Contributions
Abstract: P414
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - Genetics /Epigenetics and Pharmacogenetics
Background: Genetic and environmental risk factors and treatment paradigms overlap in multiple sclerosis (MS) and hematological malignancies. Somatic mutations have a central role in cancer but their role in MS is not known. Moreover, the general features of somatic mutations in non-cancerous cells are unclear such as mutation frequencies, clonal size (allelic fraction) and mutational hot-spots.
Aims: In this pilot study we addressed the following questions: Are somatic mutations detectable in MS patients" blood and what is their allelic fraction within a specific cell population? Are there mutational hot-spots? Do the mutations preferentially occur in a particular cell population?
Methods: We extracted peripheral blood mononuclear cells from 16 relapsing MS patients, and separated them into CD4+ T-lymphocytes, CD8+ T-lymphocytes, CD19+ B-lymphocytes and CD4/CD8/CD19 negative populations (antibody MicroBeads, Miltenyi Biotec). From these separated cell populations, we performed next-generation DNA sequencing targeting coding regions of a panel of immunologically relevant genes with a sequencing depth of 1000x (target capture by SeqCap, Roche Nimblegen; sequencing with Illumina HiSeq 2500). This depth allows the detection of somatic mutations with low allelic fractions. Potential mutations in the discovery phase were confirmed by another sequencing method (amplicon-based sequencing, Illumina MiSeq).
Results: It was possible to detect and confirm somatic mutations in MS patients´ peripheral blood. More than half of the MS patients had non-synonymous somatic mutations. The allelic fractions of the mutations within each of the cell populations were under 5%. The mutations were significantly enriched into the CD8+ T-lymphocytes (85% of mutations). There were no mutational hot-spots, each patient had a unique mutational profile. Most of the mutations were novel, while a few were previously reported in cancer databases.
Conclusions: We have outlined an approach by which next-generation sequencing can be used for screening low-frequency somatic mutations in blood. These results are among the first views into the landscape of somatic mutations in non-cancerous setting. More than half of the MS patients had somatic mutations, and the mutations were enriched in the CD8+ T-lymphocytes. Although the significance of somatic mutations in MS is presently unclear, their possible role as drivers of autoimmunity warrants further research.
Disclosure: This study has been funded by research grants from the Finnish Academy, Helsinki University Hospital, Finnish Multiple Sclerosis Foundation, University of Helsinki and Biogen Finland. Pentti Tienari has made consultations for Bayer, Biogen, Genzyme, Merck, Novartis and Teva, Others have nothing to disclose.
Abstract: P414
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - Genetics /Epigenetics and Pharmacogenetics
Background: Genetic and environmental risk factors and treatment paradigms overlap in multiple sclerosis (MS) and hematological malignancies. Somatic mutations have a central role in cancer but their role in MS is not known. Moreover, the general features of somatic mutations in non-cancerous cells are unclear such as mutation frequencies, clonal size (allelic fraction) and mutational hot-spots.
Aims: In this pilot study we addressed the following questions: Are somatic mutations detectable in MS patients" blood and what is their allelic fraction within a specific cell population? Are there mutational hot-spots? Do the mutations preferentially occur in a particular cell population?
Methods: We extracted peripheral blood mononuclear cells from 16 relapsing MS patients, and separated them into CD4+ T-lymphocytes, CD8+ T-lymphocytes, CD19+ B-lymphocytes and CD4/CD8/CD19 negative populations (antibody MicroBeads, Miltenyi Biotec). From these separated cell populations, we performed next-generation DNA sequencing targeting coding regions of a panel of immunologically relevant genes with a sequencing depth of 1000x (target capture by SeqCap, Roche Nimblegen; sequencing with Illumina HiSeq 2500). This depth allows the detection of somatic mutations with low allelic fractions. Potential mutations in the discovery phase were confirmed by another sequencing method (amplicon-based sequencing, Illumina MiSeq).
Results: It was possible to detect and confirm somatic mutations in MS patients´ peripheral blood. More than half of the MS patients had non-synonymous somatic mutations. The allelic fractions of the mutations within each of the cell populations were under 5%. The mutations were significantly enriched into the CD8+ T-lymphocytes (85% of mutations). There were no mutational hot-spots, each patient had a unique mutational profile. Most of the mutations were novel, while a few were previously reported in cancer databases.
Conclusions: We have outlined an approach by which next-generation sequencing can be used for screening low-frequency somatic mutations in blood. These results are among the first views into the landscape of somatic mutations in non-cancerous setting. More than half of the MS patients had somatic mutations, and the mutations were enriched in the CD8+ T-lymphocytes. Although the significance of somatic mutations in MS is presently unclear, their possible role as drivers of autoimmunity warrants further research.
Disclosure: This study has been funded by research grants from the Finnish Academy, Helsinki University Hospital, Finnish Multiple Sclerosis Foundation, University of Helsinki and Biogen Finland. Pentti Tienari has made consultations for Bayer, Biogen, Genzyme, Merck, Novartis and Teva, Others have nothing to disclose.