Добірка наукової літератури з теми "MonoMac and DCML syndrome"

Abstract Recent work has established that heterozygous germline GATA2 mutations predispose carriers to familial myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML), “MonoMAC” syndrome, and DCML deficiency. Here, we describe a previously unreported MDS family carrying a missense GATA2 mutation (p.Thr354Met), one patient with MDS/AML carrying a frameshift GATA2 mutation (p.Leu332Thrfs*53), another with MDS harboring a GATA2 splice site mutation, and 3 patients exhibiting MDS or MDS/AML who have large deletions encompassing the GATA2 locus. Intriguingly, 2 MDS/AML or “MonoMAC” syndrome patients with GATA2 deletions and one with a frameshift mutation also have primary lymphedema. Primary lymphedema occurs as a result of aberrations in the development and/or function of lymphatic vessels, spurring us to investigate whether GATA2 plays a role in the lymphatic vasculature. We demonstrate here that GATA2 protein is present at high levels in lymphatic vessel valves and that GATA2 controls the expression of genes important for programming lymphatic valve development. Our data expand the phenotypes associated with germline GATA2 mutations to include predisposition to primary lymphedema and suggest that complete haploinsufficiency or loss of function of GATA2, rather than missense mutations, is the key predisposing factor for lymphedema onset. Moreover, we reveal a crucial role for GATA2 in lymphatic vascular development.

Abstract Abstract 3807 Background: Somatic and germline mutations in GATA2 were recently identified in patients diagnosed with MonoMAC, the hallmarks of which include mono cytopenia, B-cell and NK-cell lymphopenia, susceptibility to opportunistic infections (e.g. MAC), and a strong propensity to develop hypocellular MDS/AML or CMML. GATA2 mutations were also recently identified in other related disorders: Emberger syndrome (primary lymphedema with myelodysplasia), Familial MDS/AML, and DCML (Dendritic Cell, Monocytes, Lymphoid Deficiency). Family members with GATA2 mutations show variable penetrance and expressivity indicating that other factors may be required for development of disease and phenotype. GATA2 mutations are thought to result in loss of function or haploinsufficiency, but the precise mechanism for the development of cytopenias, immunodeficiency, and susceptibility to MDS remains to be elucidated. MicroRNA (miR) represents a unique mechanism of post-transcriptional gene regulation. In this study we generated microRNA profiles of patient derived MonoMAC cell lines followed by functional studies to identify aberrant miRs and their targets, which could potentially cooperate with GATA2 deficiency in generating hematologic disease. Inducible deletion of Myeloid Leukemia Cell 1 (Mcl1), a member of the Bcl2 family, in mice results in the loss of hematopoietic stem cells (HSCs) and progenitors, and in development of cytopenias. Design: RNA was isolated from EBV-immortalized B cells of 10 healthy controls and 13 MonoMAC patients with MDS and defined mutations in GATA2. microRNA expression profiles were generated using the Agilent high density human microRNA array. Array data were normalized to the data point of 75th percentile signal strength and to a set of spike-in and control probes. The differences between the means of experimental groups were analyzed by Mann-Whitney rank sum test. The miRs with significant p values (p≤ 0.05) and fold change (≥ 2-fold) in both normalization methods were selected for further analysis. TargetScan was utilized to predict the mRNA targets of aberrantly expressed miRs. miR targets were validated by functional studies in the Ly8 cell line. Results: Eight miRs were significantly differentially expressed (≥ 2-fold; p ≤ 0.05) as determined by microRNA microarray profiles. Six miRs showed increased expression in monoMAC cell lines compared to controls (miR-9, −181a-2–3p, −181c, −181c-3p, −486–3p, −582–5p) while two miRs showed significantly decreased expression (miR-223, −424–3p). Among the differentially expressed miRs that were validated by quantitative RT-PCR was miR-181c, which demonstrated a 2.2 fold increase in expression in MonoMAC cell lines (p = 0.013). Among the target transcripts potentially regulated by miR-181c, MCL1 expression was significantly decreased (2 fold; p = 0.018) in monoMAC cell lines in comparison to control cell lines. Transient transfection of miR-181c in Ly8 cells resulted in 40% decrease of MCL1 mRNA level, suggesting that miR-181c negatively regulates MCL1 in MonoMAC. Conclusions: These findings indicate that MonoMAC/GATA2 deficiency is associated with significantly decreased expression of MCL1 possibly through negative regulation involving miR-181c. Deletion of Mcl1 is known to cause apoptosis, loss of HSCs, and cytopenias in murine studies. Thus, down-regulation of MCL1 seen in MonoMAC/GATA2 deficiency may similarly favor unregulated apoptosis and the depletion of hematopoietic progenitors resulting in cytopenias, immunodeficiency, and risk of MDS/AML. Disclosures: No relevant conflicts of interest to declare.

Abstract Myelodysplastic syndrome (MDS) is rare in children. Certain inherited bone marrow failure syndromes (IBMFS), such as Fanconi anemia, severe congenital neutropenia and Shwachman Diamond syndrome, markedly increase the risk of MDS during childhood. However, the genetic factor(s) underlying sporadic pediatric MDS are unknown. Germline mutations in GATA2, a hematopoietic transcription factor, explain four MDS-predisposing conditions: monocytopenia and mycobacterial infection (MonoMAC); dendritic cell, monocyte, B and NK cell lymphoid deficiency (DCML); primary lymphedema with myelodysplasia progressing to acute myeloid leukemia (Emberger syndrome); and a subset of familial MDS. Cases of pediatric MDS have been observed in some of the reported pedigrees. In addition, three individuals have been reported with large, de novo deletions encompassing GATA2 and surrounding genes and manifesting developmental delay, intellectual disability and dysmorphic features alongside their hematologic abnormalities. We identified a novel GATA2 splice site variant (c.1018-2A>C) in a teenager with MDS, WHO classification refractory cytopenia of childhood (RCC). Although he was found to have monocytopenia and B and NK cell deficiencies, he had no history of infections associated with MonoMAC or pertinent family history. We, therefore, hypothesized that mutations in GATA2 might be present in additional cases of pediatric MDS that were neither associated with an IBMFS nor relevant personal or family history. Two Baylor College of Medicine biology studies open to children with hematologic disease were queried for patients with the diagnosis of MDS. Exclusion criteria included treatment-related MDS, diagnosis of an IBMFS, prior diagnosis of severe aplastic anemia or infections suspicious for MonoMAC or DCML, and known or suspected family history of a GATA2-associated disorder. Cases lacking a pre-hematopoietic stem cell transplantation (HSCT) tissue sample available for study were also excluded. In addition to the patient described above, six children were identified who met eligibility criteria. DNA was isolated from banked peripheral blood or bone marrow cells and GATA2 sequencing performed, including upstream and intronic regulatory regions. Array comparative genomic hybridization was also performed on one sample that lacked GATA2 sequence variants, but was notable for complete absence of heterozygosity (AOH), including 6 polymorphic sites with minor allele frequencies of 0.20 or greater. Pertinent clinical and laboratory features were extracted by medical record review blinded to GATA2 status. We found heterozygous GATA2 mutations in three of the six additional patient samples. Thus, four of this seven patient, pediatric MDS cohort had mutated GATA2. Two of the newly identified mutations were splice site variants: a previously described c.1018-1G>A and a novel variant altering the exon 7 splice site acceptor (c.1114-1G>C). The third mutation was a de novo 3.1-3.3 Mb deletion encompassing the entire GATA2 locus and contiguous genes, and was established to be germline by analysis of skin fibroblasts. Notably, the patient had normal neurocognitive development and was without dysmorphic features. Their ages of presentation were 5, 9, 12 and 15 years. With the exception of the initial case, peripheral blood T and B cell phenotyping was not obtained prior to HSCT. Monocytopenia of less than 200/µL was present in five of seven patients, three of whom had a GATA2 mutation. All four GATA2 mutation cases had RCC and three of the four had monosomy 7 at diagnosis. In contrast, the three cases lacking GATA2 mutation presented with the MDS classification refractory anemia with excess blasts (RAEB-2), with either a normal karyotype, complex karyotypic changes or chromosome 13.q12q14 deletion. GATA2 mutation may explain a significant portion of sporadic, seemingly nonsyndromic pediatric MDS, particularly cases with monosomy 7. Evaluation of larger cohorts is warranted to ascertain the true prevalence. Although this cohort is small, we recommend GATA2 sequencing be performed as part of the initial evaluation of pediatric MDS as the identification of a germline mutation has critical implications for related donor selection and genetic counseling. AOH in GATA2 sequencing should prompt deletion analysis, even in cases without infections, dysmorphic features or neurocognitive impairment. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2536 Background Acute myeloid leukemia (AML) is a complex disease caused by mutations, epigenetic modifications, and deregulated expression of genes leading to increased proliferation and decreased differentiation of hematopoietic progenitor cells. Although many important molecular markers have already been discovered in AML, no known prognosis-associated cytogenetic aberrations or mutations were detectable in a subset of AML patients. In this regard, recent reports of somatic mutations affecting the C-terminal zinc finger (ZF) 2 of GATA2 are intriguing, because these GATA2 mutations were associated with the progression of chronic myeloid leukemia, whereas hereditary ZF2 of GATA2 mutations predispose to AML and myelodysplastic syndrome. GATA2 mutations were also reported as a predisposing gene of the monocytopenia, mycobacterial infection (MonoMAC) syndrome and the dendritic cell, monoctye, B and NK lymphoid deficiency (DCML) syndrome. GATA2 belongs to a family of zinc finger transcription factors, and is important for hematopoietic stem cell proliferation and normal megakaryocytic development. These findings prompted us to search for possible GATA2 mutations in pediatric AML. Methods To explore the frequency and clinical impact of GATA2 mutations, we examined 157 Japanese pediatric AML patients, including 13 with FAB-M3 and 10 with Down syndrome (DS) who were treated on different treatment protocol, by PCR following direct sequencing. As GATA2 mutations thus far reported almost exclusively involved exons 4–6 that encode zinc finger 1 and 2 domain, we confined our analysis to these exons. Results GATA2 missense mutations were found in 7 out of 157 patients (4.5%). Notably, All of GATA2 mutations were located in ZF2 in this study, although almost all of GATA2 mutations in adult AML were located in ZF1. Wild type GATA2 were found in 3 of 3 AML patients with GATA2 mutation. The other 4 patients had no history of Mono MAC syndrome, suggesting that these mutations were acquired. The zinc finger region of GATA2 is required for binding to promyelocytic leukemia zinc finger (PLZF) protein can interact with GATA2 and can modify its transactivation capacity. Interestingly, 2 GATA2 mutations were found in FAB-M3 in this study, GATA2 mutations also may be associated with acute promyelocytic leukemia. Clinical and molecular features between patients with and without GATA2 mutations were not significantly different in the clinical parameters (WBC, age, sex, etc.), and the outcome of GATA2 mutation positive patients was not poor when compared to GATA2 mutation negative patients: Two of the 7 patients received allogeneic-stem cell transplantation (Allo-SCT) and one of them died of gastrointestinal hemorrhage after SCT. The other 5 patients who did not receive the SCT were still alive. Conclusion GATA2 is a new predisposition gene for pediatric AML and shows functional changes caused by mutations within a highly conserved threonine repeat located in ZF2. Interestingly, MonoMAC and DCML syndromes were not observed in de novo AML patients with GATA2 mutations. Although further investigation is needed, our results indicated that GATA2 mutations were associated with a favorable outcome in pediatric AML. Most of the patients with GATA2 mutations have been classified into an intermediate risk group in our study, however, their favorable outcome suggests that less aggressive treatment strategy without SCT might be appropriate for AML patients carrying GATA2 mutations. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3488 Germline heterozygous mutations in GATA2 have been reported to cause familial myelodysplasia–acute myeloid leukemia (MDS/AML), monocytopenia and mycobacterial infection (monoMAC syndrome), dendritic cell, myeloid and NK cell lymphopenia (DCML), and Emberger syndrome (lymphedema and MDS). GATA2 is a zinc finger transcription factor that plays a crucial role in regulating growth of hematopoietic progenitors. In some pedigrees, patients or family members have manifestations of bone marrow failure. We hypothesized that patients with aplastic anemia (AA) may harbor mutations in GATA2. The coding regions and regulatory regions of GATA2 were sequenced in 99 patients with confirmed AA. Sequences from 100 normal individuals as well as published human genomes from unaffected individuals (dbSNP build 135 and 1000 Genomes Project) were used as controls. Genetic variants were confirmed in hematopoietic and somatic tissues. We identified 4 heterozygous mutations in regulatory regions of GATA2 in 5 patients. In two patients, a mutation at nucleotide 59T>G in exon 1 of isoform 2 was identified; both had severe AA in early adulthood refractory to immunosuppressive therapy. We noted this 59T>G mutation in two unrelated individuals with severe disseminated mycobacterial disease. We identified a mutation at nucleotide 20G>A in exon 2 of isoform 1, in a 3 year-old male with hepatitis-associated severe AA whose disease was refractory to multiple rounds of immunosuppressive therapy. Another mutation was present in 38G>A in exon 2 of isoform 1 in a 32 year old male with moderate AA and paroxysmal nocturnal hemoglobinuria (PNH). We also identified the exon 2 38G>A mutation in a patient with disseminated mycobacterial disease where reduced transcription of the mutant 38G>A allele was noted on RT-PCR. Finally, an intron 5, c.512+573 G>A variant was identified in an 18 year old male with severe AA who progressed after immunosuppressive therapy to MDS/AML. This variant, which causes a disruption of the FLI1 binding site, has also been found to be pathogenic in monoMAC syndrome. In summary, a subset of patients with AA were found to have mutations in GATA2 suggesting a role for the gene in the pathogenesis of bone marrow failure. It also may identify patients at higher risk of infectious complications, those who may have less advantageous responses to immune suppression, and command earlier bone marrow transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Mutations in the zinc finger transcription factor GATA2 are responsible for: MonoMAC, monocytopenia with nontuberculous mycobacterial (NTM) infections; DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger's syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Allogeneic hematopoietic stem cell transplant (HSCT) represents the only definitive therapy for GATA2 deficiency. Methods: Eleven patients with GATA2 deficiency received a myeloablative-conditioning regimen (2 matched related donors or MRD, 4 matched unrelated donors or URD, and 5 haploidentical related donors. MRD and URD received busulfan 3.2 mg/kg/day and fludarabine 40 mg/m2/day on days -6, -5, -4, and -3. Haploidentical related donors received cyclophosphamide 14.5 mg/kg on day's -6 and -5, fludarabine 30 mg/m2/day on day's -6 to -2, busulfan 3.2 mg/kg/day on day's -4 and -3, and 200 cGy TBI on day -1. MRD and URD recipients received tacrolimus and short course methotrexate post-transplant, while haploidentical related donor recipients received cyclophosphamide 50 mg/kg/day on days + 3 and +4 followed by tacrolimus and mycophenolate mofetil as post-transplant immunosuppression for graft-versus-host disease. Results: Ten of the 11 (91%) of patients are alive and disease-free at a mean follow-up of 12 months (range 1 mo to 24 mo). One URD recipient died from persistent acute myelogenous leukemia. Four patients developed graft-versus-host disease, one case Grade 4. All 10 patients who survived had complete reconstitution of the monocyte, NK, and B-lymphocyte compartments, the three cell compartments that were severely deficient pre-transplant. All 10 patients had reversal of the infection susceptibility phenotype. In particular, there were no recurrences of NTM infections. Importantly, all 10 patients had correction of the cytogenetic abnormalities present pre-transplant (5 patients with trisomy 8 and 1 patient with monosomy 7). Conclusions: Myeloablative HSCT in GATA2 deficiency results in uniform engraftment and reversal of the hematologic, cytogenetic, and clinical manifestations of GATA2 deficiency. There was a low regimen-related toxicity, even in this cohort of patients with considerable co-morbidities. We anticipate that with HSCT earlier in the clinical course, before significant organ damage or clonal evolution of MDS to AML or CMML occurs, the outcome of allogeneic HSCT in patients with GATA2 deficiency will continue to improve. Haploidentical related donor transplant appears to be particularly well suited for this disease, especially when the disease presents as a hypocellular myelodysplastic syndrome. Disclosures No relevant conflicts of interest to declare.

Abstract Background:Mutations in the zinc finger transcription factor GATA2 are responsible for: MonoMAC, monocytopenia with atypical mycobacterial infection (MAC); DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Allogeneic hematopoietic stem cell transplant (HSCT) represents the only definitive therapy for GATA2 deficiency. Methods: We carried out myeloablative allogeneic hematopoietic stem cell transplant (HSCT) on 24 patients (mean age 24.9 years; range 16 to 46 years) with mutations in GATA2, or the MonoMAC syndrome, during the preceding three years. Three matched related donor (MRD) recipients and 13 matched unrelated donor (URD) recipients received busulfan 3.2 mg/kg/day and fludarabine 40 mg/m2/day on day's -6, -5, -4, and -3. Eight haploidentical related donor recipients received cyclophosphamide 14.5 mg/kg on day's -6 and -5, fludarabine 30 mg/m2/day on day's -6 to -2, busulfan 3.2 mg/kg/day on day's -4 and -3, and 200 cGy TBI on day -1. The majority of the MRD and URD recipients (n=13) received tacrolimus and short course methotrexate post-transplant as graft-versus host disease (GVHD) prophylaxis; three URD, while three received post-transplant cyclophosphamide (PT/CY) on days + 3 and +4 followed by tacrolimus and mycophenolate mofetil as post-transplant immunosuppression for GVHD. All 8 haploidentical related donor recipients received PT/CY as described followed by tacrolimus/methotrexate. Results:Twenty-two of the 24 patients are alive and disease-free at a mean follow-up of 13 months (range, 2 to 36 months). One matched URD recipient died from persistent AML 110 days post-transplant, and one matched URD recipient died from GVHD 2 years post-transplant. Despite a previous history of MAC in 11 patients, including 3 patients with active MAC at the time of HSCT, there were no recurrences of MAC during or following HSCT. Similarly, 13 patients had MDS with cytogenetic abnormalities at the time of HSCT (trisomy 8, monosomy 7, 5q-, trisomy 1q), and all 13 had resolution of their cytogenetic abnormalities with no late relapses following HSCT. Three patients had lymphedema at the time of HSCT (Emberger syndrome), which was not reversed. Twenty-three of the 24 patients had complete reconstitution of the monocyte, NK, and B-lymphocyte compartments, which were severely deficient pre-transplant. One URD who received PT/CT had poor graft reconstitution and required a CD34+ donor cell boost three months post-transplant. Four of the 13 MRD and URD recipients who received tacrolimus/methotrexate developed grade III-IV acute GVHD, and 3 of the 7 who were more than one-year post-HSCT developed moderate to severe chronic GVHD. No haploidentical related donor recipient developed grade III-IV GVHD or moderate to severe chronic GVHD. Conclusions: Myeloablative HSCT in GATA2 deficiency results in uniform engraftment and reversal of the hematologic, cytogenetic, and clinical manifestations of clinical manifestations of GATA2 deficiency with low regimen-related toxicity, even in this cohort of patients with considerable co-morbidities. We are currently using PT/CY in MRD and URD recipients with normal, favorable, or intermediate cytogenetics with isolated trisomy 8 to reduce the incidence of acute and chronic GVHD. We anticipate that with HSCT earlier in the clinical course, before significant organ damage or clonal evolution of MDS to AML occurs, the outcome of allogeneic HSCT in patients with GATA2 deficiency will continue to improve. Haploidentical related donor transplant appears to be particularly well suited for this disease, especially when the disease presents as a hypocellular MDS with or without trisomy 8. Disclosures No relevant conflicts of interest to declare.

Abstract Background: GATA2 deficiency results in a clinical syndrome known variably as: MonoMAC for the lack of monocytes and atypical mycobacterial infections (MAC); DCML, dendritic cell, monocyte and lymphoid cell deficiency; Emberger's syndrome with lymphedema and monosomy 7; and familial myelodysplastic syndrome (MDS)/acute myelogenous leukemia (AML). Reconstitution of the deficient cell compartments in GATA2 deficiency with allogeneic hematopoietic stem cell transplant (HSCT) results in reversal of the infection susceptibility and represents the only curative therapy for both the myeloid disease and the virally driven malignancies seen in this disorder. However, only about one-half of patients that require HSCT will have an HLA-matched related or unrelated donor. Moreover, the use of umbilical cord blood has been reported to be suboptimal in this cohort of patients in whom infection susceptibility underlies much of the need for a transplant. Methods: We performed haploidentical, related donor HSCT with high dose post-transplant cyclophosphamide (PT/CY) in 3 patients with GATA2 deficiency in whom a suitable HLA matched donor was not available. All three patients had a first degree relative (sibling) who shared at least 1 HLA-haplotype. The first patient, a 21 year-old Chinese woman presented with Hydroa Vacciniforme like T-cell lymphoma involving her lung, bowel (resulting in multiple small bowel resections and ileostomy), and skin (with large ulcerative lesions); multiple thrombotic cerebral events; and macrophage activation syndrome requiring etoposide and high dose prednisone in the intensive care unit (ICU). The second patient, a 20 year-old Hispanic female, presented with myelodysplasia (MDS), profound neutropenia, an invasive fungal sinusits due to Fusarium sp., and recurrent bacteremias. The third patient, a 45 year-old woman, presented with Emberger’s Syndrome, multiple episodes of bacterial and fungal sepsis and hypocellular MDS. All three patients received cyclophosphamide 14.5mg/kg on day’s -6 and-5; fludarabine 30mg/m2 on day’s -6 through -2; 200cGy of total body irradiation (TBI) on day -1. Patients 2 and 3 also received busulfan 3.2 mg/kg on day’s -4 and -3 because of MDS. All 3 patients received high dose PT/CY 50mg/kg on day’s +3 and +4, and mycophenolate mofetil and tacrolimus starting on day +5. Results: All 3 patients engrafted at a mean of 18 days and all are alive at a median follow-up of 9 months (range, 5 to 13 months). All three patients achieved 100% donor myeloid and lymphoid chimerim by day +100 post-transplant. The first patient had complete resolution of her T-cell lymphoma and macrophage activation syndrome, the second patient has had complete reversal of her hematopoietic and infectious phenotype, and the third patient has had resolution of her infectious complications that required continuous IV Daptomycin for 5 years prior to transplant. In addition, all three patients had complete reconstitution of the monocyte, NK cell, and B-lymphocyte compartments that were severely deficient prior to transplant. The main toxicity was mucositis, which required intravenous narcotics in all three patients. Lastly, the first patient developed late grade I liver graft-versus-host disease (GVHD) that was responsive to cyclosporine, the second patient developed grade I acute GVHD of skin that was responsive to topical steroids, and the third patient had no evidence of acute or chronic GVHD. Conclusions: Haploidentical, related donor HSCT represents a safe and effective viable alternative for patients with GATA2 deficiency who lack an HLA-matched donor, including patients such as our first patient who was in the ICU at the time of transplant. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 405 Background: Recently, monoallelic mutations in the zinc-finger transcription factor GATA2 have been shown to be responsible for GATA2-deficiency, a syndrome characterized by opportunistic infections, frequently atypical mycobacterial infections or MAC, and a hypocellular myelodysplastic syndrome (MDS) that transforms into acute myelogenous leukemia (AML). GATA2-deficiency was previously known by several other names: MonoMAC (Monocytopenia and Atypical Mycobacterial Infection), DCML (Dendritic Cell Monocyte, Lymphoid Deficiency), Familial MDS/AML, or Emberger syndrome (lymphedema with monosomy 7). Heterogeneous genetic defects in GATA2 result in haploinsufficiency in both spontaneous and familial forms of the disease. Predicting the transformation from MDS to AML in GATA2-deficiency has clinical implications for both prognosis and the timing of hematopoietic stem cell transplantation. ASXL1, a gene related to the Drosophilia additional sex combs gene, encodes a chromatin binding/transcription repressor protein that is frequently mutated in MDS/AML. Mutations in ASXL1 are associated with reduced time to progression to AML and poor overall survival, independent of IPSS score. Methods: We sequenced the critical region of the ASXL1 gene in 20 patients with GATA2- deficiency to determine the frequency of ASXL1 mutations, and to correlate the presence of ASXL1 mutations with hematopoietic transformation. Since the ASXL1 mutations described in hematopoietic malignancies are located within the coding sequence of the two 3Õ-terminal exons (COSMIC: Catalogue of Somatic Mutations in Cancer), this ∼4.3kb region of ASLX1 was amplified by PCR and sequenced using five overlapping primer sets with substrate DNA isolated from mononuclear cell and granulocyte cell preparations from peripheral blood or bone marrow aspirates, or from extracts prepared from unfixed, unstained bone marrow aspirates. Mutations were confirmed with at least two independent PCR reactions with two unique primer sets. Results: Somatic ASXL1 mutations were detected in 8 of 20 patients with GATA2 mutations, 19/20 of whom had MDS. Five of these ASXL1 mutations have been previously associated with MDS/AML, including four independent cases of the most frequently described ASXL1 mutation (G646fs*12insG). The other four mutations were found once each; two of these were previously unreported (G652S(G>A) and L817fs*1delT). The patient cohort included two sisters with the same germline GATA2R398W mutation, but different somatic ASXL1 mutations (G464fs*12insG and R693X(C>T)). ASXL1 mutations were found in 4/5 GATA2- deficiency patients whose MDS had transformed into chronic myelomonocytic leukemia (CMML). Overall survival was lower for GATA2-deficiency patients with ASXL1 mutation (50% survival) compared to patients without ASXL1 mutation (83% survival), and was independent of IPSS score. Conclusions: ASXL1 is frequently mutated in patients with GATA2-deficiency with at least 40% of patients having a mutation in ASXL1 compared to a 10–15% mutation rate reported for all MDS/AML patients. ASXL1 mutation correlates with the development of CMML and with poor overall survival, as reported previously for MDS/AML patients. There was no correlation between the presence and type of ASXL1 mutation and the specific GATA2 mutation: the eight different ASXL1 mutation events were found in six different GATA2 mutant backgrounds. These results are directly relevant to the prognosis and the timing of hematopoietic stem cell transplantation for GATA2-deficiency. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1264 Patients with familial myelodysplastic syndrome (MDS) associated with mutations in GATA2 are at increased risk of MDS and acute myeloid leukemia (AML). Specific clinical syndromes recently found to be due to mutations in GATA2 include MonoMAC (monocytopenia and mycobacterial infection), Emberger (MDS with severe lymphedema), and DCML (defects in dendritic cells, monocytes, and B and NK lymphoid cells). Features shared by many patients with these GATA2-associated syndromes include monocytopenia, markedly decreased B and NK cells, and clinical immunodeficiency manifested as warts and mycobacteria and fungal infections. MDS and/or AML occur with multilineage dyspoieses, particularly prominent in the megakaryocyte lineage (micromegakaryocytes, small mononuclear megakaryocytes, and large megakaryocytes with multiple separated nuclei). Several reports mention family members who are “asymptomatic,” without further details. We identified mutations in GATA2 in two of three families with familial MDS. In both families, one apparently healthy parent was found to have a GATA2 mutation; only in-depth laboratory examinations uncovered subtle findings consistent with familial GATA2 mutation in these clinically silent carriers. Family 1: The proband presented at age 15 with pancytopenia, and was found to have MDS and monosomy 7; he died from post-BMT complications including aspergillosis. His brother was found to have leukopenia, neutropenia and macrocytosis at age 13 during an infection with H1N1 influenza; the leukopenia and macrocytosis persisted. Six months later, repeat bone marrow showed early refractory anemia; the next year his marrow had myeloid dyspoiesis and dysplastic megakaryocytes; FISH showed −7 in 2.3% of cells, leading to classification as MDS-RCC. In retrospect, both boys had absolute monocytopenia (<100/uL). GATA2 sequencing of samples from the surviving brother and his 51 y.o. mother identified a deleterious mutation (c.1116_1130del15, p.C373del5). The mother had breast cancer at age 50, but otherwise was asymptomatic. Closer clinical examination revealed lower limb lymphedema, while laboratory studies revealed lymphopenia (360/uL), monocytopenia (110/uL), low lymphocyte subsets, especially CD19 (3/uL) and MCV = 100fL. Her marrow did not show overt dyspoiesis in myeloid or erythroid lineages; among mostly normal megakaryocytes there were occasional atypical forms, including some with hypolobulated or separated lobes; G-banded karyotyping and interphase FISH for −7/7q- were normal. She would not have been suspected to have GATA2-related MDS based on her clinical status, and is thus a silent carrier. Family 2: Three children in this family were diagnosed with MDS. The oldest had a history of warts and pancytopenia at age 18; his marrow showed MDS with trisomy 8. His brother was a compatible transplant donor, but he had mild pancytopenia and monocytopenia; his marrow had MDS and trisomy 8. Their sister was diagnosed at age 14 with MDS and trisomy 8; she, too, had monocytopenia. All 3 were transplanted. Subsequently, a mutation - c.1187G>A, p.R396Q - was found in GATA2, in all 3 brothers and their healthy father. He had normal blood counts (monocytes 500/uL) and immunoglobulins, but low B-cells in peripheral blood (CD20 23/uL) and bone marrow. His normocellular marrow had occasional atypical megakaryocytes with separated lobes, hypolobulation, and mononuclear and micromegakaryocytes. He, too, would not have been suspected to have GATA2-related MDS, and is also a clinically silent carrier. These two families indicate that familial GATA2-related MDS is a dominantly-inherited syndrome. In our two families, dominant inheritance was not initially considered, in part because the genetically affected parent was clinically asymptomatic. It is unclear whether GATA2 MDS shows “anticipation,” in which the younger generation is more severely affected than the parental generation. It is important that GATA2 be evaluated in families with apparently inherited childhood MDS, since the variable expression might lead inadvertently to selecting an asymptomatic GATA2 mutation carrier as a stem cell transplant donor. Genetic counseling needs to be provided with regard to risk to other family members. In addition, only long-term follow-up and surveillance of the clinically silent carriers will determine whether they remain unaffected. Disclosures: No relevant conflicts of interest to declare.

We first report GATA2 mutations (heterozygous) in 4 families that are susceptible to MDS/AML (3 large families) and MDS (1 small family). Molecular analysis revealed a germline transmission of a GATA2 missense mutation (T354M) in MDS/AML families and a GATA2 deletion mutation (T355del) in MDS family. Neither germline RUNX1 nor CEBPA mutations were found in these families, in 695 non-leukemic ethnically matched controls and 268 sporadic AML samples. The mutations resided within the GATA2 zinc finger 2 domain, a critical region for DNA-binding and protein-protein interactions, but not for nuclear localization. T354M reduced DNA binding ability of GATA2; whereas, T355del bound very little, if any, to the consensus WGATAR DNA motif. T354M and T355del also significantly reduced the transactivation of GATA2 in known GATA2 responsive sequences. Moreover, co-transfection of T354M or T355del with WT reduced WT transactivation ability, suggesting that these mutants act in a dominant negative fashion. Regulatable stable promyelocytic HL- 60 cells expressing WT and mutants were generated. Forced expression of WT and T354M inhibited HL-60 cell differentiation when induced with all trans retinoic acid. However, when compared to WT, T354M enabled cell proliferation/survival while simultaneously reducing apoptosis. In contrast, T355del was a complete loss-of-function mutant. Microarray studies elucidated that both T354M and T355del significantly decreased the expression of downstream target genes. Together, our data suggest that both T354M and T355del are loss-of- function mutations with some dominant negative attributes. Recently, we and others have described GATA2 genetic lesions in other diseases. We further investigated in vitro functions of an alllelic series of GATA2 mutants representing the major disease phenotypes: MDS/AML (T354M), MDS (T355del), CML-BC (L359V), Emberger syndrome (R361L and C373R), AML-M5 and biallellic CEBPA AML (R362Q), and immunodeficiency syndrome (R398W). We showed that these GATA2 mutants (except L359V) are loss-of-function that reduce DNA binding affinity and transactivation of target genes. Nevertheless, they maintain the ability to bind to known protein binding partners. Intriguingly, T354M and C373R have an enhanced affinity for PU.1, highlighting that these mutants can influence both DNA-binding and protein-protein interaction. Preliminary transduction of Gata2 WT or mutant expression constructs into mouse whole bone marrow cells demonstrated that GATA2 mutants did not confer self-renewal capacity, but allowed specific myeloid progenitor differentiation. We further demonstrated that Gata2 is expressed in lymphatic endothelial cells and that it can bind to and transactivate a Prox1 promoter/enhancer element (PEE) region. Prox1 is required for lymphatic development and maintenance, and hence Gata2 may contribute to lymphoedema throught its action on Prox1. Intriguingly, Gata2 mutants displayed differential binding affinity to two GATA binding sites and reduced transactivation of the PEE region. Furthermore, an enhancer region 11.3kb upstream of Prox1 is activated by GATA2, FOXC2 and SOX18, but repressed by PROX1 itself suggesting that these key lymphatic TFs may cooperate to regulate Prox1 expression. In conclusion, I present the experimental work for the landmark discovery of a new MDS/AML predisposition gene. I have also characterized the molecular landscape of GATA2 mutations where each of the mutations confers specific and major effects on GATA2 function, but where there are also subtle differences between the mutants in the contexts of DNA binding and transactivation.
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2013

L’importance des modificateurs de la chromatine dans la régulation de l’hématopoïèse et des hémopathies malignes est illustrée par l’histone méthyltransférase Mixed-Lineage Leukemia (MLL) qui est essentielle au maintien des cellules souches hématopoïétiques (CSH) et dont le gène correspondant, MLL, est réarrangé dans plus de 70% des leucémies du nourrisson. Les histones déméthylases (HDM), récemment découvertes, sont aussi impliquées dans le destin des CSH et des hémopathies malignes. Le but de ce projet est d’étudier l’expression des HDM dans les cellules hématopoïétiques normales et leucémiques afin d’identifier de potentiels régulateurs de leur destin. Nous avons réalisé un profil d'expression génique des HDM par qRT-PCR et par séquençage du transcriptome (RNA-seq) dans des cellules de sang de cordon (cellules CD34+ enrichies en CSH et cellules différenciées) et des cellules de leucémie aiguë myéloïde (LAM) avec réarrangement MLL. Les deux techniques montrent une expression différentielle des HDM entre les populations cellulaires. KDM5B et KDM1A sont surexprimés dans les cellules CD34+ par rapport aux cellules différenciées. De plus, KDM4A et PADI2 sont surexprimés dans les cellules leucémiques par rapport aux cellules normales. Des études fonctionnelles permettront de déterminer si la modulation de ces candidats peut être utilisée dans des stratégies d’expansion des CSH, ou comme cible thérapeutique anti-leucémique. Nous avons aussi développé et validé un nouveau test diagnostique pour détecter les mutations de GATA2 qui code pour un facteur de transcription clé de l’hématopoïèse impliqué dans les LAM. Ces travaux soulignent l’importance des facteurs nucléaires dans la régulation de l’hématopoïèse normale et leucémique.
The importance of chromatin modifiers in regulation of hematopoiesis and hematologic malignancies is illustrated by the Mixed-Lineage Leukemia (MLL) histone methyltransferase, which is essential to maintain hematopoietic stem cells (HSC) and whose corresponding gene, MLL, is rearranged in over 70% of infant leukemia. The recently discovered histone demethylases (HDM) are also involved in HSC fate and in hematologic malignancies. The purpose of this project is to study the expression of HDM in normal and leukemic hematopoietic cells to identify potential regulators of their fate. We performed a comprehensive gene expression profile of HDM by qRTPCR and transcriptome sequencing (RNA-seq) in cord blood cells (CD34+ cells enriched in HSC and differentiated cells) and in acute myeloid leukemia (AML) cells with MLL rearrangement. Both techniques revealed differential expression of HDM between these cell populations. KDM5B and KDM1A are overexpressed in CD34+ cells compared to differentiated cells. Moreover, KDM4A and PADI2 are overexpressed in leukemic cells compared to normal cells. Functional studies will determine whether modulation of these candidates can be used in HSC expansion strategies or as anti-leukemic drug target. We have also developed and validated a new diagnostic test to detect mutations of GATA2, a gene encoding a key transcription factor involved in hematopoiesis and in AML. This work highlights the importance of nuclear factors in the regulation of normal and leukemic hematopoiesis.