Abstract 2535: Single-cell transcriptome analysis on lymphocytes of GATA2 deficiency patients
Authors: Hirvonen Elina A. , Natri Inari , Mattila Pirkko , Mattila Jenni , Pitkänen Esa , Porkka Kimmo , Kilpivaara Outi , Wartiovaara-Kautto Ulla 
Published: August 2018

Abstract
GATA2 is an essential transcription factor in the proliferation and maintenance of hematopoietic stem and progenitor cells. Germline GATA2 mutations cause hematological malignancies, especially myelodysplastic syndrome and acute myeloid leukemia. Pre-leukemic phenotype of GATA2 deficient patients varies markedly. However, peripheral blood (PB) lymphopenia is characteristic for almost all the patients and encounters most prominently B and natural killer (NK) cells. We aimed to gain understanding on the effects of GATA2 deficiency in hematopoiesis by comparing expression patterns in lymphocytes in GATA2 wild type and GATA2 deficient individuals.
We conducted single-cell transcriptomic analysis using 10x Genomics Chromium Single Cell 3′ Solution on PB samples from three healthy controls and three GATA2 deficient patients (p.Q328X, p.T354M, and one haploinsufficiency due to unidentified reason). Erythrocytes were lysed prior to washing, filtering and resuspending white blood cells in 0.04% BSA/PBS (1x106 cells/mL). The gel beads in emulsion were generated using Chromium Single Cell 3' v2 Reagent Kit. The sample libraries, prepared according to 10x Genomics instructions, were sequenced in Illumina HiSeq 2500. Cell Ranger v1.3 analysis pipelines (10x Genomics) were used to demultiplex and convert Chromium single cell 3' RNA-sequencing barcode; read data to FASTQ files; and generate align reads and gene-cell matrices. The data were analyzed using Seurat, an R package designed for single cell analysis. Cell type clusters were identified through known markers from Seurat's graph-based clustering.
Significant gene expression differences in lymphocytes (GATA2 deficient patients vs. controls) were observed (Wilcoxon rank sum test, Bonferroni-corrected p &lt;0.05). In NK cells the distinction was found e.g. in KLRB1, CD74, CCL5, STAT1, and LAG3, all linked to immune responses, and BTG1 and DDIT4, regulating cell proliferation and differentiation. Expression changes in immune system-associated genes were also noted in T cells e.g. in STAT1, IL7R, CD48, and CXCR4, as well as in NPM1, TPT1, and TXNIP, genes identified to participate in diverse cellular processes. Also, several genes encoding ribosomal proteins were differentially expressed between patients' and controls' T cells. Comparison of B cells revealed changes in e.g. GZMH, PPBP, and LCN2, which, too, play roles in the immune system.
We observed significant dissimilarities in gene expression pattern in GATA2 deficient patients' vs. controls' lymphocytes. These were reflected in several genes involved in immune system and cellular processes. Connection between our findings and patients' clinical manifestations is intriguing and may have an impact in the development of future patient care. We will extend our material and include also bone marrow sample single-cell transcriptomes and healthy mutation carriers to the analysis.
Citation Format: Elina A. Hirvonen, Inari Natri, Pirkko Mattila, Jenni Mattila, Esa Pitkänen, Kimmo Porkka, Outi Kilpivaara, Ulla Wartiovaara-Kautto. Single-cell transcriptome analysis on lymphocytes of GATA2 deficiency patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2535.