Elevated Fetal Hemoglobin Research Articles

The Current Role of Hydroxyurea in the Treatment of Sickle Cell Anemia.

Despite advancements in treatment of sickle cell disease (SCD), hydroxyurea, a ribonucleotide reductase inhibitor, remains the cornerstone of therapy. While its primary effect is the elevation of fetal hemoglobin (HbF), hydroxyurea's mechanisms of action are multifaceted. Hydroxyurea (HU) reduces leukocyte and platelet counts, decreases the expression of endothelial adhesion molecules CD36 and CD49d, and increases nitric oxide and cyclic nucleotide levels, which may facilitate vascular dilation and further HbF induction. Numerous studies have demonstrated that hydroxyurea therapy reduces the frequency of painful episodes, acute chest syndrome, and the need for erythrocyte transfusions and hospitalizations. Long-term use of hydroxyurea leads to reduced morbidity and mortality. Hydroxyurea should be initiated in children from 9 months of age, including asymptomatic individuals, and is recommended for adults experiencing pain crises that significantly interfere with daily activities or quality of life, as well as those with severe or recurrent vaso-occlusive crises, ACS, or severe symptomatic anemia. Hydroxyurea is not recommended during pregnancy or lactation due to potential teratogenic effects and transfer into breast milk. However, its use may be considered in high-risk patients, particularly during the second and third trimesters. Concerns about secondary tumor development have not been substantiated in long-term follow-up studies. Alternative therapies, including L-glutamine, crizanlizumab, and voxelotor, are not presently approved or available for clinical use in Europe.

Genetic Polymorphisms Associated with Fetal Hemoglobin (HbF) Levels and F-Cell Numbers: A Systematic Review of Genome-Wide Association Studies.

Elevated fetal hemoglobin (HbF), which is partly controlled by genetic modifiers, ameliorates disease severity in β hemoglobinopathies. Understanding the genetic basis of this trait holds great promise for personalized therapeutic approaches. PubMed, MedRxiv, and the GWAS Catalog were searched up to May 2024 to identify eligible GWAS studies following PRISMA guidelines. Four independent reviewers screened, extracted, and synthesized data using narrative and descriptive methods. Study quality was assessed using a modified version of the Q-Genie tool. Pathway enrichment analysis was conducted on gene lists derived from the selected GWAS studies. Out of 113 initially screened studies, 62 underwent full-text review, and 16 met the inclusion criteria for quality assessment and data synthesis. A total of 939 significant SNP-trait associations (p-value < 1 × 10-5) were identified, mapping to 133 genes (23 with overlapping variant positions) and 103 intergenic sequences. Most SNP-trait associations converged around BCL11A (chr.2), HBS1L-MYB, (chr.6), olfactory receptor and beta globin (HBB) gene clusters (chr.11), with less frequent loci including FHIT (chr.3), ALDH8A1, BACH2, RPS6KA2, SGK1 (chr.6), JAZF1 (chr.7), MMP26 (chr.11), COCH (chr.14), ABCC1 (chr.16), CTC1, PFAS (chr.17), GCDH, KLF1, NFIX, and ZBTB7A (chr.19). Pathway analysis highlighted Gene Ontology (GO) terms and pathways related to olfaction, hemoglobin and haptoglobin binding, and oxygen carrier activity. This systematic review confirms established genetic modifiers of HbF level, while highlighting less frequently associated loci as promising areas for further research. Expanding research across ethnic populations is essential for advancing personalized therapies and enhancing outcomes for individuals with sickle cell disease or β-thalassemia.

Novel Insights into Hb Shaare Zedek Associated with β0-Thalassemia: Molecular Characteristics, Genetic Origin and Diagnostic Approaches.

Hemoglobin Shaare Zedek (Hb SZ) is a rare structural α-Hb variant. Characterizing its genotype-phenotype relationship and genetic origin enhances diagnostic and clinical management insights. We studied a proband and six family members using high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), PCR, and sequencing to analyze α- and β-globin genes and α-globin haplotypes. Pathogenicity predictions and a rapid diagnostic method were developed. The proband, his father, grandfather, and aunt had Hb migrating to the HbH-zone on CE and elevated fetal hemoglobin (HbF) on HPLC. Direct sequencing identified an A to G mutation at codon 56 of the α2-globin gene, characteristic of Hb SZ. Additionally, the proband carried a β-globin gene mutation [HBB.52A>T]. Mild thalassemia-like changes were observed in the proband, whereas individuals with only the Hb SZ variant did not exhibit these changes. Pathogenicity predictions indicated that Hb SZ is benign. The variant can be identified using restriction fragment length polymorphism (RFLP) and allele-specific PCR. The Thai variant of Hb SZ is associated with the haplotype [- - M - - - -]. Hb SZ is a non-pathological variant that minimally affects red blood cell parameters, even when it coexists with β0-thalassemia. HPLC and CE systems cannot distinguish it from other Hbs, necessitating DNA analysis for accurate diagnosis.

Revolutionary breakthrough: FDA approves CASGEVY, the first CRISPR/Cas9 gene therapy for sickle cell disease.

Sickle cell disease (SCD) is a hereditary hemoglobinopathy resulting from a β-globin chain mutation that causes abnormal hemoglobin (HbS) polymerization and leads to severe complications. Current treatment options primarily focus on symptom management, with limited curative potential. Recently, Casgevy, the first CRISPR/Cas9-based gene therapy for SCD, has received breakthrough FDA approval. Clinical trials have shown that Casgevy administered to patients aged older than or equal to 12 years enables precise modifications in hematopoietic stem cells, resulting in elevated fetal hemoglobin (HbF) levels and a significant reduction in vaso-occlusive events. Unlike conventional treatments, this therapy offers a curative approach and eliminates the need for recurrent transfusions and transplants, thereby improving the quality of life of patients with SCD. Casgevy has emerged as a beacon of hope for SCD patients and signifies a potential paradigm shift in SCD management due to its safety, curative potential, and transformative impact, positioning it as a groundbreaking intervention. Nevertheless, ethical considerations surrounding CRISPR technology and regulatory frameworks must be addressed to ensure responsible application and equitable access to this one-time gene editing therapy. As the authors celebrate this scientific advancement, sustained interdisciplinary collaboration and ethical scrutiny are essential to navigating the evolving landscape of CRISPR technology in medicine. This review aims to provide a detailed insight into the application of Casgevy, challenges associated with its application, future prospects of this therapy, and its comparison with existing treatment options for SCD.

CRISPR-Cas9 Editing of the HBG1 and HBG2 Promoters to Treat Sickle Cell Disease

BackgroundSickle cell disease is caused by a defect in the β-globin subunit of adult hemoglobin. Sickle hemoglobin polymerizes under hypoxic conditions, producing deformed red cells that hemolyze and cause vaso-occlusion that results in progressive organ damage and early death. Elevated fetal hemoglobin levels in red cells protect against complications of sickle cell disease. OTQ923, a clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9–edited CD34+ hematopoietic stem- and progenitor-cell (HSPC) product, has a targeted disruption of the HBG1 and HBG2 (γ-globin) gene promoters that increases fetal hemoglobin expression in red-cell progeny.MethodsWe performed a tiling CRISPR-Cas9 screen of the HBG1 and HBG2 promoters by electroporating CD34+ cells obtained from healthy donors with Cas9 complexed with one of 72 guide RNAs, and we assessed the fraction of fetal hemoglobin–immunostaining erythroblasts (F cells) in erythroid-differentiated progeny. The gRNA resulting in the highest level of F cells (gRNA-68) was selected for clinical development. We enrolled participants with severe sickle cell disease in a multicenter, phase 1–2 clinical study to assess the safety and adverse-effect profile of OTQ923.ResultsIn preclinical experiments, CD34+ HSPCs (obtained from healthy donors and persons with sickle cell disease) edited with CRISPR-Cas9 and gRNA-68 had sustained on-target editing with no off-target mutations and produced high levels of fetal hemoglobin after in vitro differentiation or xenotransplantation into immunodeficient mice. In the study, three participants received autologous OTQ923 after myeloablative conditioning and were followed for 6 to 18 months. At the end of the follow-up period, all the participants had engraftment and stable induction of fetal hemoglobin (fetal hemoglobin as a percentage of total hemoglobin, 19.0 to 26.8%), with fetal hemoglobin broadly distributed in red cells (F cells as a percentage of red cells, 69.7 to 87.8%). Manifestations of sickle cell disease decreased during the follow-up period.ConclusionsCRISPR-Cas9 disruption of the HBG1 and HBG2 gene promoters was an effective strategy for induction of fetal hemoglobin. Infusion of autologous OTQ923 into three participants with severe sickle cell disease resulted in sustained induction of red-cell fetal hemoglobin and clinical improvement in disease severity. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT04443907.)

5612617 EFFICACY AND SAFETY OF A SINGLE DOSE OF EXAGAMGLOGENE AUTOTEMCEL FOR TRANSFUSION-DEPENDENT-THALASSEMIA AND SEVERE SICKLE CELL DISEASE

Background: Elevated fetal hemoglobin(HbF) is associated with improved outcomes in patients(pts) with transfusion-dependent β-thalassemia(TDT) and sickle cell disease(SCD). Exagamglogene autotemcel(exa-cel; formerly known as CTX001) is a cell therapy designed to reactivate HbF via non-viral, ex vivo CRISPR/Cas9 gene-editing at the erythroid enhancer region of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells(HSPCs). Early data from the pivotal trials CLIMB THAL-111(NCT03655678) and CLIMB SCD-121(NCT03745287) showed that a single dose of exa-cel increased HbF and total Hb sufficiently to eliminate red blood cell(RBC) transfusions(TDT) and vaso-occlusive crises(VOCs; SCD). Aims: To report efficacy and safety data from the first 75 pts dosed with exa-cel in the ongoing CLIMB THAL-111 and CLIMB SCD-121 trials. Methods: Following pharmacokinetic-adjusted busulfan myeloablation and exa-cel infusion, pts(12-35y) are monitored for engraftment, total Hb, HbF, BCL11A edited alleles, transfusions, VOCs(SCD only), and adverse events(AEs). Data presented as mean(min-max) unless noted. Results: 44 pts with TDT(age 21.3 [12-35] y) and 31 pts with SCD(age 22.5 [12-34} y) had been infused with exa-cel at data cut(follow-up 12.3 [1.2-37.2] mo and 9.6 [2.0-32.3] mo, respectively). 26/44 pts with TDT(59.1%) had β0/β0 or a β0/β0-like genotype(β0/IVS-I-110, IVS-I-110/IVS-I-110). In the 2-yr period before screening, pts with TDT received 36.0(15.0-71.0) units RBC/yr and pts with SCD had 3.9(2.0-9.5) severe VOCs/yr. After exa-cel infusion, all pts engrafted neutrophils and platelets. Median time to neutrophil and platelet engraftment was 29 and 43 days in pts with TDT and 27 and 32 days in those with SCD, respectively. Overall, 42 of 44 pts with TDT stopped RBC transfusions(duration 0.8 to 36.2 mo); 2 pts had not yet stopped transfusions but had 75% and 89% reductions in transfusion volume. By Month 3, increases in HbF and mean total Hb levels(>9 g/dL) were achieved, with mean total Hb levels increasing to >11 g/dL thereafter and were maintained. All pts with SCD(n=31) no longer had severe VOCs after exa-cel infusion(duration 2.0 to 32.3 mo). The mean proportion of HbF was >20% by Month 3, increasing to ~40% at Month 4 and was stable thereafter, with mean total Hb levels >11 g/dL after Month 3. Pts with TDT and SCD with ≥1 yr follow-up had stable proportions of edited BCL11A alleles in bone marrow CD34+ HSPCs and peripheral blood mononuclear cells. Two pts with TDT had serious AEs(SAEs) considered related to exa-cel. The first pt was previously reported. The second pt had delayed neutrophil engraftment and thrombocytopenia, which were considered related to both exa-cel and busulfan. All SAEs resolved. No pts with SCD had SAEs considered related to exa-cel. There were no deaths, discontinuations, or malignancies. Conclusions: Exa-cel infusion led to elimination of transfusions in almost all patients with TDT and elimination of VOCs in all patients with SCD, with associated clinically meaningful increases in HbF and total Hb that were maintained over time. Proportions of CRISPR/Cas9-edited BCL11A alleles remained stable after more than 1 year, indicating that long-term HSCs were successfully edited. The safety profile was generally consistent with busulfan myeloablation and autologous transplant. These results indicate exa-cel has the potential to be the first CRISPR/Cas9-based therapy to provide a one-time functional cure for TDT and severe SCD. Reference 1. Frangoul et al, N Engl J Med 2021; 384:252-260.

Evaluation of local hemoglobinopathy prevalence and promotion of accurate hemoglobin A1c testing using historical data retrieval

ObjectivesHeterozygous hemoglobin variants are known to cause method- and variant-specific interference with hemoglobin A1c (HbA1c) quantitation. Less attention has been paid to the role of other hemoglobin variants in confounding HbA1c testing. Here we evaluated the frequency with which enzymatic (ENZ) and immunoassay (IA) HbA1c quantitation methods, i.e., those unable to routinely detect the presence of hemoglobin variants, were used within our healthcare system for HbA1c analysis in patients with elevated fetal hemoglobin as well as compound heterozygous and homozygous variants. Design & MethodsThis analysis was enabled by automated review of HbA1c result history, implemented to promote detection of variants prior to HbA1c result reporting. ResultsDuring a 54-week period, 319,290 HbA1c analyses were performed. We observed 110 unique patient cases (0.03% problem identification rate) in which HbA1c testing was ordered in the presence of either a homozygous or compound heterozygous hemoglobin variant or elevated hemoglobin F beyond the tolerance of the method. Among the 110 cases identified, 55 (50%) showed a compound heterozygous or homozygous hemoglobin variant while 55 (50%) showed elevated hemoglobin F. Of those cases involving a compound heterozygous or homozygous variant, 8/55 (15%) involved patients who had one or more ENZ or IA HbA1c results reported previously within our system. Of the 55 total compound heterozygous or homozygous variants identified, 37 (67%) were hemoglobin E, 10 (18%) hemoglobin S/C, 4 (7%) hemoglobin S, 2 (4%) hemoglobin C, 1 (2%) hemoglobin Camden, and 1 (2%) unidentified variant. ConclusionsExclusive use of methods unable to routinely detect the presence of hemoglobin variants may lead to reporting of HbA1c results that are not clinically meaningful.

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

Efficacy and Safety of a Single Dose of Exagamglogene Autotemcel for Severe Sickle Cell Disease

Background: Elevated fetal hemoglobin (HbF) is associated with improved outcomes in patients with sickle cell disease (SCD). Exagamglogene autotemcel (exa-cel; formerly known as CTX001) is a cell therapy designed to reactivate HbF via non-viral, ex vivo CRISPR/Cas9 gene-editing at the erythroid enhancer region of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells (HSPCs). Recent data from the pivotal CLIMB SCD-121 (NCT03745287) trial showed that a single dose of exa-cel increased HbF and total hemoglobin (Hb) sufficiently to eliminate vaso-occlusive crises (VOCs) in patients with severe SCD. Here, we report efficacy and safety data from the first 31 patients dosed with exa-cel in the ongoing CLIMB SCD-121 trial. Methods: Patients aged 12 to 35 years with severe SCD and a history of ≥2 VOCs per year in the previous 2 years before screening were eligible. Following pharmacokinetic-adjusted, busulfan myeloablation and infusion of exa-cel, patients are monitored for engraftment, total Hb, HbF, BCL11A-edited alleles, transfusions, VOCs, and adverse events (AEs). The primary endpoint is the proportion of patients who have not experienced a severe VOC for at least 12 months after the infusion of exa-cel, starting 60 days after their last RBC transfusion. Updated efficacy and safety results on all dosed patients will be included in the presentation. Data are reported as mean (min-max), unless noted. Results: At the most recent data cut in February 2022, 31 patients with SCD (age 22.5 [12-34] years) had been infused with exa-cel (follow-up 9.6 [2.0-32.3] months), of whom 6 (19.4%) were between the ages of 12 and <18 years and 29 (93.5%) had the βs/βs genotype. In the 2-year period before screening, patients experienced 3.9 (2.0-9.5) severe VOCs per year. After exa-cel infusion, all patients engrafted neutrophils and platelets with a median time of 27 and 32 days, respectively. All patients were VOC-free at the time of the data cut (duration of follow-up 2.0-32.3 months after exa-cel infusion; Figure). Median time from exa-cel infusion to last RBC transfusion was 19 (11-52) days. The mean proportion of HbF was >20% by Month 3, with mean total Hb levels >11 g/dL on and after Month 3. All 11 patients who have at least 12 months of follow-up after exa-cel infusion have maintained HbF levels >20% while experiencing no VOCs. At Month 6, the mean proportion of edited BCL11A alleles in bone marrow CD34+ HSPCs and peripheral blood mononuclear cells was 86.6% and 76.0%, respectively. These proportions remained stable in all patients who had ≥1 year of follow-up (Figure). There were no patients who had serious AEs considered related to exa-cel. There were no deaths, discontinuations, or malignancies. Conclusions: Exa-cel infusion led to the elimination of VOCs in all patients with SCD, with associated clinically meaningful increases in HbF and total Hb that were maintained over time. Proportions of CRISPR/Cas9-edited BCL11A alleles have remained stable after ≥1 year, indicating that long-term HSCs were successfully edited. The safety profile was generally consistent with that of busulfan myeloablation and autologous transplant. Exa-cel has the potential to be the first CRISPR/Cas9-based therapy to provide a one-time functional cure for SCD. Figure. Clinical data for patients with SCD (N=31) infused with exa-cel. Patients were VOC-free after exa-cel infusion. Time (months) since exa-cel infusion is indicated by the dark blue bar. Inset depicts the percentage of BCL11A-edited alleles in bone marrow CD34+ HSPCs and peripheral blood mononuclear cells of patients with SCD infused with exa-cel over time. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Examining the Role of Individual Baf Chromatin Remodeling Complex Components in Globin Gene Regulation

Regulation of the switch from fetal (HbF, α2γ2) to adult (HbA,α2β2) hemoglobin production is an important area of study as reversal of the switch is a key therapeutic approach for treatment of sickle cell disease and β-thalassemia. While multiple specific repressors of γ-globin gene transcription have been identified, there has not been a parallel finding of a γ-globin specific transcriptional activator. The mammalian BAF (BRG1/BRM-associated factor) complex is a multi-subunit, ATP-dependent, chromatin remodeling complex of the SWI/SNF family, with diverse composition and roles in regulation of gene expression. BRG1/SMARCA4 is a core component of the complex with ATPase catalytic activity. Prior studies showed that BRG1 is essential for proper β-globin expression and regulates long-range chromatin interactions between the locus control region (LCR) and the β-globin gene promoter. Studies in K562 cells, where γ-globin expression is predominant, have similarly shown the requirement for BRG1 for transcriptional activation. To date, the role of the BAF complex in regulation of γ-globin versus β-globin gene expression has not been well characterized. Moreover, the considerable diversity in the subtypes of this complex (such as polybromo-associated BAF or PBAF and non-canonical BAF), with subunit composition and functions that differ across cell types and developmental stages, suggests the hypothesis that specific subtypes or subunits of the BAF complex have specific roles in γ-globin gene regulation. We recently identified BRG1 as a potential activator of γ-globin production in a HUDEP2 CRISPR screen targeting ATPase and helicase domains. In validation experiments in HUDEP2 and primary human erythroid cultures, loss of BRG1 led to a marked decrease in γ-globin containing F-cells by flow cytometry and γ-globin protein by western blot, along with a smaller decrease in β-globin production. Because the BAF subunits have significant heterogeneity in expression and function, we asked whether any components may have specificity for γ-globin versus β-globin gene transcription in adult erythroid cells. Using CRISPR-Cas9 targeting in a HUDEP2 sub-clone with elevated fetal hemoglobin levels, we systematically ablated 12 individual BAF/PBAF complex components and assayed fetal hemoglobin by flow cytometry, HPLC, and quantitative western blots. We identified 5 components whose loss of function led to a reduction in γ-globin relative to β-globin: BAF53A, BAF155, BAF60A, BAF60B, and BAF57. We subsequently depleted these five components in primary human erythroid cultures from three independent donors using CRISPR-Cas9 targeting. While loss of BAF53A impaired cell proliferation, loss of remaining components was well tolerated. Loss of BAF155 and BAF60B had minimal effect on globin chain production. Loss of BAF60A had the most potent effect on γ-globin, with up to 80% reduction in γ-globin with no decrease in β-globin production. BAF57 knockdown led to a more modest decrease in γ-globin, but with some decrease in total globin production. Together these data suggest that the requirements for some specific components of the BAF complex differ for activation of fetal or adult globin gene transcription. Additional studies are under way to characterize chromatin accessibility changes at the β-globin locus in response to alterations in the BAF complex, as well as the response to loss of additional BAF complex components.

COVID-19 Outcomes at a Comprehensive Sickle Cell Center: A Two-Year Report

The Georgia Comprehensive Sickle Cell (SC) center at Grady health system is the largest adult comprehensive sickle cell center in the United States and has the first 24/7 acute care unit for management of sickle cell vaso-occlusive events (VOE). in 2019, the center provided around 3000 sickle cell clinic visits and 4000 acute care visits. When the COVID-19 pandemic reached the US, the SC center had seen a precipitous drop in the number of both clinic and acute care visits as local and state regulations for lockdown were passed. On the two-year anniversary for the COVID-19 pandemic, we are submitting this report of all the COVID-19 cases at a single adult center for sickle cell disease. Since the start of the pandemic, we have been tracking any COVID-19 cases reported in the patient population the SC center follows. Out of 1343 patients currently following in the center, 173 patients contracted COVID-19 between March 2020 and February 2022, and were tracked in the clinical database under IRB approval. The results show that of the 173 patients, 85 were female and 89 were male. By genotype, 62% of patients were SS, 29% were SC and 9% were Sβ+ thalassemia or Sβ0 thalassemia. 32% of patients were on hydroxyurea for disease modification with the majority of them being of the SS genotype and 45% had elevated fetal hemoglobin determined by a percentage fetal hemoglobin above 5% by hemoglobin electrophoresis. Chronic pain was defined in the patients experiencing daily pain episodes for more than 4 days a week for the last 3 months and calculation of daily morphine equivalents was reported based on the clinic follow-ups and the narcotic database utilization. 35% of the SC patients had chronic pain and the median morphine equivalents was 90 mg daily with a range between 45 and 225mg. On evaluating the rate of emergency department (ED) visits or hospitalizations for the sickle cell patients with COVID-19, 85% of patients had either an ED visit or hospitalization. 54% of the SC patients had the reason for the visit related to VOE and 29% had the reason related to COVID-19 primarily. 15% of SC patients were not seen in any emergency setting or required any hospitalization. 22% of the patients had lung findings on imaging. The treatment utilized for the patients was most commonly Remdesivir in 33, dexamethasone in 26, red cell products in 21 and monoclonal antibody in 13 patients. Of the 3 deaths reported, 2 happened early in the pandemic. One patient had presented in June 2020 with VOE and was found to have bilateral lung opacities but was asymptomatic and was discharged home to return few days later with clinical picture of multi-organ failure (MOF) for which a red cell erythrocytapheresis was attempted to no avail. The second patient had presented in July 2020 with COVID-19 pneumonia and was treated with Remdesivir and convalescent plasma to no avail after development of MOF and ARDS. The third death occurred in August 2021 during the delta variant wave and the patient was hospitalized for COVID-19 pneumonia and had initial improvement after initial treatment but after worsened lung findings, ARDS developed followed by MOF. Several reports from different centers have been published regarding the rate of COVID-19 related mortality and morbidity in sickle cell disease. The Grady comprehensive sickle cell center experience differs in the fact that 63 out of 173 (36%) patients who had contracted COVID-19 required treatment and 3 of those 63 had died. In fact, the deaths occurred early in the course of the pandemic in June and July 2020 respectively and since then, the rate of complicated COVID-19 hospitalizations has decreased with better treatment available. No deaths were registered during the omicron wave and 1 death was registered during the delta wave. In addition, the timeline for the COVID-19 cases reported fits the population timeline of peaks respectively happening in the summer of 2020, winter of 2021, summer of 2021 and winter of 2021. During the initial peak, we have noted a decrease in the number of clinic and acute care visits respectively. This was anticipated given the statewide lockdown that was implemented. To circumvent that, the SC center resorted to virtual visits utilization to deliver the needed healthcare visits to the patients. This measure has aided in protecting patients against COVID-19 during the multiple COVID-19 peaks occurring in the last 2 years and has accordingly reduced the mortality and morbidity of the patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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

Genetic studies identify common variants within the HBS1L-MYB intergenic region (HMIP), BCL11A, and Xmn1-HBG2 as associated with elevated fetal hemoglobin (HbF) levels and other clinically important human hematological traits. Recent studies suggest HbF is a predictor of outcome in MDS/AML patients receiving decitabine. We assessed effects of HbF genetic variants on hematological traits in Myeloproliferative Neoplasm (MPN), Myelodysplastic Syndrome (MDS) and myeloma on HbF-inducing therapy to determine potential for variants predicting treatment response. Seven common HbF variants at HMIP, BCL11A, Xmn1-HGB2 loci were genotyped in 89 patients with MPN on Hydroxyurea (HU), myeloma on Lenalidomide, and MDS on Azacytidine. HbF genetic association was seen with rs9494142 (HMIP) in MPN on HU (p = 0.04) and rs1427407 (BCL11A) in myeloma on Lenalidomide (p = 0.002). HMIP variants rs9494142 and rs6920211 influenced baseline platelets (p = 0.04) and hemoglobin treatment response (p = 0.02). rs1427407 (BCL11A) was significantly associated with increased platelets (p = 0.04) negating thrombocytopenic tendency of Lenalidomide. These HbF variants showed significantly discordant minor allele frequencies in MDS/MPN/myeloma compared to wider European population data. This small study finding together suggest the implication of these variants in treatment response and disease biology in MDS/MPN/myeloma warranting larger prospective genotype-phenotype association studies.

Fetal hemoglobin regulating genetic variants identified in homozygous (HbSS) and heterozygous (HbSA) subjects from South Mexico.

Hemoglobin S is caused by a nucleotide change in HBB gene (HBB:c.20A&gt;T, p.Glu6Val), is presented in diverse forms: simple carriers (HbSA), homozygotes (HbSS) also known as sickle cell anemia, and compound heterozygotes with other β-hemoglobinopathies. It is worldwide distributed, in Mexico, is frequently observed in the southern states Guerrero, Oaxaca and Chiapas. Elevated fetal hemoglobin (HbF) is associated with mild phenotype; single-nucleotide variants (SNVs) in modifier genes, such as BCL11A, HBG2, HBBP1 pseudogene and HBS1L-MYB intergenic region, upregulate HbF synthesis. The aim of this study was to identify HbF regulating genetic variants in HbSS and HbSA Mexican subjects. We studied 39 individuals (HbSS = 24, 61%, HbSA = 15, 39%) from Chiapas (67%) and Guerrero (33%), peripheral blood was collected in ethylenediamine tetraacetic acid (EDTA) for molecular and hematological studies, DNA was isolated by salting-out technic and genotyping was performed through allelic discrimination by real time polymerase chain reaction (RT-PCR) using Taqman® probes for 15 SNV (in BCL11A: rs6706648, rs7557939, rs4671393, rs11886868, rs766432, rs7599488, rs1427407; HBS1L-MYB: rs28384513, rs7776054, rs9399137, rs4895441, rs9402686, rs1320963; HBG2: rs7482144; and HBBP1: rs10128556). The obtained data were analyzed using IMB SPSS v.22.0 software. All minor alleles were observed in frequencies over 0.05, the most frequent was rs9402686 (0.82), while the less frequent was rs101028556 (0.08). In HbSS group, the mean fetal hemoglobin was 11.9 ± 5.9% and was significantly elevated in BCL11A rs11886868 wildtype homozygotes and in carriers of HBS1L-MYB intergenic region rs7776054 (p = 0.04 and p = 0.03, respectively). In conclusion, in HbSS Mexican patients, two SNVs were observed related to increased HbF; BCL11A rs11886868 and HBS1L-MYB rs7776054.

A Rare PTPN11 Mutation in a Patient with Juvenile Myelomonocytic Leukemia: A Case Report

Background: Juvenile myelomonocytic leukemia (JMML) is a rare childhood disease characterized by hepatosplenomegaly, monocytosis, anemia, increased white blood cell count, thrombocytopenia, skin infiltration, and elevated fetal hemoglobin. Mutation in specific genes, including KRAS, NRAS, PTPN11, and NF1, can lead to the development of JMML. Case Presentation: A two-year-old boy with a history of inguinal abscess at the age of 12 months and surgery due to infectious lymphadenitis was referred to the hospital. His parents also reported a history of oral candidiasis, recurrent otitis media, and lymphadenopathy in the patient. The physical examination showed splenomegaly, macular rash, lymphadenopathy in the neck region, and rashes in the inguinal region and on the hands and feet. Laboratory and flow cytometry data showed lymphocytosis, low hemoglobin, thrombocytopenia, monocytosis, eosinophilia, and a shift to the left in the peripheral blood. The bone marrow aspiration showed a cellular marrow with myeloid hyperplasia. Whole-exome sequencing revealed a rare heterozygous ENST00000351677.2:c.1508G&gt;C, p.Gly503Ala variant in PTPN11. The patient was diagnosed with JMML but, unfortunately, passed away. Conclusion: We report a rare heterozygous mutation in the PTPN11 gene in a two-year-old boy diagnosed with JMML. This uncommon mutation should be considered in the mutational screening protocol of JMML. Management of JMML with RAS pathway targeted therapy may also have promising results and needs further investigations.

The Participation of the Organism Adaptation Mechanisms to the Lack of Oxygen According to the Assessment of the Fethemoglobin Content in the Peripheral Blood of Patients with Community-acquired Pneumonia

Introduction. Сommunity-acquired pneumonia (COP) is a global socio-medical problem. At emergence of pneumonia by any genesis, hypoxia develops. Oxygen homeostasis of the body is provided by the coordinated interaction of external respiration, circulatory system and oxygen-transport system of the blood. Hypoxia, due to the malfunction of the external respiratory system, causes the formation of compensatory changes, in the implementation of which involved components of the oxygen transport system. Molecular genetic mechanisms play an important role in the body's adaptation to oxygen deficiency. Fetal hemoglobin (FetHb), having an increased affinity for oxygen, makes a significant contribution to the body's adaptation to new conditions with altered gaseous environment in the presence of pathological processes occurring with hypoxia. In this regard, it is interest to determine FetHb in adults with COP to study its effect on the diagnosis, prognosis and outcome of the disease. The aim of the study. To determinate the participation of the organism adaptation mechanisms to the lack of oxygen according to the assessment of the content of fetal hemoglobin in the peripheral blood of patients with community-acquired pneumonia. Materials and methods. We examined 34 adult patients (18 women and 16 men) with COP, aged 18 to 80 years, who were in the therapeutic department of the City Clinical Hospital № 25 in Kharkiv. The control group was formed of 20 healthy individuals. Spirography was performed on the diagnostic complex "Valenta"; hematological examinations – on the analyzer "ADVIA 60"; measurement of pO2 and pCO2, oxygen saturation, content of fetal hemoglobin – on the device "RAPIDLAB865". Results. In patients with community-acquired pneumonia, there was a decrease of the ventilatory function of external respiration, which is confirmed by a marked decrease in partial oxygen pressure. Oxygen saturation of blood was reduced in the group of patients with COP, but the difference was not statistically significant 94.8 ± 1.0 %. This indicates the presence of compensatory mechanisms aimed at maintaining adequate blood oxygen saturation. Significant increase in pH (from 7.40 to 7.53) and decrease in standard bicarbonate (from 1.27 to 0.68 mmol/l) resulting from violation of the gas composition of the blood can be regarded as a manifestation of partially compensated respiratory alkalosis. In patients with COP, there was a reduction in the total time of hemolysis, a shift of the maximum erythrogram to the left and an increase in the maximum itself, indicating a sharp decline in erythrocyte resistance. The proportion of erythrocytes with reduced resistance was twice as large as similar forms in the control group and the number of highly resistant cells in patients with COP sharply decreased. Obviously, oxygen starvation-mediated stress erythropoiesis is accompanied by the entry into the circulation of functionally defective erythrocytes. They are subject to accelerated elimination from the vascular bed, which causes a decrease in the quantitative indicators of red blood (erythrocyte content, hemoglobin) while maintaining corpuscular parameters (Mean Corpuscular Volume, Mean Cell Hemoglobin Concentration). At the same time, the analysis of individual hemoglobin fractions revealed an increase in the proportion of fetal hemoglobin (from 2.90 ± 0.31 % in the group of healthy individuals to 5.43 ± 1.05 % in patients with COP) (p less than 0.05). Conclusions. Changes in the parameters of acid hemolysis, fetal hemoglobin in the peripheral blood of patients with community-acquired pneumonia with impaired pulmonary ventilation function indicate their participation in the mechanisms of adaptation to oxygen deficiency and they have informative potential. Elevated fetal hemoglobin in peripheral blood in these patients can be used as an indicator of hypoxia, accompanied by impaired oxygen delivery to tissues, which should be used as an additional criterion for diagnosing tissue hypoxia and justify the timely appointment of antihypoxia drugs. Keywords: hypoxia, community-acquired pneumonia, red blood cells, fetal hemoglobin.

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.

COVID-19 Infection and Outcomes at a Comprehensive Sickle Cell Center

Introduction: The Grady Comprehensive Sickle Cell (SC) center is the largest adult sickle cell center in the United States (US) and has the first 24/7 acute care unit for management of sickle cell vaso-occlusive events (VOE). In 2019, the center provided 3077 sickle cell outpatient visits and 3695 acute care visits. When the COVID-19 pandemic reached the US, the center had a precipitous drop in the number of both outpatient clinic and acute care visits as state regulations for lockdown were passed. This report follows all the COVID-19 cases at a single adult center for sickle cell disease in one year.Methods: The clinical database has been tracking COVID-19 cases reported. Out of a total of 1343 patients, 55 patients contracted COVID-19 and were tracked in the clinical database with IRB approval.Results: Of the 55 patients with COVID-19, 28 were female and 27 were male. By genotype, 64% of patients were SS, 31% were SC and 5% were Sβ+ thalassemia. 35% of patient were on hydroxyurea for disease modification with the majority of them being of the SS genotype and 31% had elevated fetal hemoglobin determined by a percentage fetal hemoglobin above 5% by hemoglobin electrophoresis. Chronic pain (defined as patients experiencing daily pain episodes for more than 4 days a week for the last 3 months) and calculation of daily morphine equivalents were reported in clinic follow-up and the narcotic database utilization. 47% of the patients had chronic pain and the median morphine equivalents was 90 mg daily (45-225mg).The rate of emergency department (ED) visits or hospitalizations for the sickle cell patients with COVID-19 was 80%. 49% of the SC patients' visits were related to VOE and 27% related to COVID-19 primarily. 20% of SC patients with COVID-19 were not seen in any emergency setting or required any hospitalization.The COVID-19 signs and symptoms experienced by the patients were as follows: 58% had pain as the main presenting symptom, followed by cough and fever (40%), dyspnea (31%), and pneumonia with chest x-ray evidence (25%). 2 patients developed acute respiratory distress syndrome (ARDS) and were intubated, and 2 patients died. 29% of the patients had lung findings on imaging and 16 of 55 patients required treatment with the use of Remdesivir in 9, dexamethasone in 8 and red cell products in 7 of the 16 patients. The 2 patients who died had both presented with COVID-19 infection in June and July 2020 respectively. One patient had presented in June 2020 with VOE and was found to have bilateral lung opacities but was asymptomatic and was discharged home to return few days later with clinical picture of multi-organ failure for which a red cell erythrocytapheresis was attempted. The second patient had presented in July 2020 with COVID-19 pneumonia and was treated with Remdesivir and convalescent plasma with development of multi-organ failure and ARDS.Discussion:Several reports were published regarding the rate of COVID-19 related mortality and morbidity in sickle cell disease. The Grady comprehensive sickle cell center experience differs in the fact that 16 out of 55 patients who had contracted COVID-19 required treatment and 2 of those 16 had died. In fact, the deaths occurred early in the course of the pandemic in June and July 2020 when 20 total cases were diagnosed (from March to Septemeber 2020). The remaining 35 cases registered zero deaths (October 2020 to March 2021) with the rate of complicated COVID-19 hospitalizations decreasing with better treatment available. In addition, the timeline for the COVID-19 cases reported fits the population timeline of 2 peaks respectively happening in the summer of 2020 and the Winter of 2021. During the initial peak, we have noted a decrease in the number of clinic and acute care visits respectively. This was anticipated given the statewide lockdown that was implemented. To circumvent that, the center adopted virtual visits to deliver healthcare needs. This measure has aided in protecting patients against COVID-19. Additionally, it is interesting that despite the second peak in the winter of 2021, there were no reported deaths among the patients who developed COVID-19. This finding can suggest that despite the concern for morbidity and mortality of sickle cell patients, their diligence and awareness to stay home during the pandemic has proven crucial in reducing morbidity and mortality and the option of virtual visits for healthcare delivery was key and should be utilized further in sickle cell care. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Updated Results of a Phase 1/2 Clinical Study of Zinc Finger Nuclease-Mediated Editing of BCL11A in Autologous Hematopoietic Stem Cells for Transfusion-Dependent Beta Thalassemia

Updated Results of a Phase 1/2 Clinical Study of Zinc Finger Nuclease-Mediated Editing of BCL11A in Autologous Hematopoietic Stem Cells for Transfusion-Dependent Beta Thalassemia

Heterozygous variants in ZBTB7A cause a neurodevelopmental disorder associated with symptomatic overgrowth of pharyngeal lymphoid tissue, macrocephaly, and elevated fetal hemoglobin

By clinical whole exome sequencing, we identified 12 individuals with ages 3 to 37 years, including three individuals from the same family, with a consistent phenotype of intellectual disability (ID), macrocephaly, and overgrowth of adenoid tissue. All 12 individuals harbored a rare heterozygous variant in ZBTB7A which encodes the transcription factor Zinc finger and BTB-domain containing protein 7A, known to play a role in lympho- and hematopoiesis. ID was generally mild. Fetal hemoglobin (HbF) fraction was elevated 2.2%-11.2% (reference value <2% in individuals > 6 months) in four of the five individuals for whom results were available. Ten of twelve individuals had undergone surgery at least once for lymphoid hypertrophy limited to the pharynx. In the most severely affected individual (individual 1), airway obstruction resulted in 17 surgical procedures before the age of 13 years. Sleep apnea was present in 8 of 10 individuals. In the nine unrelated individuals, ZBTB7A variants were novel and de novo. The six frameshift/nonsense and four missense variants were spread throughout the gene. This is the first report of a cohort of individuals with this novel syndromic neurodevelopmental disorder.

Study of Xmn-I polymorphism in β-thalassemic children in Egypt

Background β-thalassemia (βT) has a wide spectrum of clinical severity that may be attributed to the wide variations in βT gene mutations. β-Globin mutations with the Xmn-I site might be associated with elevated fetal hemoglobin levels, which in turn may affect the severity of βT phenotype. Aim To investigate the frequency of Xmn-I polymorphism (−158 C&gt;T) among Egyptian children with βT, and to examine the relationship between Xmn-I polymorphism and βT phenotypes. Patients and methods This cross-sectional study was conducted on 112 βT patients (55 males and 57 females) with a mean age of 8.34±3.71 years (2–16 years). Laboratory investigations included complete blood count, hemoglobin electrophoresis, β-globin mutation, identification of Xmn-I polymorphism by two methods: PCR–restriction fragment length polymorphism and amplification refractory mutation system. Results All patients (76 with βT major and 36 with βT intermedia) were negative for the Xmn-I SNP whether in homozygous (+/+) or heterozygous (+/−) state. Conclusion This study demonstrated that Xmn-I polymorphism was not detected in the studied patients and this supports the low frequency of this polymorphism in other Egyptian studies.