Division of Medical Genetics, Department of Medicine, University of California, San Diego, La Jolla, CA, USA; Bioinformatics and Systems Biology Program, University of California, San Diego, La Jolla, CA, USA.
Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
Cell Rep. 2024 Jul 23;43(7):114436. doi: 10.1016/j.celrep.2024.114436. Epub 2024 Jul 4.
Single-gene missense mutations remain challenging to interpret. Here, we deploy scalable functional screening by sequencing (SEUSS), a Perturb-seq method, to generate mutations at protein interfaces of RUNX1 and quantify their effect on activities of downstream cellular programs. We evaluate single-cell RNA profiles of 115 mutations in myelogenous leukemia cells and categorize them into three functionally distinct groups, wild-type (WT)-like, loss-of-function (LoF)-like, and hypomorphic, that we validate in orthogonal assays. LoF-like variants dominate the DNA-binding site and are recurrent in cancer; however, recurrence alone does not predict functional impact. Hypomorphic variants share characteristics with LoF-like but favor protein interactions, promoting gene expression indicative of nerve growth factor (NGF) response and cytokine recruitment of neutrophils. Accessible DNA near differentially expressed genes frequently contains RUNX1-binding motifs. Finally, we reclassify 16 variants of uncertain significance and train a classifier to predict 103 more. Our work demonstrates the potential of targeting protein interactions to better define the landscape of phenotypes reachable by missense mutations.
单基因错义突变的解释仍然具有挑战性。在这里，我们通过可扩展的功能筛选测序（SEUSS），一种 Perturb-seq 方法，在 RUNX1 的蛋白质界面产生突变，并量化它们对下游细胞程序活性的影响。我们评估了髓系白血病细胞中 115 个突变的单细胞 RNA 图谱，并将它们分为三个具有不同功能的组，野生型（WT）样、失活（LoF）样和功能减弱（Hypo），我们在正交试验中对其进行了验证。LoF 样变体主要存在于 DNA 结合位点，在癌症中频繁发生；然而，仅发生重现并不预示着功能影响。功能减弱的变体与 LoF 样变体具有相似的特征，但倾向于蛋白质相互作用，促进神经生长因子（NGF）反应和中性粒细胞募集细胞因子的基因表达。差异表达基因附近的可及 DNA 经常包含 RUNX1 结合基序。最后，我们重新分类了 16 个意义不明的变体，并训练了一个分类器来预测更多的 103 个变体。我们的工作表明，靶向蛋白质相互作用具有潜在的可能性，可以更好地定义错义突变可达表型的景观。
