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: The retinoblastoma tumor suppressor protein (RB) plays important roles in the control of the cell cycle, DNA-damage checkpoint, differentiation and apoptosis. It is estimated that RB is dysfunctional/inactivated in up to 40% of human leukemias. Positive as well as inhibitory signals are integrated into the phosphorylation of the RB protein to regulate the G1 to S-phase progression of the cell cycle. Despite the importance of RB in leukemia, the consequences of loss of RB on hematopoietic stem and progenitor cell (HSPC) function in vivo are still not clear and have been controversially discussed. Using Cre-enzyme expression driven by the hematopoietic specific Vav1-promotor, we generated mice that are constitutively deficient in RB (hemRb−/− animals) in HSPCs. HemRb−/− mice showed anemia with an increased number of reticulocytes in PB, consistent with a published role of RB in erythroid differentiation. In addition, the frequency of Mac-1 positive cells in BM was increased to 67% compared to 47% in control animals, whereas the frequency of B220 positive B-lymphoid cells was almost 10-fold reduced, without affecting the T-lymphoid compartment. HemRb−/− mice possessed a 3-fold enlarged spleen with a 5-fold increased number of colony-forming cells (CFCs) and severe extramedullary hematopoiesis, a phenotype also reported for animals transplanted with Rb−/− fetal liver cells. BM of hemRb−/− mice showed an almost 3-fold reduction of HSC frequency, measured by the cobblestone-area forming cell assay (CAFC) assay, but not a decrease in the number of HSCs determined by cell surface staining and flow cytometry. Upon transplantation into NOD/SCID animals or upon competitive transplantation into C57BL/6. CD45.1 animals, HSPCs from hemRb−/− mice contributed 4 to 6-fold less to hematopoiesis. HSPCs from hemRb−/− animals were neither impaired in their ability to home to the BM, nor did they show increased apoptosis. Finally, we detected a significant 4-fold decrease in stem cell function/numbers upon stress caused by 5-FU treatment in hemRB−/− mice compared to control animals. We conclude that upon transplantation/stress, HSPCs from hemRb−/− animals are impaired in their self-renewal function. HemRb−/− animals also showed a 2-fold increase in the frequency of CFCs in peripheral blood. As we detected no increased leukemia incidence in the hemRb−/− animals (now up to 1 year of age), loss of the tumor suppressor RB in hematopoietic cells might be regarded as necessary, but not sufficient for causing early onset leukemia. In summary, loss of RB results in context/localization dependent phenotypes in the hematopoietic hierarchy, influencing stem and progenitor cells in function, localization and differentiation ability.

Description: Mammalian erythropoiesis has long been established to occur within erythroblastic islands (EBIs), niches where erythroblasts differentiate in close contact with a central macrophage. While it is generally accepted that EBI macrophages play an important role in regulation of erythropoiesis, very little is known about the specific macrophage populations involved in EBI formation, the regulation that occurs within EBIs, or how this niche fits into the broader context of hematopoiesis. We analyzed native EBIs isolated from mouse bone marrow using multispectral imaging flow cytometry (Seu et. al. Front Immunol 2017). Consistent with historical observations, the EBIs were heterogeneous and many contained a number of closely CD11b+ cells in addition to erythroblasts and a central F4/80+ macrophage. Flow cytometry analysis of cells dissociated from native bone marrow EBIs indicated these niches are also enriched 2-3 fold in myeloblasts and granulocytic precursors up to metamyelocytes relative to the total bone marrow while they are depleted of mature granulocytes (bands and segmented cells). Bulk RNAseq of the CD11b+ population isolated from EBIs showed high expression of genes characteristic of the granulocytic lineage (e.g. Elane, Mpo, Gfi1, Cebpe, Camp, and Mmp9), indicating the EBI macrophages may regulate myelopoiesis along with erythropoiesis and that EBIs should really be considered as erythro-myeloblastic islands (EMBIs). To critically document the various hematopoietic cell populations that constitute EMBIs, we used the 10x Genomics Chromium system to obtain single cell gene expression data on ~3,500 total cells from isolated EMBIs along with at least 1,000 sorted cells from each of the 3 major EMBI-associated populations (F4/80+, CD71+, and CD11b+) (Fig 1a, b). The data were analyzed using 10x Genomics' Loupe cell Browser and Iterative Clustering and Guide-gene Selection (ICGS, http://www.AltAnalyze.org, Olsson et. al. Nature 2016). From the ICGS analysis, ~30% of the total EMBI-associated cells were myeloid cells that segregated into at least 3 transcriptionally distinct clusters representing granulocytic progenitors and precursors. As expected, erythroblasts with a progressive maturation pattern made up the bulk (60%) of the EMBI-associated cells, while up to 10% were a heterogeneous population of cells that exhibited expression of macrophage markers such as Csf1R and Irf8, along with genes previously described to characterize resident macrophages, such as Fn1and Fsp1/S100A4 (Fig 1c). In order to investigate the balance of myeloid cells with erythroid cells within the EMBIs, we examined the ratio of CD71+ cells to CD11b+ and how this ratio changes in models of altered granulopoiesis. While the number of myeloid cells at any island varied, the overall ratio of CD11b+ area to CD71+ within the EMBIs was relatively constant at steady state. In three different murine models of anemia of inflammation (AoI), we found that this ratio of CD11b+ to CD71+ cells within the EMBI increases dramatically indicating that the increased granulopoiesis and suppression of erythropoiesis noted in AoI is a result of altered balance of the hematopoiesis within the EMBI unit. Similarly, stimulation of granulopoiesis with GCSF also results in a shift within the EMBIs to CD11b+ myeloid cells and suppression of erythroid cells. Alternatively, in gfi1 KO mice, a model of congenital neutropenia in which granulopoiesis fails at an early stage, the ratio shifts toward CD71+ erythroid cells with paucity of the granulocytic precursors that are typically found at the EMBIs. Taken together, these data indicate that granulocyte progenitors and precursors are specifically associated with EMBI macrophages in the mouse bone marrow. The preferential localization of myeloid precursors within EMBIs suggests this niche is a site for granulopoiesis as well as erythropoiesis and production of these lineages is dynamically regulated within this niche. Our work with multiple murine models of altered granulopoiesis demonstrates that pathological expansion of one of the lineages within this niche may suppress the other and that the interactions within the EMBI could be a useful therapeutic target for AoI. These novel findings significantly broaden our understanding of the role of this hematopoietic niche in the regulated development of lineage committed erythroid and myeloid cells. Disclosures No relevant conflicts of interest to declare.

Description: Transcranial Doppler (TCD) screening in children with sickle cell anemia (SCA) identifies abnormally elevated cerebral artery flow velocities that confer an elevated risk for primary stroke. Chronic transfusions offer effective stroke prophylaxis in this setting, but must be continued indefinitely and lead to transfusional iron overload. An alternative treatment strategy that offers similar effective protection against primary stroke, and provides control of iron overload, is needed. TCD With Transfusions Changing to Hydroxyurea (TWiTCH, NCT01425307) was an NHLBI-funded Phase III multicenter randomized clinical trial comparing 24-months of standard treatment (transfusions) to alternative treatment (hydroxyurea) in children with SCA and abnormal TCD velocities. All eligible children had received at least 12 months of transfusions. TWiTCH had a non-inferiority trial design; the primary study endpoint was the 24-month TCD velocity obtained from a linear mixed model, controlling for baseline (enrollment) values, with a non-inferiority margin of 15 cm/sec. The transfusion arm maintained children at HbS

Description: Steady-state and dynamic regulation of erythrocyte production occurs by altering the balance of cell-survival versus apoptosis signaling in maturing erythroblasts. Previously, the pro-apoptotic factor Nix was identified as a critical death signal in normal erythropoietic homeostasis, acting in opposition to erythroblast-survival signaling by erythropoietin and Bcl-xl. However, the role of Nix in stress-erythropoiesis is not known. Here, by comparing the consequences of erythropoietin administration, acute phenylhydrazine-induced anemia, and aging in wild-type and Nix-deficient mice, we show that complete absence of Nix, or its genetic ablation specifically in hematopoietic cells, mimics the effects of erythropoietin (Epo). Both Nix ablation and Epo treatment increase early erythroblasts in spleen and bone marrow and increase the number of circulating reticulocytes, while maintaining a pool of mature erythroblasts as an “erythropoietic reserve”. As compared with WT, Nix null mice develop polycythemia more rapidly after Epo treatment, consistent with enhanced sensitivity to erythropoietin observed in vitro. After phenylhydrazine administration, anemia in Nix-deficient mice is less severe and recovers more rapidly than in WT mice, despite lower endogenous Epo levels. Anemic stress depletes mature erythroblasts in both WT and Nix null mice, but Nix null mice with basal erythroblastosis are resistant to anemic stress. These findings show that Nix null mice have greatly expanded erythroblast reserve and respond normally to Epo- and anemia-stimulated induction of erythropoiesis. However, the hematocrits of young adult Nix null mice are not elevated, and these mice paradoxically develop anemia as they age with decreased hemoglobin content (10g/dl) and hematocrit (36%; at 80±3 weeks of age) compared to WT mice (13g/dl and 46%; 82±5 weeks of age), inspite of persistent erythoblastosis observed in the bone marrow and spleen. Nix null erythrocytes, which are macrocytic and exhibit membrane abnormalities typically seen in immature cells or with accelerated erythropoiesis, demonstrate shorter life span with a half life of 5.2±0.6 days in the peripheral circulation by in vivo biotin labeling (as compared with a half life of 11.7±0.9 days in WT), and increased osmotic fragility as compared with normal erythrocytes. This suggests that production and release of large numbers of reticulocytes in Nix null mice can decrease erythrocyte survival. To rule out a non-hematopoietic consequence of Nix ablation that contributes to or causes increased erythrocyte fragility and in vivo consumption, such as primary hypersplenism, we undertook Tie2-Cre mediated conditional Nix gene ablation. Nixfl/fl + Tie2-Cre mice (hematopoietic-cell specific Nix null) develop erythroblastosis with splenomegaly, reticulocytosis, absence of polycythemia and increased erythrocyte fragility; suggesting that erythroblastosis and accelerated erythrocyte turnover are a primary consequence of Nix ablation in hematopoietic cells. Hence, dis-inhibition of erythropoietin-mediated erythroblast survival pathways by Nix ablation enhances steady-state and stress-mediated erythropoiesis.

Description: The Hereditary Hemolytic Anemias (HHAs) are a genetically heterogeneous group of anemias characterized by decreased red blood cell (RBC) survival because of defects in hemoglobin, RBC membrane proteins or enzymes. The diagnosis of this group of disorders is complex and challenging requiring analysis of the morphology of RBCs, hemoglobin electrophoresis, and a battery of phenotypic assays. The phenotypic analysis is often problematic in transfusion dependent patients or at times of presentation with a hemolytic crisis as transfused blood or reticulocytosis confounds diagnostic testing. Molecular genetic testing has grown in popularity in the diagnosis of hereditary hemolytic anemias as it is not affected by transfusions or other clinical variables and provides additional insight into the mechanism of the disease. We have developed a Next Generation Sequencing (NGS) panel for HHA due to RBC membrane disorders and enzymopathies and congenital dyserythropoietic anemias (CDA). CDAs, although collectively rare, are included in the panel as they are occasionally misdiagnosed as hereditary spherocytosis (HS) due to their clinical characteristics of hemolysis, increased osmotic fragility, and splenomegaly albeit with inadequate reticulocytosis We reviewed the results of 282 sequential HHA/CDA panels testing for patients with suspected HHA or CDA diagnosis, performed and interpreted at Cincinnati Children's Hospital Medical Center between 1/2013-5/2016. Forty-three samples were omitted from the final analysis due to diagnosis of other disorders, indicating that negative results were true-negatives. For the analysis of the remaining 239 panels, all results were reviewed and categorized based on the type of testing ordered: comprehensive HHA/CDA (32 genes), RBC membrane disorders (13 genes), RBC enzyme disorders (14 genes), or CDA (6 genes). The protein-coding exons plus 25 bases of exon-intron junction as well as promoter sequences were included in the design. Genomic DNA was isolated from blood and target regions were enriched using the Haloplex technology. Enriched samples were then sequenced on an Illumina MiSeq benchtop sequencer with 150 base pair, paired-end reads. Sequencing reads were aligned to the human genome reference sequence and analysis of coverage and variants was completed using NextGENe software. All positive findings were confirmed by Sanger sequencing. These 239 panels included 159 (66.5%) comprehensive HHA/CDA panels, 41 (17.2%) RBC membrane disorder panels, 10 (4.2%) RBC enzyme disorder panels, and 29 (12.1%) CDA panels. Overall, a diagnosis was confirmed or identified in 135 (56.5%) patients with specific genotype of hereditary spherocytosis in 52 patients; hereditary elliptocytosis in 15 patients; hereditary pyropoikilocytosis in 7 patients; hereditary stomatocytosis/xerocytosis in 12 patients; South East Asian Ovalocytosis in 1 patient; G6PD deficiency in 15 patients; pyruvate kinase deficiency in 17 patients; other rare RBC enzymopathies in 6 patients; and CDA in 10 patients. The clinical performance of RBC membrane disorder and RBC enzyme disorder panels were comparable between 68-70% in reaching a final diagnosis, while CDA panel confirmed final diagnosis in only 20% of suspected cases. The overall low prevalence, complexity of diagnosis with findings of dyserythropoiesis in bone marrow studies in patients with severe HHA, and evidence of locus heterogeneity in CDA might explain this result. Among patients with suspected RBC membrane disorders, approximately 14% were eventually diagnosed with hereditary xerocytosis (HX). HX diagnosis is critical to make in such patients since splenectomy is contraindicated due to the high risk of life-threatening thrombophilia complications. In more than half (56.5%) of all cases with suspected hereditary hemolytic anemia, genetic testing provided or confirmed the diagnosis and optimized patients' clinical management. Further genetic counseling and testing for other at-risk family members was made possible by achieving molecular diagnosis. Genetic testing substantially altered management in approximately 14% of cases with suspected RBC membrane disorders due to the diagnosis of HX. In conclusion, genetic testing has a significant clinical utility and may facilitate and improve diagnosis, prognosis and management considerations in patients with hereditary hemolytic or dyserythropoietic anemia. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: Maintenance of cell volume by regulated cation transport is a fundamental cellular process. The KCl cotransporter (KCC) contributes to red blood cell (RBC) volume regulation, especially in reticulocytes. Erythroid K-Cl cotransport activity is increased in sickle cells (SS RBC) and contribute to SS RBC dehydration, which potentiates sickling. Three cation cotransporter genes, SLC12A4 (KCC1), SLC12A6 (KCC3) and SLC12A7 (KCC4), and several splicing variants, mediate KCC activity in non-neuronal tissues. To determine which KCC isoform(s) predominates in human RBC we examined the quantitative expression patterns of KCC isoforms during erythroid differentiation. We developed a set of real-time RT-QPCR assays specific for KCC1, KCC1b, KCC3a, KCC3b or KCC4, over a 7-log quantitation range and sensitivity of 10 copies per reaction using multiplex amplification of GAPDH as internal controls. In human reticulocytes isolated by magnetic separation using anti-transferrin receptor coated beads, KCC3a mRNA levels were consistently the highest (4–24 fold of KCC1), while KCC4 levels varied from 1 to 7-fold of KCC1 levels (n=8). Message levels for KCC3b were relatively low (20–80% of KCC1), and for KCC1b were negligible (1–2% of KCC1). Substantial variability in the relative levels of KCC1, KCC3a, and KCC4 mRNA was observed among individual samples, but no consistent difference was apparent comparing sickle and normal reticulocytes. Western blot analysis of sickle and normal RBC ghost membranes confirmed the presence of KCC1, KCC3 and KCC4 at the protein level. To evaluate cells at various erythroid differentiation stages, human CD34+ cells were cultured under conditions favoring erythroid differentiation for 26 days. During early in vitro differentiation, KCC1 was the main mRNA species, followed by KCC4, with similar levels of KCC3a and KCC3b. RNA levels for KCC3a and KCC4 increased during maturation and became the most abundant at later stages. KCC1b mRNA remained low, and KCC3b levels decreased during erythroid development. To further define this temporal sequence of KCC expression, cells cultured for 10–17 days were sorted by FACS into four subpopulations (I to IV), characterized by immunostaining for relative expression of CD71 and glycophorin A, with enrichment of pronormoblasts basophilic normoblasts polychromatophilic normoblasts or orthrochromatic normoblasts and reticulocytes KCC1, KCC3a, KCC3b, and KCC4 were expressed in these populations, with KCC1 as the main KCC species in early precursors (I), KCC3b decreasing 〉80% during maturation, KCC4 and KCC3a becoming the most abundant in the most differentiated subpopulation (IV). In summary, we identified KCC3a as the predominant KCC isoform in human reticulocytes, followed by KCC4 and KCC1, consistent with the presence of KCC1, KCC3 and KCC4 proteins in RBC membranes. The expression of KCC3a and KCC4 increased during erythriod differentiation in vitro. Variations in relative expression of KCC species are a potential source of inter-individual differences in KCC function for volume regulation. These results also provide the foundation for the possibility of improving SS RBC hydration by reducing gene expression of the major KCC isoforms in RBC.

Description: Rac GTPases (i.e. Rac1, Rac2 and Rac3), a subfamily of Rho GTPases, control actin organization and have overlapping as well as distinct roles in cell survival, proliferation, and differentiation in various hematopoietic cell lineages (Gu et al, Science 2003, Cancelas et al, Nature Med 2005). Using conditional gene-targeting in mice, we have previously demonstrated that Rac1 and Rac2 deficiency causes anemia with abnormal erythrocyte cytoskeleton and decreased deformability (Kalfa et al, Blood 2006). In the present studies, we found by colony assays that although bone marrow (BM) BFU-E activity was unaltered from that of the wild type (WT) mice, Rac1−/−;Rac2−/− erythroid bursts had a strikingly different morphology appearing as round, small, dense colonies, likely a manifestation of motility defects associated with Rac GTPase deficiency. Total CFU-Es recovered from Rac1−/−;Rac2−/− BM were as low as 25% of that in WT mice (p

Description: To understand the role of cytoskeleton and membrane signaling molecules in erythroblast enucleation, we developed a novel analysis protocol of multiparameter high-speed cell imaging in flow. This protocol enabled us to observe F-actin and phosphorylated myosin regulatory light chain (pMRLC) assembled into a contractile actomyosin ring (CAR) between nascent reticulocyte and nucleus, in a population of enucleating erythroblasts. CAR formation and subsequent enucleation were not affected in murine erythroblasts with genetic deletion of Rac1 and Rac2 GTPases because of compensation by Rac3. Pharmacologic inhibition or genetic deletion of all Rac GTPases altered the distribution of F-actin and pMRLC and inhibited enucleation. Erythroblasts treated with NSC23766, cytochalasin-D, colchicine, ML7, or filipin that inhibited Rac activity, actin or tubulin polymerization, MRLC phosphorylation, or lipid raft assembly, respectively, exhibited decreased enucleation efficiency, as quantified by flow cytometry. As assessed by high-speed flow-imaging analysis, colchicine inhibited erythroblast polarization, implicating microtubules during the preparatory stage of enucleation, whereas NSC23766 led to absence of lipid raft assembly in the reticulocyte-pyrenocyte border. In conclusion, enucleation is a multistep process that resembles cytokinesis, requiring establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring, and coalescence of lipid rafts between reticulocyte and pyrenocyte.

Description: Hereditary spherocytosis (HS) is a genetically and phenotypically heterogeneous hemolytic anemia caused by deficiency in red blood cell (RBC) cytoskeleton proteins leading to disruptions in the vertical association of the cytoskeleton with the RBC lipid bilayer. Monoallelic mutations in the genes encoding ankyrin (ANK1), beta-spectrin (SPTB) and band 3 (SLC4A1) or biallelic mutations in the genes encoding alpha-spectrin (SPTA1), ankyrin, and protein 4.2 (EPB42) result in HS. Autosomal recessive HS due to compound heterozygous defects in SPTA1 is typically severe and diagnosis based on phenotypic assays like RBC morphology, osmotic fragility or ektacytometry is complicated by transfusion dependence resulting in most of the circulating RBCs to be of donor origin. We have developed a rapid comprehensive next-generation sequencing-based assay that evaluates 27 genes with published disease-causing mutations for RBC cytoskeletal disorders, enzymopathies, and CDAs. We describe here patients with hemolytic anemia due to SPTA1 mutations, identified utilizing this assay, and their phenotype-genotype correlation. Each of these cases, when possible, has been also evaluated with ektacytometry and immunoblotting of RBC ghosts for alpha-spectrin quantitation. Wichterle et al in 1996 had estimated that alphaLEPRA(Low Expression PRAgue) mutation (c.4339-99C〉T) occurs in SPTA1 gene in about 5% of Caucasians. This mutation leads to activation of an alternate acceptor splice site at position -70 of intron 30, causing frame shift and premature termination, thereby leading to decrease in alpha-spectrin production in this allele to about 16% of normal. We have found a cohort of three transfusion-dependent hereditary hemolytic anemia cases where a nonsense mutation in SPTA1 gene has occurred in trans to alphaLEPRA mutation, resulting in premature termination (see Table 1). Transfusion dependence was alleviated in two of these patients after splenectomy; the third one did not have splenectomy yet. RBC phenotype explored after splenectomy revealed an ektacytometry curve indicating spherocytosis (Figure 1A) and severely decreased alpha-spectrin on immunoblotting along with significant decrease of the associated beta-spectrin (Figure 1B). A patient with moderately severe form of HS, maintaining a hemoglobin value greater than 7 g/dL and requiring only occasional transfusions during periods of illness or stress, was found to have alphaLEPRA occurring in trans to an intronic splicing mutation c.1351-1G〉TG where there is substitution at nucleotide-1 of intron positioned between nucleotides 1350 and 1351 of the SPTA1 mRNA. This splicing mutation may allow for some expression of functional alpha-spectrin protein from this allele in contrast to no protein expression in the previous cases of premature termination. Alternatively, other gene mutations, not identified by the next-generation sequencing panel we used, may contribute to this patient's milder phenotype. A couple with history of two fetal losses associated with hydrops fetalis seeked genetic counseling and gave consent to have diagnostic evaluation of genes associated with non-immune hemolytic anemia using targeted next-generation sequencing. Results of the panel revealed a heterozygous frameshift SPTA1 mutation in each of the parents (c.4206delG in the father and c.4180delT in the mother). These mutations in compound heterozygous state in the offspring likely caused total absence of alpha spectrin and fatal hemolytic anemia by the time of birth. Hereditary Spherocytosis is characterized by wide phenotypic variability that will be better understood with studies of genotype-phenotype association. While complete absence of alpha-spectrin expression due to null mutations of both SPTA1 alleles is incompatible with life, a nonsense or splicing SPTA1 mutation in trans to an alphaLEPRA low expression allele causes severe or moderately severe recessive HS, respectively. Targeted next-generation sequencing can be an effective diagnostic tool particularly for patients requiring frequent transfusions that preclude meaningful phenotypical testing of their red blood cells. Figure 1. SPTA1 null mutations occurring in trans to alpha-LEPRA causing severe HS Figure 1. SPTA1 null mutations occurring in trans to alpha-LEPRA causing severe HS Figure 2. Figure 2. Disclosures Begtrup: GeneDx: Employment.