Detecting somatic mutations in lymphocytes of both healthy individuals and patients with neuroimmunological disease

Somatic mutations have a pivotal role in cancer, but recently they have been found to be causal in several rare autoimmune disorders as well, when present in lymphocytes. A similar role for somatic mutations has been speculated in more common autoimmune disorders but has not yet been shown. One example of a common autoimmune disease is multiple sclerosis (MS), a challenging disorder of the central nervous system that has several known risk factors but the exact mechanism that triggers the disease is not known, making it an interesting target to study the hypothesis. However, the detectability and prevalence of low allele fraction somatic mutations in the circulating cells of the adaptive immune system has not been well-studied and needs characterization to aid the development of suitable experiments. In this study, we recruited both MS patients and unaffected individuals and examined the presence and detectability of possibly functional low allele fraction somatic mutations in lymphocytes separated from peripheral blood. We developed custom variant calling and filtering methods to robustly detect somatic mutations in inherently noisy very deep next generation sequencing data. We performed three different sequencing experiments that form the core of the individual parts of this dissertation. For the first sequencing experiment, we recruited 16 participants with MS and 4 with other autoimmune disease. Separation of CD4+ T cells, CD8+ T cells, and CD19+ B cells and CD4-/CD8-/CD19- cells was performed from the peripheral blood of the participants using immunomagnetic beads. The purities of the lymphocyte subpopulations were checked with fluorescence-activated cell sorting. With the DNA extracted from the cell fractions, we performed hybridization-based capture of the exons of a panel of 986 immunity and cancer related genes and used Illumina sequencing that reached a depth of 700x. Somatic mutation findings were confirmed by amplicon sequencing. We found 27 verifiable nonsynonymous somatic mutations that were overwhelmingly enriched in the CD8+ T cell fraction, as 85% of the detected mutations were found in these cells. In follow-up sequencing, performed after a median time of 2.3 years, 96% of the mutations were still detectable, showing that they persist in blood over time. Having observed that detectable somatic mutations are enriched in CD8+ T cells, we concentrated on this cell fraction in the second sequencing experiment. We recruited 21 MS patients and 21 unaffected participants and used an expanded gene panel consisting of exons of 2524 immunity and cancer related genes to sequence the samples to an average depth of 2400x. With the improved sequencing depth, we detected somatic mutations in the CD8+ T cells of every participant, at a median allele fraction of 0.5%. There was no significant difference between the MS and unaffected groups in mutation prevalence. The mutations however showed significant enrichment in some genes, in particular STAT3 (p=9.0e-8), and to a lesser extent, in the SMARCA2, DNMT3A, SOCS1 and PPP3CA genes. We performed a third sequencing experiment concentrating on the STAT3 gene only, enabling a greatly increased sequencing depth of over 25000x. We recruited 94 MS patients and 99 matched controls and prepared PCR amplicons targeting the central exons that code the STAT3 SH2 domain. It was observed that 26% of the study participants carried somatic STAT3 mutations in their CD8+ T cells. The mutations were overwhelmingly nonsynonymous and contained repeating observations of previously cancer- or malignancy associated changes such as D661Y and S614R, suggesting a growth-promoting effect for the mutated clones. We detected no difference in mutation positivity between the MS and unaffected groups. However, the presence of STAT3 mutations was associated with lower neutrophil counts and increased titers of antibodies directed against the Epstein-Barr virus (EBV), a known key risk factor of MS. Collectively the results of this thesis demonstrate the existence low-allele-fraction mutant clones that are especially common in CD8+ T cells. There were no major differences in mutation prevalence between MS patients vs. controls in CD8+ T cells. An outstanding mutation hotspot in CD8+ T cells was the STAT3 SH2 domain and carriership of these mutations associated with lower neutrophil counts and higher EBV IgG levels. These results uncover new targets for research in disorders of immunity.