Transcriptome alterations in peripheral blood B cells of patients with multiple sclerosis receiving immune reconstitution therapy.

BACKGROUND: Multiple sclerosis (MS) is a chronic, inflammatory and neurodegenerative disease that leads to irreversible damage to the brain and spinal cord. The goal of so-called "immune reconstitution therapies" (IRTs) is to achieve long-term disease remission by eliminating a pathogenic immune repertoire through intense short-term immune cell depletion. B cells are major targets for effective immunotherapy in MS. OBJECTIVES: The aim of this study was to analyze the gene expression pattern of B cells before and during IRT (i.e., before B-cell depletion and after B-cell repopulation) to better understand the therapeutic effects and to identify biomarker candidates of the clinical response to therapy. METHODS: B cells were obtained from blood samples of patients with relapsing-remitting MS (n = 50), patients with primary progressive MS (n = 13) as well as healthy controls (n = 28). The patients with relapsing MS received either monthly infusions of natalizumab (n = 29) or a pulsed IRT with alemtuzumab (n = 15) or cladribine (n = 6). B-cell subpopulation frequencies were determined by flow cytometry, and transcriptome profiling was performed using Clariom D arrays. Differentially expressed genes (DEGs) between the patient groups and controls were examined with regard to their functions and interactions. We also tested for differences in gene expression between patients with and without relapse following alemtuzumab administration. RESULTS: Patients treated with alemtuzumab or cladribine showed on average a > 20% lower proportion of memory B cells as compared to before IRT. This was paralleled by profound transcriptome shifts, with > 6000 significant DEGs after adjustment for multiple comparisons. The top DEGs were found to regulate apoptosis, cell adhesion and RNA processing, and the most highly connected nodes in the network of encoded proteins were ESR2, PHB and RC3H1. Higher mRNA levels of BCL2, IL13RA1 and SLC38A11 were seen in patients with relapse despite IRT, though these differences did not pass the false discovery rate correction. CONCLUSIONS: We show that B cells circulating in the blood of patients with MS undergoing IRT present a distinct gene expression signature, and we delineated the associated biological processes and gene interactions. Moreover, we identified genes whose expression may be an indicator of relapse risk, but further studies are needed to verify their potential value as biomarkers.

Abnormal Pre-mRNA Splicing in Exonic Fabry Disease-Causing GLA Mutations.

Fabry disease (FD) is a rare X-linked disease due to a multiverse of disrupting mutations within the GLA gene encoding lysosomal α-galactosidase A (AGAL). Absent AGAL activity causes the accumulation of complex glycosphingolipids inside of lysosomes in a variety of cell types and results in a progressive multisystem disease. Known disease-associated point mutations in protein-coding gene regions usually cause translational perturbations and result in premature chain termination, punctual amino acid sequence alterations or overall altered sequence alterations downstream of the mutation site. However, nucleotide exchanges at the border between introns and exons can affect splicing behavior and lead to abnormal pre-mRNA processing. Prediction with the Human Splicing Finder (HSF) revealed an indication of a significant change in splicing-relevant information for some known FD-associated GLA mutations. To experimentally determine the extent of the change, we made use of a minigene reporter assay and verified alternative splicing events for the exonic mutations c.194G>T and c.358C>G, which led to the usage of alternative donor splice sites at exon 1 and exon 2, respectively. In addition, the mutations c.548G>T and c.638A>T led to significant exon 4 skipping. We conclude that splicing phenotype analysis should be employed in the in vitro analysis of exonic GLA gene mutations, since abnormal splicing may result in a reduction of enzyme activity and alter the amenability for treatment with pharmacological chaperone (PC).

Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpress.

Splicing is an important RNA processing step. Genetic variations can alter the splicing process and thereby contribute to the development of various diseases. Alterations of the splicing pattern can be examined by gene expression analyses, by computational tools for predicting the effects of genetic variants on splicing, and by splicing reporter minigene assays for studying alternative splicing events under defined conditions. The minigene assay is based on transient transfection of cells with a vector containing a genomic region of interest cloned between two constitutive exons. Cloning can be accomplished by the use of restriction enzymes or by site-specific recombination using Gateway cloning. The vectors pDESTsplice and pSpliceExpress represent two minigene systems based on Gateway cloning, which are available through the Addgene plasmid repository. In this review, we describe the features of these two splicing reporter minigene systems. Moreover, we provide an overview of studies in which determinants of alternative splicing were investigated by using pDESTsplice or pSpliceExpress. The studies were reviewed with regard to the investigated splicing regulatory events and the experimental strategy to construct and perform a splicing reporter minigene assay. We further elaborate on how analyses on the regulation of RNA splicing offer promising prospects for gaining important insights into disease mechanisms.

Implication of genetic variants in primary microRNA processing sites in the risk of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with a well-established genetic contribution to susceptibility. Over 200 genetic regions have been linked to the inherited risk of developing MS, but the disease-causing variants and their functional effects at the molecular level are still largely unresolved. We hypothesised that MS-associated single-nucleotide polymorphisms (SNPs) affect the recognition and enzymatic cleavage of primary microRNAs (pri-miRNAs). METHODS: Our study focused on 11 pri-miRNAs (9 primate-specific) that are encoded in genetic risk loci for MS. The levels of mature miRNAs and potential isoforms (isomiRs) produced from those pri-miRNAs were measured in B cells obtained from the peripheral blood of 63 MS patients and 28 healthy controls. We tested for associations between SNP genotypes and miRNA expression in cis using quantitative trait locus (cis-miR-eQTL) analyses. Genetic effects on miRNA stem-loop processing efficiency were verified using luciferase reporter assays. Potential direct miRNA target genes were identified by transcriptome profiling and computational binding site assessment. FINDINGS: Mature miRNAs and isomiRs from hsa-mir-26a-2, hsa-mir-199a-1, hsa-mir-4304, hsa-mir-4423, hsa-mir-4464 and hsa-mir-4492 could be detected in all B-cell samples. When MS patient subgroups were compared with healthy controls, a significant differential expression was observed for miRNAs from the 5' and 3' strands of hsa-mir-26a-2 and hsa-mir-199a-1. The cis-miR-eQTL analyses and reporter assays pointed to a slightly more efficient Drosha-mediated processing of hsa-mir-199a-1 when the MS risk allele T of SNP rs1005039 is present. On the other hand, the MS risk allele A of SNP rs817478, which substitutes the first C in a CNNC sequence motif, was found to cause a markedly lower efficiency in the processing of hsa-mir-4423. Overexpression of hsa-mir-199a-1 inhibited the expression of 60 protein-coding genes, including IRAK2, MIF, TNFRSF12A and TRAF1. The only target gene identified for hsa-mir-4423 was TMEM47. INTERPRETATION: We found that MS-associated SNPs in sequence determinants of pri-miRNA processing can affect the expression of mature miRNAs. Our findings complement the existing literature on the dysregulation of miRNAs in MS. Further studies on the maturation and function of miRNAs in different cell types and tissues may help to gain a more detailed functional understanding of the genetic basis of MS. FUNDING: This study was funded by the Rostock University Medical Center (FORUN program, grant: 889002), Sanofi Genzyme (grant: GZ-2016-11560) and Merck Serono GmbH (Darmstadt, Germany, an affiliate of Merck KGaA, CrossRef Funder ID: 10.13039/100009945, grant: 4501860307). NB was supported by the Stiftung der Deutschen Wirtschaft (sdw) and the FAZIT foundation. EP was supported by the Landesgraduiertenförderung Mecklenburg-Vorpommern.

Genetic risk variants for multiple sclerosis are linked to differences in alternative pre-mRNA splicing.

Background: Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system to which a genetic predisposition contributes. Over 200 genetic regions have been associated with increased disease risk, but the disease-causing variants and their functional impact at the molecular level are mostly poorly defined. We hypothesized that single-nucleotide polymorphisms (SNPs) have an impact on pre-mRNA splicing in MS. Methods: Our study focused on 10 bioinformatically prioritized SNP-gene pairs, in which the SNP has a high potential to alter alternative splicing events (ASEs). We tested for differential gene expression and differential alternative splicing in B cells from MS patients and healthy controls. We further examined the impact of the SNP genotypes on ASEs and on splice isoform expression levels. Novel genotype-dependent effects on splicing were verified with splicing reporter minigene assays. Results: We were able to confirm previously described findings regarding the relation of MS-associated SNPs with the ASEs of the pre-mRNAs from GSDMB and SP140. We also observed an increased IL7R exon 6 skipping when comparing relapsing and progressive MS patients to healthy subjects. Moreover, we found evidence that the MS risk alleles of the SNPs rs3851808 (EFCAB13), rs1131123 (HLA-C), rs10783847 (TSFM), and rs2014886 (TSFM) may contribute to a differential splicing pattern. Of particular interest is the genotype-dependent exon skipping of TSFM due to the SNP rs2014886. The minor allele T creates a donor splice site, resulting in the expression of the exon 3 and 4 of a short TSFM transcript isoform, whereas in the presence of the MS risk allele C, this donor site is absent, and thus the short transcript isoform is not expressed. Conclusion: In summary, we found that genetic variants from MS risk loci affect pre-mRNA splicing. Our findings substantiate the role of ASEs with respect to the genetics of MS. Further studies on how disease-causing genetic variants may modify the interactions between splicing regulatory sequence elements and RNA-binding proteins can help to deepen our understanding of the genetic susceptibility to MS.

Aberrant expression of alternative splicing variants in multiple sclerosis - A systematic review.

OBJECTIVE: Alternative splicing is an important form of RNA processing that affects nearly all human genes. The differential expression of specific transcript and protein isoforms holds the potential of novel biomarkers for complex diseases. In this systematic review, we compiled the existing literature on aberrant alternative splicing events in multiple sclerosis (MS). METHODS: A systematic literature search in the PubMed database was carried out and supplemented by screening the reference lists of the identified articles. We selected only MS-related original research studies which compared the levels of different isoforms of human protein-coding genes. A narrative synthesis of the research findings was conducted. Additionally, we performed a case-control analysis using high-density transcriptome microarray data to reevaluate the genes that were examined in the reviewed studies. RESULTS: A total of 160 records were screened. Of those, 36 studies from the last two decades were included. Most commonly, peripheral blood samples were analyzed (32 studies), and PCR-based techniques were usually employed (27 studies) for measuring the expression of selected genes. Two studies used an exploratory genome-wide approach. Overall, 27 alternatively spliced genes were investigated. Nine of these genes appeared in at least two studies (CD40, CFLAR, FOXP3, IFNAR2, IL7R, MOG, PTPRC, SP140 and TNFRSF1A). The microarray data analysis confirmed differential alternative pre-mRNA splicing for 19 genes. CONCLUSIONS: An altered RNA processing of genes mediating immune signaling pathways has been repeatedly implicated in MS. The analysis of individual exon-level expression patterns is stimulated by the advancement of transcriptome profiling technologies. In particular, the examination of genes encoded in MS-associated genetic regions may provide important insights into the pathogenesis of the disease and help to identify new biomarkers.