Elevated Fetal Hemoglobin Research Articles

Metformin Induction of Fetal Hemoglobin

Background: Elevated fetal hemoglobin (HbF, α2γ2) ameliorates the pathophysiology of sickle cell disease (SCD) by diluting HbS and inhibiting its polymerization in patient red blood cells. We identified FOXO3, a potentially “druggable” transcription factor, as a candidate HbF inducer through analysis of rare variants from whole exome sequence data from SCD patients. We then treated hematopoietic stem and progenitor cells (HSPCs) derived from patients with SCD with metformin, an FDA-approved drug used for type 2 diabetes, which is known to activate FOXO3. Metformin significantly increased HbF alone, and additively with hydroxyurea.Objective: Determine the mechanism by which metformin induces HbF in SCD patient derived HSPCs, and report preliminary clinical trial results of metformin in patients with SCD.Methods: HSPCs from patients with SCD were obtained from discarded red blood cell exchange product. 100 µM Metformin was added on day 7 of two phase primary erythroid culture, and HPLC analysis of hemoglobins or RNA extraction and RT-qPCR or RNASeq was performed on day 14 of culture. BCL11A, pAKT and pAMPK were quantified by Western blot, performed in triplicate.Patients with HbSS between the ages of 16 and 40 who produced less than 20% HbF at maximum tolerated dose of hydroxyurea or were not taking hydroxyurea were recruited to participate in an IRB approved pilot clinical trial of metformin in patients with hemoglobinopathies (Fetal Hemoglobin Induction with Metformin, NCT02981329). RNA was collected from CD45+ can CD71+ cells collected from their peripheral blood at study entry and after 3 months at 1500 mg metformin, and analyzed by RNASeq.Results: Metformin significantly increased HbF production in 80% of the HSPCs derived from unique SCD patients, in contrast with hydroxyurea, which failed to increase HbF in 33% of patient derived HSPCs. When FOXO3 is knocked out in HSPCs, metformin no longer induces HbF. Expression of known gamma globin regulators BCL11A, KLF1, and MYB are not altered by metformin treatment of patient derived HSPCs, nor are they altered by metformin treatment in patient derived RNA from CD45+ or CD71+ cells. Metformin treatment of SCD patient derived HSPCs causes FOXO3 to accumulate in the nucleus, reduces the active, phosphorylated form of AKT, a FOXO3 inhibitor, and increases the active, phosphorylated form of AMPK, a FOXO3 activator. Our clinical trial is ongoing, has enrolled 10 patients to date; results will be presented at the American Society of Hematology meeting.Conclusions: Metformin induction of HbF in SCD patient derived HSPCs requires FOXO3 expression, and affects the activity of known FOXO3 activator AMPK and FOXO3 inhibitor AKT. Metformin does not alter expression levels of known regulators of HbF either in treated HSPCs from patients with SCD or in samples from clinical trial subjects treated with metformin compared to their trial entry samples. We conclude that metformin induces HbF through the FOXO3-AMPK-AKT pathway, and may be a viable pharmacologic HbF inducer alone or in combination with hydroxyurea. DisclosuresNo relevant conflicts of interest to declare.

Common Fetal Hemoglobin Variants Modify Hematological Phenotypes in MDS/MPN/Myeloma Patients Receiving Cytotoxic Drugs

Background: Genetic studies have identified common variants within the HBS1L-MYB intergenic polymorphism (HMIP) region on chromosome 6q, BCL11A on chromosome 2p, and Xmn 1- HBG2 (rs748214) as associated with elevated fetal hemoglobin (HbF) levels and other clinically important human hematological traits. A recent study suggested that HbF is a predictor of outcome in myelodysplastic syndrome (MDS)/acute myeloid leukemia patients receiving decitabine. We set out to evaluate the genotypic significance of HbF and its effect on hematological traits in patients with myeloproliferative neoplasm (MPN), myeloma and MDS treated with HbF-inducing cytotoxic agents to determine potential of the HbF-associated variants for prediction of treatment response in these conditions.Methods: 89 patients - 44 with MPN on Hydroxyurea (HU), 33 with myeloma on Lenalidomide, and 12 with MDS on Azacytidine were recruited from hematology clinics at Brighton & Sussex NHS Trust, UK. Blood samples were obtained from patients with written consent and analyzed for HbF/hematological parameters. 7 common HbF variants (rs6545816 and rs1427407 on BCL11A, rs9494142, rs6920211, rs9494145, and rs66650371 in HMIP, and rs748214) were genotyped on DNA extracted from peripheral blood using Taqman procedure. A subset of the patients (N = 16) (7 MPN, 6 myeloma and 3 MDS patients) had baseline values before starting therapy and had repeat values 3 months after cytotoxic agent initiation. The study was approved by the local and national UK NHS Research Ethics Committee (REC) (reference 14/WA/1078). Statistical analysis to test and graph genetic association of blood traits was performed with linear regression (IBM SPSS Statistics 24) with a genotypic genetic model adjusting for age and sex as covariates and analysis of variance determining genotype effect and mean HbF. Hardy-Weinberg test was performed on all genotype results for quality assurance. Hematological parameters which did not satisfy assumptions of normality were log-transformed.Results: HbF variants genotyped in our hematological malignancy study population had significantly discordant minor allele frequencies (MAF) differing in comparison to MAF from wider British/European population data derived from 1000 Genome project with MDS having greatest MAF discordance (see Fig. 1). Amongst the total hematological malignancy group, HMIP variants were statistically associated with higher baseline and induced HbF levels as well as lower WBC (see Table 1). Amongst MPN patients on HU, rs9494142 (HMIP) was observed as significantly associated with induced HbF levels (p = 0.04) in addition to increased baseline platelet count (p = 0.04) (see Table 1). These variants were seen to affect different hematological parameters at different stages of treatment as observed with HMIP variants rs9494142 and rs6920211 shifting from their regulation of baseline platelet levels to the regulation of induced hemoglobin levels (p = 0.02) 3 months post cytotoxic agent initiation. Amongst the Myeloma group, rs6920211(HMIP) was significantly associated with baseline HbF (p < 0.001). BCL11A variant rs1427407 was strongly associated with induced HbF levels with Lenalidomide (p = 0.002). rs1427407 was also significantly associated with increased platelet count (p = 0.04) negating the thrombocytopenic tendency of Lenalidomide (see Table 1). Xmn 1- HBG2 (rs748214) was not observed to have any statistically significant association with HbF but was associated with decreased hemoglobin (p = 0.03) in Myeloma and increased levels of hemolysis markers (reticulocytes (p = 0.003) and lactate dehydrogenase (p = 0.001)) in MPN patients on HU.Conclusion: Although our sample size is small,the data suggests that the common HbF variants influence HbF and hematological traits in MDS, MPN, and myeloma and that the variants may potentially be involved in disease biology and treatment response in these conditions. The study also suggests HbF acts as a marker for the presence of the genetic variants that influences the hematological response in MDS/AML in the previous study. We hope that this study stimulates a larger prospective genotype-phenotype association studies of these disorders with the HbF variants. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MBD2-NuRD but Not MBD3-NuRD Mediates Strong Silencing of Fetal γ-Globin Gene Expression in Human Adult Erythroid Cells

Elevated fetal hemoglobin (HbF) levels are effective in ameliorating the underlying pathophysiologic defects in β-thalassemia and sickle cell anemia. Studies aimed at a full understanding of the mechanisms that enforce silencing of HbF expression in adult erythroid cells offer the promise of targeted molecular therapy. Here we show that depletion of Methyl CpG Binding Domain Protein 2 (MBD2) or disruption of MBD2-Nucleosome Remodeling and Deacetylose Complex (NuRD) interactions results in high levels of HbF in HUDEP-2 cell progenitor derived primary erythroblasts and CD34 + progenitor derived primary adult erythroid cells.We have previously shown that depletion of MBD2 causes a 10-20 fold increase in γ-globin gene expression in adult erythroblasts of β-YAC transgenic mice. To determine the effect of total depletion of MBD2 in human erythroid cells, CRISPR/Cas9 mediated knockout was carried out in HUDEP-2 cells. The absolute level of γ-globin gene expression increased by > 100 fold and the γ/ γ+β mRNA ratio increased from 1-3% in CRISPR/Cas9 scrambled guide control cells to 50-60% in MBD2 knockout HUDEP-2 cells after erythroid differentiation, equivalent to the highest levels reported with knockout of other silencing factors. In contrast CRISPR/Cas9 mediated knockout of Methyl CpG Binding Domain Protein 3 (MBD3) had no significant effect on γ-globin gene expression levels in HUDEP-2 cells after erythroid differentiation.Partial depletion of several strong γ-globin gene silencers, including BCL11A, LRF and KLF-1 has been shown to relieve silencing in CD34+ progenitor derived primary adult erythroid cells, but differences in the differentiation states reported in individual studies make comparisons of the relative effects of these silencer factors difficult. We show here that knockdown of MBD2 by shRNA in CD34+ progenitor derived primary adult erythroid cells consistently increased γ/ γ+β mRNA rations by 7-10 fold across different stages of differentiation without affecting the level of differentiation as compared to shRNA scrambled control cells. MBD2 knockdown led to ~30-40% γ-globin mRNA levels compared to 3%-4% in scrambled shRNA control cells, a level comparable to that typically observed in sickle cell patient reticulocytes. A comparable increase in HbF levels was observed in the MBD2 shRNA knockdown CD34+ progenitor derived erythroid cells compared to scrambled shRNA controls.We have previously shown that MBD2 interacts with the CHD4 chromatin remodeling component of the NuRD complex through a coiled-coil domain, and with the histone deacetylase component of the complex via an intrinsically disordered region (IDR) within MBD2. To test the effects of disrupting these domains in a human adult erythroid cell model, MBD2 knockout HUDEP-2 cells were infected with lentiviral expression vectors containing either wild type MBD2, coiled-coil mutant MBD2, or IDR mutant MBD2. Wild type MBD2 expression resulted in significant silencing of γ-globin gene expression while neither the coiled-coil mutant nor the IDR mutant MBD2 had any silencing effect.These results establish MBD2-NuRD as a major silencer of fetal hemoglobin expression in adult human erythroid cells. Given the minimal phenotype of MBD2 knockout mice, these results suggest that the coiled-coil and IDR domains of the MBD2-NuRD complex are potential targets for therapeutic agents aimed at increasing HbF levels in patients with β-hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

CRISPR/Cas9 Gene-Edited Hematopoietic Stem Cell Therapy for Sickle Cell Disease

Sickle cell disease (SCD) is a life-threatening hereditary disorder that affects thirty million individuals worldwide. The disease results from a single amino acid change in the β-globin gene, which causes polymerization of hemoglobin and deformation of red blood cells, leading to vaso-occlusion, severe pain crisis, multi-organ dysfunction, and ultimately reduction of lifespan in affected individuals. Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative therapy for SCD, yet its success is limited by donor availability and high risk of graft-versus-host disease. Increasing expression of fetal γ-globin in adults is an accepted therapeutic strategy for treating SCD. Supporting this approach are GWAS studies that have identified genetic loci, including within the BCL11A gene, that are associated with elevated fetal hemoglobin (HbF) and mitigated disease severity. Leveraging CRISPR/Cas9 genome editing technology, we aim to develop a safe and curative therapy by autologous, genome-modified HSCT leading to sustained generation of HbF-containing, functional red blood cells. Guide RNAs (gRNAs) targeting the erythroid-specific enhancer region of the BCL11A gene were assayed in CD34+ hematopoietic stem and progenitor cells (HSPCs), and one gRNA was selected for in vivo evaluation based on editing efficiency and functional potency. We achieved approximately 80% target site editing in human CD34+ HSPCs and 65% in CD34+CD38-CD90+CD45RA-CD49f+ defined LT-HSCs. Potential off-target editing sites were identified by two approaches: in silico prediction and unbiased oligo insertion method. These sites were then directly interrogated in HSPCs by targeted PCR amplification and NGS analysis, and to date our selected gRNA had zero validated off-target sites. In addition, HSPC composition was not altered following electroporation of ribonucleoprotein complex, and cells were capable of in vitro expansion and multi-lineage differentiation as measured by CFU assays. Upon in vitro differentiation into the erythroid lineage, edited cells demonstrated ~40% reduction in BCL11A mRNA with a corresponding two-fold increase in γ-globin transcript, translating to a 30-40% increase in HbF positive cells. Most importantly, when CD34+ cells derived from SCD patients were gene-edited and differentiated into erythroid cells, a similar editing efficiency and increase in HbF positive cells was observed. To evaluate whether gene editing alters the self-renewal, engraftment or lineage differentiation capacity of HSCs, gene-edited HSPCs were transplanted into NOD- scid IL2rγnull (NSG) mice to assess reconstitution of the hematopoietic system. Mice displayed 30-40% human CD45+ cell engraftment and multi-lineage reconstitution at 16 weeks following transplantation. When long-term engrafted bone marrow cells were harvested from the recipients and differentiated into erythroid cells in vitro, gene-edited bone marrow cells produced significantly higher numbers of HbF positive cells compared to mock-edited bone marrow cells. These data support the development of autologous genome-modified HSPC transplantation as a novel cell therapy for SCD. DisclosuresYu:Novartis Institutes for BioMedical Research: Employment. Liu:Novartis Institutes for BioMedical Research: Employment. Curran:Novartis Institutes for BioMedical Research: Employment. Zhang:Novartis: Employment. Snead:Novartis: Employment. Schmedt:Novartis: Employment. Yang:Novartis Institutes for BioMedical Research: Employment. Lin:Novartis: Employment. Tschantz:Novartis Institutes for BioMedical Research: Employment. Quinn:Novartis Institutes for BioMedical Research: Employment. Russ:Novartis: Employment. Clarkson:Novartis: Employment. Janiak:Novartis: Employment. Stewart:Intellia Therapeutics: Employment. Mulumba:Intellia Therapeutics: Employment. Lescarbeau:Intellia Therapeutics: Employment. Murray:Intellia Therapeutics: Employment. Seitzer:Intellia Therapeutics: Employment. Strapps:Intellia Therapeutics: Employment. Huang:Intellia Therapeutics: Employment. Sloan:Intellia Therapeutics: Employment. Mickanin:Novartis: Employment. Klickstein:Novartis: Employment. Stevenson:Novartis: Employment.

Fetomaternal hemorrhage: evaluation of recurrence within a large integrated healthcare system

Fetomaternal hemorrhage is associated with severe fetal morbidity and mortality. The recurrence risk of fetomaternal hemorrhage is unknown. We sought to establish the recurrence rate of fetomaternal hemorrhage in a large integrated healthcare system over a 10-year period. In this retrospective study within the Kaiser Permanente Northern California medical system, cases of fetomaternal hemorrhage were defined by either an elevated fetal hemoglobin level as determined by flow cytometry for a concerning pregnancy outcome (preterm delivery, perinatal demise, neonatal anemia, or transfusion within the first 2 days of life) or by perinatal demise with autopsy findings suggestive of fetomaternal hemorrhage. The outcomes of subsequent pregnancies were reviewed for features of recurrence. Within the 2008 to 2018 birth cohort of 375,864 pregnancies, flow cytometry testing for fetal hemoglobin levels was performed in 20,582 pregnancies. We identified 340 cases of fetomaternal hemorrhage (approximately 1 in 1100 births). Within the cohort of 340 affected pregnancies, perinatal loss was recorded for 80 (23.5%) pregnancies and 50 (14.7%) pregnancies delivered neonates who required transfusion. The affected patients had 225 subsequent pregnancies of which 210 were included in the analysis. Of these, 174 (82.9%) advanced beyond the threshold of viability and were delivered within our healthcare system. There was 1 case of recurrent fetomaternal hemorrhage identified. The recurrent case involved a spontaneous preterm delivery of an infant who was noted to have an elevated reticulocyte count but was clinically well. Within our large integrated healthcare system, approximately 1 in 1100 pregnancies was affected by fetomaternal hemorrhage within a 10-year period, which is comparable with previous studies. We identified 1 case of recurrence, yielding a recurrence rate of 0.5%. This infant did not have features of clinically important fetomaternal hemorrhage. This information can inform counseling of patients with affected pregnancies.

Hypoxia-Induced Alpha-Globin Expression in Syncytiotrophoblasts Mimics the Pattern Observed in Preeclamptic Placentas.

Preeclampsia (PE) is a pregnancy disorder associated with placental dysfunction and elevated fetal hemoglobin (HbF). Early in pregnancy the placenta harbors hematopoietic stem and progenitor cells (HSPCs) and is an extramedullary source of erythropoiesis. However, globin expression is not unique to erythroid cells and can be triggered by hypoxia. To investigate the role of the placenta in increasing globin levels previously reported in PE, flow cytometry, histological and immunostaining and in situ analyses were used on placenta samples and ex vivo explant cultures. Our results indicated that in PE pregnancies, placental HSPC homing and erythropoiesis were not affected. Non-erythroid alpha-globin mRNA and protein, but not gamma-globin, were detected in syncytiotrophoblasts and stroma of PE placenta samples. Similarly, alpha-globin protein and mRNA were upregulated in normal placenta explants cultured in hypoxia. The upregulation was independent of HIF1 and NRF2, the two main candidates of globin transcription in non-erythroid cells. Our study is the first to demonstrate alpha-globin mRNA expression in syncytiotrophoblasts in PE, induced by hypoxia. However, gamma-globin was only expressed in erythrocytes. We conclude that alpha-globin, but not HbF, is expressed in placental syncytiotrophoblasts in PE and may contribute to the pathology of the disease.

Long noncoding RNA HBBP1 enhances γ-globin expression through the ETS transcription factor ELK1

β-Thalassemia is an autosomal recessive genetic disease caused by defects in the production of adult hemoglobin (HbA, α2β2), which leads to an imbalance between α- and non-α-globin chains. Reactivation of γ-globin expression is an effective strategy to treat β-thalassemia patients. Previously, it was demonstrated that hemoglobin subunit beta pseudogene 1 (HBBP1) is associated with elevated fetal hemoglobin (HbF, α2γ2) in β-thalassemia patients. However, the mechanism underlying HBBP1-mediated HbF production is unknown. In this study, using bioinformatics analysis, we found that HBBP1 is involved in γ-globin production, and then preliminarily confirmed this finding in K562 cells. When HBBP1 was overexpressed, γ-globin expression was increased at the transcript and protein levels in HUDEP-2 cells. Next, we found that ETS transcription factor ELK1 (ELK1) binds to the HBBP1 proximal promoter and significantly promotes its activity. Moreover, the synthesis of γ-globin was enhanced when ELK1 was overexpressed in HUDEP-2 cells. Surprisingly, ELK1 also directly bound to and activated the γ-globin proximal promoter. Furthermore, we found that HBBP1 and ELK1 can interact with each other in HUDEP-2 cells. Collectively, these findings suggest that HBBP1 can induce γ-globin by enhancing ELK1 expression, providing some clues for γ-globin reactivation in β-thalassemia.

Contribution of Xmn I Polymorphism in the Variation of Hemoglobin F in Thalassemia Intermedia Patients in Pakistan

Background: The −158 (C→T) nucleotide change, known as Xmn I polymorphism, occurs in G γ-globin gene promoter and may result in elevated fetal hemoglobin (HbF). However some studies did not find any association of HbF with the mutation. This study was taken to confirm the influence of Xmn-I polymorphism on HbF production in thalassemia intermedia patients in Pakistani population. Methods: Blood samples from 100 known thalassemia intermedia patients were collected and analyzed for Xmn -I polymorphism and levels of hemoglobin F. Results: High levels of HbF were found in Thalassemia intermedia patients being heterozygous and homozygous for Xmn -I polymorphism. Conclusion: Xmn I polymorphism (C-T) of the G γ-globin gene promoter is associated with increased expression of the G γ-globin gene, higher production of HbF and lesser clinical severity.

International Consensus Definition of DNA Methylation Subgroups in Juvenile Myelomonocytic Leukemia.

Known clinical and genetic markers have limitations in predicting disease course and outcome in juvenile myelomonocytic leukemia (JMML). DNA methylation patterns in JMML have correlated with outcome across multiple studies, suggesting it as a biomarker to improve patient stratification. However, standardized approaches to classify JMML on the basis of DNA methylation patterns are lacking. We, therefore, sought to define an international consensus for DNA methylation subgroups in JMML and develop classification methods for clinical implementation. Published DNA methylation data from 255 patients with JMML were used to develop and internally validate a classifier model. Accuracy across platforms (EPIC-arrays and MethylSeq) was tested using a technical validation cohort (32 patients). The suitability of both methods for single-patient classification was demonstrated using an independent cohort (47 patients). Analysis of pooled, published data established three DNA methylation subgroups as a de facto standard. Unfavorable prognostic parameters (PTPN11 mutation, elevated fetal hemoglobin, and older age) were significantly enriched in the high methylation (HM) subgroup. A classifier was then developed that predicted subgroups with 98% accuracy across different technological platforms. Applying the classifier to an independent validation cohort confirmed an association of HM with secondary mutations, high relapse incidence, and inferior overall survival (OS), while the low methylation subgroup was associated with a favorable disease course. Multivariable analysis established DNA methylation subgroups as the only significant factor predicting OS. This study provides an international consensus definition for DNA methylation subgroups in JMML. We developed and validated methods which will facilitate the design of risk-stratified clinical trials in JMML.

High Adenosine Deaminase Level Among Healthy Probands of Diamond Blackfan Anemia (DBA) Cosegregates With the DBA Gene Region on Chromosome 19q13

AbstractPhenotypic characterization of Diamond Blackfan Anemia (DBA) patients and their relatives was performed in 54 families. Complete blood count, fetal hemoglobin level, erythrocyte i antigen expression, and erythrocyte adenosine deaminase (eADA) activities were quantitated in patients and relatives. eADA was elevated in 28 of 34 transfusion-independent DBA patients, whereas persistence of erythrocyte i antigen was noticed in only 10 of 20 DBA patients. High eADA activities were also found in 14 of 149 healthy family members, allowing us to identify an isolated high eADA phenotype in these families. In contrast, increase in erythrocyte i antigen expression, elevated fetal hemoglobin levels, and macrocytosis were much less frequently noted in nonaffected members of the DBA families studied. Importantly, isolated high eADA phenotype was found to be significantly associated with genetic markers on chromosome 19 that segregate with the DBA phenotype. Isolated high eADA phenotype thus seems to reflect a silent phenotype of DBA in affected families. These findings suggest that elevated eADA activity in unaffected individuals needs to be taken into account during genetic assessment of DBA families and could be used for accurate assessment of mode of inheritance.

JMML Fetal Identity Results Either from Retention of a Physiologic Signature or Aberrant Activation of Master Oncofetal Regulators

Objectives: Juvenile myelomonocytic leukemia (JMML) is a rare but aggressive myeloproliferative/myelodysplastic neoplasm affecting infants and young children. The narrow age-window of onset suggests that a prenatal environment is needed for JMML oncogenesis. In search of a transcriptional reminiscence of embryo-fetal characteristics that would confirm this hypothesis, we investigated how the gene expression profile of JMML hematopoietic progenitors compared to their healthy counterpart isolated at different stages of ontogeny. Methods: Hematopoietic stem cell and progenitor cell (HSPC) fractions of JMML (n=16), bone marrow (BM) of healthy children (n=7), fetal liver (FL; n=3) and fetal BM (FBM; n=2) were phenotyped and sorted using signatures validated in the fetal and adult BM (Notta et al, Science 2011). RNAseq was performed using the TruSeq® Stranded Total RNASample preparation kit. Unsupervised hierarchical clustering analysis was done with the Bioconductor edgeR package. Differentially expressed genes were identified with the Bioconductor limma package. Results: To eliminate the impact of variations in the HSPC distribution, the JMML transcriptome was assessed on FACS-sorted common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP) and megakaryocyte-erythroid progenitor (MEP) cell fractions from 16 JMML and compared to healthy counterparts at different stages of ontogeny (FL, FBM, age-matched children BM). Unsupervised hierarchical clustering separated the samples into 4 groups (C1-4), primarily according to ontogeny, with 14/15 embryo-fetal fractions in C1 and all healthy post-natal progenitors in C2 (CMP, MEP) or C3 (GMP). Most JMML fractions clustered either with the prenatal fractions (C1; 17/47 fractions from 8/16 patients) or in a separate group containing no healthy sample (C4; 23/47 samples from 10/16 patients). Two groups were defined accordingly: one with JMML resembling embryo-fetal samples ('Fetal-JMML'; n=6/16), and a JMML-specific group ('Onco-JMML'; 8/16). Patients with Onco-JMML tended to be older, with a more severe presentation and elevated fetal hemoglobin levels. All PTPN11-mutated JMML were in this group whereas 5/6 Fetal-JMML had NRAS or KRAS mutations. Analysis of differential gene expression between Fetal and Onco-JMML highlighted 344 up-regulated genes versus 19 up-regulated genes in Onco-JMML. Surprisingly, LIN28B and WT1, both known to activate fetal pathways, were the most up-regulated genes in Onco-JMML. These key transcription factors were deregulated as early as the hematopoietic stem cell (HSC) compartment. Gene Set Enrichment Analysis (GSEA) confirmed enrichment in LIN28B and WT1-related signatures and showed enrichment in an AML signature in Onco-JMML. On the other hand, Fetal-JMML showed striking overexpression of monocytic /dendritic cell markers and inflammasome and innate immunity components. GSEA confirmed the strong monocyte identity of Fetal-JMML progenitors compared to onco-JMML or healthy postnatal progenitors. Part of the monocytic markers 'aberrantly' expressed in JMML progenitors was expressed in healthy fetal progenitors. Analysis of the HSC and multipotent progenitor (MPP) fractions showed that up regulation of monocytic markers was limited to the JMML progeny compartments. As we were able to confirm the transcriptional and functional identity of the sorted progenitors, these data suggest an early monocytic priming in these JMML progenitors, reminiscent of the monocyte-biased myelopoiesis characterizing physiologic fetal hematopoiesis. Conclusion: Our findings give a striking example of how ontogeny-related features are involved in childhood oncogenesis. They highlight a strong but complex link beween JMML and development, with a fetal identity resulting either from retention of a physiologic fetal monocytic signature or from aberrant (re)activation of master oncofetal regulators. Intriguingly, although LIN28B is thought to reprogram hematopoietic progenitors into a fetal-like state, its activation does not lead to an overall fetal profile in JMML, suggesting a regulatory mechanism distinct from that of physiological development. These two ontogeny-based signatures are likely to uncover the biology underlying previous classifiers based on AML-like profile or DNA methylation and suggest that a subset of JMML patient may benefit from immunomodulating therapies. Disclosures Dalle: Bellicum: Consultancy, Honoraria; bluebird bio: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi-Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Medac: Consultancy, Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; AbbVie Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Orchard: Consultancy, Honoraria; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees. Baruchel:Jazz Pharmaceuticals: Consultancy, Honoraria; Celgene Corporation: Consultancy, Honoraria; Astra Zeneca: Consultancy; Servier: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Shire: Research Funding; Bellicum: Consultancy.

Significance of heme oxygenase-1(HMOX1) gene on fetal hemoglobin induction in sickle cell anemia patients

Though the patients with sickle cell anemia (SCA) inherit same genetic mutation, they show considerable phenotypic heterogeneity. It has been observed that patients with elevated fetal hemoglobin (HbF) levels have a relatively mild clinical course. There is sparse literature on the association of higher HbF levels leading to reduction in the oxidative stress in SCA patients. Hence in this study, the significance between the HMOX1 gene polymorphisms and the HbF levels has been studied. Preliminary screening was carried out. Genotyping of 3 variants in the HMOX1 gene was performed in 90 SCA patients and 50 healthy controls by PCR–RFLP, GeneScan and direct DNA sequencing. It was observed that SCA patients with higher HbF levels, showed improved hematological indices with an inverse effect on HbS levels. The TT genotype of rs2071746 (A→T) polymorphism was found to be associated with elevated HbF levels (P: 0.012). Also, the long form (> 25 GT repeats) of rs3074372 (GT)n repeats was found to be linked with increased HbF levels. We could not find any association of rs2071749 (A→G) polymorphism with the HbF levels. As, the sickle cell anemia patients show significant oxidative stress due to hemolysis, the study of polymorphisms in the HMOX1 gene may act as a potential independent marker for elevated HbF levels.

Manufacturing Scale-up and Preclinical Development of ET-01, Autologous CD34+ Cells with the BCL11A Erythroid Enhancer Edited By CRISPR/Cas9, for Patients with β-Thalassemia Major

In China, it is estimated that there are over 50,000 patients with β-thalassemia major, of which the standard care requires regular red blood cell transfusions along with iron chelation, often associated with iron overload and organ damage. The only curative therapy, allogeneic hematopoietic stem cells (HSCs) transplantation, is primarily available to a fraction of young patients with HLA-matched sibling donors. A study has estimated that 1% of patients with β-thalassemia major in China expect to live beyond age 20, representing severe unmet needs. Recently, transplantation of genetically modified autologous HSCs is emerging as an attractive therapeutic alternative for β-thalassemia. Epidemiology data have shown that in some patients with β-thalassemia, manifested clinical symptoms could be ameliorated by elevated fetal hemoglobin (HbF). As BCL11A is one of the main transcription factors known to play critical roles to repress expression of γ-globin, a critical element of HbF, we are developing ET-01, in which autologous CD34+ cells are edited by CRISPR/Cas9 to disrupt the BCL11A erythroid enhancer and reactivate the developmentally silenced γ-globin, to treat patients with β-thalassemia. Our data have shown that in ET-01, the BCL11A enhancer can be edited with high precision and efficiency, resulting in HbF levels above a potential therapeutic threshold. We have developed and scaled up the manufacturing process under the cGMP standard. IND-enabling preclinical studies, including GLP studies using ET-01 manufactured at the clinical scale, are on-going. Overall our data support continuing development of ET-01 as a potentially safe and efficacious therapy for patients with β-thalassemia major To disrupt the BCL11A enhancer, Cas9 mRNA and synthetic sgRNA were delivered into CD34+ HSCs from healthy and β0/β0 donors using electroporation. Indels frequencies (i.e., gene editing efficiency) were approximately 80%, consistent for both types of donors. Efficiency of in vitro erythroid differentiation was essentially identical between ET-01 and unedited CD34+ HSCs. HbF+ cells and γ-globin mRNA levels were both significantly elevated in ET-01 compared to unedited CD34+ HSCs. Importantly, in ET-01 made from cells of β0/β0 donors, the level of γ-globin, plus that of β-globin, reached above a threshold with potentially clinically meaningful effect. Meanwhile, data from the colony-forming unit assay have shown colony types and total numbers of colonies were comparable between those of ET-01 and unedited CD34+ HSCs, suggesting that the colony-forming capability was not significantly affected in ET-01. To evaluate the specificity of gene-editing methodology used in ET-01, we generated a signature panel consisting of 42 sites with highest probability of off-target editing by in silico prediction and unbiased DiGenome-seq. These sites were deeply interrogated via targeted PCR amplification and next generation sequencing analysis. The data showed no significant off-target events at these sites, suggesting minimal off-target effects for ET-01. Moreover, to evaluate the in vivo engraftment potential of ET-01, we transplanted ET-01 and unedited CD34+ HSCs into immunodeficient NPG mice. Both types of cells produced similarly rapid and efficient engraftment in all mice, and similar multi-lineage reconstitution of human cells in multiple hematopoietic and immune organs. Importantly, the Indels efficiencies of ET-01 were maintained at similar levels in NPG mice after 4 months transplantation and after 2nd transplantation. These findings suggest that ET-01 retained in vivo long-term hematopoietic repopulating and differentiation capabilities. No tumorigenicity was observed in these in vivo studies. ET-01 manufacturing process has been successfully scaled up to 9.69x108 CD34+ HSC produced using mobilized leukopak from healthy donors. This upper bound is limited by the amount of starting materials obtained, not the actual process capacity. High cell viability, CD34+ purity and Indels frequencies were maintained throughout the manufacturing process and achieved in all batches. In vitro erythroid differentiation efficiency, HbF+ cells and γ-globin mRNA levels were similar to those observed at research scale. IND-enabling preclinical studies, including GLP studies using ET-01 manufactured at the clinical scale, are ongoing. Disclosures Fang: EdiGene Inc.: Employment. Yuan:EdiGene, Inc.: Employment. Yu:EdiGene, Inc.: Employment. Yang:EdiGene, Inc.: Employment. Liu:EdiGene Guangzhou Inc.: Employment. Shi:EdiGene Guangzhou Inc.: Employment. Zhang:EdiGene Inc.: Employment. Zhang:EdiGene Inc.: Employment. Zhao:EdiGene Inc.: Employment. Li:EdiGene Inc.: Employment. Wei:EdiGene, Inc.: Employment.

Durable and Robust Fetal Globin Induction without Anemia in Rhesus Monkeys Following Autologous Hematopoietic Stem Cell Transplant with BCL11A Erythroid Enhancer Editing

Elevated fetal hemoglobin (HbF, α2γ2) levels are clinically beneficial for patients with β-hemoglobinopathies. Editing of the erythroid-specific BCL11A enhancer induces HbF, inhibiting sickling and restoring globin chain balance in erythroid cells derived from hematopoietic stem and progenitor cells (HSPCs) from SCD and β-thalassemia patients respectively, without detectable genotoxicity or adverse effects on hematopoietic stem cell (HSC) function (Wu Y, Nat Med, 2019). Here, we sought to evaluate engraftment and HbF induction potential of erythroid-specific BCL11A enhancer edited CD34+ HSPCs in a non-human primate transplantation model in which hemoglobin switching is conserved. We targeted the erythroid-specific +58 DNAse I hypersensitive site of BCL11A, which has identical human and rhesus sequences at the spacer and protospacer adjacent motif (PAM) of the potent #1617 sgRNA. Ribonucleoprotein complex (RNP) composed of 3x-NLS SpCas9 protein and either BCL11A enhancer targeting (#1617) or AAVS1 targeting sgRNA was electroporated into rhesus CD34+ HSPCs (n=3). Following erythroid differentiation, substantial γ-globin expression (54-77%, p&lt;0.01) was observed in BCL11A edited cells (81-85% indels) as compared to 19-25% and 15-24% for non-electroporated and AAVS1 edited cells, respectively, with no significant difference in red blood cell (RBC) enucleation efficiency (44-47%) among groups. We tested BCL11A enhancer editing with autologous HSC transplant in two cohorts, with two macaques per cohort. For cohort 1, we performed competitive engraftment of BCL11A enhancer and AAVS1 edited HSPCs to test long-term reconstitution. For cohort 2, we evaluated BCL11A enhancer editing alone to evaluate HbF induction and hematopoietic reconstitution. For each cohort, purified CD34+ HSPCs were electroporated with RNP one day after G-CSF and plerixafor mobilization and cultured for two days prior to cryopreservation. HSPCs were thawed and infused following 2×5 Gy total body irradiation. For cohort 1 (n=2, ZL25 and ZL22, 1.34-1.39×106 CD34+ HSPCs/kg), we observed reduced indel frequencies (8-41%) at early post-infusion time points compared to cell products (18-49%), suggesting indels in unfractionated HSPCs may overestimate those in engrafting cells and/or hematopoietic ablation was incomplete. From weeks 6 to 83, stable indel frequencies were detected in both BCL11A (~3-18%) and AAVS1 (~10-45%), suggesting no selective advantage for BCL11A enhancer edited, AAVS1 edited, or non-edited HSCs. For cohort 2 (BCL11A enhancer editing alone (n=2, ZM17 and ZM26, 1.78-6.06×106 CD34+ cells/kg), cell products showed improved editing with ~95% indels and ~65-78% γ-globin protein after in vitro erythroid culture. Animals engrafted with typical kinetics and displayed stable indel ratios up to 28 weeks post-transplantation. A significant correlation was detected between γ-globin level and indel frequency comparing all 4 transplanted animals and unedited controls (R2=0.76, p&lt;0.01). In both edited and unedited animals γ-globin levels peaked in the first two months after transplantation and subsequently declined and plateaued. In ZM17 (~70% BCL11A enhancer indels at ~24 weeks), ~12% γ-globin was observed in peripheral blood (PB) at last measurement (compared to 0.5% γ-globin in RBC prior to transplant). In the same animal, editing ranged from 78-81% across all PB and bone marrow (BM) lineages (excluding CD3+ T-cells with 63% indels), including B-lymphoid, myeloid, erythroid, and HSPCs (in particular including 78% indels in CD71+ CD45- erythroblasts). Hemoglobin, hematocrit, and reticulocyte counts and peripheral smear appearance were all normal, suggesting no erythroid toxicity. Colony-forming ability of BM-derived mononuclear cells was similar in edited and control animals. In summary, we evaluated the clinical potential of autologous BCL11A erythroid enhancer editing in rhesus macaques. BCL11A enhancer edited HSCs can persist for at least 83 weeks post-transplant and provide therapeutic levels of HbF in peripheral RBCs without anemia or other apparent hematologic toxicity. Furthermore, these results emphasize input CD34+ HSPC dose and conditioning intensity as critical variables that influence gene editing following autologous HSCT. Overall, these findings support BCL11A erythroid enhancer genome editing as a promising strategy for therapeutic HbF induction. Disclosures Weiss: GlaxoSmithKline: Consultancy; Cellarity INC: Consultancy; Esperian: Consultancy; Beam Therapeutics: Consultancy; Rubius INC: Consultancy.

Fetal hemoglobin and F-cell variance in mobilized CD34+ cell-transplanted rhesus monkeys

Elevated fetal hemoglobin (HbF) is associated with reduced severity of sickle cell disease. Therefore, γ-globin protein levels and F-cell (HbF-positive red blood cell) percentages are used for estimation of clinical benefit. Here, we monitored transplantation-related changes in HbF and F-cell percentages for rhesus macaques (Macaca mulatta) following total body irradiation or busulfan conditioning prior to CD34+ cell transplantation. HbF protein expression peaked in the first 4-9 weeks posttransplant (0.99%-2.53%), and F-cells increased in the first 6-17 weeks posttransplant (8.7%-45.3%). HbF and F-cell ratios gradually decreased and stabilized to levels similar to those of control animals (1.96 ± 1.97% for F cells and 0.49 ± 0.19% γ-globin expression) 4-7 months post-transplant. These findings confirm and expand on previous reports of transient induction in HbF and F-cell percentages in rhesus macaques following CD34+ cell transplantation, an observation that must be taken into consideration when evaluating therapeutic strategies that aim to specifically elevate HbF expression, which are currently in clinical development.

PF352 TUNISIAN FANCONI ANEMIA REGISTRY (TFAR) REPORT, THE 2018 UPDATE

Background:Fanconi anemia (FA) is a genetically and phenotypically heterogeneous inherited disease. Many groups have established FA registers. In Tunisia, we created, in collaboration with the Tunisian Fanconi Anemia Study Group (TFASG), the Tunisian Fanconi Anemia Registry (TFAR), already initiated in 1998, by retro and prospective registration of FA. The first report was published in 2012.Aims:We present here the update of Tunisan Fanconi Anemia Registry on 2018.Methods:All hematology and pediatrics departments include their FA patients diagnosed between January 1983 and April 2018. The diagnosis was based on the following criteria: before 1998, a progressive bone marrow failure, the malfomative syndrome (IFAR score greater than 2), spontaneous chromosomal instability +/‐ family history of FA. After 1998, it is based on chromosomal instability on exposure to mitomycin C (MMC) greater than 70% of mitoses. We analyzed epidemiological, clinical, biological features, treatment and evolution of the 232 patients included until 2018.Results:232 patients belonging to 157 families were included. The annual incidence was 6 cases / year. The mean age at diagnosis was 10 years 6 months, the median was 9 years (extreme: prenatal diagnosis to 41 years). The Geographic distribution shows predominance in the south and center of Tunisia (38% each) compared to the north (24%). The circumstances of discovery were anemic syndrome in 63% of patients, bleeding syndrome in 37% of patients, acute leukemia in 2% cancer in 0.8% of patients, and family survey in 9.5% of patients. There was consanguinity in 80%. Malformative syndrome in 78%: stature ponderal retardation in 71%, « café au lait » spots in 72%, facial dysmorphy in 85%, bone malformations 53%, thumb column anomalies 37% and renal malformations 57%. Pancytopenia at diagnosis was found in 56%: Thrombocypenia is the most constant hematologic anomaly 96%, anemia in 80% and leuconeutropenia is found in 73%. Elevation of fetal hemoglobin is found in 91% of cases.Among 232 patients, 58 patients have been allo grafted from matched geno identical donors: 56 brodherhood, 1 fadher and 1 grand fadher 68% of patients with donor. Five patients without geno identical donor, have been allografted: from pheno identical donors in four cases in France and one from phenoidentical cord blood in Germany.Androgen treatment was given in 141 mainly Norethandrolone. 79% of those patients were good responders at 12 months. Evolution to a myelodysplastic syndrome is observed in 26 patients 11% and to acute leukemia in 26 patients 11%: 21 AML and 5 ALL. Seven cancers is diagnosed in 6 patients 2.5%. The median survival age in all patients is 14 years 3 months, it is significantly better in allo grafted patients (19 years vs 13 years 6 months, p &lt; 0.00001). All cases of acute leukemia and cancer have died.Summary/Conclusion:FA seems frequent in Tunisia, which is in part explained by the high consanguinity and endogamy in our country. It is often severe or moderate and requires androgen treatment or bone marrow transplantation. It must concern all patients with matched related donor. We have not access to cord blood bank or international file of volunteer bone marrow donors to do phenoidentical allograft. We project to introduce the haplo identical related donor allograft for FA patients. All patients with solid tumor or acute leukemia have died. We must do prevention and early detection of cancer.

Targeted deletion of BCL11A gene by CRISPR-Cas9 system for fetal hemoglobin reactivation: A promising approach for gene therapy of beta thalassemia disease

Hemoglobinopathies, such as β-thalassemia, and sickle cell disease (SCD) are caused by abnormal structure or reduced production of β-chains and affect millions of people worldwide. Hereditary persistence of fetal hemoglobin (HPFH) is a condition which is naturally occurring and characterized by a considerable elevation of fetal hemoglobin (HbF) in adult red blood cells. Individuals with compound heterozygous β-thalassemia or SCD and HPFH have milder clinical symptoms. So, HbF reactivation has long been sought as an approach to mitigate the clinical symptoms of β-thalassemia and SCD. Using CRISPR-Cas9 genome-editing strategy, we deleted a 200bp genomic region within the human erythroid-specific BCL11A (B-cell lymphoma/leukemia 11A) enhancer in KU-812, KG-1, and K562 cell lines. In our study, deletion of 200bp of BCL11A erythroid enhancer including GATAA motif leads to strong induction of γ-hemoglobin expression in K562 cells, but not in KU-812 and KG-1 cells. Altogether, our findings highlight the therapeutic potential of CRISPR-Cas9 as a precision genome editing tool for treating β-thalassemia. In addition, our data indicate that KU-812 and KG-1 cell lines are not good models for studying HbF reactivation through inactivation of BCL11A silencing pathway.

KLF1 Mutations Are Not Common in Israel but Can Explain Occasional Cases of Elevated HbA2 or Very Elevated Fetal Hemoglobin

Introduction: Mutations in KLF1 (Kruppel Like Factor1) have been noted to cause a number of different phenotypes of erythrocyte abnormalities, as KLF1 is known as a master regulator of many genes expressed in red blood cells. One important manifestation caused by KLF1 mutations is the upregulation of γ- or δ-globin genes, with associated microcytosis, thus mimicking beta thalassemia (β-thal) trait. Such findings cause difficulty in counseling couples referred for prenatal diagnosis of β-thal. KLF1 mutations have been reported to be more frequent in geographic regions where β-thal is common. Therefore we undertook to analyze for the presence of KLF1 mutations in Israeli individuals.Materials and Methods: We selected 100 individuals for analysis belonging to one of 4 different groups: 1. Individuals with isolated elevated HbA2 (n=14), or isolated elevation of HbF (n=13) or elevation of both HbA2 and HbF (n=6), who are not carriers of a β-thal mutation by sequence analysis. 2. Individuals with β-thal trait (n=19) who have a higher HbF (and/or HbA2) than is expected for β-thal trait, who do not have β-thal intermedia on clinical criteria (blood count, peripheral smear, spleen size). These individuals carried one of 8 mutations known to cause β-thal in our region 3. Two patients (pts) with a history of transfusions (one with massive splenomegaly and sickle trait, suspected to have coinherited CDA, and one with β-thal intermedia with HbF elevation unexpectedly high for his mutation (TATA box -28 A to C; HBB:c.-78A>C). This patient had HbF levels ranging from 23-33% and HbA2 ranging from 7.8-8.9% over years of followup from age 39-50 years. 4. Anonymous controls (n=46) who are pts with a hematological malignancy not suspected of carrying a hemoglobinopathy. KLF1 (exons 1, 2 and 3) was amplified using PCR (exon 1: 564 base pair product and exons 2 and 3: 1703 base pair product). PCR products were subjected to DNA sequence analysis using an Applied Biosystems ABI apparatus. The sequence obtained was analyzed using BLAST alignment and deviations from the published sequence were analyzed using the Mutation Taster program.Results: Two pts were found to carry substitutions with possible or proven clinical significance. One pt is heterozygous for a substitution at c.972C>A (codon 324 exon 3, E324D, aspartic acid to glutamine) which has not been previously reported. According to Mutation Taster this substitution may have clinical significance. This pt, who has normal hematological parameters, had isolated HbA2 elevation (4.1%) with no β-thal mutation identified. The second pt was found to be heterozygous for a substitution at c.901C>T (codon 301 exon 2, R301C, arginine to cysteine). This substitution has been reported by Gallienne et al, 2012, to be associated with elevated Hb F. This young pt was found to have 28% Hb F with a low HbA2 level of 1.4% and MCV of 63.8. His father also had 28% HbF. No β-thal mutation was identified in the pt or his father but the patient was found to be homozygous for a single alpha globin gene deletion. In addition to these 2 substitutions, many pts and controls carried known polymorphisms in KLF1. These polymorphisms are: c.304T>C, exon 2 residue 102 (S102P) (found in 24 pts and 19 controls); -148(G>A), in the upstream noncoding region (found in 11 pts and 8 controls); c.544T>C codon 182 exon 2 (F182L) (found in 3 pts and 4 controls); and c.115A>C, codon 39 in exon 2, M39L (found in 1 pt and 3 controls). One rare, previously unreported substitution was found in heterozygous form in the thal intermedia pt, located at c.259C>G codon 87 exon 2, P87A (proline to alanine). This was not found in the controls, however according to the location in the gene, and to Mutation Taster, this is not suspected to be a functional variant.Conclusions: Functional variants in KLF1 were rarely found in this group of patients. One out of 14 (7%) of individuals with isolated elevated HbA2 and 1/13 patients with isolated HbF elevation (7.7%) were identified as carrying heterozygous KLF1 functional variants. Two novel substitutions were found: E324D which is suspected to have clinical significance and P87A which is not presently suspected to be a functional variant. We conclude that KLF1 analysis may explain occasional individuals with high HbA2, or marked HbF elevation, in the absence of β-thal trait in our patient population. Larger studies are needed to confirm these findings. DisclosuresNo relevant conflicts of interest to declare.

Precision Medicine for Sickle Cell Disease through Whole Genome Sequencing

▪Although sickle cell disease (SCD) is a monogenic disorder, the severity and specific organ dysfunction and failure are strongly influenced by genetic modifiers. Rapid identification of all modifiers in patients and well-phenotyped cohorts will better define the impact of relevant variants on clinical status, inform disease biology, and identify new therapeutic strategies. We created the Sickle Genome Project (SGP), a whole genome sequencing (WGS) strategy, to define genomic variation and modifiers of SCD. We performed WGS on 871 African American SCD patients from St. Jude Children's Research Hospital who participated in the Sickle Cell Clinical Research and Intervention Program (SCCRIP, Hankins et al. Pediatr Blood Cancer, 2018) and Texas Children's Hospital Hematology Center (TCHC). We developed robust pipelines for accurate detection of single nucleotide polymorphisms (SNPs), identification of structural variants and data retrieval/sharing via the St. Jude Cloud platform (to be described elsewhere). Notable findings include:1) Confirmed associations of common genetic modifiers with SCD phenotypes, including levels of fetal hemoglobin (BCL11A, HBS1L-MYB, HBB), bilirubin (UGT1A1), and microalbuminuria (APOL1). Additional associations approaching genome-wide significance require further investigation, including replication in independent samples.2) Improved determination of the SCD modifier α-thalassemia. The most common α-thalassemia mutations in SCD are 3.7 kb or 4.2 kb deletions (-α3.7 and -α4.2 alleles), which arose from recombination between homologous HBA1 and HBA2 genes and are difficult to map using standard WGS reads. Three independent crossover events are described for -α3.7 and one for -α4.2 in SCD cohorts. We developed a novel approach to identify α-globin gene deletions by local de novo assembly of WGS data and coverage depth analysis. We identified 5 -α3.7 alleles (frequencies 0.77-32.12%) and 7 -α4.2 alleles (frequencies 0.19-5.77%). Collectively, the frequency of all -α alleles was 57%, reflecting at least 12 distinct recombination events, greatly exceeding previously published counts. These findings better define the evolution of α-globin genes to allow improved understanding of their regulation and influence on SCD.3) Characterization of β0-thalassemia alleles. Mutations in the extended β-globin locus influence SCD phenotypes. Five SGP patients had large β-globin (HBB) deletions associated with elevated fetal hemoglobin, which ameliorates symptoms of SCD. Twenty-three patients had HbSβ0-thalassemia, which reduces the severity of some SCD phenotypes. Overall, 48.6% (18/37) of patients clinically designated as HbSβ0 -thalassemia had no identified β-thalassemia mutation. Moreover, 4/680 patients (0.6%) designated HbSS were identified to be β0-thalassemia heterozygotes. The MCV, RBC and %HbA2 distributions overlapped substantially in correct vs. incorrect genotype assignments. Improved discrimination of HbSβ0 vs HbSS genotypes by WGS will better define associated phenotype differences to impact clinical care.4) Determination of a genetic variant linked to vaso-occlusive crisis (VOC). Previously, a single GWAS study linked rs3115229, located 63.7 kb 5′ upstream of the KIAA1109 gene, with VOC at borderline significance (P = 5.63 × 10−8) (Chaturvedi et al, Blood 130, 2017). Using WGS data for 327 SGP participants (HbSS or HbSβ0-thalassemia) enrolled in the SCCRIPP study, we found strong association (p = 7 x 10-5) between the onset of VOC and a 4-SNP diplotype within an adjacent LD block of the KIAA1109-TENR-IL2-IL21 region (chr4: 122.8Mb - 123.8Mb) which has been previously associated with numerous inflammatory disorders. We validated this association using imputed genome-wide array data in an independent group of SCD patients (Sleep and Asthma Cohort, n= 181 patients, p = 0.05) (Cohen et al, Ann Am Thorac Soc, 2016). This works provides confirmation that the region surrounding KIAA1109 is associated with pain crisis in SCD.Our studies provide new information on the genomic architecture of SCD patients and delineate a consolidated approach for future applications of precision medicine. [Display omitted] DisclosuresHankins:Novartis: Research Funding; Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy. Estepp:Global Blood Therapeutics: Consultancy, Research Funding; ASH Scholar: Research Funding; NHLBI: Research Funding; Daiichi Sankyo: Consultancy.

Elevated fetal haemoglobin levels are associated with decreased incidence of retinopathy in adults with sickle cell disease

Among the many vascular complications of sickle cell disease (SCD), retinopathy is the most prevalent and represents a leading cause of blindness. Hydroxycarbamide therapy ameliorates many symptoms of SCD, and high fetal haemoglobin (HbF) levels have been shown to protect against the development of retinopathy in children with HbSS. Its effect on adults with SCD, who are at a much higher risk of developing retinopathy, has not been studied. We aimed to investigate the effect of hydroxycarbamide use and HbF level on sickle cell retinopathy development in adults. We performed a retrospective cross-sectional study and collected demographics, comorbidities, and ocular and haematological data from 300 adult sickle cell subjects examined at the Henkind Eye Institute at Montefiore Medical Center during a 5-year period, from October 2012 to November 2017. The cohort was comprised mainly of Black and Hispanic subjects with all SCD genotypes, aged 18-71years. Results show that in HbSS patients treated with hydroxycarbamide, those with retinopathy had significantly lower HbF levels compared to patients without retinopathy (P=0·018). Our study identified the optimal HbF cut-off point for retinopathy protection to be 14·87%. Thus, a HbF level of 15% appears to be the threshold above which the odds for developing retinopathy in SS patients are reduced by 50%.