; Jang, H.R.; Bagnasco, S.M.; Ko, G.-J.; Agreda, P.; Satpute, S.R.; Crow, M.T.; King, L.S.; Rabb, H. Foxp3+ regulatory T cells participate in repair of ischemic acute kidney injury. Kidney Int. 2009,76, 717–729. [CrossRef] [PubMed] 85. Park, J.-E.; Jang, J.; Choi, J.-H.; Kang, M.-S.; Woo, Y.-J.; Seong, Y.-R.; Choi, C.-B.; Lee, H.-S.; Bae, S.-C.; Bae, Y.-S. DC-Based Immunotherapy Combined with Low-Dose Methotrexate Effective in the Treatment of Advanced CIA in Mice. J. Immunol. Res. 2015,2015, 834085. [CrossRef] [PubMed] 86. Guggino, G.; Giardina, A.; Ferrante, A.; Giardina, G.; Schinocca, C.; Sireci, G.; Dieli, F.; Ciccia, F.; Triolo, G. The in vitro addition of methotrexate and/or methylprednisolone determines peripheral reduction in Th17 and expansion of conventional Treg and of IL-10 producing Th17 lymphocytes in patients with early rheumatoid arthritis. Rheumatol. Int. 2014,35, 171–175. [CrossRef] 87. Stamp, L.K.; Chapman, P.T.; O’Donnell, J.L.; Zhang, M.; James, J.; Frampton, C.; Barclay, M.L.; Kennedy, M.A.; Roberts, R.L. Polymorphisms within the folate pathway predict folate concentrations but are not associated with disease activity in rheumatoid arthritis patients on methotrexate. Pharmacogenetics Genom. 2010,20, 367–376. [CrossRef] 88. D’cruz, L.G.; McEleney, K.G.; Tan, K.B.C.; Shukla, P.; Gardiner, P.V.; Connolly, P.; Conway, C.; Cobice, D.; Gibson, D.S. Clinical and Laboratory Associations with Methotrexate Metabolism Gene Polymorphisms in Rheumatoid Arthritis. J. Pers. Med. 2020 ,10, 149. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.