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Somatic mutations have a central role in cancer, but there are also a few rare autoimmune diseases in which somatic mutations play a major role. We have recently shown that nonsynonymous somatic mutations with low allele fractions are preferentially detectable in CD8+ cells and that the STAT3 gene is a promising target for screening. Here, we analyzed somatic mutations in the STAT3 SH2 domain in peripheral blood CD8+ cells in a set of 94 multiple sclerosis (MS) patients and 99 matched controls. PCR amplicons targeting the exons 20 and 21 of STAT3 were prepared and sequenced using the Illumina MiSeq instrument with 2x300bp reads. We designed a novel variant calling method, optimized for large number of samples, high sequencing depth (>25,000x) and small target genomic area. Overall, we discovered 64 STAT3 somatic mutations in the 193 donors, of which 63 were non-synonymous and 77% have been previously reported in cancer or lymphoproliferative disease. The overall median variant allele fraction was 0.065% (range 0.007-1.2%), without significant difference between MS and controls (p = 0.82). There were 26 (28%) MS patients vs. 24 (24%) controls with mutations (p = 0.62). Two or more mutations were found in 9 MS patients vs. 2 controls (p = 0.03, pcorr = 0.12). Carriership of mutations associated with older age and lower neutrophil counts. These results demonstrate that STAT3 SH2 domain is a hotspot for somatic mutations in CD8+ cells with a prevalence of 26% among the participants. There were no significant differences in the mutation prevalences between MS patients and controls. Further research is needed to elucidate the role of antigenic stimuli in the expansion of the mutant clones. Furthermore, the high discovered prevalence of STAT3 somatic mutations makes it feasible to analyze these mutations directly in tissue-infiltrating CD8+ cells in autoimmune diseases.

Somatic mutations have a central role in cancer, but there are also a few rare autoimmune diseases in which somatic mutations play a major role. We have recently shown that nonsynonymous somatic mutations with low allele fractions are preferentially detectable in CD8+ cells and that the STAT3 gene is a promising target for screening. Here, we analyzed somatic mutations in the STAT3 SH2 domain in peripheral blood CD8+ cells in a set of 94 multiple sclerosis (MS) patients and 99 matched controls. PCR amplicons targeting the exons 20 and 21 of STAT3 were prepared and sequenced using the Illumina MiSeq instrument with 2x300bp reads. We designed a novel variant calling method, optimized for large number of samples, high sequencing depth (>25,000x) and small target genomic area. Overall, we discovered 64 STAT3 somatic mutations in the 193 donors, of which 63 were non-synonymous and 77% have been previously reported in cancer or lymphoproliferative disease. The overall median variant allele fraction was 0.065% (range 0.007–1.2%), without significant difference between MS and controls (p = 0.82). There were 26 (28%) MS patients vs. 24 (24%) controls with mutations (p = 0.62). Two or more mutations were found in 9 MS patients vs. 2 controls (p = 0.03, pcorr = 0.12). Carriership of mutations associated with older age and lower neutrophil counts. These results demonstrate that STAT3 SH2 domain is a hotspot for somatic mutations in CD8+ cells with a prevalence of 26% among the participants. There were no significant differences in the mutation prevalences between MS patients and controls. Further research is needed to elucidate the role of antigenic stimuli in the expansion of the mutant clones. Furthermore, the high discovered prevalence of STAT3 somatic mutations makes it feasible to analyze these mutations directly in tissue-infiltrating CD8+ cells in autoimmune diseases.

Somatic mutations have a central role in cancer but their role in other diseases such as common autoimmune disorders is not clear. Previously we and others have demonstrated that especially CD8+ T cells in blood can harbor persistent somatic mutations in some patients with multiple sclerosis (MS) and rheumatoid arthritis. Here we concentrated on CD8+ cells in more detail and tested (i) how commonly somatic mutations are detectable, (ii) does the overall mutation load differ between MS patients and controls, and (iii) do the somatic mutations accumulate non-randomly in certain genes? We separated peripheral blood CD8+ cells from newly diagnosed relapsing MS patients (n = 21) as well as matched controls (n = 21) and performed next-generation sequencing of the CD8+ cells’ DNA, limiting our search to a custom panel of 2524 immunity and cancer related genes, which enabled us to obtain a median sequencing depth of over 2000x. We discovered nonsynonymous somatic mutations in all MS patients’ and controls’ CD8+ cell DNA samples, with no significant difference in number between the groups (p = 0.60), at a median allelic fraction of 0.5% (range 0.2–8.6%). The mutations showed statistically significant clustering especially to the STAT3 gene, and also enrichment to the SMARCA2, DNMT3A, SOCS1 and PPP3CA genes. Known activating STAT3 mutations were found both in MS patients and controls and overall 1/5 of the mutations were previously described cancer mutations. The detected clustering suggests a selection advantage of the mutated CD8+ clones and calls for further research on possible phenotypic effects.

Somatic mutations have a central role in cancer but their role in other diseases such as common autoimmune disorders is not clear. Previously we and others have demonstrated that especially CD8+ T cells in blood can harbor persistent somatic mutations in some patients with multiple sclerosis (MS) and rheumatoid arthritis. Here we concentrated on CD8+ cells in more detail and tested (i) how commonly somatic mutations are detectable, (ii) does the overall mutation load differ between MS patients and controls, and (iii) do the somatic mutations accumulate non-randomly in certain genes? We separated peripheral blood CD8+ cells from newly diagnosed relapsing MS patients (n = 21) as well as matched controls (n = 21) and performed next-generation sequencing of the CD8+ cells' DNA, limiting our search to a custom panel of 2524 immunity and cancer related genes, which enabled us to obtain a median sequencing depth of over 2000x. We discovered nonsynonymous somatic mutations in all MS patients' and controls' CD8+ cell DNA samples, with no significant difference in number between the groups (p = 0.60), at a median allelic fraction of 0.5% (range 0.2-8.6%). The mutations showed statistically significant clustering especially to the STAT3 gene, and also enrichment to the SMARCA2, DNMT3A, SOCS1 and PPP3CA genes. Known activating STAT3 mutations were found both in MS patients and controls and overall 1/5 of the mutations were previously described cancer mutations. The detected clustering suggests a selection advantage of the mutated CD8+ clones and calls for further research on possible phenotypic effects.