MicroRNA signature associated with treatment response in myasthenia gravis: A further step towards precision medicine.

Myasthenia gravis (MG) is an autoimmune disorder affecting neuromuscular transmission currently treated with chronic immunosuppression. Inter-subject variation in treatment response and side effects highlight the need for personalized therapies by identification of biomarkers predictive of drug efficacy in individual patients, still lacking in MG. MicroRNAs (miRNAs) play a key role in immune response and drug metabolism modulation. This study, part of an Italian-Israeli collaborative project, aimed to identify specific miRNAs as biomarkers associated with immunosuppressive treatment response in MG patients. Whole miRNome sequencing, followed by miRNA validation by real-time PCR, was performed in peripheral blood from Italian MG patients (n = 40) classified as responder and non-responder to immunosuppressive therapies. MiRNA sequencing identified 41 miRNAs differentially expressed in non-responder compared to responder Italian MG patients. Validation phase pointed out three miRNAs, miR-323b-3p, -409-3p, and -485-3p, clustered on chromosome 14q32.31, the levels of which were significantly decreased in non-responder versus responder patients, whereas miR-181d-5p and -340-3p showed an opposite trend. ROC curve analysis showed sensitivity and specificity performance results indicative of miR-323b-3p, -409-3p, and -485-3p predictive value for responsiveness to immunosuppressive drugs in MG. Validated miRNAs were further analyzed in blood from responder and non-responder MG patients of the Israeli population (n = 33), confirming a role for miR-323b-3p, -409-3p, -485-3p, -181d-5p and -340-3p as biomarkers of drug efficacy. Gene Ontology enrichment analysis, mRNA target prediction, and in silico modeling for function of the identified miRNAs disclosed functional involvement of the five miRNAs, and their putative target genes, in both immune (i.e. neurotrophin TRK and Fc-epsilon receptor signaling pathways) and drug metabolism processes. Our overall findings thus revealed a blood "miR-323b-3p, -409-3p, -485-3p, -181d-5p, and -340-3p" signature associated with drug responsiveness in MG patients. Its identification sets the basis for precision medicine approaches based on "pharmacomiRs" as biomarkers of drug responsiveness in MG, promising to improve therapeutic success in a cost/effective manner.

A novel ABCC6 haplotype is associated with azathioprine drug response in myasthenia gravis.

OBJECTIVE: We investigated the association of single nucleotide polymorphisms (SNPs) in drug-metabolizing enzymes and transporters (DMETs) with the response to azathioprine (AZA) in patients affected by myasthenia gravis (MG) to determine possible genotype-phenotype correlations. PATIENTS AND METHODS: Genomic DNA from 180 AZA-treated MG patients was screened through the Affymetrix DMET platform, which characterizes 1931 SNPs in 225 genes. The significant SNPs, identified to be involved in AZA response, were subsequently validated by allelic discrimination and direct sequencing. SNP analysis was carried out using the SNPassoc R package and the haploblocks were determined using haploview software. RESULTS: We studied 127 patients in the discovery phase and 53 patients in the validation phase. We showed that two SNPs (rs8058694 and rs8058696) found in ATP-binding cassette subfamily C member 6, a subfamily member of ATP-binding cassette genes, constituted a new haplotype associated with AZA response in MG patients in the discovery cohort (P=0.011; odds ratio: 0.40; 95% confidence interval: 0.20-0.83) and in the combined cohort (P=0.04; odds ratio: 1.58). CONCLUSION: These findings highlight the role that the ATP-binding cassette subfamily C member 6 haplotype may play in AZA drug response. In view of the significant effects and AZA intolerance, these novel SNPs should be taken into consideration in pharmacogenetic profiling for AZA.

Toll-like receptors 7 and 9 in myasthenia gravis thymus: amplifiers of autoimmunity?

Pathogen infections and dysregulated Toll-like receptor (TLR)-mediated innate immune responses are suspected to play key roles in autoimmunity. Among TLRs, TLR7 and TLR9 have been implicated in several autoimmune conditions, mainly because of their ability to promote abnormal B cell activation and survival. Recently, we provided evidence of Epstein-Barr virus (EBV) persistence and reactivation in the thymus of myasthenia gravis (MG) patients, suggesting an involvement of EBV in the intrathymic pathogenesis of the disease. Considerable data highlight the existence of pathogenic crosstalk among EBV, TLR7, and TLR9: EBV elicits TLR7/9 signaling, which in turn can enhance B cell dysfunction and autoimmunity. In this article, after a brief summary of data demonstrating TLR activation in MG thymus, we provide an overview on the contribution of TLR7 and TLR9 to autoimmune diseases and discuss our recent findings indicating a pivotal role for these two receptors, along with EBV, in driving, perpetuating, and/or amplifying intrathymic B cell dysregulation and autoimmune responses in MG. Development of therapeutic approaches targeting TLR7 and TLR9 signaling could be a novel strategy for treating the chronic inflammatory autoimmune process in myasthenia gravis.

Increased expression of Toll-like receptors 7 and 9 in myasthenia gravis thymus characterized by active Epstein-Barr virus infection.

Considerable data implicate the thymus as the main site of autosensitization to the acetylcholine receptor in myasthenia gravis (MG), a B-cell-mediated autoimmune disease affecting the neuromuscular junction. We recently demonstrated an active Epstein-Barr virus (EBV) infection in the thymus of MG patients, suggesting that EBV might contribute to the onset or maintenance of the autoimmune response within MG thymus, because of its ability to activate and immortalize autoreactive B cells. EBV has been reported to elicit and modulate Toll-like receptor (TLR) 7- and TLR9-mediated innate immune responses, which are known to favor B-cell dysfunction and autoimmunity. Aim of this study was to investigate whether EBV infection is associated with altered expression of TLR7 and TLR9 in MG thymus. By real-time PCR, we found that TLR7 and TLR9 mRNA levels were significantly higher in EBV-positive MG compared to EBV-negative normal thymuses. By confocal microscopy, high expression levels of TLR7 and TLR9 proteins were observed in B cells and plasma cells of MG thymic germinal centers (GCs) and lymphoid infiltrates, where the two receptors co-localized with EBV antigens. An increased frequency of Ki67-positive proliferating B cells was found in MG thymuses, where we also detected proliferating cells expressing TLR7, TLR9 and EBV antigens, thus supporting the idea that EBV-associated TLR7/9 signaling may promote abnormal B-cell activation and proliferation. Along with B cells and plasma cells, thymic epithelium, plasmacytoid dendritic cells and macrophages exhibited enhanced TLR7 and TLR9 expression in MG thymus; TLR7 was also increased in thymic myeloid dendritic cells and its transcriptional levels positively correlated with those of interferon (IFN)-ß. We suggested that TLR7/9 signaling may be involved in antiviral type I IFN production and long-term inflammation in EBV-infected MG thymuses. Our overall findings indicate that EBV-driven TLR7- and TLR9-mediated innate immune responses may participate in the intra-thymic pathogenesis of MG.

A novel infection- and inflammation-associated molecular signature in peripheral blood of myasthenia gravis patients.

Myasthenia gravis (MG) is a T-cell dependent autoimmune disorder of the neuromuscular junction, characterised by muscle weakness and fatigability. Autoimmunity is thought to initiate in the thymus of acetylcholine receptor (AChR)-positive MG patients; however, the molecular mechanisms linking intra-thymic MG pathogenesis with autoreactivity via the circulation to the muscle target organ are poorly understood. Using whole-transcriptome sequencing, we compared the transcriptional profile of peripheral blood mononuclear cells from AChR-early onset MG (AChR-EOMG) patients with healthy controls: 178 coding transcripts and 229 long non-coding RNAs, including 11 pre-miRNAs, were differentially expressed. Among the 178 coding transcripts, 128 were annotated of which 17% were associated with the 'infectious disease' functional category and 46% with 'inflammatory disease' and 'inflammatory response-associated' categories. Validation of selected transcripts by qPCR indicated that of the infectious disease-related transcripts, ETF1, NFKB2, PLK3, and PPP1R15A were upregulated, whereas CLC and IL4 were downregulated in AChR-EOMG patients; in the 'inflammatory' categories, ABCA1, FUS, and RELB were upregulated, suggesting a contribution of these molecules to immunological dysfunctions in MG. Data selection and validation were also based on predicted microRNA-mRNA interactions. We found that miR-612, miR-3654, and miR-3651 were increased, whereas miR-612-putative AKAp12 and HRH4 targets and the miR-3651-putative CRISP3 target were downregulated in AChR-EOMG, also suggesting altered immunoregulation. Our findings reveal a novel peripheral molecular signature in AChR-EOMG, reflecting a critical involvement of inflammatory- and infectious disease-related immune responses in disease pathogenesis.

Up-regulation of neural and cell cycle-related microRNAs in brain of amyotrophic lateral sclerosis mice at late disease stage.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motor neuron degeneration in motor cortex, brainstem and spinal cord. microRNAs (miRNAs) are small non-coding RNAs that bind complementary target sequences and modulate gene expression; they are key molecules for establishing a neuronal phenotype, and in neurodegeneration. Here we investigated neural miR-9, miR-124a, miR-125b, miR-219, miR-134, and cell cycle-related miR-19a and -19b, in G93A-SOD1 mouse brain in pre-symptomatic and late stage disease. RESULTS: Expression of miR-9, miR-124a, miR-19a and -19b was significantly increased in G93A-SOD1 whole brain at late stage disease compared to B6.SJL and Wt-SOD1 control brains. These miRNAs were then analyzed in manually dissected SVZ, hippocampus, primary motor cortex and brainstem motor nuclei in 18-week-old ALS mice compared to same age controls. In SVZ and hippocampus miR-124a was up-regulated, miR-219 was down-regulated, and numbers of neural stem progenitor cells (NSPCs) were significantly increased. In G93A-SOD1 brainstem motor nuclei and primary motor cortex, miR-9 and miR-124a were significantly up-regulated, miR-125b expression was also increased. miR-19a and -19b were up-regulated in primary motor cortex and hippocampus, respectively. Expression analysis of predicted miRNA targets identified miRNA/target gene pairs differentially expressed in G93A-SOD1 brain regions compared to controls. CONCLUSIONS: Hierarchical clustering analysis, identifying two clusters of miRNA/target genes, one characterizing brainstem motor nuclei and primary motor cortex, the other hippocampus and SVZ, suggests that altered expression of neural and cell cycle-related miRNAs in these brain regions might contribute to ALS pathogenesis in G93A-SOD1 mice. Re-establishing their expression to normal levels could be a new therapeutic approach to ALS.

Altered miRNA expression is associated with neuronal fate in G93A-SOD1 ependymal stem progenitor cells.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motoneuron loss in the CNS. In G93A-SOD1 mice, motoneuron degeneration is associated with proliferative restorative attempts of ependymal stem progenitor cells (epSPCs), usually quiescent in the spinal cord. The aims of the study were to demonstrate that epSPCs isolated from the spinal cord of G93A-SOD1 mice express neurogenic potential in vitro, and thus gain a better understanding of epSPC neural differentiation properties. For this purpose, we compared the ability of epSPCs from asymptomatic and symptomatic G93A-SOD1 and WT SOD1 transgenic mice to proliferate and differentiate into neural cells. Compared to control cells, G93A-SOD1 epSPCs differentiated more into neurons than into astrocytes, whereas oligodendrocyte proportions were similar in the two populations. G93A-SOD1 neurons were small and astrocytes had an activated phenotype. Evaluation of microRNAs, specific for neural cell fate and cell-cycle regulation, in G93A-SOD1 epSPCs showed that miR-9, miR-124a, miR-19a and miR-19b were differentially expressed. Expression analysis of the predicted miRNA targets allowed identification of a functional network in which Hes1, Pten, Socs1, and Stat3 genes were important for controlling epSPC fate. Our findings demonstrate that G93A-SOD1 epSPCs are a source of multipotent cells that have neurogenic potential in vitro, and might be a useful tool to investigate the mechanisms of neural differentiation in relation to miRNA expression whose modulation might constitute new targeted therapeutic approaches to ALS.