Platelet RNAseq data for SLFN14 K219N patients

Pathogenic missense variants in SLFN14, which encodes an RNA endoribonuclease protein that regulates rRNA degradation, are known to cause inherited thrombocytopenia with impaired platelet aggregation and ATP secretion. Despite rather mild laboratory defects, these patients display an obvious bleeding phenotype. The function of SLFN14 in megakaryocyte (MK) and platelet biology is unknown. This study aims to characterize the platelet transcriptome in patients with a SLFN14 K219N variant and model the disease in the immortalized megakaryocyte cell line imMKCL. Total platelet RNA was sequenced for two patients and 19 healthy controls. Differential gene expression analysis yielded a total of 2999 and 2888 significantly (|log2FC|>1, FDR<0.05) up- and downregulated genes, respectively. Remarkably, these downregulated genes were not enriched for any biological pathway while upregulated genes were enriched for pathways involved in (mitochondrial) translation and transcription with a significant upregulation of 134 ribosomal protein genes (RPG). Heterozygous and homozygous SLFN14 K219N imMKCL showed a defect in MK differentiation that aggravated during cell passaging of undifferentiated cells and in proplatelet formation. SLFN14 defective platelets and MK showed signs of rRNA degradation while this was absent in undifferentiated imMKCL cells. Upregulation of RPG through increased mTOR signaling in SLFN14 K219N MK seems to be a compensatory response for rRNA degradation. Indeed, mTOR inhibition with rapamycin resulted in further enhanced rRNA degradation in SLFN14 K219N MK. Taken together, our study indicates dysregulation of mTOR-regulated ribosomal biogenesis as the disease mechanism for SLFN14-related thrombocytopenia.EGA study EGAS00001006339

SLFN14编码一种调控核糖体RNA（ribosomal RNA, rRNA）降解的核糖核酸内切酶（RNA endoribonuclease），其致病错义变异已知会引发遗传性血小板减少症，伴随血小板聚集与ATP分泌功能受损。尽管实验室检测缺陷相对轻微，但此类患者会出现明显的出血表型。目前SLFN14在巨核细胞（megakaryocyte, MK）与血小板生物学中的功能尚不明确。本研究旨在对携带SLFN14 K219N变异的患者的血小板转录组进行特征分析，并在永生化巨核细胞系imMKCL中构建该疾病模型。本研究对2名患者与19名健康对照者的血小板总RNA进行了测序。差异基因表达分析共筛选得到2999个显著上调基因与2888个显著下调基因，筛选标准为|log2FC|>1且错误发现率（False Discovery Rate, FDR）<0.05。值得注意的是，下调基因未富集到任何生物学通路，而上调基因则富集于（线粒体）翻译与转录相关通路，且134个核糖体蛋白基因（ribosomal protein genes, RPG）呈现显著上调。杂合与纯合型SLFN14 K219N imMKCL细胞均表现出巨核细胞分化缺陷，该缺陷会随未分化细胞的传代培养以及前血小板形成过程而进一步加重。SLFN14功能缺陷的血小板与巨核细胞呈现核糖体RNA降解异常，而未分化的imMKCL细胞中并未观察到此现象。SLFN14 K219N巨核细胞中通过雷帕霉素靶蛋白（mechanistic Target of Rapamycin, mTOR）信号通路上调核糖体蛋白基因，似乎是针对核糖体RNA降解的代偿性反应。进一步实验证实，使用雷帕霉素抑制mTOR通路，会进一步加剧SLFN14 K219N巨核细胞的核糖体RNA降解。综上，本研究表明mTOR调控的核糖体生物发生失调是SLFN14相关血小板减少症的致病机制。本研究数据存档于EGA数据库，研究编号为EGAS00001006339