Precision medicine in rheumatoid arthritis:role of alternative splicing in methotrexate response

After specifying the content of the thesis chapters (chapter 1), the main research topic that is investigated in the thesis is outlined in chapter 2; i.e., the role of the enzyme folylpolyglutamate synthetase (FPGS) in the polyglutamylation process of methotrexate (MTX) - the anchor drug in rheumatoid arthritis (RA) treatment - in white blood cells of RA patients. The formation and intracellular accumulation of MTX-polyglutamates (MTX-PGs) is crucial for prolonged intracellular retention and conferring enhanced inhibitory effects on key enzymes in folate and purine metabolism. In RA, studies so far involved analyses of MTX-PG levels in red blood cells (RBCs) as a potential indicator for therapy response. However, immune cells in circulation and inflamed synovium are more representative for disease pathogenesis, thus examination of FPGS activity and MTX-PG accumulation in these cells, beyond RBCs, warrant investigation. The development and validation of a sensitive LC-MS/MS-based assay to measure FPGS catalytic activity in small numbers of blood cell samples is presented in chapter 3. The assay was applied to determine FPGS activity in peripheral blood mononuclear cells (PBMCs) of RA patients and clinical acute leukemic specimens. FPGS activity was markedly higher (50-100-fold) in leukemia samples as compared to PBMCs of RA patients. Next, we researched the impact of aberrant pre-mRNA splicing of FPGS in whole blood of 36 RA patients at baseline for MTX-responsiveness after 3- and 6-months therapy (chapter 4). Particularly, the expression of a FPGS splice variant, a partial retention of FPGS intron 8 (8PR) over wild type (WT) FPGS, was explored. Intriguingly, higher baseline ratios of 8PR/WT were significantly associated with higher DAS44 scores at 3 and 6 months MTX therapy. These were the first data in RA linking aberrant FPGS splicing to reduced MTX-responsiveness. Multiple computational tools to investigate splicing are available for researchers, but their mutual performance is unclear. The performance of 3 ...