Deciphering molecular mechanisms of multiple sclerosis-targeting HLA variants with applied epigenetics

Abstract

Multiple Sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of the central nervous system affecting approximately 3.1 million people worldwide and predominantly women. The major genetic risk factor for the disease is the Human Leukocyte Antigen (HLA) locus, while environmental and epigenetic factors have been found to contribute to the disease, increasing the overall risk. The HLA-DR15 haplotype, which comprises the HLA-DRB1*15:01 and HLA-DRB5*01:01 alleles, is the most common haplotype in Caucasians with MS. This thesis investigated the regulation of the HLA locus by epigenetic changes conferred by DNA methylation. Owing to the complexity of the HLA locus, its epigenetic regulation remains poorly understood. To address this, we developed and applied complementary approaches including epigenome editing tools, targeted library sequencing, RNA interference, and in vivo animal models. A custom capture-based sequencing approach was established to simultaneously decipher the genetic and epigenetic landscape of the HLA locus and other immune-related genes under inflammatory conditions. Bisulfite-converted (BS) libraries provided greater sensitivity for detection of DNA methylation compared with enzymatic methylation libraries (EM), while enabling accurate allele calling. Long-read ONT sequencing was further employed to fully reconstruct the HLA haplotypes in THP-1 cells, allowing haplotype-specific methylation analysis. We found that inflammation induces early but subtle DNA methylation changes across the HLA locus, which increase over time. Notably, exonic and intronic regions of the HLA genes showed changes in methylation upon inflammation, and were targeted by the dCas9-DNMT3A complex, suggesting that these regions may contribute to HLA gene regulation. In vivo targeting of the promoter of the murine HLA-DRB1 ortholog, H2-Ab1, showed that despite increased and site- specific promoter methylation, no transcriptional changes could be detected. These findings pinpoint the complexity of DNA methylation-mediated gene regulation and suggest that further studies are required to identify the functionally relevant CpGs within the locus. Apart from DNA methylation, other epigenetic mechanisms, such as microRNAs (miRNAs), play a role in MS pathogenesis. Among these, miR-150 has been found to be upregulated in cerebrospinal fluid (CSF) cells of MS patients in relapse. In this thesis, we demonstrated that miR-150 presence exacerbates the disease and is linked with T regulatory cell function. Genetic ablation of miR-150 ameliorated disease manifestations in mice and enhanced T regulatory cell functions, identifying miR-150 as a potential target for future therapeutic opportunities in MS. In conclusion, this thesis shows that epigenetic mechanisms, such as DNA methylation and microRNAs, play a role in the pathogenesis of Multiple Sclerosis. DNA methylation changes arise following inflammation in putative regulatory regions of the HLA, while miR-150 has a particular role in regulatory T cell biology. Together, these findings advance our understanding of epigenetic regulation in MS and identify potential targets for future therapeutic intervention. List of scientific papers I. Pahlevan Kakhki, M., Rangani, F., Ewing, E., Starvaggi Cucuzza, C., Zheleznyakova, G., Kalomoiri, M., Kenny, L., Raghavan, A., Rao Prakash, C., van den Hoeven, G., Venkata S. Badam, T., Covacu, R., Andreou, I., Needhamsen, M., Kular, L., Jagodic, M., 2026. Comprehensive profiling of CRISPR/dCas9 epigenome editors indicates a complex link between on and off target effects. Genome Biology, 2026 Jan 31;27(1):52. https://doi.org/10.1186/s13059-026-03967-6 II. Kalomoiri, M., Rao Prakash, C., Lagström, S., Hauschulz, K., Ewing, E., Shchetynsky, K., Kular, L., Needhamsen, M., Jagodic, M., 2023. Simultaneous detection of DNA variation and methylation at HLA class II locus and immune gene promoters using targeted SureSelect Methyl-Sequencing. Front Immunol, 2023 Aug 24;14:1251772. https://doi.org/10.3389/fimmu.2023.1251772 III. Kalomoiri, M., van den Hoeven, G., Rao Prakash, C., Ewing, E., Gvojić, N., Athipotta Variem, A., Martin, R., Kakhki Pahlevan, M., Needhamsen, M., Kular, L., Jagodic, M. Characterization of the epigenetic regulation of the HLA-DR15 haplotype. [Manuscript] IV. Kalomoiri, M., Sorini, C., Vos, S., Camargo, A., Tsalikou, A., Krstic, A., Rao Prakash, C., Svenningsson, P., Needhamsen, M., Pahlevan Kakhki, M., Kular, L., Jagodic, M. Transgenic dCas9-DNMT3A mouse lines enable ex vivo and in vivo methylation editing with locus-dependent transcriptional effects. bioRxiv, 2026.02.26.708277. https://doi.org/10.64898/2026.02.26.708277 V. Piket, E#., Kalomoiri, M#., Sorini, C., Beckers, V., Finardi, A., N'diaye, M., Williams, L., Olofsson, A., Ewing, E., Han, Y., Pahlevan Kakhki, M., Venkata Satya Badam, T., Zeitelhofer Adzemovic, M., Lavrnja, I., Hallén, N., Rangani, R., Nyberg, WA., Furlan, R., Espinosa, A., Needhamsen, M., Ortlieb Guerreiro-Cacais, A., Kular, L., Jagodic, M. MicroRNA-150 controls regulatory T cells and modulates neuroinflammation in experimental autoimmune encephalomyelitis and multiple sclerosis. [Manuscript] (#equal contribution)