Pluripotent cell models of Fanconi anemia identify the early pathological defect in human hemoangiogenic progenitors
ABSTRACT: Fanconi anemia (FA) is a disorder of genomic instability characterized by progressive bone marrow failure (BMF), developmental abnormalities and an increased susceptibility to cancer. Although various consequences in hematopoietic stem/progenitor cells have been attributed to FA-BMF, the quest to identify the initial pathological event is still ongoing. To address this issue, we established induced pluripotent stem cells (iPSCs) from fibroblasts of six FA patients with FANCA mutations. An improved reprogramming method yielded iPSC-like colonies from all patients, and iPSC clones were propagated from two patients. Quantitative evaluation of the differentiation ability demonstrated that the differentiation propensity toward the hematopoietic and endothelial lineages is already defective in early hemoangiogenic progenitors. The expression levels of critical transcription factors were significantly downregulated in these progenitors. These data indicate that the hematopoietic consequences in FA patients originate from the early hematopoietic stage, and highlight the potential usefulness of iPSC technology for elucidating the pathogenesis of FA-BMF. The investigation of the RNA-seq analysis of iPSC-derived HAPCs.
Project description:Fanconi anemia (FA) is a disorder of genomic instability characterized by progressive bone marrow failure (BMF), developmental abnormalities, and an increased susceptibility to cancer. Although various consequences in hematopoietic stem/progenitor cells have been attributed to FA-BMF, the quest to identify the initial pathological event is still ongoing. To address this issue, we established induced pluripotent stem cells (iPSCs) from fibroblasts of six patients with FA and FANCA mutations. An improved reprogramming method yielded iPSC-like colonies from all patients, and iPSC clones were propagated from two patients. Quantitative evaluation of the differentiation ability demonstrated that the differentiation propensity toward the hematopoietic and endothelial lineages is already defective in early hemoangiogenic progenitors. The expression levels of critical transcription factors were significantly downregulated in these progenitors. These data indicate that the hematopoietic consequences in FA patients originate from the early hematopoietic stage and highlight the potential usefulness of iPSC technology for elucidating the pathogenesis of FA-BMF.
Project description:Fanconi anemia (FA) is an inherited DNA repair deficiency syndrome. FA patients undergo progressive bone marrow failure (BMF) during childhood, which frequently requires allogeneic hematopoietic stem cell transplantation. The pathogenesis of this BMF has been elusive to date. Here we found that FA patients exhibit a profound defect in hematopoietic stem and progenitor cells (HSPCs) that is present before the onset of clinical BMF. In response to replicative stress and unresolved DNA damage, p53 is hyperactivated in FA cells and triggers a late p21(Cdkn1a)-dependent G0/G1 cell-cycle arrest. Knockdown of p53 rescued the HSPC defects observed in several in vitro and in vivo models, including human FA or FA-like cells. Taken together, our results identify an exacerbated p53/p21 "physiological" response to cellular stress and DNA damage accumulation as a central mechanism for progressive HSPC elimination in FA patients, and have implications for clinical care.
Project description:Fanconi anemia (FA) is an inherited DNA repair disorder characterized by progressive bone marrow failure (BMF) from hematopoietic stem and progenitor cell (HSPC) attrition. A greater understanding of the pathogenesis of BMF could improve the therapeutic options for FA patients. Using a genome-wide shRNA screen in human FA fibroblasts, we identify transforming growth factor-? (TGF-?) pathway-mediated growth suppression as a cause of BMF in FA. Blocking the TGF-? pathway improves the survival of FA cells and rescues the proliferative and functional defects of HSPCs derived from FA mice and FA patients. Inhibition of TGF-? signaling in FA HSPCs results in elevated homologous recombination (HR) repair with a concomitant decrease in non-homologous end-joining (NHEJ), accounting for the improvement in cellular growth. Together, our results suggest that elevated TGF-? signaling contributes to BMF in FA by impairing HSPC function and may be a potential therapeutic target for the treatment of FA.
Project description:Fanconi anemia (FA) is a DNA repair disorder resulting from mutations in genes encoding for FA DNA repair complex components and is characterized by variable congenital abnormalities, bone marrow failure (BMF), and high incidences of malignancies. FA mosaicism arises from reversion or other compensatory mutations in hematopoietic cells and may be associated with BMF reversal and decreased blood cell sensitivity to DNA-damaging agents (clastogens); this sensitivity is a phenotypic and diagnostic hallmark of FA. Uncertainty regarding the clinical significance of FA mosaicism persists; in some cases, patients have survived multiple decades without BMF or hematologic malignancy, and in others hematologic failure occurred despite the presence of clastogen-resistant cell populations. Assessment of mosaicism is further complicated because clinical evaluation is frequently based on clastogen resistance in lymphocytes, which may arise from reversion events both in lymphoid-specific lineages and in more pluripotent hematopoietic stem/progenitor cells (HSPCs). In this review, we describe diagnostic methods and outcomes in published mosaicism series, including the substantial intervals (1-6 years) over which blood counts normalized, and the relatively favorable clinical course in cases where clastogen resistance was demonstrated in bone marrow progenitors. We also analyzed published FA mosaic cases with emphasis on long-term clinical outcomes when blood count normalization was identified. Blood count normalization in FA mosaicism likely arises from reversion events in long-term primitive HSPCs and is associated with low incidences of BMF or hematologic malignancy. These observations have ramifications for current investigational therapeutic programs in FA intended to enable gene correction in long-term repopulating HSPCs.
Project description:BACKGROUND:Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome with 22 related genes identified. The ALDH2 rs671variant has been proved related to accelerate the progression of BMF in FA patients. The phenotype and genetic basis of Chinese FA patients have not been investigated yet. METHODS:We analyzed the 22 FA-related genes of 63 BMF patients suspected to be FA. Clinical manifestations, morphological and cytogenetic feathers, ALDH2 genotypes, treatment, and outcomes of the definite cases were retrospectively studied. RESULTS:A total of 21 patients were confirmed the diagnosis of FA with the median age of BMF onset was 4-year-old. The number of patients manifested as congenital malformations and growth retardation were 20/21 and 14/21, respectively. BM dysplasia and cytogenetic abnormalities were found in 13/20 and 8/19 patients. All the patients with abnormal karyotypes also manifested as BM dysplasia or had evident blasts. Thirty-five different mutations were identified involving six genes and including twenty novel mutations. FANCA mutations contributed to 66.67% of cases. Eight patients harboring ALDH2-G/A genotype have a significantly younger age of BMF onset (p?=?0.025). Within the 19 patients adhering to continuous follow-up, 15 patients underwent hematopoietic stem cell transplantations (HSCTs). During the 29?months of follow-up, 8/19 patients died, seven of which were HSCT-related, and one patient who did not receive HSCT died from severe infection. CONCLUSIONS:The phenotypic and genetic spectrum of Chinese FA patients is broad. Bone marrow dysplasia and cytogenetic abnormalities are prevalent and highly consistent. The overall outcome of HSCTs is disappointing. Nationwide multicenter studies are needed for the rarity and adverse outcome of this disease.
Project description:Fanconi anemia (FA) is a recessive genetic disease characterized by congenital abnormalities, chromosome instability, progressive bone marrow failure (BMF), and a strong predisposition to cancer. Twenty FA genes have been identified, and the FANC proteins they encode cooperate in a common pathway that regulates DNA crosslink repair and replication fork stability. We identified a child with severe BMF who harbored biallelic inactivating mutations of the translesion DNA synthesis (TLS) gene REV7 (also known as MAD2L2), which encodes the mutant REV7 protein REV7-V85E. Patient-derived cells demonstrated an extended FA phenotype, which included increased chromosome breaks and G2/M accumulation upon exposure to DNA crosslinking agents, ?H2AX and 53BP1 foci accumulation, and enhanced p53/p21 activation relative to cells derived from healthy patients. Expression of WT REV7 restored normal cellular and functional phenotypes in the patient's cells, and CRISPR/Cas9 inactivation of REV7 in a non-FA human cell line produced an FA phenotype. Finally, silencing Rev7 in primary hematopoietic cells impaired progenitor function, suggesting that the DNA repair defect underlies the development of BMF in FA. Taken together, our genetic and functional analyses identified REV7 as a previously undescribed FA gene, which we term FANCV.
Project description:Fanconi anemia (FA) is a rare inherited syndrome characterized by progressive bone marrow failure (BMF), abnormal skin pigmentation, short stature, and increased cancer risk. BMF in FA is multifactorial and largely results from the death of hematopoietic stem cells due to genomic instability. Also, inflammatory pathology in FA has been previously reported, however the mechanism is still not clear. In literature, decreased NK-cell count and/or impaired NK-cell activity, along with other immunological abnormalities have been described in FA-patients (1). However, to the best of our knowledge, this is the first report showing a defective degranulation mechanism leading to abnormal NK-cell cytotoxicity in FA-patients, which may explain the development of a hyperinflammatory response in these patients. This may predispose some patients to develop Hemophagocytic lymphohistiocytosis (HLH) which manifests with prolonged fever, progressive cytopenias and organomegaly. Early diagnosis and initiation of immunosuppressive therapy in these patients will help to better manage these patients. We also propose FA genes to be listed as a cause of familial HLH.
Project description:Fanconi anemia (FA) is a severe hereditary disorder with defective DNA damage response and repair. It is characterized by phenotypes including progressive bone marrow failure (BMF), developmental abnormalities, and increased occurrence of leukemia and cancer. Recent studies in mice have suggested that the FA proteins might counteract aldehyde-induced genotoxicity in hematopoietic stem cells. Nearly half of the Japanese population carries a dominant-negative allele (rs671) of the aldehyde-catalyzing enzyme ALDH2 (acetaldehyde dehydrogenase 2), providing an opportunity to test this hypothesis in humans. We examined 64 Japanese FA patients, and found that the ALDH2 variant is associated with accelerated progression of BMF, while birth weight or the number of physical abnormalities was not affected. Moreover, malformations at some specific anatomic locations were observed more frequently in ALDH2-deficient patients. Our current data indicate that the level of ALDH2 activity impacts pathogenesis in FA, suggesting the possibility of a novel therapeutic approach.
Project description:Hematopoietic stem cell (HSC) attrition is considered the key event underlying progressive BM failure (BMF) in Fanconi anemia (FA), the most frequent inherited BMF disorder in humans. However, despite major advances, how the cellular, biochemical, and molecular alterations reported in FA lead to HSC exhaustion remains poorly understood. Here, we demonstrated in human and mouse cells that loss-of-function of FANCA or FANCC, products of 2 genes affecting more than 80% of FA patients worldwide, is associated with constitutive expression of the transcription factor microphthalmia (MiTF) through the cooperative, unscheduled activation of several stress-signaling pathways, including the SMAD2/3, p38 MAPK, NF-?B, and AKT cascades. We validated the unrestrained Mitf expression downstream of p38 in Fanca-/- mice, which display hallmarks of hematopoietic stress, including loss of HSC quiescence, DNA damage accumulation in HSCs, and reduced HSC repopulation capacity. Importantly, we demonstrated that shRNA-mediated downregulation of Mitf expression or inhibition of p38 signaling rescued HSC quiescence and prevented DNA damage accumulation. Our data support the hypothesis that HSC attrition in FA is the consequence of defects in the DNA-damage response combined with chronic activation of otherwise transiently activated signaling pathways, which jointly prevent the recovery of HSC quiescence.
Project description:Aplastic Anemia (AA) is a bone marrow failure (BMF) disorder, resulting in bone marrow hypocellularity and peripheral pancytopenia. Severe aplastic anemia (SAA) is a subset of AA defined by a more severe phenotype. Although the immunological nature of SAA pathogenesis is widely accepted, there is an increasing recognition of the role of dysfunctional hematopoietic stem cells in the disease phenotype. While pediatric SAA can be attributable to genetic causes, evidence is evolving on previously unrecognized genetic etiologies in a proportion of adults with SAA. Thus, there is an urgent need to better understand the pathophysiology of SAA, which will help to inform the course of disease progression and treatment options. We have derived induced pluripotent stem cell (iPSC) from three unaffected controls and three SAA patients and have shown that this in vitro model mimics two key features of the disease: (1) the failure to maintain telomere length during the reprogramming process and hematopoietic differentiation resulting in SAA-iPSC and iPSC-derived-hematopoietic progenitors with shorter telomeres than controls; (2) the impaired ability of SAA-iPSC-derived hematopoietic progenitors to give rise to erythroid and myeloid cells. While apoptosis and DNA damage response to replicative stress is similar between the control and SAA-iPSC-derived-hematopoietic progenitors, the latter show impaired proliferation which was not restored by eltrombopag, a drug which has been shown to restore hematopoiesis in SAA patients. Together, our data highlight the utility of patient specific iPSC in providing a disease model for SAA and predicting patient responses to various treatment modalities.