Sphingosine -1- phosphate receptor 1 polymorphism as a cause of Fingolimod unresponsiveness and Multiple Sclerosis initiation

Background: Alot of immune cells have sphingosine -1phosphate receptor (SIPR) which regulates its migration from lymph node to blood circulation. The SIPR polymorphisms have been the focus the focus of some studies as genetic risk factor for multiple sclerosis and drug responsiveness. Aim: This study was aimed to show if the genetic variations of the S1P1 have a role in Fingolimod/Gilenya (FTY720) unresponsiveness and if S1P1polymorphisms were considered as genetic predisposition factor for MS. Materials and methods: This Case control study involved; sixty-six (66) with multiple sclerosis (MS), their ages were range from 14 to 69 years. They attended to seek treatment in the MS out patient’s clinic at Medical CityBaghdad Teaching Hospital in the period, which extended from December 2018 to March 2020. Patient was divided into two group resistant group (34) and response group (32) to fingolimod (Gelyinia) treatment. Results: The SIPR 1 gene sequencing revealed two SNPs rs3737577andrs3737578without significant differences between control group and patients, responder patients and non-responder patients on the level of genotype and allele frequency. A comparison between responsive and resistant patients revealed that the haplotype block TT was more frequent among nonresponder patients (30.88%) than responder patients (15.63%) with a significant difference (OR= 2.41, 95%CI=1.03-5.64, p= 0.042). Conclusion: There is no relationship between SIPR1 gene polymorphismand the responsiveness to the fingolimod in MS patients, but the TT haplotype block in this gene may have a negative effect on response to that medication also this gene polymorphisms cannot considered as genetic risk factor for MS initiation. Keyword: Multiple Sclerosis, SIPR1 and Fingolimod (Gelyinia) How to cite this article: Khaliel AH, Abbas AA-H, et al (2021): Sphingosine-1 phosphate receptor 1 polymorphism as a cause of fingolimod unresponsiveness and multiple sclerosis initiation, Ann Trop Med & Public Health; 24(S2): SP24204. DOI: http://doi.org/10.36295/ASRO.2021.24204 Introduction Multiple sclerosis (MS) is a chronic, inflammatory immune-mediated, neurodegenerative disorder. It is the leading nontraumatic cause of disability in young adults and affect three times women than men (1,2) .The deep heterogeneity of MS is not restricted to the symptoms, but to histologic appearances of lesions, neuroradiologic and response to therapy (3) Over the past two decades, different novel disease-modifying drugs for multiple sclerosis (MS) have been approved. However, there are high differences in the patient response to the available medications, which is hypothesized to be partly attributed to genetics (4) . Sphingosine-1-phosphate (S1P) is a normal bioactive lipid molecule and a commonfirst Khaliel et al (2021): Genetic variation of S1P1 in FTY720 and MS Feb 2021 Vol. 24 Issue 2 Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2021.24204 or second messenger in the immune systems and cardiovascular. By binding with its receptors, S1P can work as mediator of signaling during celldifferentiation, migration, proliferation and apoptosis (5) .Until 2010, there was not oral drug for MS patients. In 2010, FDA approved Fingolimod (Gilenya) for the treatment ofrelapsing multiple sclerosis (MS) andmarketed as the first effective oral alternative to injection therapy (6) . It is a sphingosine 1-phosphate receptor modulator, impairing egress of peripheral T and B cells from secondary lymphoid tissue into blood, which lead to reducing access to the central nervous system (CNS). Some studies suggesting neuroprotective effect (7) Few studies suggest that individual genetic variationsof sphingosine -1phosphate receptor can influencereceptor function and, therefore,infer differential disease susceptibility and interaction withsphingosine -1phosphate receptor targetedTherapeutics.Human S1PR1 variants have been reported to have functional heterogeneity in vitro, suggesting that S1PR1 function may influence fingolimod efficacy (8) .There is a limitation in the references that referred to the polymorphisms in the SIPR1, its relationship with multiple sclerosis and interaction with the fingolimod treatment. Allot of studies investigated the polymorphisms this gene with another diseases like Graft-versus-host disease, asthma, acute respiratory destress syndrome ARDSlung diseases. The S1PR modulators are currently under clinical trials for diverse pathophysiological conditions. There aresignificant Trials in targeting various components of S1P signaling for different diseases (9-11) . So, this study was aimed to show if the genetic variations of the S1P1 have a role in Fingolimod/Gilenya (FTY720) unresponsiveness and if S1P1polymorphisms were considered as genetic predisposition factor for MS. Materials and methods This Case control study involved; sixty six with multiple sclerosis (MS) their ages were range from 14 to 69 years. They were attended for seeking treatment of fingolimod in the MS out patient’s clinic at Medical CityBaghdad Teaching Hospital in the period, which extended from December 2018 to March 2020. The diagnosis of each case was established according to MC Donald criteria done by a neurologist and confirmed by MRI and certain cases by oligoclonalband test in the CSF. Patients were subjected to questionnaire about name, age, sex, smoking, family history, medication, type of medication, number of relapses in the last year and first clinical signs during diagnosis. Patients were divided into two groupsof responder and non-responder, the responsiveness to treatment was detected according to Rio criteria. The ethical committee of College of Medicine/Al-Nahrain University approved this study, and all samples were obtained with informed consent in accordance with the Ministry of Health declaration .Sixty volunteers after explaining the objective of the current study and agreed to accession of the study, their sex and ages were matched with patients group were included in this work as control. All of them received no treatment with no complaint of other chronic or systemic diseases; not suffering from any neurological signs in the last 2 years their age range was (16-68) years. Inclusion criteria Multiple sclerosis patients on fingolimod for more than 1 year Exclusion criteria Patients whom not adhere for treatment and have a period of treatment discontinuous Deoxyribonucleic acid extraction and SIPR1 Gene Sequencing: Two ml of venous blood were drawn from patients and controls in EDTA tube for DNA extraction which used in gene sequencing for 66 patients and 60 control, the DNA kept in Eppendorf tube -20°C till used. We used DNA extraction Khaliel et al (2021): Genetic variation of S1P1 in FTY720 and MS Feb 2021 Vol. 24 Issue 2 Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2021.24204 Kit (Geneaid – Tiwan) and PCR Kit (Bioneer /Korea). A specific pair of primers (table 1) was used to amplify S1PR1 gene (EDG1) with an expected amplicon of 1331 bp. Gel electrophoresis of PCR products was done. Table (1): SIPR1 primer sequence The coding sequence of the human S1PR1 gene was amplified by PCR using the human genomic DNAs as templates. The master mix which used is ready master mix (AccuPowerTM PCR premix/ Korea). One micro of each primer (foreword and reverse as in table 1) and three microliter of template DNA were added to the master mix tube. The final volume was adjusted to 25μlwith free nuclease distal water. The mixture was then vortexed for 10 seconds and put in thermocycler (Bioneer / Korea) which was previously programmed as shown in table 2. Table (2): SIPR1 PCR amplification programme PCR product was sent for Sanger sequencing using ABI3730XL, automated DNA sequence, by Macrogen Corporation – Korea. The results were received by email then analyzed using genius software. Statistical analysis For statistical analysis, the Statistical Package for the Social sciences V26 (SPSS Inc., Chicago, USA) was used. The polymorphisms were tested for deviation from Hardy Weinberg Equilibrium (HWE) by comparing the observed and expected frequencies (Chi-square test). The association between genotype and risk of Multiple sclerosis and drug responsiveness was estimated by calculation of Odds ratio (OR) with 95% confidence interval (95%CI). Statistical significance was set at a p value < 0.05. Results The results which presented in this study were built on the analysis of 66 patients with MS on fingolimod, in comparison with 60 apparently healthy individuals considered as controls. Overall, there were no significant differences between patients and control in the frequency of different age group. Younger age group (<30 years) was the most affected group with MS representing 37% of the patients; while the older age group (≥50 years) was the least frequent, the mean age of patients group was 35.6±10.7(13-58) and in control group was 34.9 ±10.3(18-53) . Similarly, the groups were comparable regarding Sex distribution (Table 3) Primer Sequence Product length Method forward 5 ′ -TCTCCAGCCAAGGAAAAGC-3 ′ 1331 bp Conventional PCR Reveres 5 ′ -AGTAAAGAGCGCTTCCGG-3 ′ Step Temperature Time Cycle Initial denaturation 95C° 5 mints 1X Denaturation 94C° 45 second 30 X Annealing 62C° 30 second Extension 72C° 30 second Final extension 72C° 7 mints 1 X Khaliel et al (2021): Genetic variation of S1P1 in FTY720 and MS Feb 2021 Vol. 24 Issue 2 Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2021.24204 Table 3:-Demographic Characteristics of the Study Population Characteristics Controls (60) MS patients teared With Gelyinia (66) P-value Age, years <30 30-39 40-49 ≥50 24(40%) 12(20%) 18(30%) 6(10%) 25(37.88 %) 19(28.79%) 16(24.24%) 6(9.09%) 0.749 Sex Male Female 24(40%) 36(60%) 28(42.42%) 38(57.58%) 0.783 Polymorphisms in SIPR1 gene were investigated for their association with the development of MS as well as with the drug resistance.