BCL11A Loci Research Articles

Genotyping the BCL11A Single Nucleotide Polymorphism and Associated Levels of Fetal Hemoglobin in Mauritanian Sickle Cell Patients.

Sickle cell disease (SCD) is a major heritable genetic disease in sub-Saharan Africa, including Mauritania. Fetal hemoglobin (HbF) can affect the pathophysiology, moderate the clinical course, and offer prospects for curative treatment of SCD. This study aimed to investigate the influence of single nucleotide polymorphisms (SNPs) in the BCL11A gene on the levels of HbF and hematological parameters in Mauritanian sickle cell (HbSS) patients. Complete blood count was assessed in 565 patients suspected to have SCD. Polymerase chain reaction (PCR)-restriction fragment length polymorphism was performed to identify the HbSS, and sequencing was used for genotyping three SNPs: rs4671393 (A>G) and rs11886868 (C>T) in the intron 2 and rs1052520 (G>A) in the 3'UTR regions of the BCL11A gene in 50 sickle cell patients. The prevalence of HbSS among the study population was 8.8% (50/565), and the mean (± standard deviation) of HbF level was 15.0% (± 6.0%). Sequencing showed the presence of three genotypes: AA (13.6%), AG (46.6%), GG (39.6%) in rs4671393; CC (17.6%), CT (48.7%), and TT (33.6%) in rs11886868. All samples from HbSS individuals displayed a wild-type genotype in the rs1052520 allele. The prevalence of minor alleles A (rs4671393) and C (rs11886868) were 37% and 39%, respectively. There was a statistically significant association (p = 0.034) between rs4671393 SNP and elevated HbF (mean 12.72 ± 6.26%). The study of three SNPs in the BCL11A locus in Mauritanian patients with SCD showed a significant association of rs4671393 allele with the HbF level. Further research is needed to explore additional SNPs in the BCL11A locus and investigate other genetic markers reported to modulate HbF levels, such as HBS1L-MYB and Xmn1-HBG2, to improve the management of this potentially life-threatening condition in Mauritania.

Tetramer Formation of BCL11A Is Required for Both Protein Stability and Fetal Hemoglobin Silencing

The zinc-finger (ZnF) repressor protein BCL11A regulates the switch from fetal (HbF, α 2γ 2) to adult hemoglobin (HbA, α 2β 2) through direct binding at the γ-globin promoter. Clinical trials of gene-based reduction of BCL11A with ex vivo modified hematopoietic stem cells (HSCs) have yielded transformative outcomes in patients with sickle cell disease and β-thalassemia, establishing BCL11A as a therapeutic target. Unless in vivo methods for delivery of gene-based therapies become efficient enough to modify a substantial fraction of host HSCs, small molecule drugs will be required to reduce overall disease burden. With the goal of targeting BCL11A with small molecules, we have characterized its native state. The protein is largely unstructured, except for 7 ZnFs, numbered 0-6. Previously we showed that BCL11A protein appears stable in cells with a T 1/2 of ~ 24 hrs. Here we report the unexpected finding that the protein assembles and functions as a tetramer. Recombinant BCL11A behaves as a tetramer in biophysical assays. Gene editing of the endogenous BCL11A locus in CD34-derived, immortal HUDEP2 cells has been employed to correlate protein structure and expression with consequences of a given modification. We found that BCL11A lacking ZnF0 is unstable, leading to protein loss and derepression of γ-globin. Recombinant ZnF0 self-assembles into a stable tetramer, whose X-ray structure reveals a hydrophobic interface between subunits. Mutation of residues in the hydrophobic core disrupts tetramerization and markedly reduces steady-state protein in cells. Reciprocal immunoprecipitation in cells reveals that ZnF0 is both necessary and sufficient to mediate BCL11A mutlimer assembly. Collectively, these results indicate that BCL11A tetramerization is required for protein stability. In cells expressing BCL11A lacking ZnF0, the steady-state protein level is partially restored by addition of proteasome inhibitors, suggesting that mutant protein is actively degraded by the ubiquitin-proteasome system. In contrast to findings with ZnF0, discrete removal of ZnF1 within the context of full-length BCL11A results in a marked increase in steady-state protein in cells. In vitro purified ZnF1 forms a dimer and only dimeric ZnF1 can be stably expressed in cells. These findings re consistent with a model in which assembly of the BCL11A tetramer is required to mask a degron in ZnF1 that becomes obscured on its dimerization. Masking of a degron in ZnF1 predicts that discrete removal of both ZnF0 and ZnF1 within full-length BCL11A will lead to production of a monomer. Indeed, HUDEP2 cells expressing BCL11A lacking both ZnFs exhibit an elevated level of steady-state, monomeric protein, consistent with the degron masking model. However, despite an elevated level, cells expressing monomeric BCL11A are defective in HbF silencing, indicating that the tetramer structure is critical for γ-globin repression. Our findings provide novel insights into BCL11A action and suggest new strategies for therapeutic targeting. Combining structural, biochemical, and cellular approaches, we have shown that ZnF-mediated tetramer formation shields BCL11A from proteolytic degradation, and that the tetramer state itself is required for effective γ-globin repression. Of relevance to BCL11A as a therapeutic target, the tetramer of ZnF0 constitutes a structured, stable domain in an otherwise largely unstructured protein, and thereby offers possibilities for small molecule targeting. The multimer state of BCL11A is a vulnerability for HbF repression.

Responses of β-thalassemia and compound heterozygote of Sickle/βthalassemia of BCL11A Gene Polymorphism in Pakistani Patients.

Beta-thalassemia major (β-Thal) and compound heterozygote of Sickle β-thalassemia (S-β Thal) are hereditary autosomal recessive disorders resulting from mutations or deletion in β-globin gene cluster. Patients with increased HbF levels having polymorphism at BCL11A site loci have shown clinical significance. The present study aimed to assess the frequency of BCL11A gene polymorphism in a study population of β-Thal, S-β Thal & Controls using Sanger sequencing leading to plot the HbF response of polymorphism with reference to wild type. The sample size of the study is n=180, groups were divided in Controls, β-thal & S-β thal. One ml blood was drawn from patients and controls to extract DNA for PCR amplification and BCL11A locus genotyping using Sanger sequencing. This study was carried out at Dow Research Institute of Biotechnology and Biomedical Sciences, for one year from March 2021 to February 2022. The HbF response of three groups is hyperbolic with 83 for β-Thal, 16 for S-β Thal and close to zero for controls. The frequency of heterozygous variant GA of BCL11A gene polymorphism is 51%. The frequency of homozygous variant GG is 49%. Complete absence of wild type AA in patient group. The frequency of BCL11A polymorphism in control group was 43% (with male 18% and female 21%) showing wild type status of 57%. The patient groups of SCD and Beta thalassemia are devoid of wild type status. The wild type status of BCL11A is 57% even in control population. Higher level of HbF in B-thalassemia and SCD and B Thalassemia is a cost-effective screening marker before switching to an expensive genotyping testing.

Interim Safety and Efficacy Results from a Phase 1/2 Study of Zinc Finger Nuclease-Modified Autologous Hematopoietic Stem Cells for Sickle Cell Disease (PRECIZN-1)

Introduction Sickle cell disease (SCD) is caused by pathologic variants in both alleles of the β-globin gene. Elevated fetal hemoglobin (HbF) levels inhibit sickle cell formation and ameliorate symptoms and improve survival in patients with SCD (Hebert, Am J Hematol, 2020;95:1235-45). BIVV003 is a novel therapeutic product comprising autologous CD34+ hematopoietic stem precursor cells (HSPC) modified ex vivo by zinc finger nucleases (ZFN) targeting specifically the BCL11A gene erythroid-specific enhancer (ESE) to increase endogenous HbF production in erythrocytes (RBC). Methods PRECIZN-1 (NCT03653247) is an ongoing first-in-human, open label, single arm, multi-center study evaluating safety and tolerability of BIVV003 (n=8; aged 18-40 years) with severe SCD. Eligible subjects underwent mobilization and apheresis with plerixafor 240 ug/kg/day for up to 3 days to collect autologous CD34+HSPCs for manufacturing of BIVV003. HSPCs were transfected ex vivo with ZFN mRNAs targeting the ESE region of the BCL11A locus to manufacture BIVV003. After myeloablative conditioning with busulfan 3.2 mg/kg/day intravenously (IV) for 4 days with dose adjustment based on pharmacokinetics, subjects received BIVV003 as a single minimum dose of 3× 106 CD34+ HSPC/kg IV. Subjects were monitored for stem cell engraftment and hematopoietic recovery, adverse events (AEs), clinical and laboratory hemolysis markers, total hemoglobin (Hb) and HbF, RBC containing HbF (F cells), and SCD-related events for up to 104 weeks post-BIVV003 infusion. Results As of the cutoff date of 03 May 2022, 4 subjects have received BIVV003 infusions up to 125 weeks of follow-up. A fifth subject was dosed with BIVV003 on 04 May 2022 and has engrafted. Among the 5 subjects, the male:female ratio is 3:2, the age range is 18-35 years, and 4 have genotype HbSS and 1 HbS-β0. No subject has required HSPC rescue; all subjects had hematopoietic reconstitution without requiring RBC or platelet transfusions for more than one month post-BIVV003 infusion. The average time to neutrophil and platelet engraftment was 23 days and 33 days, respectively. In bone marrow aspirates 26 weeks post-infusion, the frequency of cells containing indels at the BCL11A ESE target locus was in the range of 17.1-34.1%. No clonal dominance has been observed in bone marrow or peripheral blood cells. By Week 26, all 4 subjects reached a protective level of ≥ 10 pg HbF/F cell that inhibits HbS polymerization (Steinberg, Blood, 2014;123:481-485), and this level was sustained in 3 out of 4 subjects through the most recent assessment at Week 104, 78, and 52, respectively, along with F cell levels of 90% or higher. The change in total Hb from baseline (at screening) to the latest visit was -0.3-3.1 g/dL, and the HbF fraction increased to 12.2-41.2% (Figure 1). Three subjects have had no recurrence of severe vaso-occlusive crises (VOCs) post-infusion, compared to experiencing 6 to 22 severe VOCs in the 2 years pre-study. The one subject who did not sustain a HbF/F level ≥ 10 pg experienced 2 severe VOCs at 9 and 16 months post-infusion. The adverse events (AEs) reported were consistent with plerixafor mobilization and busulfan myeloablative conditioning. No AEs related to BIVV003 were reported. Conclusions The interim safety and efficacy results reported here confirm the potential therapeutic value of ZFN-mediated modification of the BCL11A ESE region and BIVV003 infusion to address current unmet needs of patients with SCD. BIVV003 has been well tolerated in the 5 subjects infused to date, with no AEs related to BIVV003. Three of the 4 subjects infused and with over 52 weeks of follow-up have had stable engraftment of ZFN-modified HSPCs resulting in sustained elevated HbF levels and absence of severe VOCs post-BIVV003 administration. A fifth subject received BIVV003 manufactured using an improved process that has shown in internal experiments to increase the number of ZFN-modified long-term progenitors in the drug product. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Authority of Gene Modifiers in β-Thalassemia Major and Its Relationship to the Pathophysiology of the Disease

β-Thalassemia, an inherited red blood cell disorder, presents a significant health problem worldwide and is caused by defects in the β-globin gene, resulting in the reduction or absence of β-globin chain synthesis. That leads to blood transfusion dependency with its terrible complications. Polymorphisms at position -158 of XmnI-HBG2 on chromosome 11 and BCL11A site on chromosome 2p16 might be linked with elevated hemoglobin F (HbF) appearance, which may, in turn, improve β-thalassemia sternness. This study aims to walk around the amending effects of XmnI and BCL11A loci on HbF levels in Egyptian β-thalassemia patients. Material and Methods. A prospective case-control study of 70 multi-transfused β-thalassemia major patients and 22 controls was performed in the Paediatric hematology unit of Assiut university hospital from June 2019 till April 2021. PCR-RFLP was used to detect single nucleotide polymorphisms at XmnI and BCL11A site loci. Results. XmnI Polymorphism was detected in 9 of 70 patients and associated with higher mean HbF levels (53.48%) than patients without polymorphism (mean Hb level was 42.23%)(P-Value=0.035). The frequency of CT heterozygous genotype was 8 (11.4%), TT homozygous genotype was (1.4%), while the wild genotype CC was detected in 61 (87.1%) of the cases. While BCL11A Polymorphism detected in 21 of 70 patients did not affect either Hb or HbF levels (P-Value =0.26). The TT genotype frequency was 49 (70%), and TC heterozygous genotype was detected in 21 (30 %) of patients. The CC genotype was absent. Conclusion: XmnI -158Gγ polymorphism, but not BCL11A polymorphism, has a modifying effect on both Hb and HbF levels in Egyptian β-thalassaemia major patients. Our research goal is to investigate the frequency of XmnI and BCL11A polymorphism in thalassemia major and investigate the HbF level difference according to the XmnI and BCL11A polymorphism of phenotype group in thalassemia major.

Epigenomic analysis of KLF1 haploinsufficiency in primary human erythroblasts

Haploinsufficiency for the erythroid-specific transcription factor KLF1 is associated with hereditary persistence of fetal hemoglobin (HPFH). Increased HbF ameliorates the symptoms of β-hemoglobinopathies and downregulation of KLF1 activity has been proposed as a potential therapeutic strategy. However, the feasibility of this approach has been challenged by the observation that KLF1 haploinsufficient individuals with the same KLF1 variant, within the same family, display a wide range of HbF levels. This phenotypic variability is not readily explained by co-inheritance of known HbF-modulating variants in the HBB, HBS1L-MYB and/or BCL11A loci. We studied cultured erythroid progenitors obtained from Maltese individuals in which KLF1 p.K288X carriers display HbF levels ranging between 1.3 and 12.3% of total Hb. Using a combination of gene expression analysis, chromatin accessibility assays and promoter activity tests we find that variation in expression of the wildtype KLF1 allele may explain a significant part of the variability in HbF levels observed in KLF1 haploinsufficiency. Our results have general bearing on the variable penetrance of haploinsufficiency phenotypes and on conflicting interpretations of pathogenicity of variants in other transcriptional regulators such as EP300, GATA2 and RUNX1.

Preliminary Safety and Efficacy Results from Precizn-1: An Ongoing Phase 1/2 Study on Zinc Finger Nuclease-Modified Autologous CD34+ HSPCs for Sickle Cell Disease (SCD)

▪IntroductionSickle cell disease (SCD) is caused by pathologic variants in both alleles of the β-globin gene, affecting ~100,000 patients in the US (Strouse. Handb Clin Neurol 2016;138:311-24). Elevated fetal hemoglobin (HbF) levels ameliorate symptoms and improve survival in patients with SCD (Hebert. Am J Hematol 2020;95:1235-45). SAR445136 (BIVV003) is a novel therapeutic product comprising autologous CD34+ HSPCs modified ex vivo by zinc finger nucleases (ZFN) and targeting the BCL11A gene erythroid-specific enhancer (ESE) to increase endogenous HbF production.MethodsPRECIZN-1 (NCT03653247) is an ongoing first-in-human, open label, single arm, multi-site study evaluating safety and tolerability of SAR445136 (n=8; aged 18-40 years), with severe SCD across 6 US sites. Eligible subjects underwent mobilization and apheresis with plerixafor 240 ug/kg/day for up to 3 days to collect autologous CD34+ HSPCs to achieve a minimum of 10 × 10 6 CD34+ HSPC/kg for manufacturing of the SAR445136 dose. Additional apheresis cycles were allowed to achieve the minimum cell dose and rescue aliquots. Autologous HSPCs were transfected ex vivo with ZFN mRNAs targeting the ESE region of the BCL11A locus to manufacture SAR445136. A single IV infusion of 3-20 × 10 6 CD34+ HSPC/kg was administered at least 72 hours after the final busulfan myeloablation dose. Subjects were monitored for stem cell engraftment and hematopoietic recovery, adverse events (AEs), clinical and laboratory hemolysis markers, total Hb and HbF, percentage of F cells and sickle-cell related events post-SAR445136 infusion. Health-related quality of life (HRQoL) was assessed via the PROMIS-57 survey at screening, Weeks 26 and 52, and early termination visit.ResultsOf the 7 subjects that underwent mobilization and apheresis to date (25 June 2021), 5 achieved successful target yields ranging from 3.4-13.8 x 10 6 CD34+ HSPC/kg per apheresis day (mean: 6.49 x10 6 CD34+ HSPC/kg per apheresis day) in one apheresis cycle (4.45-10.9 x 10 6 CD34+ HSPC/kg per 2-day cycle). One subject failed to mobilize; one discontinued due to intercurrent cholangitis. Baseline patient characteristics of the 4 patients infused are in Table 1.Pre-apheresis peripheral blood WBC ranged from 23.2-36.9 x 10 3/μL (mean: 28.7 x 10 3/μL) and % CD34+ was 0.09-0.36% (0.22%) with absolute CD34+ counts of 20-80/μL (mean: 60/μL). Four of the mobilized subjects were successfully infused with SAR445136 at a single dose ranging from 3.2-9.7 x 10 6 CD34+ HSPC/kg (mean: 5.17 x 10 6 CD34+ HSPC/kg). All 4 subjects engrafted with a median time to neutrophil and platelet recovery of 21.5 and 24.5 days, respectively. No rescue doses were required.All 4 patients improved clinically since SAR445136 infusion, with no recurrence of previous vaso-occlusive crises (VOCs). Total Hb stabilized at 9-10 g/dL by week 26 post SAR445136 infusion along with improvements in the clinical markers of hemolysis in all 4 subjects. Percent HbF levels were 1-11% at screening, increasing to 15-29% by Week 13 in all 4 subjects, to 14-39% by Week 26 in the 3 subjects with at least 26 weeks follow up; and persisting at 35% in 1 subject with 65 weeks follow up (Figure 1). Percent F cells increased to 49-94% in 3 subjects with at least 26 weeks follow up, persisting at 90% in 1 subject with 65 weeks follow up. The fourth subject had 87.5% F cells at 13 weeks follow up.Although preliminary, a trend of improvement exceeding the proposed minimally clinically important difference in all PROMIS-57 HRQoL domains except sleep disturbance was observed in the 3 subjects with 26 weeks follow up, whose scores were “worse” than the norm at baseline (≤2 points per domain).SAR445136 was generally well tolerated with no infusion related reactions. The AEs reported were consistent with plerixafor mobilization and busulfan myeloablation therapy. No AEs or SAEs were reported as related to SAR445136.ConclusionsAs of 25 June 2021, these preliminary proof-of-concept efficacy and safety results confirm the potential therapeutic value of ZFN-mediated modification of the BCL11A ESE region and SAR445136 infusion to address current unmet needs of patients with SCD. All 4 infused patients had no SCD related events including VOCs following SAR445136 infusion, as well as increases in total Hb, HbF, and %F cells, and clinical improvements in PROMIS-57 domains. SAR445136 is generally well tolerated in the 4 subjects infused to date, with no related AEs or SAEs reported. [Display omitted] DisclosuresAbedi: BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Seattle Genetics: Speakers Bureau; Abbvie: Speakers Bureau. Galeon: Sanofi: Current Employment. Reiner: Sanofi: Current Employment. Smith: Sanofi: Current Employment. Wang: Sanofi: Current Employment. Ramezi: Sanofi: Current Employment. Rendo: Sanofi: Current Employment, Other: May hold shares and/or stock options . Walters: AllCells, Inc: Consultancy; Vertex pharmaceuticals: Consultancy; Ensoma, Inc.: Consultancy; BioLabs, Inc: Consultancy.

LMO2 is essential to maintain the ability of progenitors to differentiate into T-cell lineage in mice.

Notch signaling primarily determines T-cell fate. However, the molecular mechanisms underlying the maintenance of T-lineage potential in pre-thymic progenitors remain unclear. Here, we established two murine Ebf1-deficient pro-B cell lines, with and without T-lineage potential. The latter expressed lower levels of Lmo2; their potential was restored via ectopic expression of Lmo2. Conversely, the CRISPR/Cas9-mediated deletion of Lmo2 resulted in the loss of the T-lineage potential. Introduction of Bcl2 rescued massive cell death of Notch-stimulated pro-B cells without efficient LMO2-driven Bcl11a expression but was not sufficient to retain their T-lineage potential. Pro-B cells without T-lineage potential failed to activate Tcf7 due to DNA methylation; Tcf7 transduction restored this capacity. Moreover, direct binding of LMO2 to the Bcl11a and Tcf7 loci was observed. Altogether, our results highlight LMO2 as a crucial player in the survival and maintenance of T-lineage potential in T-cell progenitors via the regulation of the expression of Bcl11a and Tcf7.

Haemoglobin switching modulator SNPs rs5006884 is associated with increased HbA2 in β-thalassaemia carriers.

IntroductionHaemoglobin A2 (HbA2), the tetramer of α- and δ-globin chains, is used as a diagnostic biomarker for β-thalassaemia carriers. The HbA2 levels are regulated by the presence of HPFH, δ-thalassaemia, HbA1/2 gene triplication, and variants of KLF1, β-globin gene, and HbF regulating QTLs. Saudi Arabia has a high incidence of borderline HbA2 levels, thereby making it difficult to classify the haemoglobinopathies. This study aims to investigate the association of known HbF enhancer QTL gene SNPs with HbA2 levelsMaterial and methods14 Specific SNPs in BCL11A, HMIP, OR51B6, HBBP1, and HBG2 loci were genotyped in 164 Saudi β-thalassaemia carriers by TaqMan assay to validate their role as regulators of HbA2 levels. HbA2 levels were determined using the Variant II β-Thalassemia Short Program Recorder kit. The non-random association of these SNPs was tested using HaploView software. Protein interaction was assessed using 3D structure modelling for OR51B6 (rs5006884), comparative energy minimisation, and root-mean-square deviation (RMSD) prediction.ResultsElevated HbA2 levels were associated with SNPs in HBBP1, OR51B6, and TCT haplotype from HBG2 promoter region. The bioinformatics modelling and prediction revealed that the exonic rs5006884 had RMSD value deviations and significantly varied binding energy minimisation. α-globin variations were found in 57.92% of individuals but were not associated with elevated HbA2.ConclusionsThe haemoglobin switching modulators rs2071348, rs7482144, and rs5006884 are involved in regulation of HbA2 level with rs5006884 influencing the tetramer formation. Screening for haemoglobinopathies should take these SNPs into consideration, specifically in borderline HbA2 cases. Assiduous analysis of rs5006884 as HbA2 modulator for amelioration of disease severity is recommended.

Unique Polymorphisms at BCL11A, HBS1L-MYB and HBB Loci Associated with HbF in Kuwaiti Patients with Sickle Cell Disease.

Patients with sickle cell disease (SCD) in Kuwait have elevated HbF levels ranging from ~10–44%; however, the modulating factors are unclear. We investigated the association of single nucleotide polymorphisms (SNPs) at BCL11A, HBS1L-MYB and HBB with HbF levels in 237 Kuwaiti SCD patients, divided into 3 subgroups according to their HbF levels. Illumina Ampliseq custom DNA panel was used for genotyping and confirmed by arrayed primer extension or Sanger sequencing. In the BCL11A locus, the CC genotype of rs7606173 [χ2 = 16.5] and (GG) of rs10195871 [χ2 = 15.0] were associated with Hb-F1 and HbF-2 subgroups, unlike rs1427404-T [χ2 = 17.3], which showed the highest association across the three subgroups. HBS1L-MYB locus revealed 2 previously-described SNPs (rs66650371 [χ2 = 9.5] and rs35795442 [χ2 = 9.2]) and 2 previously-unreported SNPs, (rs13220662 [χ2 = 6.2] and rs1406811 [χ2 = 6.7]) that were associated with the HbF-3 subgroup, making this the key locus elevating HbF to the highest levels. HBB cluster variants were associated with lower levels of HbF (β = −1.1). We report four previously-unpublished variants showing significant association with HbF. Each of the three quantitative trait loci affects HbF levels differently; unique SNPs, especially in HBS1L-MYB, elevate HbF to the highest levels.

Modulatory effect of single nucleotide polymorphism in Xmn1, BCL11A and HBS1L-MYB loci on foetal haemoglobin levels in β-thalassemia major and Intermedia patients.

To evaluate the influence of certain genetic modifiers on foetal haemoglobin levels in thalassemia major and thalassemia intermedia. The cohort study was conducted from November 2018 to August 2019, at Department of Haematology, University of Health Sciences, Lahore and comprised beta thalassemia intermedia and thalassemia major patients who were referred by various healthcare facilities across Punjab, Pakistan. Foetal haemoglobin was quantified by high performance liquid chromatography. Primary mutation analysis and single nucleotide polymorphisms were done by amplification refractory mutation system-based polymerase chain reaction. Data was analysed using SPSS 20. Of the 116 patients, 52(45%) had beta thalassemia intermedia and 64(55%) had thalassemia major. Foetal haemoglobin levels were primarily influenced by alleles of the HBG2 (rs7482144) and BCL11A (rs766432) genes, but single nucleotide polymorphism of HBS1L-MYB (rs9399137) had no significant role (p>0.05). The rs7482144 single nucleotide polymorphism explained 8.3% of the variation in the foetal haemoglobin levels, while 5% of trait variation was explained by rs766432. There was found a clear association between foetal haemoglobin level and single nucleotide polymorphisms in HBG2 (rs7482144) and BCL11A (rs766432) genes. This correlation was additive and was seen both in thalassemia major and thalassemia intermedia cohorts.

Novel Genetic Loci That Influence Fetal Hemoglobin Expression in Children with Sickle Cell Anemia

Introduction: Elevated levels of fetal hemoglobin (HbF) are known to ameliorate both the morbidity and mortality of sickle cell anemia (SCA). Sustained post-natal HbF expression is heritable and regulated by multiple quantitative trait loci. Previous genomic studies have identified three major gene loci (BCL11A, HBS1L-MYB, and HBG2) that account for ~40% of HbF variation in SCA, but additional genetic modifiers remain to be discovered. We performed a genome wide association study (GWAS) using DNA collected from multiple cohorts of children with SCA, to identify novel genes and variants involved in HbF expression. Methods: We analyzed genomic DNA from 1009 children with SCA and pre-treatment steady-state HbF levels who enrolled in prospective research trials from the United States (HUSTLE, SWiTCH, TWiTCH), the Caribbean (SACRED) or sub-Saharan Africa (REACH, NOHARM). Whole blood DNA was first genotyped using the H3Africa SNP array (Illumina) that identifies over 2.2 million single nucleotide variants (SNVs) across the genome. Most samples also underwent whole exome sequencing (WES) using NimbleGen VCRome 2.1 capture reagents and the Illumina HiSeq2500 platform analysis, which identifies coding variants in all known exons. Square root transformed HbF values were the continuous variable for association testing using single-locus mixed model (EMMAX) adjusted for population stratification, with both age and sex as co-variates. The GWAS approach included 3 distinct steps. First, we performed two independent GWAS discovery steps using distinct African populations; these were designated Discovery I (N=211) and Discovery II (223). Second, only SNVs that were significant (p<0.05) in both datasets were then selected for two independent replication steps; these were designated either African-American (N=157) or African (N=269). Third, the SNVs that were significant in both dual discovery and at least one of the replication cohorts were then verified using an additional Caribbean cohort (N=149) with TaqMan techniques for genotyping specific variants. Through this multistep process, we searched for genomic loci with consistent HbF associations across multiple cohorts. Results: From the combined SNP and WES dataset, 8 BCL11A variants passed genome wide significance (p<10-8) in the discovery analysis, and 1,048 additional variants were identified with nominal HbF association (p<0.001). We found that 173 of these novel variants had sustained association in at least one of the replication cohorts (p<0.05). We selected 20 variants with the strongest and most consistent associations with HbF from the discovery and replication analyses for further verification (Table 1). Expected HbF associations with BCL11A (rs1427407) and HBS1L-MYB (rs4895441) were identified. Among other 18 novel candidate variants, the rs77737207 variant (allele frequency ~0.10) near the RUNX1T1 locus was strongly associated with lower HbF levels, while coding variant rs2279587 (allele frequency ~0.03) in the ITGA1 gene approached statistical significance (p<0.08) in the final verification cohort and was associated with higher levels of HbF. Conclusions: Our large GWAS of HbF with diverse global cohorts of children with SCA from Africa, the United States, and the Caribbean validated the strong associations of HbF with common genetic variants near the BCL11A and HBS1L-MYB gene loci. We also identified two novel gene loci, ITGA1 and RUNX1T1, that have statistical associations with HbF expression. The RUNX1T1 gene is a broad transcriptional corepressor known to impact myeloid differentiation in hematopoiesis, while ITGA1 encodes the integrin alpha subunit of a cell-surface receptor involved in cell-cell adhesion and inflammation. Both of these genes represent novel loci that may be involved in the regulation of HbF expression in children with SCA and should be investigated further using cellular and animal models. Disclosures No relevant conflicts of interest to declare.

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.

PB2406 THE INFLUENCE OF XMNI AND BCL11A GENE MODIFIERS ON HEMOGLOBIN F LEVELS IN EGYPTIAN β‐THALASSEMIA MAJOR PATIENTS

Background:β‐thalassemia major (β‐TM) is an autosomal recessive disorder in which β‐globin chain synthesis is severely decreased or absent which lead to blood transfusion dependency with its catastrophic complications. Polymorphisms at position ‐158 of XmnI‐HBG2 on chromosome 11 and BCL11A site on chromosome 2p16 might be associated with elevated hemoglobin F (HbF) expression which may,in turn,improve β‐thalassemia severityAims:This study aims to explore the modifying effects of XmnI and BCL11A loci on HbF levels in Egyptian β‐thalassemia patientsMethods:A prospective case‐control study of 70 multi‐transfused β‐thalassemia major patients and 22 controls was conducted in Paediatric hematology unit of Assiut university hospital from January 2016 till April 2017. PCR‐RFLP was used for detection of single nucleotide polymorphisms at XmnI and BCL11A site loci.Results:XmnI Polymorphism detected in 9 of 70 patients and associated with higher mean HbF levels (53.48%) than patients without polymorphism (meanHbF level was 42.23%)(P‐Value = 0.035).The frequency of CT heterozygousgenotype was 8(11.4%), TT homozygous genotype was (1.4%) while the wild genotype CC detected in 61 (87.1%) of the cases. While BCL11A Polymorphism detected in 21 of 70 patients had no effect on either Hb or HbF levels (P‐Value = 0.26). The TT genotype frequency was 49 (70%) and TC heterozygous genotype was detected in 21 (30 %) of patients. The CC genotype was absent.Summary/Conclusion:The presence of XmnI polymorphism rather than BCL11A polymorphism has a modifying effect on HbF production and increased Hb levels between β‐TM patients and so, it helps in identifying patients’ benefits from HU therapy.image

Quantitative Trait Loci Influencing Hb F Levels in Southern Thai Hb E (HBB: c.79G>A) Heterozygotes

Variation of fetal hemoglobin (Hb F) expression in heterozygous Hb E (HBB: c.79G>A) individuals is associated with several genetic modifiers and not well understood. This study was undertaken in order to determine the effect of single nucleotide polymorphisms (SNPs), including XmnI Gγ (rs7482144), rs766432 on the BCL11A gene and rs9376074 on the HBS1L gene, on Hb F levels in Southern Thai heterozygous Hb E individuals. A total of 97 Southern Thai subjects carrying heterozygous Hb E were selected for the hematological study. After excluding the samples with α-thalassemia (α-thal) interaction or moderate anemia, because both conditions can affect the hematological parameters, the remaining 74 samples were submitted to SNP analysis. Hematological parameters were measured using an automated hematology analyzer and high performance liquid chromatography (HPLC). The results show that rs766432 was strongly associated with increased Hb F levels and rs7482144 was associated with Hb F levels in each subgroup (genotype) of rs766432. This study suggested that the BCL11A locus has a major effect on Hb F levels compared with the XmnI polymorphism in Hb E heterozygotes. This association of Hb F levels with SNPs is useful for the interpretation of hemoglobin (Hb) typing in heterozygous Hb E samples with high Hb F levels. Future research will need to address the better understanding of the mechanisms of the SNPs that regulate Hb F production without stress erythropoiesis in Hb E heterozygotes.

A Nonhuman Primate Transplantation Model to Evaluate Hematopoietic Stem Cell Gene Editing Strategies for β-Hemoglobinopathies

Reactivation of fetal hemoglobin (HbF) is a promising approach for the treatment of β-hemoglobinopathies and the targeting of genes involved in HbF regulation is under intensive investigation. Here, we established a nonhuman primate (NHP) transplantation model to evaluate hematopoietic stem cell (HSC)-based gene editing strategies aimed at reactivating HbF. We first characterized the transient HbF induction to autologous HSC transplantation in pigtailed macaques, which was comparable in duration and amplitude to that of human patients. After validating function of the HbF repressor BCL11A in NHPs, we transplanted a pigtailed macaque with CD34+ cells electroporated with TALE nuclease mRNA targeting the BCL11A coding sequence. In vivo gene editing levels were low, but some BCL11A deletions were detected as late as 200 days post-transplantation. HbF production, as determined by F-cell staining and γ-globin expression, was slightly increased in this animal as compared to transplant controls. We also provided proof-of-concept results for the selection of edited NHP CD34+ cells in culture following integration of the P140K/MGMT cassette at the BCL11A locus. In summary, the NHP model described here will allow the testing of novel therapeutic approaches for hemoglobinopathies and should facilitate clinical translation.

Genetic Variants at BCL11A and HBS1L-MYB loci Influence Hb F Levels in Chinese Zhuang β-Thalassemia Intermedia Patients

Increased Hb F levels can ameliorate the symptoms of β-thalassemia (β-thal). Due to the genetic heterogenicity of β-thal, the relationship between genetic variants in modifier genes and Hb F level has been studied in different populations. The Chinese Zhuang has the second largest population in China and has 6.78% prevalence of β-thal. However, the effects of these single nucleotide polymorphism (SNP) variants on the Hb F levels of β-thal intermedia (β-TI) patients in this population have not been reported. To explore the association between modifier loci (β-globin gene cluster, HBS1L-MYB intergenic region and BCL11A) and Hb F levels in Chinese Zhuang β-TI patients, 96 unrelated β-TI patients (50 males and 46 females) with different Hb F levels were recruited and genotyped by mass spectrometry. A total of 13 SNPs were confirmed to be in a significant relationship with Hb F levels in this population. Of these, high-risk genotypes of six Hb F-associated SNPs, rs9376090, rs7776054, rs9399137, rs9389268, rs9402685 in the HBS1L-MYB intergenic region and rs189984760 in the BCL11A locus, showed association with high Hb F levels, especially for SNPs in linkage disequilibrium. One novel Hb F-associated SNP, rs189984760, was identified in our study. Our findings will be of valuable reference for correlation between modifier genes and Hb F in Chinese Zhuang populations and may lead to better understand the modifying mechanisms for β-thal.

758. A Nonhuman Primate Transplantation Model to Evaluate Gene Editing Strategies Aimed at Inducing Fetal Hemoglobin Production for the Treatment of Hemoglobinopathies

Fetal hemoglobin (HbF) is the major form of hemoglobin present in newborns but is almost completely replaced by adult hemoglobin after birth, where it constitutes less than 1 percent of total hemoglobin. Hereditary persistence of HbF is linked to mutations at multiple genetic loci that regulate the switch from fetal to adult hemoglobin. The targeting of these genes using site-specific nucleases thus constitutes a promising approach for the treatment of hemoglobinopathies by increasing HbF production.We have developed a nonhuman primate (NHP) model to investigate gene editing strategies aimed at inducing HbF production following hematopoietic stem cell (HSC) transplantation. As proof of principle, we focused on the transcription factor B-cell lymphoma/leukemia 11A (BCL11A), which functions as suppressor of HbF in humans. We disrupted the Bcl11a coding region using Transcription Activator-Like Effector Nucleases (TALENs) and achieved on average 30% gene editing by electroporation of mRNA in NHP CD34+ cells. Erythroid differentiation of these cells in culture confirmed that HbF expression was increased in Bcllla-edited cells as compared to control cells. To determine if Bcl11a-edited HSCs could engraft and give rise to HbF-producing erythrocytes, we transplanted a NHP with autologous CD34+ electroporated with Bcl11a TALEN mRNA following conditioning by total body irradiation. Using next generation sequencing, we detected about 1 % disruption in vivo one week after transplant, to reach a set point of about 0.3% over the course of the experiment. We were able to track several clones that persisted at least 200 days post transplantation based on their mutation signatures, suggesting engraftment of Bcllla-modified cells. HbF production was monitored in this animal by flow cytometry analysis of peripheral blood and was compared with three transplanted controls and one untransplanted control. In all transplanted animals, we observed a rapid increase in the frequency of F cells, reaching 10% to 40%, and lasting for about 140 days. In contrast, F cell production in the untransplanted control remained constant and minimal (<0.5%). After returning to basal levels, we found significantly higher HbF levels (1-1.5%) in the animal transplanted with Bcl11a-edited cells as compared to all other transplanted animals. These findings were confirmed by real-time PCR analysis of hemoglobin transcripts, which showed a 5-to 10-fold increase in gamma to beta globin ratio in the animal transplanted with Bcl11a-edited cells as compared to all controls. We also initiated work demonstrating the targeted integration of the chemoselection cassette P140K/MGMT at the Bcl11a locus in NHP HSCs by co-delivery of TALEN mRNA with a donor template carried on an adeno-associated viral vector, offering the potential for in vivo selection of modified cells. In summary, our experiments establish the NHP as pre-clinical model to evaluate therapeutic gene editing strategies for the treatment of hemoglobinopathies.

Abstract PR01: Understanding the role of BCL11A in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) has poor prognostic outcome compared to other subtypes of breast cancer. The molecular and cellular mechanisms underlying TNBC pathology are not fully understood. We analyzed both METABRIC and TCGA data found the transcription factor BCL11A is overexpressed in TNBC including basal-like breast cancer (BLBC) and that its genomic locus is amplified in up to 38% of BLBC tumors. Moreover, analysis of the TCGA data revealed that the BCL11A locus is hypo-methylated almost exclusively in BLBC tumors but not other subtypes. We show that exogenous BCL11A overexpression promotes tumor formation, whereas its knockdown in TNBC cell lines suppresses their tumorigenic potential in xenograft models. In the mouse DMBA-induced tumor model, Bcl11a deletion substantially decreases tumor formation, even in p53-null cells and inactivation of Bcl11a in established tumors causes their regression. At the cellular level, Bcl11a deletion causes a reduction in the number of mammary epithelial stem and progenitor cells. Mechanistically, proteomics studies have revealed that BCL11A interacts with a specific set of chromatin modifiers in tumor cells but not in normal cells - the significance of which is currently under investigation. This study highlights the potential for targeting BCL11A as an approach for TNBC-targeted therapy. To this end we are currently using the Crispr/Cas9 system to generate endogenous reporter cell lines for high-throughput drug screens to identify potential inhibitors of BCL11A. Citation Format: Kyren Lazarus, Carlos Caldas, Pentao Liu, Walid T. Khaled. Understanding the role of BCL11A in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr PR01.

Clonal Analysis of Human Bone Marrow CD34+ Cells Edited By BCL11A-Targeting Zinc Finger Nucleases Reveals Clinically Relevant Levels of Fetal Globin Expression in Edited Erythroid Progeny

Sickle cell disease (SCD) is one of the most common inherited blood disorders and is caused by a mutation at the adult beta globin gene resulting in substitution of valine for glutamic acid at position 6 in the encoded protein. While SCD can be cured by hematopoietic stem cell transplant (HSCT), complete donor chimerism is not required to achieve clinical benefits. Stable mixed chimerism of 10-15% in bone marrow or peripheral blood nucleated cells with >70% donor-derived RBCs has been reported to achieve transfusion independence and a symptom-free state in a SCD patient. It has also been proposed that SCD can be treated by reactivating developmentally silenced fetal gamma globin to form fetal hemoglobin (alpha2gamma2, HbF), which inhibits polymerization of HbS. The effect of HbF is predicted to be maximal when HbF content per cell exceeds 10 pg (~30% of total Hb). Furthermore, pathology is prevented when protective F cells (>30% HbF per cell) constitute >70% of total RBCs. We hypothesize that in a gene therapy setting, if >15% of SCD patients' autologous HSCs are programmed to produce protective F cells during erythropoiesis, it will translate into >70% protective F cells in circulation and provide significant alleviation of clinical symptoms.Genome wide association studies have identified BCL11A as a major modifier of HbF levels. Subsequent studies have shown that BCL11A plays a critical role in the fetal to adult globin developmental switch and in repressing fetal globin expression in adult erythroid cells. Conditional inactivation of BCL11A in adult erythroid cells leads to high levels of pan-cellular fetal globin expression and correction of hematologic and pathologic defects in a humanized SCD mouse model. Previously, we have reported that zinc finger nucleases (ZFNs) targeting BCL11A either in the coding region or the GATAA motif in the erythroid-specific enhancer efficiently disrupt the BCL11A locus in human primary CD34+ cells following electroporation of ZFN-encoding mRNA. Elevated fetal globin expression in bulk erythroid cultures was observed following disruption. To determine what percentage of HSPCs have been modified and whether the HbF/F cell content has reached the hypothesized therapeutic level, we analyzed erythroid cells clonally derived from ZFN-transfected CD34+ cells. Genotype of each clonal culture was determined by deep sequencing and globin production was analyzed by a highly sensitive UPLC method. We found that up to 80% of the BFU-Es had both BCL11A alleles edited, half of which had KO/KO alleles (either out of frame mutations for coding region or elimination of the GATAA motif in the enhancer). BCL11A coding KO/KO cells expressed on average 79.1% ± 12.2% fetal globin (Mean ± SD) whereas GATAA motif enhancer region KO/KO cells expressed approximately 48.4% ± 14.1% fetal globin, in comparison with 14.5% ± 9.6% in WT/WT cells . These levels of fetal globin should be sufficiently high to confer protection against HbS polymerization in sickle cells. WT/KO cells in both coding and enhancer editing experiments showed an intermediate phenotype with fetal globin averaging 26.9%± 9.9% and 25.79% ± 12.6%, respectively. Interestingly, when background (WT/WT) fetal globin level was subtracted, the fetal globin levels in WT/KO cells are comparable to those observed in patients with BCL11A haploinsufficiency, which average 14.6%± 10.3%. Together, our data demonstrate that genome editing of BCL11A using highly efficient ZFNs can lead to clinically relevant levels of fetal globin expression in KO/KO erythroid cells. If the frequency of KO/KO BFU-Es we observed in vitro reflects the frequency of KO/KO HSCs in bone marrow after autologous transplantation, genome editing of BCL11A has the potential to provide significant clinical benefit for patients with SCD. DisclosuresChang:Biogen: Employment, Equity Ownership. Sullivan:Biogen: Employment, Equity Ownership. Liu:Biogen: Employment, Equity Ownership. Yang:Biogen: Employment, Equity Ownership. Sun:Biogen: Employment, Equity Ownership. Vieira:Biogen: Employment, Equity Ownership. Zhang:Biogen: Employment. Hong:Biogen: Employment, Equity Ownership. Chen:Biogen: Employment, Equity Ownership. Smith:Biogen: Employment, Equity Ownership. Tan:Biogen: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Urnov:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Rebar:Sangamo BioSciences: Employment. Danos:Biogen: Employment, Equity Ownership. Jiang:Biogen: Employment, Equity Ownership.