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3'-TARGET-seq: a novel method for high-sensitivity single-cell mutational analysis and parallel high throughput RNA-sequencing [Figures 5 and 6, Figure S7]

ABSTRACT: We developed 3'-TARGET-seq, a single-cell genotyping and RNA-seq method, which allows accurate detection of multiple mutations within single-cells from genomic and coding DNA in parallel with high throughput 3'-biased whole transcriptome analysis, providing a powerful tool to link transcriptional and genetic tumor heterogeneity. Single cell whole transcriptome sequencing of 2798 Lineage-CD34+ HSPC (Hematopoietic Stem and Progenitor Cells) from patients with JAK2-V617F mutant myelofibrosis and normal controls using 3'-TARGET-seq reveals distinct molecular signatures associated with the presence of somatic mutations in single cells as well as distinct transcriptional profiles of WT cells from patient samples as compared with normal controls.

ORGANISM(S): Homo sapiens

PROVIDER: GSE122198 | GEO | 2019/02/11

REPOSITORIES: GEO

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Project description:Even though mutations in epigenetic regulators frequently occur in myeloproliferative neoplasms, their effects on the epigenome have not been well studied. Furthermore, even though primary myelofibrosis (PMF) has a markedly worse prognosis compared to essential thrombocytosis (ET) or polycythemia vera (PV), the molecular distinctions between these subgroups are not well elucidated. We performed the HELP (HpaII tiny fragment enriched by LM-PCR) assay to study genome-wide methylation in PV, ET and PMF samples compared with healthy controls. We determined that PV and ET are characterized by aberrant promoter hypermethylation while PMF is an epigenetically distinct subgroup characterized by both aberrant hyper and hypomethylation. Aberrant hypomethylation in PMF was seen to occur in non CpG island loci, demonstrating further qualitative differences between the disease subgroups. The differentially methylated genes in PV and ET were involved predominantly in cell signaling pathways and were enriched for binding sites of GATA1 and other transcription factors. In contrast, aberrantly methylated genes in PMF were involved in inflammatory pathways and were enriched for NF1 (NFI), LEF1 and other transcription factors. Within the PMF subgroup, cases with ASXL1 disruptions formed an epigenetically distinct subgroup with relatively increased methylation. Cases of MPNs with TET2 mutations demonstrated decreased levels of hydroxymethylation and distinct set of hypermethylated genes. In contrast, the JAK2V617F mutation did not drive epigenetic clustering within MPNs. Finally, the significance of aberrant methylation was demonstrated by sensitivity of MPN derived cell lines to decitabine. These results demonstrate epigenetic differences between PMF and PV/ET and reveal methylomic signatures of ASXL1 and TET2 mutations. The study population consisted of 26 MPN patients (6 cases of ET, 8 cases of PV and 12 cases of PMF) and 3 healthy controls. Individual HpaII restriction digest profiles were compared to an internal MspI digest control, to yield differentially methylated fragments for every sample. The purpose of this study was to assess genome wide patterns of DNA methylation across the MPN stratified by disease class.

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3'-TARGET-seq: a novel method for high-sensitivity single-cell mutational analysis and parallel high throughput RNA-sequencing [Figures 5 and 6, Figure S7]

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