Hereditary Persistence Of Fetal Hemoglobin Research Articles

Molecular epidemiology and hematologic characterization of \u03b4\u03b2-thalassemia and hereditary persistence of fetal hemoglobin in 125,661 families of greater Guangzhou area, the metropolis of southern China

BackgroundIndividuals with δβ-thalassemia/HPFH and β-thalassemia usually present with intermedia or thalassemia major. No large-scale survey on HPFH/δβ-thalassemia in southern China has been reported to date. The purpose of this study was to examine the molecular epidemiology and hematologic characteristics of these disorders in Guangzhou, the largest city in Southern China, to offer advice for thalassemia screening programs and genetic counseling.MethodsA total of 125,661 couples participated in pregestational thalassemia screening. 654 subjects with fetal hemoglobin (HbF) level ≥ 5% were selected for further investigation. Gap-PCR combined with Multiplex ligation dependent probe amplification (MLPA) was used to screen for β-globin gene cluster deletions. Gene sequencing for the promoter region of HBG1 /HBG2 gene was performed for all those subjects.ResultsA total of 654 individuals had hemoglobin (HbF) levels≥5, and 0.12% of the couples were found to be heterozygous for HPFH/δβ-thalassemia, including Chinese Gγ (Aγδβ)0-thal, Southeast Asia HPFH (SEA-HPFH), Taiwanese deletion and Hb Lepore–Boston–Washington. The highest prevalence was observed in the Huadu district and the lowest in the Nansha district. Three cases were identified as carrying β-globin gene cluster deletions, which had not been previously reported. Two at-risk couples (0.0015%) were required to receive prenatal diagnosis. We also found 55cases of nondeletional-HPFH (nd-HPFH), including 54 with Italian nd-HPFH and one with the Aγ-197C-T heterozygous state. It is difficult to discriminate between Chinese Gγ (Aγδβ)0-thal and Italian nd-HPFH carriers using hemoglobin (Hb) analysis.ConclusionsThis study is the first to describe the familial prevalence of HPFH/δβ-thalassemia and the high-risk rate in Greater Guangzhou Area, and the findings will support the implementation of thalassemia screening for three common deletions by gap-PCR. We also presented a systematic description of genotype-phenotype relationships which will be useful for genetic counseling and prenatal diagnostic services for β-thalassemia intermedia.

Application Value of Capillary Electrophoresis in Screeningβ-Thalassemia of Children

to explore the value of capillary electrophoresis in screening β- thalassemia of children, and to establish the cutoff values of HbA2 and HbF in our laboratory. The data of hemoglobin capillary electrophoresis and genetic diagnosis of β- thalassemia from 886 examined children were retrospectively analyzed. The cutoff values of HbA2 and HbF were determined by ROC curve. The cutoff value of HbA2 screening minor β- thalassemia was 3.65%, the specificity was 0.996, and the sensitivity was 0.995. The cut-off value of HbF for screening minor β- thalassemia was 1.45%, specificity was 0.751 and sensitivity was 0.675. Thus, 1 case with codon5 (CCT→C) mutation, 1 case with SEA -HPFH β deletion, 1 case with - 28 (A→G) merger IVS-Ι-128 (T→G) double heterozygous mutations yet were found out, 1 case with 47 bp β gene missing has not yet been reported in literature. Capillary electrophoresis has more high sensitivity and specificity in the screening of β- thalassemia in children, especially for the detection of rare β- thalassemia.

High Hemoglobin F in Sickle Cell Disease: Waning Protection with Age

Background: Sickle cell disease (SCD) is a monogenetic inherited red cell disorder with pleomorphic clinical manifestations. Hemoglobin F (HbF) concentration is the major genetic modifier of clinical expression and levels between 10% and 20% have been found to improve survival and decrease vaso-occlusive complications (VOCs). As survival for patients with SCD has increased dramatically in the past 20 years, we hypothesized that SCD-related complications may increase with age, even in "high F" individuals. Recent data suggest that individuals with the Arab-Indian haplotype and/or HbS-δβ thalassemia, who have HbF of 15% to 25%, still suffer from VOCs. SCD patients with HBS and deletional hereditary persistence of fetal hemoglobin (HPFH) have high levels of HbF (>30%) in a pancellular distribution, and absence of symptoms as suggested by prior studies. Here, we report on a cohort of patients with HbSS/Sβ0, with HbF >14%, either hydroxyurea naïve or treated for ≤ 6 months. Methods: We report on a cohort of 11 adults with SCD and HbF levels > 14%, who had never been on hydroxyurea, or were treated for ≤6 months. Charts were reviewed for VOCs, opioid therapy, acute chest syndrome (ACS), avascular necrosis (AVN), retinopathy, pneumonia, leg ulcers, splenomegaly/infarction, stroke, pulmonary hypertension, cardiomyopathy/infarction, chronic kidney disease, priapism and miscarriage. All events were confirmed by supportive laboratory or imaging data. HbF levels for each patient are represented as lifetime average values. In 10/11 patients, cellular distribution of HbF by flow cytometry (CytoFlex, Beckman Coulter, CA) was performed following fixation, permeabilization, and incubation of red blood cells with a monoclonal antibody against Hb-F, directly conjugated with FITC (Cat # MHFH01, Life Technologies, MD). Beta globin dosage genetic analysis was performed on a subset of patients (8/11) which used multiplex-polymerase chain amplification of fragments specific for portions of the epsilon, gammaG, gammaA, delta, and beta globin genes, which is commonly associated with deletional variant of HPFH (Quest Diagnostics, VA). Results: Our cohort consisted of 11 patients, 3 males, age range 23 -72 years, one of which (age 72) is deceased. Genotypes included 10 HbSS, four with alpha thalassemia trait, and one HbS/β0. Mean HbF levels ranged from 14 % to 29.50%, SD ± 4.76. Table 1 shows patients ordered in ascending age with clinical complications for each patient. Notably, all patients experienced VOCs regardless of age or HbF level, 91% were prescribed opioids, 46% of whom were on chronic opioid therapy, 40 % had leg ulcers 36% had AVN and 27% had ACS . The number of complications per patient increased with age, despite maintenance of high HbF. Available flow cytometry and genetic data revealed heterocellular distribution of HbF in 8 patients, and pancellular distribution in 2 patients: patient (#2) and patient (#10). Patient (#2) is positive for deletional HPFH with genetic confirmation of heterozygous positive deletion in the beta globin gene cluster. Genetic data is not available for patient (#10) at this time. Interestingly, both patients still exhibited VOCs, complicated by yearly hospitalizations, required intermittent opioid therapy and with age, developed AVN. Laboratory data are notable for minimal levels of hemolysis. Discussion: Most individuals with SCD living in high resources countries now reach adulthood. Therefore the disease profile that was previously studied and reported mostly in the pediatric population is evolving. High levels of HbF appear to delay the onset of complications, but do not completely prevent them, perhaps due to a heterocellular distribution of HbF or non-deletional HPFH. In our cohort, two patients with confirmed pancellular distribution of HbF, one of which with confirmed deletional HPFH, were not fully protected from the clinical complications of SCD, suggesting alternative pathogenic mechanisms for VOCs may be at play. Therefore, individuals with HbF concentrations of as high as ~30 % may still suffer from pain, hospitalizations for VOC's, and vasculopathic changes, such as leg ulcers. The number of complications increases with age despite maintenance of high HbF. Therefore, additional therapy and close vigilance is necessary when treating sickle cell patients with high levels of HbF, especially with increasing age. Disclosures Reyes Gil: Albert Einstein College of Medicine: Patents & Royalties: Patent application pending for NETs AI software. Ogu:Vertex Pharmaceuticals: Consultancy. Minniti:Doris Duke Foundation: Research Funding.

CRISPR-Cas9 Genome Editing of γ-Globin Promoters in Human Hematopoietic Stem Cells to Induce Erythrocyte Fetal Hemoglobin for Treatment of β-Hemoglobinopathies

Induction of fetal hemoglobin (HbF, α2γ2) via genome editing-mediated disruption of DNA regulatory elements that repress expression of γ-globin genes (HBG1 and HBG2) is a promising therapeutic strategy for b-hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia. Optimal technical approaches and safety profiles are yet to be fully defined. We used CRISPR/Cas9 to target a DNA repressor element near the distal CCAAT box of the HBG1/HBG2 promoters. This region contains a "TGACC" motif recognized by BCL11A, a transcriptional repressor protein that regulates γ-to-β globin switch after birth. Rare germline variants at or near this motif are associated with hereditary persistence of fetal hemoglobin, a benign genetic condition that alleviates the clinical manifestations of co-inherited b-hemoglobinopathies. Previously, we showed that transduction of human CD34+ cells with lentiviral vector encoding Cas9 and guideRNA (gRNA) targeting the HBG1/HBG2 promoter caused induction of HbF in red blood cell (RBC) progeny generated in vitro (Traxler et. al, Nature Medicine v22,2016). Here we present a clinically tractable approach for disrupting the HBG1/HBG2 BCL11A binding site in human hematopoietic stem cells (HSCs). Electroporation of Cas9:gRNA ribonucleoprotein (RNP) complex into healthy or SCD donor CD34+ cells resulted in up to 80% on-target insertion-deletion (indel) mutations and 35% HbF in erythroid progeny generated in vitro. Sixteen to 17 weeks after transplantation of gene edited CD34+ cells into immunodeficient NBSGW mice, up to 75% of donor CD34+ cells in recipient bone marrow contained on-target indels, demonstrating efficient modification of repopulating human HSCs. No differences in CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages were observed between edited and control cells. Moreover, up to 78% of gene edited erythroid cells stained with anti-HbF antibody ("F-cells") compared to 15% in control erythroid cells, suggesting a "pan-cellular" pattern of HbF expression after editing. Strikingly, human donor-derived erythroid cells in recipient bone marrow expressed up to 40% HbF compared to 3% HbF in controls. Although the editing frequencies of HBG1 and HBG2 promoters varied between different donor CD34+ cells, an engineered variant of Cas9 containing 3 nuclear localization sequences (Wu et. al,Nature Medicine v25, 2019) edited repopulating HSCs more efficiently and consistently than conventional Cas9 with two nuclear localization signals. Simultaneous on-target RNP-induced DSBs at both HBG1 and HBG2 can result in the deletion of the intervening 4.9-kb region, leaving a single hybrid gene with HBG2 promoter sequences fused to the downstream HBG1 gene. We detected this deletion in approximately 30% of edited cells, with no associated decline in HbF expression determined by clonal analysis of erythroid colonies. No off-target mutations were detected by targeted sequencing of the 26 top candidate sites identified by CIRCLE-seq, an in vitro genome-scale method for detecting Cas9 activity. Analysis of gene edited human donor cells purified from mouse bone marrow showed no chromosomal rearrangements by G-banding (n=20) or fluorescence in situ hybridization with a probe located distal to the HBG1/HBG2 loci (n=225). Taken together, our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a CRISPR-Cas9 genome editing approach to induce HbF for treating hemoglobinopathies. Figure. Gene editing of the HBG1/HBG2 promoters in HSCs and HbF induction of erythroid progeny in vivo. Plerixafor-mobilized CD34+ cells from an individual with SCD were edited with RNP and transplanted into NBSGW mice, which were analyzed 16-17 weeks later. A. On-target indel frequency before (Pre) and after bone marrow transplantation (BM). The black bars represent a 13-nucleotide deletion associated with human hereditary persistence of fetal hemoglobin. B. Human erythroblasts and reticulocytes derived from RNP-edited and non-edited Control CD34+ donor cells. Scale bar = 10 mm. C. HbF immunostaining control and RNP edited erythroid cells in recipient bone marrow assessed by flow cytometry. D. %HbF protein in hemolysates of control (C) and RNP edited erythroid cells assessed by ion-exchange HPLC. n= 3 biological replicates. **** P < 0.0001. Figure Disclosures Metais: MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Sharma:Doris Duke Foundation: Research Funding; Vertex Pharmaceuticals: Other: Study PI. Weiss:Beam Therapeutics: Consultancy; Rubius INC: Consultancy; GlaxoSmithKline: Consultancy; Cellarity INC: Consultancy; Esperian: Consultancy.

EDIT-301: An Experimental Autologous Cell Therapy Comprising Cas12a-RNP Modified mPB-CD34+ Cells for the Potential Treatment of SCD

Sickle cell disease (SCD) is an inherited blood disorder affecting approximately 100,000 individuals in the United States. Fetal hemoglobin (HbF) is a major modifier of SCD severity. Studies showed that individuals with compound heterozygosity for sickle hemoglobin (HbS) and hereditary persistence of fetal hemoglobin (HPFH) that expressed approximately 30% HbF did not show features of SCD. Therefore, we are developing EDIT-301, an experimental autologous cell therapy comprising CD34+ cells genetically modified using a Cas12a RNP (ribonucleoprotein) to promote HbF expression to treat SCD. Several HPFH mutations have been reported at the HBG locus. In particular, disruption of the distal CCAAT-box region of the HBG1/2 promoters was associated with elevated levels of HbF, suggesting that this region is a relevant genome editing target for the treatment of SCD. Genotype to phenotype analysis at the distal CCAAT-box region of the HBG1/2 promoters identified the mutations leading to elevated HbF expression. Indels disrupting more than 3 nucleotides were generally associated with elevated HbF expression while smaller indels had lower to no impact. We evaluated editing at this site using several RNP configurations, based on either SpCas9 or Cas12a (also known as Cpf1). Cas12a RNP resulted in larger deletions and a higher frequency of productive indels than SpCas9 RNP. Furthermore, productive indels generated with SpCas9 RNP relied predominantly on microhomology mediated end joining (MMEJ) mechanism. As the MMEJ repair mechanism is not frequently used by hematopoietic stem cells (HSC), productive editing may be low in vivo. In contrast, Cas12a RNP generated more productive indels at the HBG1/2 promoters target site irrespective of the DNA repair mechanism. We therefore postulated that editing at the distal CCAAT-box region with Cas12a would better support long-term persistence of productive indels and robust HbF expression. Consistent with this hypothesis, 80-90% editing was observed after electroporation of mobilized peripheral blood CD34+ cells (mPB-CD34+ cells) from healthy donors with a Cas12a RNP targeting the HBG1/2 promoters, resulting in approximately 40% of HbF expression in their erythroid progeny. Infusion of the modified CD34+ cells into NBSGW mice resulted in long-term multi-lineage reconstitution at 16 weeks post-infusion, with no reduction in editing levels compared to those at the time of infusion. A diverse on-target editing profile was observed in all animals, indicative of a polyclonal engraftment. Erythroid cells purified from the bone marrow of animals that received Cas12a-RNP treated cells demonstrated robust HbF expression averaging 40-50% compared to ~5% observed in the vehicle-treated control group. Off-target activity of the Cas12a RNP was also evaluated. A set of candidate off-target sites was first determined using orthogonal methods, including: in-silico prediction, Digenome-Seq and GUIDE-Seq. Each candidate site was then analyzed for editing by targeted PCR-NGS in electroporated CD34+ cells. No off-target editing was verified, demonstrating the specificity of this Cas12a RNP. Taken together, we identified a specific Cas12a RNP that efficiently edited the distal CCAAT-box region at the HBG1/2 promoters in CD34+ cells. These edited CD34+ cells led to long-term polyclonal multilineage engraftment and therapeutically meaningful levels of 40-50% HbF in vivo. Based on these results, IND-enabling activities have been initiated for EDIT-301: an experimental autologous cell therapy comprising Cas12a-RNP modified mPB-CD34+ cells for the potential treatment of SCD. Disclosures De Dreuzy: Editas Medicine Inc.: Employment, Equity Ownership. Heath:Editas Medicine Inc.: Employment, Equity Ownership. Zuris:Editas Medicine Inc.: Employment, Equity Ownership. Sousa:Editas Medicine Inc.: Employment, Research Funding. Viswanathan:Editas Medicine Inc.: Employment, Equity Ownership. Scott:Editas Medicine Inc.: Employment, Equity Ownership. Da Silva:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Capehart:Editas Medicine Inc.: Equity Ownership. Wang:Editas Medicine Inc.: Employment, Equity Ownership. Fernandez:Editas Medicine Inc.: Employment, Equity Ownership. Myer:Editas Medicine Inc.: Employment, Equity Ownership. Albright:Editas Medicine Inc.: Employment, Equity Ownership. Wilson:Editas Medicine Inc.: Employment, Equity Ownership. Teixeira:Editas Medicine Inc.: Employment, Equity Ownership. Chang:Editas Medicine Inc.: Employment, Equity Ownership.

Complementary Base Editing Approaches for the Treatment of Sickle Cell Disease and Beta Thalassemia

Sickle cell disease (SCD) and Beta thalassemia are disorders of beta globin production and function that lead to severe anemia and significant disease complications across a multitude of organ systems. Autologous transplantation of hematopoietic stem cells engineered through the upregulation of fetal hemoglobin (HbF) or correction of the beta globin gene have the potential to reduce disease burden in patients with beta hemoglobinopathies. Base editing is a recently developed technology that enables precise modification of the genome without the introduction of double strand DNA breaks. Gamma globin gene promoters were comprehensively screened with cytosine and adenine base editors (ABE) for the identification of alterations that would derepress HbF. Three regions were identified that significantly upregulated HbF, and the most effective nucleotide residue conversions are supported by natural variation seen in patients with hereditary persistence of fetal hemoglobin (HPFH). ABEs have been developed that significantly increase the level of HbF following nucleotide conversion at key regulatory motifs within the HBG1 and HBG2 promoters. CD34+ hematopoietic stem and progenitor cells (HSPC) were purified at clinical scale and edited using a process designed to preserve self-renewal capacity. Editing at two independent sites with different ABEs reached 94 percent and resulted in up to 63 percent gamma globin by UPLC. The levels of HbF observed should afford protection to the majority of SCD and Beta thalassemia patients based on clinical observations of HPFH and non-interventional therapy that links higher HbF dosage with milder disease (Ngo et al, 2011 Brit J Hem; Musallam et al, 2012 Blood). Directly correcting the Glu6Val mutation of SCD has been a recent goal of genetic therapies designed for the SCD population. Current base editing technology cannot yet convert mutations like those that result from the A-T transversion in sickle beta globin; however, ABE variants have been designed to recognize and edit the opposite stranded adenine residue of valine. This results in the conversion of valine to alanine and the production of a naturally occurring variant known as Hb G-Makassar. Beta globin with alanine at this position does not contribute to polymer formation, and patients with Hb G-Makassar present with normal hematological parameters and red blood cell morphology. SCD patient fibroblasts edited with these ABE variants achieve up to 70 percent conversion of the target adenine. CD34 cells from healthy donors were then edited with a lead ABE variant, targeting a synonymous mutation in an adjacent proline that resides within the editing window and serves as a proxy for editing the SCD mutation. The average editing frequency was 40 percent. Donor myeloid chimerism documented at these levels in the allogeneic transplant setting exceeds the 20 percent that is required for reversing the sickle phenotype (Fitzhugh et al, 2017 Blood). These next generation editing approaches provide a promising new modality for treating patients with Beta thalassemia and SCD. Disclosures No relevant conflicts of interest to declare.

Combining HPFH Mutations in Human Adult HSCs to Enhance Reactivation of Fetal Hemoglobin

Despite the significant advances in developing novel therapies for hemoglobinopathies, developing a universal and robust therapeutic approach, able to achieve an event-free result in all β0/β0 thalassemic and sickle cell patients is a work in progress. It is known that increase of Fetal hemoglobin (HbF) can lead to an ameliorated clinical picture up to transfusion independency. Inactivation via genome editing of γ-globin suppressors or introduction of Hereditary Persistence of Fetal Hemoglobin (HPFH) mutations in the promoter of hemoglobin gamma (HBG) have been shown to significantly increase the endogenous HbF expression. Here we studied the effect in HbF reactivation by targeting the binding site of the two main HbF silencers, BCL11a and LRF and how it compares to the well-established editing of the BCL11a erythroid enhancer. Furthermore, in order to achieve higher levels of fetal hemoglobin, we assayed the effect of all combinations of these cis and trans acting mutations. We first observed that all single knock outs had similar effects in HbF reactivation. Interestingly, by targeting both sites in the gamma globin promoter we observed a decrease in HbF induction compared to each single edit, suggesting a possible binding of a gamma globin activator in the in-between region. However, editing of the BCL11a erythroid enhancer and either of the two silencer binding sites yielded maximum levels of fetal hemoglobin, with pancellular HbF expression, both in vitro and in vivo following xeno-transplantation in NSGW mice. Collectively, our data suggest that the combination of two fetal globin reactivation mutations, one cis and one trans, have the potential to significantly increase HbF both totally and on per cell basis. This strategy has the potential to induce higher levels of HbF reactivation with a clinical benefit in patients with beta globin disorders. Disclosures No relevant conflicts of interest to declare.

Editing a γ-Globin Repressor Binding Site Restores Fetal Hemoglobin Synthesis and Corrects the Phenotype of Sickle Cell Disease Erythrocytes

Sickle cell disease (SCD) is a severe, life-threatening disorder caused by a single amino acid change (β6Glu→Val) in the adult hemoglobin (Hb) β-chain that causes Hb polymerization with consequent red blood cell (RBC) rigidity, anemia, vaso-occlusive crises, organ damage and reduced life expectancy. The co-inheritance of genetic mutations causing a sustained fetal γ-globin chain production in adult life (hereditary persistence of fetal hemoglobin, HPFH) decreases sickling and significantly reduces the clinical severity of SCD. HPFH mutations cluster at several loci in the promoters of the two γ-globin genes, HBG1 and HBG2, and, disrupt binding sites for transcriptional repressors (e.g., BCL11A and LRF), leading to elevated levels of fetal hemoglobin (HbF) representing up to 40% of total hemoglobin tetramers. In addition, SNPs at position -158 of both γ-globin promoters are associated with enhanced γ-globin expression and may identify a putative transcriptional repressor binding site. Here, we used the CRISPR/Cas9 system to mimic HPFH mutations in the HBG promoters by generating insertion and deletions (InDels) leading to disruption of known and putative binding sites for transcriptional repressors via non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) DNA repair mechanisms. Efficient editing of the LRF binding site (≥ 3 γ-globin promoters/cell in >70% of cells) in SCD patient-derived hematopoietic stem/progenitor cells (HSPCs) resulted in a robust, virtually pancellular HbF reactivation and a concomitant reduction in βS-globin levels, recapitulating the phenotype of asymptomatic SCD-HPFH patients. Of note, in RBCs derived from edited HSPCs, HbF levels exceeded 40% of total Hb tetramers, suggesting that CRISPR/Cas9 mediated disruption of the LRF binding site is even more potent than naturally occurring HPFH point mutations in reactivating HbF expression. RBCs derived from edited HSPCs displayed HbF levels sufficient to substantially correct the SCD cell phenotype (~80% of non-sickling RBCs). Although larger deletions (in part generated likely by MMEJ) disrupt more efficiently the LRF binding site, shorter InDels mainly produced by NHEJ, the most active repair mechanism in bona fide hematopoietic stem cells (HSCs), were still able to induce a potent γ-globin reactivation and correct the SCD cell phenotype. Interestingly, upon targeting of the -158 site, we observed HBG de-repression, which, however, was mild, indicating that this region contains a sequence that moderately inhibits γ-globin expression in adult cells, consistently with the mild increase in HbF associated with the -158 SNP. To evaluate the efficiency and the safety of this approach in long-term repopulating HSCs, untreated and edited HSPCs were injected into NSG immunodeficient mice. 16 weeks after transplantation, the engraftment of control and HBG-edited cells was comparable, as analyzed in bone marrow, spleen, thymus and blood. Edited HSCs were capable to differentiate into multiple hematopoietic cell types, with no skewing towards any particular lineage. Importantly, xenotransplantation of HSPCs edited using the gRNAs targeting the LRF binding site demonstrated a high editing efficiency in long-term repopulating HSCs (up to 75%) and a modest reduction in the frequency of deletions likely generated by MMEJ. Overall, this study identifies the binding site for the LRF repressor as a novel, potent and safe target for a genome editing-based treatment of SCD. Disclosures El Nemer: Hemanext: Other: Other. Cavazzana:SmartImmune: Other: Founder.

Identification of Novel Variants Associated with Fetal Hemoglobin Levels in Healthy Donors (the INTERVAL study)

Sickle cell disease (SCD) affects more than five million people worldwide, predominantly in sub-Saharan Africa. Hereditary persistence of fetal hemoglobin (HbF) is an uncommon genetic condition in which production of HbF in early life is not suppressed. SCD symptoms are reduced in patients carrying this condition, suggesting that increased HbF levels may be a promising therapeutic strategy to ameliorate the symptoms of SCD. This is exemplified by the HbF-raising compound hydroxyurea, currently the most commonly used US Food and Drug Administration approved drug treatment for SCD. However, hydroxyurea is only effective in ~70% of patients and carries a black box warning for carcinogenicity, hence there is a need for additional SCD treatments. Previous genome-wide association studies (GWAS) have identified four loci robustly associated with HbF levels, including variants in the BCL11A region. These initial genetic discoveries have led to promising ex vivo gene-editing approaches to silence or reduce levels of BCL11A, which are currently being tested in clinical trials. To identify novel potential therapeutic targets to raise HbF levels, we conducted the largest GWAS of HbF levels in ~11,000 healthy blood donors from the INTERVAL study among whom HbF was measured in whole blood using a mass spectrometry approach. We ran linear mixed models accounting for age, sex, blood group, technical effects and 10 principal components of ancestry for 14,910,742 variants either directly genotyped using the Affymetrix UK Biobank array or imputed from a combined 1000 Genomes/UK10K reference panel. In addition to confirming previously reported signals at the BCL11A, HBS1L-MYB and HBB loci, stepwise conditional analysis identified six novel genomic regions at genome-wide significance (p<5x10-8). At some loci we identified multiple independent association signals, including two at BCL11A, three at HBS1L-MYB, and two at HBB. Genetic fine-mapping resolved some loci to highly likely causal variants (posterior probability>0.5), including a rare (frequency=0.2%) variant near GRIK2. To identify likely causal genes and mechanisms, we integrated our results with relevant transcriptomic and epigenomic datasets. Preliminary results suggest that a common (frequency=26%) 29 base-pair indel upstream of CHAC2 is highly likely (posterior probability=0.77) to be the causal variant at this locus. The variant maps to a site of active chromatin and GATA1 transcription factor binding specifically in erythroblasts, and the insertion allele contains a GATA motif suggesting that this variant regulates HbF levels by the presence or absence of a second GATA1 binding site. Gene expression data across a range of hematopoietic cells revealed a restricted expression pattern of CHAC2 in erythroblasts, but not for other genes in the region, suggesting CHAC2 as the likely causal gene. CHAC2 encodes the glutathione-specific gamma-glutamylcyclotransferase 2, an enzyme that catalyzes the cleavage of glutathione into 5-oxo-L-proline and a Cys-Gly dipeptide. This finding adds support to the idea that altered erythrocyte glutathione levels play a role in SCD pathogenesis, potentially via HbF modulation. Ongoing work includes characterizing the likely causal mechanisms of the six novel loci, and providing target validation of CHAC2 using genome editing and deep phenotyping of erythroid cells. In summary, our expanded GWAS has identified new loci associated with HbF levels, providing novel potential therapeutic targets for SCD. Disclosures Lessard: Sanofi: Employment. Peters:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment.

Recent Progress in Gene Therapy and Other Targeted Therapeutic Approaches for Beta Thalassemia.

Beta-thalassemia is a genetic disorder characterized by the impaired synthesis of the betaglobin chain of adult hemoglobin. The disorder has a complex pathophysiology that affects multiple organ systems. The main complications of beta thalassemia are ineffective erythropoiesis, chronic hemolytic anemia and hemosiderosis-induced organ dysfunction. Regular blood transfusions are the main therapy for beta thalassemia major; however, this treatment can cause cardiac and hepatic hemosiderosis - the most common cause of death in these patients. This review focuses on unique future therapeutic interventions for thalassemia that reverse splenomegaly, reduce transfusion frequency, decrease iron toxicity in organs, and correct chronic anemia. The targeted effective protocols include hemoglobin fetal inducers, ineffective erythropoiesis correctors, antioxidants, vitamins, and natural products. Resveratrol is a new herbal therapeutic approach which serves as fetal Hb inducer in beta thalassemia. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for beta thalassemia major and is preferred over iron chelation and blood transfusion for ensuring long life in these patients. Meanwhile, several molecular therapies, such as ActRIIB/IgG1 Fc recombinant protein, have emerged to address complications of beta thalassemia or the adverse effects of current drugs. Regarding gene correction strategies, a phase III trial called HGB-207 (Northstar-2; NCT02906202) is evaluating the efficacy and safety of autologous cell transplantation with LentiGlobin. Advanced gene-editing approaches aim to cut DNA at a targeted site and convert HbF to HbA during infancy, such as the suppression of BCL11A (B cell lymphoma 11A), HPFH (hereditary persistence of fetal hemoglobin) and zinc-finger nucleases. Gene therapy is progressing rapidly, with multiple clinical trials being conducted in many countries and the promise of commercial products to be available in the near future.

Construction and Analysis of a Long Non-Coding RNA (lncRNA)-Associated ceRNA Network in β-Thalassemia and Hereditary Persistence of Fetal Hemoglobin.

BackgroundHigher fetal hemoglobin (HbF) levels can ameliorate the clinical severity of β-thalassemia. The use of integrative strategies to combine results from gene microarray expression profiling, experimental evidence, and bioinformatics helps reveal functional long noncoding RNAs (lncRNAs) in β-thalassemia and HbF induction.Material/MethodsIn a previous study, a microarray profiling was performed of 7 individuals with high HbF levels and 7 normal individuals. Thirteen paired samples were used for validation. lncRNA NR_001589 and uc002fcj.1 were chosen for further research. The quantitative reverse transcription-PCR was used to detect the expression levels of 2 lncRNAs. The Spearman correlation test was employed. The nuclear and cytoplasmic distribution experiment in K562 cells was used to verify the subcellular localization of 2 lncRNAs. Potential relationships among lncRNAs, predicted microRNAs (miRNAs), and target gene HBG1/2 were based on competitive endogenous RNA theory and bioinformatics analysis.ResultsAverage expression levels of NR_001589 and uc002fcj.1 were significantly higher in the high-HbF group than in the control group. A positive correlation existed between NR_001589, uc002fcj.1, and HbF. The expression of NR_001589 was in both the cytoplasm and the nucleus, mostly (77%) in the cytoplasm. The expression of uc002fcj.1 was in both the cytoplasm and the nucleus; the cytoplasmic proportion was 43% of the total amount. A triple lncRNA-miRNA-mRNA network was established.ConclusionsNovel candidate genetic factors associated with the HBG1/2 expression were identified. Further functional investigation of NR_001589 and uc002fcj.1 can help deepen the understanding of molecular mechanisms in β-thalassemia.

Hemoglobin Phenotypes in Nigeria: Data From a National Reference Laboratory

Abstract On a global scale, 5% to 7% of the population carries an abnormal hemoglobin. With a sickle hemoglobin (HbS) carrier prevalence of 25% to 40%, Nigeria bears the greatest burden of sickle cell disorder worldwide. Until recent times, detection of other clinically significant hemoglobin variants associated with HbS has been unavailable, resulting in missed/wrong diagnosis, weak national data, and suboptimal control of hemoglobin disorders. This study aimed to review hemoglobin assays over a 2-year period at a national reference laboratory in Nigeria, in order to describe the prevalent hemoglobin phenotypes for health planning. The study was a retrospective, laboratory data-based survey. Sociodemographics and hemoglobin assay results of 6,851 specimens between January 2016 and December 2017 were analyzed. Study protocol was approved by the Institutional Review Board. Participants aged less than 1 year and who had recent transfusions and a history of hydroxyurea therapy were excluded. At the hemoglobin laboratory, specimens were analyzed using cation exchange high-performance liquid chromatography (CE-HPLC) (Bio Rad D10, California). When indicated, further analysis of suspected abnormal hemoglobins was confirmed using a second method, capillary electrophoresis (SEBIA Capiflex II, France). The distribution of the identified hemoglobin phenotypes was determined as a proportion of the total number of participants. The mean (SD) age of the participants was 31.8 (14.9) years. The most frequent hemoglobin phenotypes were AA (59.4%), AS (23.3%), and SS (13.3%). Hemoglobin phenotypes AC (2.1%), SC (0.9%), AGPhiladelphia (0.2%), and CC (0.2%) were less frequent. Least occurring phenotypes were ADIbadan, AE, AGPhiladelphia, AOArab, DD, and hereditary persistence of fetal hemoglobin (HPFH). In the Nigerian context, accurate diagnosis of hemoglobin variants using quantitative, high-resolution hemoglobin assays, compared to zone (cellulose acetate) electrophoresis at alkaline pH will provide data for health planning, better access to genetic counseling, informed reproductive health choices, and secondary prevention of hemoglobinopathies.

FUNCTIONALLY DISTINCT UTILIZATION OF CIS-REGULATORY ELEMENTS IN HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN

Hereditary persistence of fetal hemoglobin (HPFH) mutations are rare, naturally-occurring variations which reduce the severity of beta-hemoglobinopathies by increasing postnatal expression of the gamma-globin genes (HBG1/2). Genome editing to inhibit erythroid transcriptional silencers or alter cis-elements in the HBG1/2 promoters recapitulates HPFH and should be tractable clinically. To define mechanisms involved with reactivation of fetal hemoglobin (HbF), we performed Cas9-mediated homology-directed repair to introduce HPFH mutations at the -115 distal CCAAT box cluster of the HBG1/2 promoters in an immortalized human erythroid cell line. HPFH mutations at positions -117, -114, -113, -110, and a 13-nucleotide (nt) deletion significantly elevated HbF levels compared to controls. Previous data suggest HPFH mutations either block repressor binding or create new binding sites for activators, such as GATA1. Using Cleavage Under Targets and Release Using Nuclease (CUTR and 2) an intact proximal CCAAT box is required for HBG1/2 de-repression by HPFH mutations in the distal CCAAT box. Elucidating the HBG1/2 cis-elements that control the perinatal gamma-to-beta globin switch should facilitate the development of optimal precision medicine-based treatments for beta-hemoglobinopathies.

Homozygous Deletion Alpha-Thalassemia and Hereditary Persistence of Fetal Hemoglobin, Two Genetic Factors Predictive the Reduction of Morbidity and Mortality During Pregnancy in Sickle Cell Patients. A Report from the Democratic Republic of Congo.

Objectiveto determine the beneficial role of Fetal Hemoglobin (FHb) and alpha-thal on fetal and maternal morbidity during pregnancy in sickle cell patients.Study sitethe study was conducted at the sickle cell center of Kinshasa between 2008 and 2018Setting and study populationthis is a documentary and analytical study that included 980 deliveries of homozygous sickle cell patients.Methodsthe diagnosis of SCD and the quantification of FHb were performed with the capillary electrophoresis technique. The molecular test confirmed the diagnosis of SCD. The diagnosis of alpha-thal was made with the multiplex ligation-dependent probe amplification (MLPA) technique. Sickle cell pregnancies were followed according to the protocol of care in force in the University of Kinshasa Hospital service. The variables of interest were: hematological variables, sickle cell crises during pregnancy, maternal and fetal complications.Statisticsstatistical analyses were performed with SPSS 20.0 software. Means and standard deviations were compared with the Student’s t and ANOVA tests. The value of p <0.05 was considered the significance level.Resultsthe Hb-SS / alpha-thal and HbSS / HPFH genotypes were observed in 101 and 121 women, respectively. Otherwise, 758 women had HbSS genotype. The morbidity related to sickle cell complications in the mother and fetus were less frequent in the Hb-SS / alpha-thal and HbSS / HPFH groups than in HB-SS group. The differences were statistically significant.Conclusionthis study showed a significant protective effect of alpha-thal and HPFH during pregnancy in sickle-cell pregnant women.

PS1518 GENOME EDITING OF HBG1/2 PROMOTER LEADS TO ROBUST HBF INDUCTION IN VIVO, WHILE EDITING OF BCL11A ERYTHROID ENHANCER RESULTS IN ERYTHROID DEFECTS

Background:Induction of fetal hemoglobin (HbF) is an attractive approach to ameliorate disease symptoms in beta hemoglobinopathies. Gene editing by zinc finger or CRISPR‐Cas nucleases to disrupt BCL11A expression, a known mediator of fetal globin silencing, via targeting BCL11A erythroid‐enhancer, is one approach to reactivate fetal globin expression. Alternatively, the genetics of hereditary persistence of fetal hemoglobin (HPFH) suggest that disruption of certain cis‐regulatory elements in the beta‐globin locus may also be a viable strategy. A previous lenti‐CRISPR mediated saturated mutagenesis screen identified multiple HbF‐inducing genomic domains concentrated at the gamma‐globin (HBG1/2) promoters.Aims:To compare editing of HBG1/2 promoters versus BCL11A erythroid‐enhancer in human hematopoietic stem and progenitor cells (HSPCs) using CRISPR‐Cas technologies as potential treatments for beta hemoglobinopathies.Methods:Ribonucleoprotein (RNP) complexes were designed to target either BCL11A erythroid‐enhancer GATAA motif or several HbF‐inducing genomic domains in the HBG1/2 promoters. These RNPs were transfected into mobilized peripheral blood (mPB) CD34+ HSPCs from healthy volunteers or peripheral blood mononuclear cells (PBMCs) from patients with sickle cell disease. RNPs demonstrated to have high HbF‐inducing potentials were used to edit HSPCs which were then infused into immunocompromised NOD.Cg‐KitW−41J Tyr+ Prkdcscid Il2rgtm1Wjl/ThomJ (NBSGW) mice to assess editing in SCID‐repopulating cells (SRC) and their multilineage reconstitution potential.Results:Following transfection of RNPs targeting HBG1/2 promoters into CD34+ HSPCs or sickle PBMCs, &gt;70% editing was achieved. Erythroid progeny derived from edited HSPCs or PBMCs had increased levels of HbF compared to those derived from control cells (up to 40% vs. 10% respectively). When infused into NBSGW mice, HSPCs edited with RNPs targeting either HBG1/2 promoters or BCL11A erythroid‐enhancer both reached &gt;70% human chimerism (hCD45+/hCD45+mCD45). Comparable monocytes, granulocytes, B lymphocytes, and HSPCs reconstitution were attained with both approaches. However, the erythroid lineage output in the BCL11A‐edited group was approximately 80% less than that in the control group. In addition, a 20% reduction in editing levels, compounded by a 2.5‐fold higher nonproductive indel frequency, was observed in the erythroid compartment compared to unfractionated bone marrow (BM) or other human lineages from the same animal in the BCL11A‐edited group. In contrast, HBG1/2 promoter‐edited cells had similar editing levels and indel patterns across all lineages with no significant lineage skewing. Importantly, robust long‐term induction of HbF was observed in human erythroid cells either sorted from chimeric BM or obtained from ex vivo culture of BM from HBG1/2 promoter‐edited group compared to the control group up to 16‐weeks post‐transplant.Summary/Conclusion:Our data suggest that sickle PBMCs, bulk CD34+ cells, and long‐term hematopoietic stem cells can be successfully edited using RNPs targeting BCL11A erythroid‐enhancer or HBG1/2 promoters. However, HBG1/2 promoter‐edited cells exhibited a more favorable preclinical profile than BCL11A‐edited cells, as BCL11A‐edited HSPCs displayed erythroid‐specific defects in the NBSGW mouse model. Long term induction of HbF by HBG1/2 promoter‐edited cells without lineage skewing suggests that this editing strategy has favorable clinical potential for the treatment of beta hemoglobinopathies.

Analysis of deletional hereditary persistence of fetal hemoglobin/δβ-thalassemia and δ-globin gene mutations in Southerwestern China.

BackgroundDeletional hereditary persistence of fetal hemoglobin (HPFH)/δβ‐thalassemia and δ‐thalassemia are rare inherited disorders which may complicate the diagnosis of β‐thalassemia. The aim of this study was to reveal the frequency of these two disorders in Southwestern China.MethodsA total of 33,596 subjects were enrolled for deletional HPFH/δβ‐thalassemia, and positive individuals with high fetal hemoglobin (Hb F) level were diagnosed by multiplex ligation‐dependent probe amplification (MLPA). A total of 17,834 subjects were analyzed for mutations in the δ‐globin gene. Positive samples with low Hb A2 levels were confirmed by δ‐globin gene sequencing. Furthermore, the pathogenicity and construction of a selected δ‐globin mutation were analyzed.ResultsA total of 92 suspected cases with Hb F ≥5.0% were further characterized by MLPA. Eight different deletional HPFH/δβ‐thalassemia were observed at a frequency of 0.024%. In addition, 195 cases suspected to have a δ‐globin gene mutation (Hb A2 ≤2.0%) were characterized by molecular analysis. δ‐Globin gene mutation was found at a frequency of 0.49% in Yunnan. The pathogenicity and construction for a selected δ‐globin mutation was predicted.ConclusionScreening of these two disorders was analyzed in Southwestern China, which could define the molecular basis of these conditions in this population.

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.

A Case of Hereditary Spherocytosis Caused by a Novel Homozygous Mutation in the SPTB Gene Misdiagnosed as β-Thalassemia Intermedia Due to a KLF1 Gene Mutation

We report a rare case of hereditary spherocytosis (HS) and hereditary persistence of fetal hemoglobin (Hb) (HPFH) complicated with a β-thalassemia (β-thal) trait and a Krüppel-like factor 1 (KLF1) gene mutation misdiagnosed as β-thal intermedia (β-TI) due to a high percentage of Hb F. The proband presented with pale skin, jaundice and splenomegaly. Analysis of the thalassemia gene indicated βcodon 17/βA (HBB: c.52A>T), while Hb analysis showed significantly increased Hb F levels. The proband was diagnosed to carry β-TI, and a blood transfusion regimen together with iron chelation treatment was recommended. Due to the difference between the phenotype and genotype, next generation sequencing (NGS) was performed and the proband was found to carry a homozygous mutation on the SPTB gene combined with a heterozygous mutation in KLF1. An eosin-5-maleimide binding test (EMA-BT) showed that the mean fluorescence intensity decreased by 47.1%. The proband was finally diagnosed with HS and HPFH complicated with a β-thal trait and the high percentage of Hb F was believed to be ascribed to the KLF1 gene mutation, which is frequent in areas where thalassemia is prevalent. For patients with a β gene mutation accompanying significantly high percentage of Hb F, the diagnosis of β-TI could be warranted, and the influence of the KLF1 gene mutation should be carefully excluded to avoid misdiagnosis of other types of hereditary hemolytic diseases.

A natural regulatory mutation in the proximal promoter elevates fetal globin expression by creating a de novo GATA1 site

Abstractβ-hemoglobinopathies, such as sickle cell disease and β-thalassemia, result from mutations in the adult β-globin gene. Reactivating the developmentally silenced fetal γ-globin gene elevates fetal hemoglobin levels and ameliorates symptoms of β-hemoglobinopathies. The continued expression of fetal γ-globin into adulthood occurs naturally in a genetic condition termed hereditary persistence of fetal hemoglobin (HPFH). Point mutations in the fetal γ-globin proximal promoter can cause HPFH. The −113A>G HPFH mutation falls within the −115 cluster of HPFH mutations, a binding site for the fetal globin repressor BCL11A. We demonstrate that the −113A>G HPFH mutation, unlike other mutations in the cluster, does not disrupt BCL11A binding but rather creates a de novo binding site for the transcriptional activator GATA1. Introduction of the −113A>G HPFH mutation into erythroid cells using the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) system increases GATA1 binding and elevates fetal globin levels. These results reveal the mechanism by which the −113A>G HPFH mutation elevates fetal globin and demonstrate the sensitivity of the fetal globin promoter to point mutations that often disrupt repressor binding sites but here create a de novo site for an erythroid activator.

Wide range of F cell levels in healthy Thai adults: Influence of Swiss-type hereditary persistence of foetal haemoglobin & β-haemoglobinopathy.

Background & objectives:Swiss-type hereditary persistence of foetal haemoglobin (HPFH) has been shown to be responsible for the wide range of F cell levels in healthy Thai adults. However, a survey for F cells in healthy Thai adults has not been performed. This study was conducted to determine the F cell distribution in adult Thai blood donors and to assess the possible involvement of β-thalassaemia and haemoglobin E (HbE) carriers in increased HbF levels.Methods:Thai blood donors (n=375, 205 males and 170 females) were included in the study. Blood samples were collected for measuring haemoglobin (Hb) concentration and haematocrit (Hct) and F cell levels. Hb and Hct levels were determined by automated blood counter, while F cells were quantified by flow cytometric analysis of F cells stained by fluorescein isothiocyanate-conjugated anti γ-globin monoclonal antibody. Finally, F cell levels were compared between blood samples having mean corpuscular volume (MCV) <80 fl and ≥80 fl as well as between β-haemoglobinopathies (HbE and β-thalassaemia carriers) and normal adults.Results:F cell levels varied markedly spanning 0.80-39.2 per cent with a positively skewed distribution. Thirty two per cent of these individuals had F cell levels more than the 4.5 per cent cut-off point. F cell levels in females were significantly higher than those in males (P<0.05). F cell levels in individuals having MCV <80 fl were significantly higher than those having MCV ≥80 fl (P<0.05). β-haemoglobinopathy (HbE and β-thalassaemia carriers) had significantly higher F cell levels than normal individuals (P<0.05).Interpretation & conclusions:The present results showed that besides Swiss-type HPFH, the β-haemoglobinopathy was expected to be involved in increased F cell levels in adult Thais. Thus, influence of β-haemoglobinopathy must be considered in interpreting F cell levels in area endemic of this globin disorder.