Single-cell RNA Sequencing To Explore The Pathogenesis Of Myelodysplastic Syndromes

Objective:Myelodysplastic syndrome(MDS)is a group of heterogeneous clonal diseases,and the pathogenesis has not been fully elucidated.The origin of clonality and abnormal differentiation of hematopoietic stem and progenitor cells(HSPCs)play an important role in the pathogenesis of MDS.Our group has explored the mechanism of clonal hematopoiesis,immune abnormality and hemocytopenia in MDS previously.In this study,single-cell RNA sequencing(sc RNA-seq)technique was applied to draw the cell landscape and trajectory of HSPCs differentiation and development in MDS,to identify the abnormal expression markers of HSPCs,and investigate the functions,to explore the mechanism of clonal proliferation and abnormal differentiation of HSPCs.It is an in-depth study on the pathogenesis of stem cells in MDS.Methods:Newly diagnosed MDS patients,acute myeloid leukemia(AML)patients and controls in General Hospital of Tianjin Medical University from October 2019 to January 2021 were enrolled in our study.Part I Data on sc RNA-seq of 5 patients with MDS[2 low-risk(LR-MDS)patients and 3 high-risk(HR-MDS)ones]and 2AML patients with dysplasia were studied.Lineage negative(Lin~-)cells from bone marrow were sorted by immunomagnetic beads.Data of 17 healthy donors were from Gene Expression Omnibus(GEO)database.Bioinformatics analysis included quantification of gene expression,clustering,analysis on differentially expressed genes,function enrichment,Pseudotime analysis.Part II To verify the aberrant expression of genes related to ribosome protein and mitochondria in patients with MDS.Bone marrow samples from 23 controls,43 patients with MDS and 27 patients with AML were collected,and CD34~+HSPCs were sorted by immunomagnetic beads.The mRNA of genes related to ribosome protein and mitochondria which were shown aberrant expression in MDS patients in part I were verified by q PCR,and the correlation between mRNA and clinical characteristics of MDS patients was analyzed.Results:Part Ⅰ The gene expression patterns of patients with MDS and AML were significantly different from those of healthy donors.There is also significant heterogeneity in gene expression patterns between and within patients.The proportion of cellular component was significantly aberrant in patients with MDS and AML.The proportion of granulocytes(67.76%vs 7.26%)was significantly increased in patients,while frequency in erythroid(8.22%vs 61.75%)and lymphoid lineage(1.08%vs 20.12%)of patients were decreased.Hematopoietic stem cells(HSCs)/multipotent progenitors(MPPs)were expanded in HR-MDS(4.76%)and AML(28.85%)patients compared to healthy donors(2.27%).Similarly,myeloid progenitors were also expanded in HR-MDS(21.88%)and AML(5.7%)patients compared to healthy donors(3.42%).The proportion of mitochondrial genes in MDS patients was significantly higher than that in healthy donors,and even more than 50%in some patients,with the highest proportion in granulocytes.Function enrichment of differentially expressed genes showed that,compared with healthy donors,the up-regulated genes in HSCs/MPPs were mainly enriched in mitochondrial inheritance,electron transport chain,abnormal mitochondrial metabolism,neutrophil degranulation,oxidative phosphorylation in patients with MDS and AML.While the down-regulated genes were mainly enriched in ribosome,protein localization to cell membrane,nonsense-mediated decay,translation,r RNA processing,metabolism of RNA,cellular amide metabolic process,metabolism of proteins,gene expression,cellular biosynthetic process in HSCs/MPPs of patients with MDS and AML.The enriched functions of differentially expressed genes in HSCs/MPPs of MDS patients compared with healthy donors were similar.The pseudotime analysis showed that the expression trajectories of transcription factors GATA1,KLF1,HIF1A and TAL1 were significantly aberrant in the process of erythroid differentiation,and the expression trajectory of HIF1A was significantly aberrant in the process of granulocytic differentiation.Part Ⅱ The mRNA of RPL31(0.44±0.53,0.58±0.59,0.49±0.33 vs 1.00±0.57,P<0.01),RPL21(0.38±0.32,0.45±0.40,0.40±0.28 vs 1.00±1.03,P<0.01)and RPS17(0.37±0.44,0.43±0.35,0.44±0.34 vs 1.00±0.72,P<0.01)of CD34~+cells in patients with LR-MDS,HR-MDS and AML were significantly lower than those in controls.The RPS26 mRNA of CD34~+cells in LR-MDS and AML patients was significantly lower than that in controls(0.30±0.39,0.37±0.45 vs 1.00±1.05,P<0.01),and the RPS26 mRNA in HR-MDS patients was lower than that in controls,but the difference was not significant(0.52±0.50 vs 1.00±1.05,P=0.1340).The RPL31mRNA of CD34~+cells in patients with MDS was positively correlated with hemoglobin(HB)level(r=0.377,P=0.020)and platelet(PLT)counts(r=0.367,P=0.023),but not correlated with white blood cell(WBC)counts,absolute neutrophil counts(ANC)or the proportion of bone marrow blasts(P>0.05).The RPL21 mRNA of CD34~+cells in patients with MDS was also positively correlated with HB level(r=0.357,P=0.030)and PLT counts(r=0.330,P=0.046),but not correlated with WBC,ANC or the proportion of bone marrow blasts(P>0.05).The mRNA of MT-CO1(3.40±2.61、2.65±2.49、2.34±1.94 vs 1.00±0.67,P<0.01),MT-CYB(2.03±0.98、2.88±3.75、3.61±4.69 vs 1.00±0.47,P<0.05)of CD34~+cells in LR-MDS,HR-MDS and AML patients were significantly higher than those in controls.The mRNA of MT-CO1 and MT-CYB were not significantly correlated with HB,PLT,WBC or ANC.Conclusions:(1)Significant abnormalities were shown on gene expression patterns in patients with MDS and AML,and obvious heterogeneity of gene expression patterns were between and within patients.In patients with MDS and AML,the proportion of cellular component was significantly aberrant.The proportion of granulocytes was significantly increased,while the proportion of erythroid and lymphocytic lineage were significantly decreased.HSCs/MPPs and myeloid progenitor cells were expanded in HR-MDS and AML.(2)The proportion of mitochondrial genes in MDS patients was significantly increased,and even more than 50%in some patients,with the highest proportion in granulocytes.(3)Expression of genes related to mitochondrial metabolism,oxidative phosphorylation were up-regulated and those related to ribosome,translation,protein metabolism,RNA processing and gene expression were down-regulated in HSCs/MPPs in patients with MDS and AML.(4)The trajectories of some transcription factors were significantly abnormal in the process of erythroid and granulocytic differentiation.(5)The mRNA of genes related to ribosome protein in CD34~+HSPCs of patients with MDS and AML was down-regulated and correlated with the characteristics of blood routine,while the expression of genes related to mitochondria was up-regulated.