Modeling GATA2 deficiency in mice: the R396Q mutation disrupts normal hematopoiesis

GATA2 deficiency is an autosomal dominant germline disorder of immune dysfunction and bone marrow failure with a high propensity for leukemic transformation in adolescents, present in up to 7% of pediatric myelodysplastic syndrome (MDS) and 15% of advanced MDS cases. While sequencing studies have identified several secondary mutations thought to contribute to malignancy, the mechanisms of disease progression have been difficult to identify due to a lack of disease-specific experimental models. Here, we generated a murine model of one of the most common GATA2 mutations associated with leukemic progression in GATA2 deficiency, Gata2R396Q/+. While mutant mice exhibit mild defects in peripheral blood output throughout life, they display significant hematopoietic abnormalities in the bone marrow (BM), including a reduction in hematopoietic stem cell (HSC) function and intrinsic biases toward specific stem cell subsets that differ from previous models of GATA2 loss. Supporting this observation, single-cell RNA sequencing of BM hematopoietic progenitors revealed a loss of HSC stemness, myeloid-bias, and accelerated ageing phenotype. Importantly, we show that Gata2R396Q/+ exerts effects early in hematopoietic development, as mutant mice generate fewer HSCs in the aorta gonad mesonephros, and fetal liver HSCs have reduced function. This reduced pool of HSCs and aged phenotype could be potential contributors to leukemic transformation in patients, and our model provides a useful tool to study the mechanisms of malignant transformation in GATA2 deficiency. Bone marrow LSK cells were isolated from WT and mutant mice for single cell RNA-seq via the 10X Genomics 3' v3.1 kit.GATA2缺陷是一种常染色体显性生殖系疾病（autosomal dominant germline disorder），以免疫功能异常（immune dysfunction）与骨髓衰竭（bone marrow failure）为核心特征，青少年患者发生白血病转化（leukemic transformation）的风险极高，在儿童骨髓增生异常综合征（pediatric myelodysplastic syndrome, MDS）患者中占比可达7%，在进展期MDS病例中占比达15%。尽管已有测序研究（sequencing studies）鉴定出若干被认为可促进恶性肿瘤（malignancy）发生的继发突变（secondary mutations），但由于缺乏疾病特异性实验模型（disease-specific experimental models），疾病进展的具体分子机制仍难以阐明。本研究构建了一种与GATA2缺陷患者白血病进展最常见突变之一相关的小鼠模型（murine model）：Gata2R396Q/+。该突变小鼠在整个生命周期中仅表现出轻微的外周血生成（peripheral blood output）缺陷，但其骨髓（bone marrow, BM）却存在显著的造血异常（hematopoietic abnormalities），包括造血干细胞（hematopoietic stem cell, HSC）功能受损，以及相较于既往GATA2缺失模型，其特定干细胞亚群呈现出更为特异的内在功能偏向性。对骨髓造血祖细胞开展的单细胞RNA测序（single-cell RNA sequencing）分析佐证了这一观察：突变小鼠的造血干细胞干性丢失、髓系偏向性增强，并呈现加速衰老表型。值得注意的是，Gata2R396Q/+的致病效应早在造血发育早期即可显现：突变小鼠主动脉-性腺-中肾区（aorta gonad mesonephros）产生的造血干细胞数量减少，且胎肝造血干细胞（fetal liver HSCs）功能下降。上述造血干细胞池缩减与衰老表型，或可解释患者的白血病转化风险；而本研究构建的模型，也为探究GATA2缺陷的恶性转化机制提供了实用工具。本研究通过10X Genomics 3'端v3.1测序试剂盒（10X Genomics 3' v3.1 kit），对野生型（wild type, WT）与突变小鼠的骨髓LSK细胞（LSK cells）进行了单细胞RNA测序。