ALBERT

Description: The Congenital Dyserythropoietic Anemia Registry (CDAR, ClinicalTrials.gov Identifier: NCT02964494) was created to investigate the natural history, biology, and molecular pathogenetic mechanisms of CDA. To date, there are 6 genes known to cause CDA (CDAN1, C15orf41, SEC23B, KIF23, KLF1, GATA1). However, 57% of patients registered in CDAR so far (17 out of 33 patients) have an unidentified genetic cause. We have utilized whole exome sequencing (WES) in family-trio design to search for novel candidate gene mutations that may be responsible for the disease. Three unrelated patients with dyserythropoiesis, hemolytic anemia, and neurodevelopmental delay were found to have missense mutations in the gene VPS4A which encodes an ATPase that participates with the ESCRT III machinery in endosomal vesicle trafficking, centrosome localization, and the abscission step of cytokinesis. It has been shown to play an essential role in division of HeLa cells in vitro where it concentrates at the spindle poles during mitosis and at the midbody during cytokinesis. The aim of this work is to validate the pathogenetic role of these VPS4A variants in CDA and further investigate the role of VPS4A in erythropoiesis. Patients 1 and 3 had de novo mutations (R284W and G203A) and transfusion-dependent anemia with presence of binucleated erythroblasts in the bone marrow resembling CDA type I. Of note, the patients' erythroblasts exhibited cytoplasmic bridges (Figure 1A) rather than the nuclear chromatin bridges observed in CDA-I. Patient 2, offspring of consanguineous parents, presented with hemolytic anemia and was found to have a homozygous mutation (A28V) in a highly conserved alanine residue in the microtubule-interacting domain (MIT) of VPS4A. She had rare evidence of dyserythropoiesis with fewer than 3% binucleated erythroblasts in bone marrow studies. All three patients had significant neurodevelopmental delay with axial hypotonia and appendicular hypertonia. Flow cytometry analysis of peripheral blood from each of these patients revealed a unique cell population which is negative for RNA (by thiazole orange) but still CD71 positive suggesting that loss of VPS4A function also impacts reticulocyte maturation, likely because of defective endosomal vesicle trafficking. Using CD34+ cells in ex vivo erythropoiesis cultures, we first confirmed that VPS4A is expressed in human erythroblasts and localizes at the spindle poles and midbody during mitosis and cytokinesis in these cells. RNA isolated from reticulocytes from patients 1 and 2 was assessed for expression of VPS4A and the paralogous VPS4B. Samples from patient 1 had reduced expression of VPS4A (

Description: GFI1 is a zinc-finger transcriptional repressor and master regulator of growth, differentiation and survival. Elevated GFI1 expression is observed in many cancer types, and targeted depletion of GFI1 in experimental models of some cancers leads to tumor regression. GFI1 confers growth and survival advantages to cells in which it is expressed. Its impact can be attributed at least in part to repression of cell cycle checkpoint proteins such as p21 and to inhibition of the p53-induced DNA damage response. Understanding determinants of GFI1-mediated transcriptional repression may allow control over normal cell and tissue homeostasis and enable rationally conceived opportunities for cancer therapy. GFI1 is comprised of an N-terminal, 20 amino acid SNAG domain, a C-terminal concatemer of six zinc fingers and a linker that separates them. The GFI1 SNAG domain is responsible for recruitment of LSD1 to GFI1-regulated genes while its zinc fingers recognize and bind a response element found within their promoters. LSD1 recruitment by GFI1 is essential for GFI1 function as a transcriptional repressor, yet the molecular features that enable regulation of the GFI1-LSD1 relationship to control cell fate are not known. We used primitive erythropoiesis in Zebrafish and a chromatinized luciferase reporter system in human cells to interrogate details of the GFI1-LSD1 relationship in hematopoiesis and transcriptional control. We show that GFI1-LSD1 binding via the SNAG domain is required for primitive erythropoiesis and that LSD1 depletion phenocopies Gfi1 loss. In parallel, we show the SNAG domain is the dominant transcriptional repression motif in GFI1, that transcriptional repression attributable to the GFI1 SNAG domain requires LSD1 recruitment and is accompanied by demethylation of histone H3K4 at the reporter locus. SNAG domain derivatives devoid of LSD1 binding fail to repress gene expression. To gain additional mechanistic insights, we carefully examined the SNAG domain primary structure. We show that the GFI1 SNAG domain harbors a primary structural motif, -8KSKK11-, similar to that observed in p53 (-370KSKK373-) where it is known to alter p53 transcriptional activity through site-specific methylation. Lysine (K) 370 monomethylation (K370me) deactivates p53, while dimethylation (K370me2) activates it. Notably, LSD1 can demethylate both K370me and K370me2. This suggests an activation-inactivation cycle for p53 controlled by LSD1 and p53-K370 protein lysine methyltransferases (PKMTs). Like p53, we find GFI1's SNAG domain is methylated, and when dimethylated (me2) on K8, dramatically enhances LSD1 binding. However, unlike K370me1/2 in p53, K8me2 in GFI1 is not a substrate for LSD1, and K8me2-SNAG peptide is a potent competitive inhibitor of LSD1-mediated lysine demethylation in vitro. Mutation of K8 in GFI1 to leucine (GFI1-K8L) abolishes LSD1 binding and GFI1-mediated transcriptional repression. Identical results are observed when analyzing transcriptional repression by the SNAG domain in isolation. In Zebrafish primitive erythropoiesis, GFI1-K8L expression fails to complement the Gfi1 depletion phenotype, while injection of K8me2-SNAG peptide phenocopies GFI1 or LSD1 depletion in this same assay. We then screened p53 -370KSKK373- PKMTs, SMYD2, SETD7 and G9a for their specificity toward lysine residues in the GFI1 -8KSKK11- motif. We find PKMT specificity toward GFI1's -8KSKK11- motif analogous to that reported for p53. Our findings indicate that SNAG domain methylation modulates transcriptional repression and cell fate determination functions of GFI1, and suggest methylation-dependent integration of GFI1 and p53 actions in cell growth and survival through a shared structural motif. Disclosures Sharma: Salarius Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; TheraTarget: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals Inc., Cambridge, MA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Research Funding; ConverGene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Congenital dyserythropoietic anemias (CDA) are rare hereditary diseases of abnormal erythropoiesis. The CDA Registry of North America (CDAR) (NCT02964494) was opened in 2016 to investigate the natural history and molecular biology of CDA. CDA type I (CDA-I) is a recessive form of CDA characterized by macrocytic anemia, hemolysis with inadequate reticulocytosis, and iron overload. The bone marrow shows binucleated erythroblasts with chromatin bridges by light microscopy and spongy heterochromatin in erythroblasts by electron microscopy. The phenotypic heterogeneity in presentation and course of CDA-I is remarkable. Most CDA-I cases are caused by biallelic mutations in CDAN1or C15orf41, and 10-20% do not have an identifiable mutation. Non-hematological features, especially skeletal features, were historically reported in 10-20% of patients (Wickramasinghe, 1998). Due to the rarity of CDA-I and its clinical overlap with several disorders, the diagnosis is often missed or delayed by up to 17 yrs (median) (Roy, 2019). We describe in this study the characteristics and clinical course of CDA-I patients due to CDAN1 mutations enrolled in CDAR. Patients with a phenotypic diagnosis of CDA and their family members were enrolled in CDAR. Clinical and demographic data were gathered from participants at study entry and updated periodically thereafter. Participants elect to give blood, bone marrow, and DNA samples to the biorepository associated with CDAR. Participants with a phenotypic diagnosis of CDA-I and confirmed mutations in CDAN1 were included in this study. Six participants had a diagnosis of CDA-I due to biallelic CDAN1 mutations, comprising 18% (6/33) of affected CDAR participants. CDAN1 mutations were found in 75% of cases diagnosed phenotypically as CDA-I. All six participants presented early in life with a variable degree of non-immune hemolysis, and the diagnosis was confirmed within a median of 2 years from presentation. The characteristics of participants are summarized in table 1. Two had family history of stillbirth or fetal demise in older siblings due to hydrops fetalis. One participant presented prenatally with fetal anemia and started intrauterine transfusions at 24 weeks of gestation; 2 presented with severe anemia and signs of hydrops, pulmonary hypertension, transaminitis, severe hyperbilirubinemia, and thrombocytopenia at birth; and 3 presented with neonatal jaundice and moderate anemia. All participants required blood transfusions in the neonatal period. Three had spontaneous improvement and did not require transfusions after the first year of life. One remained transfusion-dependent at last follow up at the age of 4 yrs. One became transfusion-independent after starting interferon-alpha at 1 yr of age and did not need further transfusions even after discontinuation at 3 yrs of age. One had splenectomy at 11 y.o because he was misdiagnosed to have a membrane disorder but presented in adulthood with hemolytic anemia and pulmonary hypertension and was diagnosed at that time with CDA-I by genetic sequencing. All participants had one or more non-hematological manifestations, including hypertrophic skin folds, onychocryptosis, curved toenails, syndactyly, café-au-lait spots, macrocephaly, spinal fusion, scoliosis, and short stature. One participant suffered a thalamic stroke in the postnatal period, 2 had transient neonatal pulmonary hypertension in the setting of severe anemia, and one had pulmonary hypertension post-splenectomy in adulthood. Ferritin was high in all participants at last follow up, and 4 received chelation therapy. In summary, mutations in CDAN1 are the most common identified mutations in CDAR. CDA-I causes early-onset macrocytic anemia, which may present prenatally, with variable severity of hemolysis ranging from hydrops to mild neonatal jaundice and anemia. Non-hematological manifestations, mainly skeletal, nail and skin abnormalities are more common in CDA-I than previously reported, and their presence in infants with unexplained anemia should raise suspicion for the diagnosis. The availability of molecular testing has significantly accelerated the diagnosis. Management of patients with CDA-I requires multidisciplinary approach from an early age to improve outcome. Collaboration between clinicians, scientists, patients, and families is needed to advance the understanding and treatment of this rare disease. Disclosures Chonat: Alexion: Other: advisory board; Agios Pharmaceuticals, Inc.: Other: advisory board. Kalfa:Agios: Other: local PI of clinical research trial; FORMA: Other: sponsored research agreement.