Clonal Evolution Of Fanconi Anemia In Patient And The Pathogenesis Of MLASA Anemia In Mice

ObjectiveFanconi anemia(FA)is a hereditary bone marrow failure disease that is prone to develop to hematological myeloid malignancies.The development of FA is a process of accumulation of genetic mutations.Increased genomic instability leads to the randomness and complexity of mutations in this process,which is the main reason for the heterogeneity of the disease.Thus,the pathogenesis,genetic variation and the evolution of bone marrow failure require further in-depth research.Clonal hematopoiesis(CH)refers to HSCs(Hematopoietic stem cell)with molecular genetic mutation characteristics,through multi-lineage hematopoietic differentiation,to form terminally differentiated mature blood cells carrying reproducible biological markers.CH is a nonmalignant expansion of hematopoietic model,which has the advantage of competitive clonal expansion and maintains the ability of multi-lineage hematopoietic differentiation.CH clones have a higher risk of evolving into myeloid malignant clones.In recent years,somatic mutations of driver genes have been found in hereditary bone marrow failure diseases(Inherited bone marrow failure syndrome,IBMFS,including FA)that have not yet developed leukemia,suggesting that clonal hematopoiesis may also play an important role in the malignant transformation of such diseases.Therefore,by using consecutive clinical samples of the same FA patient at different stages of disease,in combination with whole-genome sequencing and single-cell targeted genome sequencing technology,we conducted a detailed analysis of clonal evolution of FA to investigate the path and pattern of its malignant evolution including the mechanism of clone initiation,clone selection and clone evolution in the process.On the basis of in-depth analysis of the molecular mechanism,the early intervention targeted therapy will be explored,and the scientific basis for the treatment of Fanconi anemia,primary bone marrow failure diseases and myeloid malignancies will be provided.Methods1.Dissect the clonal changes in the malignant progression of FA(three stages of FA,MDS,and AML)and predict and screen driver mutant genes.(1)Extract genomic DNA from bone marrow and peripheral blood mononuclear cells of the same FA patient at different stages of progression,and extract genomic DNA from the patient’s nails and hair.(2)Sequence the genomic DNA collected from bone marrow and peripheral blood mononuclear cells,and use the Illumina sequencing platform for whole genome sequencing analysis.The sequencing depth is more than 60×,and bioinformatics analysis is carried out to clarify the gene mutation spectrum of the same patient in different disease progression stages;(3)Compare mutant genes and clonal structures in different disease stages of the same patient,and screening for driver genes;(4)Design targeted primers for these mutations,and verify primer specificity by PCR.Single-cell targeted genome sequencing was then performed on samples at different stages to determine the evolutionary patterns of malignant clones.2.In the collected samples of FA patients who progressed to MDS,compare the clonal structure and mutated genes evolved on the basis of different FA mutations.(1)Isolate mononuclear cells of bone marrow and peripheral blood and extract genomic DNA;(2)Identify the mutant genes of these samples using whole genome sequencing;(3)Compare the mutational pattern,mutational burden,and mutated genes of different FA patients progressing to MDS.ResultsThrough whole-genome sequencing of bone marrow and peripheral blood samples from the same patient at different stages,the mutant genes,clonal structure and clonal evolution were analyzed.The G>A/C>T nucleotide mutations were significantly higher than the rest of the mutation patterns,and the proportion of G>A/C>T mutations gradually increased with disease progression.It indicates that cytosine deamination also played a role in the evolution of Fanconi anemia to hematological myeloid malignancies.Moreover,the comparative analysis of the bone marrow and peripheral blood samples of the patient at the same disease stage found that the VAF of mutations in the bone marrow and peripheral blood samples showed a high correlation,indicating that in the process of evolution,it is possible that mutations in hematopoietic stem progenitor cells of bone marrow drive this process.Analysis of the clonal status of the patient at different stages found that in the MDS stage,the cell fraction(CCF)of SF3B1 and RUNX1 clones gradually increased,while UBASH3A gradually disappeared.In the later stage of MDS,the CCF of ASXL1 and IDH2 mutations began to appear and CCF increased in the AML stage,suggesting that ASXL1 and IDH2 mutations may play an important role in the transition from MDS to AML disease in this patient.In addition,we performed wholegenome sequencing analysis on the bone marrow samples of 3 FA-MDS patients who carried different mutations of Fanconi anemia-related genes such as FANCA,FANCL,and FANCD2,respectively.We found that,consistent with the consecutive samples,patients of FA-MDS all had a high transition ratio,in which G>A/C>T nucleotide mutations were significantly higher than others.Moreover,3 FA-MDS patients acquired different mutations during progression of disease,indicating that the mechanism of progression to myeloid malignancies in patients with different FA mutations is heterogeneous.ConclusionThrough whole-genome sequencing and single-cell targeted genome sequencing of sequential samples from FA to MDS/AML,our study demonstrates the existence of a polyclonal pattern of clonal evolution during the accumulation of driver mutations in the progression of FA to MDS/AML,and SF3B1,ASXL1,IDH2 and other genes have played an important role in the process of clonal evolution.In addition,the driver mutations in patients with different FA gene mutations are heterogeneous.This study provides new insight into the mechanism of clonal evolution in FA patients.ObjectiveMitochondrial myopathy,lactic acidosis,and sideroblastic anemia(MLASA)is a rare autosomal recessive disorder characterized by congenital sideroblastic anemia,mitochondrial dysfunction,and elevated lactate levels.Mutations in PUS1,YARS2,MTATP6 have been reported to cause MLASA.PUS1 encodes a pseudouridine synthase that converts uridine to pseudouridine and plays an important role in tRNA function and stabilization of the secondary and tertiary structure of a variety of RNAs.Currently,how PUS1 affects the hematopoietic system and its underlying molecular mechanisms are still unclear.Previous studies have suggested that the mutation in PUS1 gene affects erythroid differentiation by causing mitochondrial oxidative respiration disorder,and leads to the ring sideroblastic anemia.However,these studies are limited to genetic mutation identification and clinically phenotypic descriptions in limited clinical cases without functional experiments and inner mechanism studies.At present,there is no effective treatment for patients with mitochondrial myopathy combined with sideroblastic anemia,and only blood transfusion,iron removal therapy and other systematic supportive treatment.Therefore,dissecting the role and mechanism of mitochondria-related metabolic pathways in erythroid differentiation can provide new ideas for the treatment of some patients with sideroblastic anemia,and reveal the functional role of mitochondria in erythroid development.Based on this,we constructed a mouse model with the most reported PUS1 R144W mutant in MLASA patients,and a mouse model consistent with the PUS1 P175fs mutation site identified in an MLASA patient in our hospital to investigate the mechanism of sideroblastic anemia and the potential intervention strategy.MethodsWe identified the mutation sites of the mouse genome corresponding to MLASA patients(mouse R110W corresponds to human R144W,mouse S142fs corresponds to human P175fs),and with CRISPR-Cas9 technology constructed these two Pus1 mutant mouse models.The peripheral blood routine,erythroid development of bone marrow and spleen,the proportion and absolute number of cells in each stage of hematopoiesis were analyzed.Continuous competitive transplantation experiments were used to identify the ability of hematopoietic stem progenitor cells to differentiate into erythroid cells.Futhermore,the oxidative phosphorylation of erythroid cells was detected by Searhorse experiment,and the activities mitochondrial complex units was determined by ELISA.Then,the mitochondrial membrane potential,mitochondrial mass,mitochondrial superoxide and cellular Ros levels of erythroid cells were measured by flow cytometry.In order to study the effect of this gene mutation on tRNA and translation,tRNA PCR Array were used to determine the changes of tRNA expression in mutant mouse erythroid cells and the translation level of mutant mouse erythroid cells by RNA sequencing and Ribosome profiling.To clarify the effects of mitochondrial OXPHOS,cytoplasmic translation and mitochondrial translation on erythroid development,an in vitro erythroid differentiation system were established to address this question.ResultsBoth Pus1 R110W and S142fs homozygous mutant mice developed anemia at the age of four weeks,and the proportion of basophilic erythroblast and polychromatophilic erythroblast in the bone marrow and spleen increased.These results suggest that the erythroid development of the bone marrow and spleen is blocked in Pus1 mutant mice.The effect of Pusl R110W mutation on the ability of hematopoietic stem progenitor cells to differentiate into erythroid cells was further clarified through serial competitive transplantation experiments.Although compared with the control group,the proportion and number of hematopoietic stem progenitor cells in homozygous mutant mice were not significantly different,but its ability to differentiate into erythroid cells was significantly impaired.Further investigation of mitochondrial dysfunction showed that the oxygen consumption rate of Ter 119+cells in Pus1 R110W and S142fs homozygous mutant mice was significantly decreased.The results of tRNA PCR Array on bone marrow erythroid cells in Pus1 R110W mutant mice showed that the expression of mtIle-GAT tRNA significantly decreased and the expression of mtTyr-GTA tRNA increased in mutant mice respectively.Most of cytoplasmic tRNAs were significantly up-regulated and only two down-regulated in Pus1 R110W mutant mice.In addition,Ribosome profiling results showed that the translation efficiency of some genes was significantly changed.The number of genes whose translation efficiency(TE)is down-regulated in Pus1 R110W mutant mice is higher than that of genes whose TE is increased,while the decreased TE genes are mainly enriched in metabolism and ribosome-related pathways.By analyzing the proportion of down-regulated cytoplasmic tRNA(Pro-GGG)corresponding to codon CCC in ribosomal protein genes,it was found that the translation efficiency of genes containing a higher proportion of CCC codons decreased significantly.Taken together,these results suggest that changes in tRNA expression levels can affect translational changes in related genes,and may further affect cytoplasmic translation through effects on ribosomal protein gene translation.Consistent with this result,the hemoglobin level in bone marrow erythroid cells of R110W homozygous mutant mice decreased,suggesting that cytoplasmic translation of erythroid cells is impaired in Pus1 R110W mutant mice.Our in vitro erythroid differentiation experiments further suggest that inhibitors of mitochondrial OXPHOS complex and cytoplasmic translation inhibitors can significantly inhibit the process of erythroid differentiation.However,the effect of mitochondrial translation inhibitor on erythroid differentiation was not obvious.ConclusionThis study investigated the role of pseudouridine modification and mitochondrial function in erythroid development.Using the Pus1-mutant mouse model,we systematically studied the role of mitochondrial oxidative respiration-related pathways and translation in erythroid differentiation and the related molecular biological mechanism.Our data provided a theoretical basis for clinical treatment of patients with related diseases.