The Clinical And Basic Exploration Of Resistance To Mycophenolate Mofetil In Neuromyelitis Optica Spectrum Disorder

Background:Neuromyelitis optica spectrum disorder(NMOSD)is a demyelinating disease of the central nervous system.Its main characteristics are the simultaneous or sequential attacks of optic neuritis(ON)and transverse myelitis(TM).It can also manifest as brain,thalamus,and brainstem syndrome.NMOSD is widely distributed in the world,with a prevalence of only 0.52~10/100,000,but the disease has a poor prognosis and can cause serious consequences such as blindness and disability.Therefore,clinicians still attach great attention to it.Autoantibodies against aquaporin 4(AQP4-Ig G)can be detected in the serum of more than 80% of patients with NMOSD,which are abundantly expressed on the foot processes of astrocytes.It is currently believed that AQP4-Ig G is involved in the pathogenesis of NMOSD.AQP4-Ig G in the peripheral blood passes through the blood-brain barrier and binds to AQP4 on the foot processes of astrocytes,and is accompanied by the deposition of complement C9 and the activation of terminal complement pathways,resulting in Membrane attack complex(MAC)formation,causing damage to astrocytes,and secondary damage to oligodendrocytes and neurons.The current treatment of NMOSD aims at prolonging the time of recurrence and reducing the number of recurrences.High-dose glucocorticoids and plasma exchange(PE)or immunoadsorption(IA)are the main methods for the treatment of acute NMOSD,while other immunosuppressants such as rituximab,azathioprine,and mycophenolate mofetil are used as treatment in the recurrent phase.However,despite the use of immunosuppressive therapy,there are still some patients who have repeated attacks during the medication,which seriously affects survival and prognosis.In order to improve the understanding of drug resistance mechanisms,and to explore the risk factors that lead to drug resistance,and guide the rational use of drugs in clinical practice,this study targets patients who use mycophenolate mofetil to explore the occurrence of resistance to mycophenolate mofetil in NMOSD and its underlying mechanism.This study will explain the characteristics of patients with mycophenolate mofetil resistance from clinical,molecular and genetic aspects,and discuss its mechanism.Part Ⅰ:Clinical risk factors of resistance to mycophenolate mofetil in patients with neuromyelitis optica spectrum disorders.Objective:To analyze the incidence of drug resistance of mycophenolate mofetil in patients with neuromyelitis optica spectrum disorders,as well as the clinical characteristics of the patients,and the risk factors for drug resistance.Methods:NMOSD patients who were treated in the Department of Neurology of West China Hospital of Sichuan University and took MMF for more than 1 year from were recruited from December 2010 to December 2019.Demographic,clinical,and magnetic resonance imaging(MRI)characteristics of patients were collected including age of onset,gender,course of disease,duration from first episode to start of MMF,serum AQP4 antibody results,symptoms and history of onset,spinal cord enhanced MRI lesions,baseline EDSS score,previous and current treatment,and specific and non-specific autoimmunity serological test results of antibodies(such as rheumatoid factor,anti-nuclear antibodies,SSA antibodies,anti-double-stranded DNA antibodies,SSB antibodies,anti-microsomes and anti-thyroglobulin antibodies).During follow-up,a detailed neurological examination and EDSS score were performed on the patient.The above indicators were analyzed using a logistic regression model to evaluate the relationship between each factor and MMF resistance.Results:78 patients were eventually recruited,among them,29 were MMF resistant and49 were MMF effective.The average age of onset was 39.93±13.32 years old,and the average course before immunosuppressive treatment was 48.28±76.35 months.The average course of disease before treatment for MMF-resistant patients was59.60±68.71 months,and the average course of disease before treatment for patients in the effective group was 41.58±80.46 months.There was a statistically significant difference in the course of disease before treatment between the two groups.The course of disease before treatment in the MMF-resistant group was significantly longer(P=0.008).However,there was no significant difference in the average annually relapsing rate before treatment between the two groups.The pre-treatment EDSS score was 3.34±2.04 for the MMF-resistant patients and 2.74±2.39 for the effective group,while the difference was not statistically significant.There was a statistically significant difference in the incidence of medication delays for more than half a year between the two groups,and patients in the MMF-resistant group were more likely to have treatment delays more than 6 months(P=0.004).22 cases in the MMF-resistant group and 19 cases in the effective group experienced pre-treatment severe attacks.The MMF-resistant group experienced a greater probability of severe attacks before treatment(P=0.002).The prognosis of MMF-resistant patients was significantly worse than that of the effective group,which is reflected in the higher EDSS score.The average EDSS after treatment in the MMF-resistant group was3.93±2.38,and in the effective group was 2.09±2.43(P=0.002).There were no significant differences in age of onset,sex ratio,type of first symptoms,positive rate of autoimmune antibodies,and length of spinal cord lesions before treatment between the two groups.Multivariate logistic regression showed that severe relapses before treatment and more than 6 months of delay before MMF treatment were risk factors for MMF resistance.Conclusion:Higher pre-treatment severe relapse possibility,longer of treatment delay are clinical characteristics of patients with MMF resistance.Logistic regression proved that severe attack and delay of treatment were independent risk factors for MMF resistance in NMOSD patients.Part Ⅱ:Phenotypic and functional differences of T lymphocytes in patients with mycophenolate-resistant neuromyelitis optica spectrum disorders.Objective:To analyze the phenotypic characteristics and cell function of T lymphocytes in patients with mycophenolate-resistant neuromyelitis optica spectrum disorders and the differences to patients with effective medications.Methods:MMF-resistant patients and patients with good drug efficacy,as well as healthy people were recruited.Peripheral blood T cell subsets and CD8-positive T cell function were measured by flow cytometry.Peripheral blood pro-inflammatory cytokine levels were measured by ELISA and RT-PCR.ANOVA test was performed on the percentage of cell subpopulations in each group measured by flow cytometry.When P≤0.2,paired T test was performed on each two of the three groups.An ANOVA test was performed on the relative expression of RNA,and a T test was performed on the serum cytokine concentration.The above-mentioned T test is considered to be statistically different when P<0.05.Results:Flow cytometry were conducted with peripheral blood of 10 healthy volunteers,10 patients in the effective group and 10 patients in the MMF-resistant group.CD8-positive T cells in the MMF-resistant group were significantly higher than the effective group(P=0.019),and were significantly higher than the control group(P=0.036).The CD4-positive T-cells in the MMF-resistant group were significantly lower than the effective group(P=0.007),and were significantly lower than the control group(P=0.002).The effective group has an increasing trend of CD4-positive T cells compared with the control group(P=0.094).The CD4/CD8 ratio of the MMF-resistant group was significantly lower than that of the effective group(P=0.009),which was significantly lower than that of the control group(P=0.002).Compared with the control group,the effective group had a decreasing trend of CD4/CD8 ratio(P=0.056).The percentage of naive T cells differed among the three groups.The naive T cells of the MMF-resistant group were significantly lower than those of the effective group(P=0.021).There was no significant difference in the percentage of naive T cells between the MMF-resistant group and the control group,and the effective group and the control group.There was no significant difference in the percentage of effect memory T cells between the control group,the effective group and the MMF-resistant group.The memory precursor T cells of the drug resistance group were significantly lower than the effective group(P=0.024).There was no significant difference in the percentage of memory precursor T cells between the drug resistance group and the control group,and the effective group and the control group.There was no significant difference in the percentage of effector T cells among the control group,the effective group and the MMF-resistant group.There was no significant difference in the percentage of Th1 cells among the three groups.The percentage of Th2 cells in the MMF-resistant group was higher than that in the effective group(P=0.036),and there was a tendency to be higher than that in the control group(P=0.065).There was no significant difference in the percentage of Treg cells among the three groups.There was no significant difference in the function of CD8-positive T cells among the three groups.There was no significant difference in the relative expression level of RNA IFN-γ among effective group and MMF-resistant groups.The relative expression level of RNA IL-6 of MMF-resistant group was lower than effective group(P=0.025).The serum IFN-γ protein content of the MMF-resistant group and the effective group had no significant difference,and the IFN-γ protein content of patients in the acute phase was consistent.Conclusion:Compared with patients with effective MMF treatment,patients with MMF resistance has a specific feature of T cell phenotype.Part ⅢCorrelation analysis of TYK2 gene polymorphism and resistance to MMF in NMOSD patients.Objective:Using whole-exome sequencing methods to explore the genetic factors that cause differences in cell subpopulations,to explore the mechanism of MMF resistance,and to screen potential biomarkers for predicting MMF resistance.Methods:A total of 47 patients who were diagnosed as NMOSD and used MMF in the Department of Neurology of West China Hospital of Sichuan University were recruited.Among them,25 patients were resistant to MMF and 22 patients were responsive to MMF.Peripheral blood of patients was collected,and DNA in peripheral blood was extracted and purified.After determining the concentration and purity,the DNA is fragmented so that most of the DNA fragments are between100-500 bp in length.After modification of the 5’end and 3’end,fragment size screening and PCR amplification were performed.Use probes to capture all exons and use high-fidelity polymerase to amplify the original library.Finally,the Illumina Hiseq platform was used to perform high-throughput sequencing in a 2×150 bp paired-end sequencing mode,and Fast Q data was obtained and analyzed.Results:Whole-exome sequencing and SNP association analysis were performed on the DNA of 25 MMF-resistant patients and 22 MMF-responsive patients,and 1100 differential genes with 1471 differential SNP loci were screened out.According to the results of the second part of this study,a total of 59 genes were retrieved from the KEGG database for genes related to Th1/2 cell differentiation and CD8 cell development.Finally,the differential genes obtained in the SNP association analysis were compared with the genes retrieved by KEGG,and two differential genes were screened out,namely TYK2 and IFNGR2.TYK2,IFNGR2 and their SNP sites may be related to MMF resistance in NMOSD patients.HWE analysis was performed on the SNP sites of TYK2 and IFNGR2,except for rs2304256 in the MMF resistant group,all were consistent with HWE.After adjusting the gender and age of the two groups of patients,a logistic regression analysis was performed.The rs9808753 locus of IFNGR2 was statistically different between the two groups.The rs2304256(A:A),rs2304256(A:C),and rs280519(G:G),rs280519(G:A)increase the risk of patients with resistance to MMF.ConclusionThe rs2304256(A:A),rs2304256(A:C),rs280519(G:G),and rs280519(G:A)of the TYK2 gene are significantly related to MMF resistance in NMOSD patients.