Identification of Possible Driver Gene in Patients with Acquired Pure Red Cell Aplasia

OBJECTIVE: Acquired pure red cell aplasia (aPRCA) is a disorder characterized by normocytic anemia associated with reticulocytopenia and an absence of erythroblasts. The pathogenesis of aPRCA is believed to be the induction of autoimmunity, although many underline mechanisms are still not known. In this study, we aimed to identify the gene mutations in aPRCA patients and its possible effects on the origin of the disease.

METHODS: Blood genome DNA extracted from ten newly diagnosed patients with aPRCA was checked with whole-exome sequencing (WES). Candidate gene (nonsynonymous, splicing single-nucleotide variants (SNVs) or insertion/deletions (INDELs)) which met the criteria of high frequency in aPRCA but rare in 1000 Genomes were first confirmed by Sanger sequencing and checked the mRNA level in PRCA patients by quantitative real-time polymerase chain reaction. Next the pGreen-CMV-puro system was employed to generate lentivirus expressing short hairpin RNAs to target the candidate genes, and stable transfected K562 cell lines with silenced candidate genes was constructed. Finally the erythroid and megakaryocytic differentiation was evaluated for the transfected K562 cell lines either by benzidine staining hemoglobin, CD235a or CD41 expression on the cell surface.

RESULTS: MKI67, SEC22B and STK10 genes who met the selection criteria were selected for further study. Of the three genes, mRNA level were down-regulated in the mutated patients. The mRNA expression of the three genes was all found decreased in the transfected K562 cell lines. Only STK10 silenced K562 cell lines showed significantly reduction in number of benzidine-positive cells and expression of CD235a stimulated by hemin compared with non-transfected cell lines. However, CD41 expression was similar in STK10 silenced K562cells to control K562 cells after inducted by phorbol 12-myristate 13-acetate. No difference was found between the transfected and non-transfected cell lines with MKI67 or SEC22B gene either in erythropoiesis or megakaryocyte hematopoiesis.

CONCLUSION: STK10 gene mutation is common in patients with aPRCA and causes the reduction of mRNAs expression in the mutated patients. Silence of STK10 gene in K562 cell lines might inhibit erythroid, but not megakaryocytic differentiation. STK10 might be one of the driver genes for the abnormal erythropoiesis in patients with aPRCA.