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FRIMED2-FRIPRO forskerprosjekt, medisin og helse

Identification of molecular mechanisms and biomarkers of Multiple Sclerosis

Alternative title: Molekylære mekanismer og biomarkører ved multippel sklerose

Awarded: NOK 8.7 mill.

Our primary objective is to identify molecular mechanisms and novel biomarkers of MS, paving the road for improved personalized medicine for MS patients. This project extends from ongoing research performed by the MS research group at OUS/UiO led by prof. Harbo in collaboration with local, national and leading international research groups. This project has three main branches: 1) Genome wide genetic, epigenetic, transcriptomic and proteomic characteristics of immune cells of MS patients and healthy controls; 2) Molecular and functional characterization of MS-associated genes and 3) MRI-studies to improve phenotyping of the MS patients. Sub-Project 1: In this project, we aim to analyze genetic, epigenetic (DNA methylation), transcriptomic (RNAseq) and proteomic (mass spectrometry) data from purified human immune cells (CD4+ and CD8+ T cells). We have included only treatment-naïve, female MS patients that were recently diagnosed and have no other autoimmune disorders and age- and sex matched healthy controls. In total, we have now purified cells from 55 MS patients and 55 matched controls. These have been genotyped, and profiled for DNA methylation, and on a selection RNA-sequencing and mass spectrometry analyses have been completed. a) In 2015, we published a pilot study on DNA methylation in T cells from MS patients and healthy controls. These data has been combined with new DNA methylation data from T cells collected by us as well as a collaborator from Australia. This work was published in PLoS One in October 2018. Ina S Brorson and her main supervisor, Dr. Steffan D Bos led the project on behalf of the Norwegian MS research group. We were able to replicate findings from the pilot study in 2015 and identified differential DNA methylation in the SLFN12 gene in samples from MS patients b) Researcher Steffan Bos published two papers on the differential gene expression analysis of CD4+ and CD8+ T cell RNA-sequencing. In both papers, PhD student Anna Eriksson has performed the pathway analyses. Results from these studies are the foundation for further experiments in related projects within our group. c) Researcher Steffan Bos has established a collaboration with the research group of Prof. Sergio Baranzini at the University of California, San Francisco. Prof. Baranzini has been leading gene pathway analysis work resulting in a publication in Nature Communications. Several group members including this NFR projects' leader Hanne Harbo and Steffan Bos are co-authors on this paper. DNA methylation and RNA-sequencing data generated in our project can be processed using the pipeline published in this paper, in order to add a level of depth in the analyses performed so far. This analysis has been delayed as a result of the Covid-19 pandemic, which resulted in limited resources at out collaboration partner. This analysis remains to be performed. d) In a subset of the samples, we have performed mass spectrometry analyses to characterize the proteomic profiles of T cells from MS patients and healthy Controls in collaboration with Professor Frode Berven at the University of Bergen. We show that proteins in T cell specific pathways are differentially expressed between T cells from MS patients and healthy controls. This study, where PhD student Anna Eriksson is the second author, was published in Clinical Proteomics in May 2019. We have extended these analyses and collected mass spectrometry fra resting and in vitro activated CD4+ T cells. The dynamic response upon T cell activation at the protein Level is compared between samples from MS and healthy Controls. We are completing a manuscript, with Eriksson as second author and Harbo and Bos as central co-authors, to be submitted early this year. Sub-project 2: The main focus for the PhD studies of Anna Eriksson is to study single proteins encoded by selected MS susceptibility genes in immune cells. A manuscript concerning the role of CLEC16 A is currently under revision in Scandinavian Journal of Immunology. Here, Eriksson has used Jurkat cells as model system to show that CLEC16A is localized to recycling endosomes With a potential impact on T cell receptor recycling. Erikssons second main work, is molecular analyese of DEXI, encoded by another MS Associated gene. We have identified two protein interaction partners for DEXI, and we are using cell lines to study the effect DEXI and its interaction partners have on immune cell responses in vitro. The data collection is completed and a mansucript will be finalized this semester. Eriksson will submit her PhD thesis January this year. Sub-project 3:We have during the last year completed the third examination of our cohort of approximately 70 MS patients that we follow longitudinally with detailed MR-examinations, clinical assessments and collection of blood samples. These data are now being analysed in collaboration with our partners.

Vår studie av genekspresjon av CD4 + T-celler har resultert i nye studier der immuncellepopulasjonen er kartlagt i detalj. Genekspresjonen identifisert i CD8 + T-celler styrker hypotesen om at immunceller i perifert blod er potensielle biomarkører for sykdom og muligens sykdomsprogresjon. Våre DNA-metyleringsstudier gir ytterligere bevis for at immunceller er viktige i sykdomsprosesser ved MS. De longitudinelt innsamlede DNA-, RNA- og proteinprøvene er svært viktige for videre studier. Våre funksjonelle studier har karakterisert immunologiske mekanismene som er bakenforliggende for MS og bidrar til å forklare funn gjort i de genom-vide gen-screeningene. Våre MR-studier viser at MR kan benyttes om en biomarkør for subgrupper av MS-pasienter. Prosjektet har altså resultert i viktige vitenskapelige funn, har lagt grunnlaget for forskerkarrieren til flere ph.d. stipendiater og forskere og har videreutviklet forskningsgruppens sentrale rollen i ledende internasjonale forskningsnettverk.

This is a cutting-edge translational study of multiple sclerosis (MS), a devastating, inflammatory disease of the central nervous system (CNS) that affects 10,000 adults in Norway. Our primary objective is to identify molecular mechanisms and novel biomarkers of MS, paving the road for improved personalized medicine for MS patients. The proposal extends from ongoing research performed by the MS research group in Oslo led by the applicant, in collaboration with international as well as Norwegian leading experts. Taking advantage of our recent breakthrough in MS genetics (Nature 2011, Nature Genetics 2013) as well as our unique sample collection and established expertise and networks, we will: 1) Identify MRI phenotypes in updated MS patient collections including detailed clinical and MRI analyses. Our current collection of 30 yet untreated MS patients and 30 controls (already genome-wide genotyped and DNA-methylation mapped in whole blood and immune cell subsets) will be expanded with 30 new patients and 30 controls. The patients will undergo detailed clinical and MRI analyses twice (year 1 and year 3) in local (n=60) as well as international (n=500) studies. 2) Evaluation of RNA sequencing data from CD4+ T cells as potential biomarkers for MS, using patients and data collected under objective 1), combined with genome-wide genotyping and DNA-methylation data (n=60): We will perform a) RNA sequencing of CD4+ T cells, b) Analysis of RNA sequencing data for overall gene expression, splicing differences and allelic differences, c)Validation and replication of lead signals, and d) Molecular characterization of selected lead signals. 3) Identify the molecular function of the DEXI and other MS susceptibility genes: We will a) Identify the subcellular localization of DEXI in immune cells, b) Identify protein interaction partners for DEXI and c) Perform functional characterization of DEXI and gene X, defined from the RNA sequencing project.

Funding scheme:

FRIMED2-FRIPRO forskerprosjekt, medisin og helse

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