Beta-globin Gene Mutations Research Articles

Reemergence of Hemoglobin Yukuhashi

Abstract The abnormal hemoglobin, Hb Yukuhashi, identified in Japan in 1963, exhibits slower migration compared to Hb A and is associated with target cells on peripheral smear. A similar hemoglobin, Hb Dhofar, is found in an Omani tribesman and is defined as a substitution of arginine for proline at the 58th amino acid position of the beta-globin chain. Despite both Yukuhashi and Dhofar seemingly arising from the same exonic point mutation, variations in expression were clarified through beta-globin gene amplification. Hb Dhofar, unlike Yukuhashi, was linked to a splice-site mutation at codon 29, explaining its reduced expression. Despite these hemoglobins’ structural alterations, their functional properties on a molecular level, including oxygen affinity and heme-heme interaction, remained largely unaffected. While mutations at codon 58 have been observed alongside other mutations in Hb Dhofar and Hb C-Ziguinchor, this case represents the first sequencing and report of a beta-globin gene mutation solely at codon 58, as only Yukuhashi’s protein coding region had been sequenced, not the whole gene. “Hemoglobin D” was detected on newborn screening of a male child with no other significant medical history or physical abnormalities. Capillary Electrophoresis and Cation-Exchange HPLC showed abnormal hemoglobin fractions, with 20.1% in the S Zone and 25.7% in the D-window, respectively. This led to a presumptive diagnosis of Hemoglobin Dhofar trait; however, discrepancies in hematologic parameters and relative hemoglobin levels were noted. Genetic testing of the Beta Globin gene confirmed a C to G substitution at codon 58 but lacked the characteristic codon 29 mutations associated with Hemoglobin Dhofar. The patient, was discovered to possess a mutation at codon 58 on the beta globin gene, a mutation not previously reported in isolation. In contrast to previous studies on Hb Dhofar, where variant Hb levels were lower and accompanied by abnormal RBC indices indicative of beta thalassemia trait, this case exhibited higher variant Hb levels with normal RBC indices, suggestive of a typical beta globin variant. The mutation at codon 58 did not significantly alter intermolecular interactions of the globin which may mean the variant is clinically asymptomatic in nature. Continued follow-up is necessary to confirm this suspicion, although initial assessments at 3 and 6 months of age showed normal RBC parameters. This case represents the first instance of an isolated mutation at codon 58 of the beta globin gene, emphasizing the need to consider this mutation in interpreting hemoglobinopathy screening results.

Beta globin genemutation in beta thalessemia can be treated with stem cell therapy followed by circulating innate immune cells transfusion

Beta globin genemutation in beta thalessemia can be treated with stem cell therapy followed by circulating innate immune cells transfusion

Alpha globin gene alterations modifying the phenotype of homozygous beta thalassaemia.

The phenotype of β-thalassemia varies widely. The primary determinant is the type of beta-globin gene mutation; however, there are secondary and tertiary modifiers also as associated alpha mutations, polymorphisms, as well as coinheritance of mutations affecting other related systems. Co-inheritance of alpha thalassemia mutations is known to ameliorate the severity of HbE-β thalassemia. However, the role of alpha globin gene alterations (deletions and triplication) is not well illustrated in homozygous β-thalassemia. Here we evaluated the role of alpha globin gene alterations in 122 β-thalassemia patients having IVS1-5 (G>C) homozygous mutation. β-thalassemia mutations were detected by ARMS PCR and alpha mutations by GAP-PCR. Gene expression by qRT-PCR. Out of 122 cases, 15 patients had alpha 3.7 triplications (ααα3.7anti), 24 had alpha 3.7kb deletion (-α3.7) mutation and three patients had 4.2kb deletion (-α4.2). Patients were divided into two groups, requiring less than 8 units (NTDT) and more than 8 units (TDT) of blood transfusion per year (≥8U BT/year). The percentage of alpha deletion was significantly (p=0.0042) high in NTDT (42.1%) as compared with TDT (13.2%). Conversely, the proportion of alpha triplication is high in the TDT as compared with NTDT. Even mean serum ferritin level was found to be significantly high in patients having alpha triplication as compared with those having alpha deletions (p=0.0184) and normal alpha gene (p=0.0003). α/β globin ratio was highest in TDT patients with alpha triplication and lowest in NTDT patients with alpha-del. The results show that concurrent inheritance of alpha gene alterations influences the phenotypic severity of homozygous β-thalassemia.

Genotype-Phenotype Profile of Beta-thalassemia.

Beta thalassemias are extremely heterogenous hereditary monogenic blood disorders and preventable genetic hemolytic anemia caused by >200 mutations in HBB gene. In Nepal, it is more prevalent in Tharu tribe but it seen in other communities as well. Out of more than 200 mutations of beta globin gene, approximate 20 different alleles are responsible for >80% of the mutations. Mutations vary in different geographic population and are responsible for manifestation of different phenotypes. This study was done to find common mutations of HBB gene in Nepal which were responsible for different phenotypic manifestations and to know clinical severity according to the mutations. This was a descriptive, cross sectional study conducted in the pediatric and medicine department of Nepalgunj Medical College and Bheri Zonal Hospital, Nepalgunj from January 2020 to December 2020. The genotype and phenotype profiles of thalassemia cases were reported. The data was analyzed by SPSS 20. The results obtained showed that clinical presentation differed with different β-globin gene mutations present. Individuals with HBB:c.47G>A and HBB:c.20A>T/ c.79G>A mutations showed milder presentation than those with HBB:c.47G>A/-619del and HBB:c.20A>T/c.47G>A. Therefore, these findings can be used to predict clinical severity so that we can take appropriate measures by early genotype identification for prenatal diagnosis of beta thalassemia.

Multi-Organ Morphological Findings in a Humanized Murine Model of Sickle Cell Trait.

Sickle cell disease (SCD) is caused by the homozygous beta-globin gene mutation that can lead to ischemic multi-organ damage and consequently reduce life expectancy. On the other hand, sickle cell trait (SCT), the heterozygous beta-globin gene mutation, is still considered a benign condition. Although the mechanisms are not well understood, clinical evidence has recently shown that specific pathological symptoms can also be recognized in SCT carriers. So far, there are still scant data regarding the morphological modifications referable to possible multi-organ damage in the SCT condition. Therefore, after genotypic and hematological characterization, by conventional light microscopy and transmission electron microscopy (TEM), we investigated the presence of tissue alterations in 13 heterozygous Townes mice, one of the best-known animal models that, up to now, was used only for the study of the homozygous condition. We found that endothelial alterations, as among which the thickening of vessel basal lamina, are ubiquitous in the lung, liver, kidney, and spleen of SCT carrier mice. The lung shows the most significant alterations, with a distortion of the general tissue architecture, while the heart is the least affected. Collectively, our findings contribute novel data to the histopathological modifications at microscopic and ultrastructural levels, underlying the heterozygous beta-globin gene mutation, and indicate the translational suitability of the Townes model to characterize the features of multiple organ involvement in the SCT carriers.

Rare mutations in the beta-globin gene and their clinical phenotypes

Objectives: Beta-thalassemia is one of the most common inherited genetic disorders and the repertoire of mutations in the beta-globin gene is ever-expanding. Sequencing for beta-globin gene mutations, is done, where phenotype-high-performance liquid chromatography discrepancies exist or where amplification refractory mutation system – polymerase chain reaction (ARMS-PCR) cannot identify common mutations, and often leads to the discovery of rare and novel mutations. Materials and Methods: This is a retrospective data analysis of 160 patients of beta-thalassemia and other hemoglobinopathies where some patients were found to have unexplained clinical features. Comprehensive genetic diagnosis was done on these patients by ARMS-PCR, gap-polymerase chain reaction, and sequencing. Results: Out of the total, 124 cases were homozygous/compound heterozygous for beta-thalassemia; 26 cases had heterozygous beta mutations with coexistent alpha-triplications and four patients (with unique clinical features) were found to harbor five rare mutations. The mutations detected were hemoglobin (Hb) Monroe (co-occurring with beta nt-42 mutation), beta-globin mutation −90(C>T), Hb Randwick, and Hb-M-Saskatoon (a variant hemoglobin causing methemoglobinemia and cyanosis). The spectrum of common mutations detected, in our study, was similar to that published in the literature. The unique clinical features of the patients were conclusively explained by the sequencing results. Conclusion: This study emphasizes the role of sequencing in the genetic diagnosis of beta-thalassemia. As next-generation sequencing increasingly finds use in routine diagnostics, newer clinically significant mutations will continue to be added to the large palette of mutations in beta-thalassemia.

Impact of hydroxyurea on psychological health of beta thalassemia patients with moderating role of healthcare professionals’ performance

Background: Beta –thalassemia (β- thalassemia) has more than 300 different mutations of beta globin gene. The complexity of its physical composition is caused by the imbalance ratio of β-globin which classified into blood transfusion- dependent (TDT) and non-transfusion dependent (NTDT). Modern treatment approaches considered for TDT patients is monthly transfusions to maintain hemoglobin (Hb) levels ranges between 9-10 g/dL. The objectives of this study are to seek out the impact of hydroxyurea (HU) on improving psychological health problems of Quality of Life (QoL), patients’ satisfaction with the use of HU and moderating role of healthcare professionals in satisfying the gap between the use of medicine in managing β- thalassemia disease in a tertiary care hospital. Methods: A cross-sectional, non-probability study and purposive- judgment sampling was conducted after ethical approval. The World Health Organization (WHO) sample size calculator 2.0 was used to recruit TDT patients on HU therapy while visiting the outpatient (OPD) and daycare department of National Institute of Blood Diseases & Bone Marrow Transplantation (NIBD & BMT) hospital in Karachi. A well -structured electronic questionnaire as web form was used for data collection by using Likert scale, with a range of 1-5 and 1-7. The data was analyzed using IBM Statistical Package for Social Sciences (SPSS) version 23.0 and moderation process done by using Process Hayes for SPSS version 3.5.2. Results: We enrolled 290 patients. This study revealed that the impact of HU on improving the psychological health problems of β- thalassemia patients are not evident (r=0.078; p=0.185). Similarly, by inducing moderating role of healthcare professional performance, the impact of HU on psychological health problems were a weak positive non- significant with (r=0.050; p=0.397) correlation observed. Moreover, no significant improvement was observed in psychological health problems of QoL by moderating role of healthcare professional performance (p=0.0835 and 0.6764, respectively). Conclusion: The psychological health problems in β- thalassemia patients are not dependent on HU therapy to have better QoL. In addition, the introduction of healthcare professionals' performance as a moderator does not improve the correlation between HU and psychological domain of QoL.

Association of Predisposing Haplotype Snp-Markers of HBB Gene in Thalassaemia

Most of the beta thalassaemia cases are heterogeneous at the molecular level. The genetic variation arises due to the mutation of a single beta-globin gene (HBB) on chromosome 11p15.5. Eventhough, the basis and natural history of this disorder are still not well understood and the clinical presentation in certain patients was not always correlated well with the single gene diagnosis [1].
 
 The study aimed to evaluate the predisposing effect of HBB polymorphisms and their haplotypes in conferring beta-thalassaemia in Malaysia using a bioinformatics tool. Archived genotype data for five tagging SNPs of HBB gene assigned as c.9T/C, IVS2-16G/C, IVS2-74T/G, 3'UTR +314G/A and 3'UTR +316A/C across thalassaemia patient group (n=433) and normal control group (n=794) controls were retrieved from the year 2011 to 2016 at Institute for Medical Research (IMR). The genotype data were analysed for individual SNP-based association analysis and haplotype-based association analysis using SHEsis online software.
 
 The study in all races in Malaysia showed significant association in four SNPs of the HBB gene (c.9T/C, P-value = 0.014; IVS2-16G/C, P-value = 0.017; 3'UTR+314G/A, P value = 2.08x108; 3'UTR+316A/C, P-value = 0.021). However, IVS2-74T/G did not show significant association with a P-value of 0.763 (Table 1). We further revealed two susceptible haplotypes in all races (haplotype TGTAA, P-value = 1.68x10-7, OR = 2.382 [1.707~3.325]; haplotype CCTGC, P-value = 0.003, OR = 1.300 [1.089~1.551]). Meanwhile, one protective haplotype observed was haplotype TGTGA (P-value = 2.99x10-7, OR = 0.625 [0.522~0.749]) (Table 2).
 In conclusion, this study suggests the potential susceptible and protective haplotypes of the HBB gene in predisposing to beta-thalassaemia in Malaysia

Cell-free fetal DNA as a non-invasive method using pyrosequencing in detecting beta-globin gene mutation: A pilot study from area with limited facilities in Indonesia.

BackgroundThalassemia is a monogenic, autosomal recessive, inherited disorder of the red blood cells caused by mutations or deletions in the globin gene. Approximately 6–10% of the Indonesian population carries the β-globin gene mutation; however, premarital screening is rarely conducted, and antenatal screening is optional. We explored the use of cell-free fetal DNA (cffDNA) as a potential non-invasive method of detecting the fetal β-globin gene mutation prenatally in pregnant women.Materials and methodsPregnant mothers (n = 10), who were known carriers of thalassemia and who had a history of having borne a baby with thalassemia major, and their carrier husbands (n = 4) were recruited after providing consent. EDTA blood was drawn, and maternal DNA, including cffDNA, and paternal DNA were isolated. Maternal contamination tests were conducted using the variable number tandem repeat test for ApoB and D1S80 loci. Allele quantification was performed by pyrosequencing. Known mutations from the bio-archived DNA of patients with thalassemia major (n = 16) were run alongside as a control.ResultsIn total, 7 out of 10 cffDNA successfully passed the maternal contamination test. The results of the allele quantification showed that six fetuses were predictive carriers of IVS1nt5 and one was predictive normal, in line with the allele quantification for the bio-archived DNA from patients with thalassemia major. The minimum threshold percentage for mutant A allele at cd26 was 32%, mutant T allele at IVS1nt1 was 23%, and mutant C allele at IVS1nt5 was 39%.ConclusionTaking cffDNA from the mother’s blood proved useful as a non-invasive means of detecting the β-globin gene mutation using pyrosequencing allele quantification. This non-invasive method is of great interest for prenatal diagnosis in settings with limited facilities, as it minimizes the risk of abortion. Further study of other mutations of the β-globin gene is needed.

Frequency of consanguineous marriage among the thalassaemia major patients in Bangabandhu Sheikh Mujib Medical University

Thalassaemia is a commonly occurring hereditary disorder. There is a high prevalence of thalassaemia disease in South-East Asia as well as Bangladesh. It is an autosomal recessive disorder, so consanguineous marriage is a very important factor for this disease. Mutated beta globin gene of haemoglobin from both parents is responsible for this disease to occur. But when the number of thalassaemia carriers is miserably high, then only avoidance of consanguineous marriage can’t prevent the birth of children with thalassaemia major. So, this cross sectional study was conducted among 120 diagnosed (by haemoglobin electrophoresis or high performance liquid chromatography) thalassaemia major patients in the Department of Haematology, Bangabandhu Sheikh Mujib Medical University, Dhaka from July 2019 to May 2020. History from each patient was taken and blood samples were collected from their parents to confirm carrier state by haemoglobin electrophoresis. Blood samples were also collected from patients and their parents toperform complete blood count and peripheral Blood Film. This study showed the mean age of the participants was 15±9.34 and 73 (60.8%) patients were male and 47 (39.2%) were female. About, 71% of the study population lived in urban area, 81 (67%) patients were transfusion dependent thalassaemia patients. 15% thalas- saemia major patients had the history of consanguinity of their parents. Among the parents of the thalassaemia major patients, 62.5% were Beta thalassaemia trait (heterozygous state), 25.83% parents were Hb E trait ( heterozygous state), 7.08% parents were suffering from Beta Thalassaemia Major(homozygous state) and 11 (4.58%) parents were suffering from Hb E/Beta Thalassaemia which is a compound heterozygous state. Though consanguinity is a very influential factor, but the most important causative factor is the presence of high number of thalassaemia carrier in the population. So, it’s a burning issue for the nation to perform a routine carrier screening for all rather than focusing on consanguinity only, to lessen the burden of thalassaemia disease in Bangladesh. BSMMU J 2022; 15(1): 25-28

Characterization of Common β-Thalassemia Major Mutations in Southwest Iran with Respect to Biochemical Parameters, Oxidative Status and Complications

Background & Objective: Beta-thalassemia are among the most common autosomal recessive genetic disorders in Iran, especially in Khuzestan province. Beta-thalassemia exhibits significant phenotype heterogeneity and there are currently more than 200 known mutations in this region. Oxidative stress exacerbates multiple disorders, including thalassemia, an inherited hemolytic anemia caused by globin gene mutations. We aim to characterize significant mutations of widespread β-thalassemia in south-western Iran with respect to biochemical parameters, oxidative status and complications of diseases. Material & method: Forty-five patients, aged between 15-35 years with β-thalassemia major were selected. The patients were receiving regular blood transfusion and chelation therapy and have been previously characterized to bear beta globin gene mutations. The subjects’ medical histories were documented by review of previous medical records. We also determined biochemical parameters including glycemic and iron indices, hepatic and renal function tests, oxidative stress markers and levels of advanced glycation end product species (Carboxy methyl lysine and Pentosidin). Results: The most common mutation was found to be CD36/37(28.9%) followed by IVSII-1, and IVSI-110. Values of iron indices were significantly different in various mutation groups. Carboxy methyl lysine and pentosidine were found to be higher in the β-thalassemia patients with IVSII-1 and IVSI-110, respectively. Also sLOX-1 was found to be significantly higher in IVSI-110 group. Complications of the disease were differently presented in mutation groups and hemochromatosis, hepatomegaly, and diabetes were among the most common problems. Conclusion: About 72 % of β-thalassemia major cases in southwest Iran result form 3 common mutations with different clinical and laboratory presentations. Molecular genetic testing can be helpful to evaluate the patients’ situation.

Studies on molecular spectrum of beta thalassemia among residents of Chennai

<abstract> <p>Beta thalassemia is caused by a mutation in the human beta globin gene. More than 400 causative mutations have been characterized in the Hemoglobin Subunit Beta (HBB) gene. These causative mutations are present in the beta globin gene or the regulatory region. Though more than 400 causative mutations of HBB region have been described, rare and novel mutations are being reported in studies indicating the need for characterization of mutations in all regions and information regarding the same should be made available for successful implementation of prenatal diagnosis. The study aims to characterize the spectrum of beta thalassemia mutations in beta thalassemia heterozygous among residents of Chennai. A total of 5,207 cases were screened for beta thalassemia heterozygous by HPLC method. 387 beta thalassemia heterozygous identified by HPLC method were subjected to molecular DNA analysis by ARMS PCR technique and DNA Sanger sequencing for the characterization of causative beta thalassemia mutations. In the present study molecular characterization of beta thalassemia mutations revealed 30 different mutations with a high prevalence of IVS 1-5 (G-C) mutation, five new rare mutations viz., IVS II-1 (G>T), CD 37 TGG-TGA, IVS II 781 (C-G), CD114 CTG-CCG and Poly A (A-G) were diagnosed and reported first in India. One novel beta thalassemia mutation HBB.c319DelC was detected in the study. The diagnostic outcome of detecting the causative mutations for beta thalassemia imposes strong resources for developing easy and cheaper methods for prenatal diagnosis which will reduce the burden of disease.</p> </abstract>

Spectrum of Genetic Mutation in Beta Globin Gene in Various Type of Thalassaemia in Bangladesh

Background: Hb-E/Beta thalassaemia is a congenital haemoglobin disorder which is a compound heterozygous state consists of qualitative disorder like Hb E variant & quantitative Hb disorder caused by genetic mutation of Beta chain. Objective: The aim of the study was to identify the beta gene mutation in Hb E/Beta thalassaemia. Method: A total of 32 diagnosed Hb E/Beta thalassaemia patients were included in this cross-sectional study from May 2019 to July 2020. Genetic analysis was done by sanger sequencing. Results: In this observational study, we found 13 different types of Beta gene mutations. Heterozygous for IVS 1-5(G>C) mutation was most frequent (53.1%). Conclusion: Genetic mutation is the confirmatory diagnosis for thalassaemia as well as one of the main factors for clinical expression. Mutation pattern also varies according to the geographical distribution. So, this study shows the frequently found mutation in Bangladesh and should carry out routinely to point out phenotypic expression.

Sickle Cell Hemoglobin Induces Autophagy in Human Macrophages

BACKGROUND: Autophagy plays an important role in multiple cell processes including elimination of misfolded proteins and damaged organelles, clearance of intracellular microbes and regulation of innate immunity. Autophagic activity is significantly up regulated in diseases characterized by proteins aggregation such as Alzheimer's, Huntington's, and Parkinson's diseases. Sickle cell anemia (SCA) is associated with E6V mutation in beta-globin gene that induces hemoglobin (Hb) polymerization under low oxygen or acidic conditions. SCA is characterized by chronic presence of low-level plasma Hb (3-10 µM). Hb is cleared from circulation by macrophage's endocytosis, resulting in degradation of Hb in lysosomes. Acidic lysosome environment may induce Hb polymerization, lysosome dysregulation and autophagy activation.HYPOTHESIS: We hypothesize that endocytosis of HbS induces autophagy in human macrophages.METHODS: The study was approved by Howard University review board (IRB) and all subjects provided written inform consent prior the sample collection. Whole blood samples were obtained from three SCA patients and healthy individuals. Human THP-1 promonocytic cells were differentiated into macrophages with 25 nM PMA for 72 hrs and treated with either purified HbS or HbA (5 µM, Sigma-Aldrich). Total RNA was depleted of cytoplasmic and mitochondrial ribosomal RNA, and strand-specific libraries were constructed using the Illumina ® TruSeq Stranded Total RNA Gold kit. Libraries were sequenced on an Illumina ® NextSeq 500 using 75 bp paired-end sequencing on two v2.5 150 cycle High-Output kits, generating 40-50 million paired-end reads per sample. The sequencing data were mapped to human reference genome using Dragen RNA v.3.8.4 software (Illumina). Comparisons were conducted using Dragen differential expression software v. 3.6.3 software (Illumina). Transmission electron microscopy (TEM) imaging was performed at Talos 200X transmission electron microscope (Thermo Fisher Scientific). CYTO-ID Autophagy detection kit (Enzo Life Science) was used for detection of autophagy in live cells by fluorescence microscopy, and fluorescent microplate assay.RESULTS: In THP-1-derived macrophages treated with HbS compared to HbA, 28362 genes were detected including 322 differentially expressed genes (1.1%, 1.5-fold difference, 187 down and 135 up) at 5% FDR. In THP-1-differentiated macrophages treated with SCD RBC lysate compared to control RBC lysate, 30840 genes were detected including 256 differentially expressed genes (0.83%, 1.5-fold difference, 117 down and 139 up) at 5% FDR.We focused our analysis on 33 autophagy-related genes. Among these 33 genes, 30 genes were up-regulated (1.2-1.7-fold) and only 3 genes were down-regulated in macrophages treated with HbS compared to HbA (Fig.1). These changes were statistically significant for 20 genes (p<0.01). No significant differences were found in expression of autophagy-related genes in macrophages treated either with HbS or SCD RBC lysate.Autophagy activation was validated by CYTO-ID Autophagy detection kit, demonstrating significant autophagy activation in HbS treated macrophages compared to HbA treatment (1.2-fold, p=0.0019). Transmission electron microscopy demonstrated accumulation of autophagosomes in the cells treated with HbS compared to non-treated cells and cells treated with HbA.CONCLUSION: Treatment of macrophages with sickle cell hemoglobin or lysate of red blood cells collected from SCD patients up-regulates expression of autophagy-related genes and increased autophagosome formation.ACKNOWLEDGMENTS: We thank Drs. Castle Raley and Keith Crandall, George Washington University SPH Genomics Core, for sequencing and guidance in data analysis; Dr. Christine Brantner, GW Nanofabrication and Imaging Center, for TEM imaging; and students Kavita Pandya, Gretchen Whitney High School, Cerritos, CA, and Tharindi Jayatilake, Montgomery Blair High School, Silver Spring, MD, for help in analysis of autophagy-related gene expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Benefits Trial of PB-04 to Evaluate Safety, PK, and Preliminary Efficacy in Inducing Fetal Globin Expression in Beta Thalassemia Intermedia and Sickle Cell Disease

Increased expression of fetal globin (HBG) has been shown to reduce clinical severity of beta-globin disorders and increase survival in sickle cell disease (SCD), through improved globin chain balance in beta thalassemia and reduced HbS polymerization. Pharmacologic approaches are considered most feasible for a majority of patients. Both proportions of F-cells and quantity of hemoglobin F (HbF)/cell are important for therapeutic effects. PB-04 was identified in a high-throughput screen of US and EU-approved drugs to activate HBG gene transcription, without cytotoxicity, to induce fetal globin expression (gamma globin mRNA, F-cells, HbF/cell, HbF), and to suppress or displace 4 repressors of the fetal globin gene promoter (BCL11A, LSD-1, KLF-1, and HDAC-3) in hemoglobinopathy patients' erythroid progenitors. PB-04 enhanced HbF in progenitors from hydroxyurea (HU)-treated patients with sickle cell disease. In in vivo studies, PB-04 induced fetal globin expression >20-fold with oral dosing in anemic baboons, and by 3.5-fold in mice transgenic for 2 copies of the human β-globin gene locus. This agent has been approved and in broad use for Parkinson's Disease treatment for 5 decades in Europe and Canada, to enhance the PK of an active Parkinson's agent, solely in a combination formulation. Because of its safety with chronic dosing up to 1300 mg/day, PB-04 is therefore of interest for repurposing in the treatment of thalassemia and SCD.This Ph1b trial (NCT004432623) will evaluate safety, tolerability, PK, and preliminary efficacy (fetal globin expression) with at least 3 escalating dose cohorts in up to 24 beta thalassemia intermedia (BTI) patients and 12 patients with sickle cell disease, > 18 years, both genders, with 12 weeks of treatment and 4 weeks of follow-up. Doses to be explored were selected from safe and active human equivalent doses in preclinical toxicology and efficacy studies in 2 species. Primary inclusion criteria include clinical diagnosis of BTI, including HbE beta thalassemia, with at least one beta globin gene mutation, or diagnosis of HbSS or HbSb thalassemia (after dose selection); total Hb levels 6-10 gm/dL. Exclusion criteria include red blood cell (RBC) transfusion within 2 months prior to administration of study medication, receiving regular transfusions, and hepatic or renal function > 3 x institutional ULN.Primary endpoints include occurrence, severity, and duration of adverse events by CTCAE v5.0 criteria and PK parameters. Secondary endpoints include change from baseline in assays of HbF expression, total Hb, markers of hemolysis . Exploratory endpoints include select polymorphisms associated with basal fetal globin expression and in vitro responses to the study drug. Statistical analysis will assess the rate of adverse events, PK parameters by dose, and changes in F-cells, F-reticulocytes, HbF/cell, HbF (% and total), compared to 2 averaged baseline values using the Wilcoxon signed rank test. Changes will be analyzed by mixed effect models during the 12-week dosing and 4-week follow-up period. Currently 3 dose cohorts with BTI are enrolled. This study, if successful, will provide a rational basis for definitive trials of an established safe oral therapeutic, with no overlapping toxicities with other experimental or approved agents, for combined use with HU and other therapeutics which enhance red cell viability in the globin disorders.The expertise and contributions of Jim Cradock of NCATS TRND to this work before his passing are gratefully acknowledged. DisclosuresKuo: Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Apellis: Consultancy; Bluebird Bio: Consultancy; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Honoraria; Alexion: Consultancy, Honoraria. Sheth: CRISPR: Consultancy; Bluebird bio: Consultancy; Bristol Myers Squibb: Consultancy, Research Funding; Chiesi: Consultancy; Imara: Research Funding; Agios: Consultancy; Dispersol: Research Funding. Al-Samkari: Moderna: Consultancy; Amgen: Research Funding; Rigel: Consultancy; Argenx: Consultancy; Novartis: Consultancy; Dova/Sobi: Consultancy, Research Funding; Agios: Consultancy, Research Funding. Pace: Imara Inc.: Consultancy. Kuypers: Forma Therapeutics, Inc.: Research Funding. Nouraie: Phoenicia BioScience Inc.: Consultancy. Perrine: Agios Pharmaceuticals: Consultancy; Emmaus Medical: Consultancy.

Beta-thalassemia mutation types and the relationship with the demographic factors in Sanliurfa, Turkey

Introduction: Beta-thalassemia is an autosomal recessive disease that occurs as a result of a disorder in the (β-globin chains synthesis), and the gold standard method for diagnosis is genetic mutation analysis. It is important to know the distribution of mutations according to regions and races. The aim of this study is to document the mutations in the beta-globin gene of beta-thalassemia major and intermedia patients who were followed and treated in Sanliurfa province, and to examine the relationships between these mutations by defining them according to gender, nationality, consanguineous marriage, history of disease in siblings and blood type.Methods: The files of 272 patients diagnosed with beta-thalassemia major and intermedia followed up in the Pediatric Hematology-Oncology outpatient clinic of Sanliurfa Training and Research Hospital between August 2016 and August 2017 were retrospectively reviewed and mutation analyzes were documented. Coding exons and exon-intron junction regions of beta globin-HBB gene were amplified by PCR method and then DNA sequencing was performed. Gender, nationality, consanguineous marriage, sibling history and blood type information were recorded.Results: Out of 272 patients, 94.1% were thalassemia major and the others were thalassemia intermedia. Approximately one third of the patients (30.1%) were foreign nationals. A total of 27 different mutations in the beta-globin gene were detected. The most common mutation is IVS-I-110 c.93-21 (G&amp;gt; A) (23.1%), which is followed by IVS-I-1 c.92 + 1 (G&amp;gt; A) (15.8%) and Codon 39 c.118 (C&amp;gt; T) (11.5%). One hundred and forty-two individuals (52.2%) had no sibling history, while 103 (37.9%) had one sibling and 27 (9.9%) had two siblings with thalassemia disorder. First-degree, second degree and third degree consanguineous marriages were present in 42.6% (n = 116), 8.1% (n = 22), 11% (n = 30) of parents, respectively.Conclusions: Beta-thalassemia disease is a common hematological condition in Sanliurfa. Approximately one-third of the patients who apply are foreign nationals. Correct identification of beta-globin gene mutations will guide genetic counseling and preventing prenatal disease. This data can contribute to the national thalassemia prevention program in Turkey.

High-Resolution CRISPR Functional Genomics Reveals Critical Vulnerabilities of the NuRD Complex for Fetal Hemoglobin Control

Hemoglobinopathies resulting from mutations of the adult beta-globin gene affect millions worldwide. Persistently elevated levels of fetal hemoglobin (HbF) are without consequence in healthy individuals but convey a major clinical benefit in patients with sickle cell disease and beta-thalassemia. Fetal globin silencing relies on interactions between DNA binding factors and chromatin readers, writers, and erasers acting within multiprotein nuclear complexes. The nucleosome remodeling and deacetylase (NuRD) complex is an epigenetic chromatin modifier, including ATP-dependent nucleosome remodeling and histone deacetylase activities, that has been implicated in HbF control. BCL11A and ZBTB7A, two key transcription factors required for HbF repression in adult erythroid cells, physically interact with NuRD complex members. In order to identify potential target sites within the NuRD complex with maximal HbF de-repression and minimal cellular toxicity potential we set out to dissect the function of the NuRD complex in adult erythroid precursors by dense mutagenesis. We performed CRISPR-Cas9 mediated saturating mutagenesis of all coding regions of human NuRD complex members in human umbilical cord blood-derived erythroid progenitor (HUDEP-2) adult erythroid cells with HbF expression as the primary readout and cell fitness as a counter-screen. This approach distinguished four classes of NuRD complex members: those required for HbF repression but dispensable for cellular fitness (such as MTA2, MBD2, HDAC2, and GATAD2A), those required for both HbF repression and cell fitness (such as CHD4), those only required for cell fitness (such as RBBP4), and many not essential for either HbF repression or cell fitness. The NuRD paralogs essential for HbF repression correlated with the abundance of peptides retrieved by MTA2 immunoprecipitation followed by mass spectrometry, suggesting a NuRD sub-complex that silences fetal globin. We demonstrated that this tiling CRISPR screen approach identifies residues at which short in-frame deletions result in loss of function, including at catalytic residues of CHD4 and HDAC2, additional NuRD domains and conserved regions of unknown significance. By mapping functional data onto existing structures we detected sequences essential for HbF repression within the ELM2 and BAH domains of MTA2 that mediate key protein-protein interactions with HDAC1/2 and with chromatin. At CHD4, we found numerous critical positions where in-frame alleles resulted in robust HbF elevation to a greater magnitude than disruption of any other NuRD member. However nearly all of these HbF-inducing perturbations at CHD4 were inversely correlated with cell fitness. In contrast, we observed that in-frame deletions within the CHDCT2 domain of CHD4 resulted in profound elevation of HbF level but spared the negative impact on cell growth. Overall, these results demonstrate a sub-complex of NuRD essential for HbF silencing and identify key interfaces including at the potent HbF repressor CHD4 which could serve as selective small molecule development targets for therapeutic HbF re-induction. DisclosuresOrkin:Epizyme Inc.: Consultancy; Bioverativ: Consultancy.

The Spectrum of Beta-Thalassemia Mutations in Syrian Refugees and Turkish Citizens.

Introduction and objectivesNeighboring the border between Turkey and Syria, Sanliurfa is one of the Turkish provinces with the highest number of Syrian refugees in our country. We aimed to find out the spectrum of beta-globin gene mutations in adult Turkish citizens and Syrian refugees with beta-thalassemia major.ResultsOf the participants, 35 patients (70%) were Turkish citizens and 15 patients (30%) were Syrian. The most common mutation in Turkish patients was found to be IVS-I-110 (G>A) with a frequency of 28.8%, followed by IVS-I-6 (T>C) with a frequency of 15.5%. Other common mutations were IVS-I-1 (G>A) and codon 39 (C>T) with frequencies of 11.1%. These four mutations accounted for 65.5% of all mutations in the Turkish cohort. The most common mutations in Syrian refugee patients were IVS-I-1 (G>A), IVS-II-1 (G>A), IVS-I-5 (G>C), and codon 5 (-CT), all with a frequency of 15.7%, accounting for 62.8% of all mutations in the Syrian patients. In the analysis, codon 5 (-CT) mutation (15.7% vs 0%, p=0.023) was found significantly higher in Syrian refugees compared to Turkish citizens.Discussion and conclusionsA wide spectrum of mutations was detected in beta-thalassemia major patients living in the Sanliurfa region. Mutational profiles in Turkish and Syrian patients were found to be significantly different from each other. Because marriages between Syrian refugees and Turkish citizens are increasing in our region, the genetic findings and the mutational profiles in Turkish and Syrian patients obtained in this study are thought to become useful for future prenatal molecular diagnostic tests.

Frequency of hereditary hemochromatosis gene mutations and their effects on iron overload among beta thalassemia patients of Chennai residents

&lt;abstract&gt; &lt;p&gt;Hereditary Hemochromatosis (HH) is an autosomal recessive disorder of iron metabolism associated with &lt;italic&gt;HFE&lt;/italic&gt; gene mutations, characterized by increased iron absorption and accumulation leading to multi-organ damage caused by iron overload toxicity. Beta thalassemia is caused by a mutation in the human beta globin gene. Imbalanced production of globin chain results in beta thalassemia, where the unpaired alpha chains precipitates in red cell precursors leading to ineffective erythropoiesis and reduced RBC survival. Both HH and beta thalassemia condition results in rapid accumulation of iron lead to iron overload in tissues and organs. The study aims to analyze the frequency of &lt;italic&gt;HFE&lt;/italic&gt; variants among beta thalassemia cases and their effect on iron overload. The frequency of three &lt;italic&gt;HFE&lt;/italic&gt; variants C282Y, H63D, S65C was analyzed by PCR RFLP method among Beta Thalassemia Trait (BTT) (n = 203), Beta Thalassemia Major (BTM) (n = 19) and age and sex-matched control samples (n = 200). The present study furnished allele frequency of H63D variant in BTT, BTM and controls 8.13, 15.8 and 6% respectively. Ten out of 33 heterozygous H63D variants exhibited iron overload with higher ferritin levels indicating &lt;italic&gt;HFE&lt;/italic&gt; variant might aggravate the absorption of iron. The C282Y variant was present in heterozygous state in 1 case among beta thalassemia carriers. The C282Y variant was absent among BTM and control cases. S65C &lt;italic&gt;HFE&lt;/italic&gt; variant was absent in the present study. Iron overload was completely absent in the control cases among all three &lt;italic&gt;HFE&lt;/italic&gt; genotypes. Hence it is inferred from the present investigation, analysis of &lt;italic&gt;HFE&lt;/italic&gt; genes and iron status will remarkably help to reason out the probable reason behind the iron status and support in proper management of beta thalassemia cases.&lt;/p&gt; &lt;/abstract&gt;

Evaluation of Beta Globin Gene Mutations in Beta Thalassemia Carrier Children in Aydın Province and its Environment

Objective: Beta thalassemia carriers (BTC) in Turkey is observed with a frequency of 2.1%, and it is the most common cause of anemia after iron deficiency. There are few studies showing the effect of genotype on phenotype in beta thalassemia carrying children. The aim of this study is to determine the mutation diversity of these children in and around Aydın and evaluate the effects of these mutations on complete blood count parameters and hemoglobin electrophoresis. Methods: This study included mutation analysis of 65 patients who were diagnosed as BTC in Adnan Menderes University, Faculty of Medicine, Department of Child Health and Diseases, Pediatric Hematology Outpatient Clinic between 01.01.2014 and 01.08.2019. Complete blood count, hemoglobin electrophoresis and mutation analysis results were obtained from the computer data system and patient files. Research data were evaluated by using SPSS 21.0 statistics program. Results: 39 (60.0%) of the sixty-five patients were male, 26 (40.0%) were female, and the mean age was 8.34±4.94 years. When full blood count parameters were analyzed, 87.7% had anemia, 100% microcytosis and high red cell distribution width (RDW) level, 49.2% hypochromia and 87.7% high red blood cell (RBC) count. RDW level was ≥16 in 66.2% of the cases. Seventeen different mutations were detected. The mutations most frequently occurred in “intron 1” gene region (66.1%). The most common ones were IVS I-110 (44.11%), IVS I-1 G&gt;A Heterozygotes (8.8%,) IVS I-6 T&gt;C Heterozygotes (7.5%) and IVS II-745 (7.5%). RDW level was ≥16 in 66.2% of the cases. Seventeen different mutations were detected. The mutations most frequently occurred in “intron 1” (66.1%) gene region. IVS I-110 (44.11%), IVS I-1 G&gt;A Heterozygotes (8.8%,) IVS I-6 T&gt;C Heterozygotes (7.5%) and IVS II-745 (7.5%) were observed most commonly. In patients with IVS I-110 mutation, mean Hb level was 10.55 gr/dL, MCV level was 58.44 fL, RDW level was 16.51, RBC level was 5680x10⁹/L, HbA2 level was 4.77%, HbF level was 2.34%. Mutations detected in 12 patients with HbF level above 5% were as IVS I-110 (5 people), IVS I-6 T&gt;C Heterozygotes, Codon 39 C&gt;T Heterozygotes, IVS I-116, c.25-26 del AA (plys9Valfs) Heterozygotes, c.27dupG (pSer10valfs*14), c316-373 (IVSII-478 C&gt;A Heterozygotes), -87 C&gt;T Heterozygotes. Mentzer index was calculated as &gt;13 in six patients (9.2%). The mutations seen in these patients were IVS-I 110, c.27dupG (p.Ser10valfs*14), c316-373 (IVS II-478 C&gt;A Heterozygotes), -87 C&gt;T Heterozygotes. There were four patients (6.1%) with a RDW index &gt;220. Two of these patients had c.27dupG (p.Ser10valfs*14) and others had c316-373 (IVS II-478 C&gt;A Heterozygotes) and -87 C&gt;T Heterozygotes mutations. Mutations detected in four patients with HbF levels in the range of 9.48-15.67 were IVS I-116 T&gt;G Heterozygotes, IVS I-110 G&gt;A Heterozygotes, c.25-26 del AA (p.lys59valfs) Heterozygotes, c27 dupG (pSer10 valfs*14) and three of these mutations carrying β° mutation type were located in exon 1 and one of them carrying β⁺ mutation type (IVS I-110) was in intron 1. Conclusion: The same mutations detected in patients with beta thalassemia carriers have different effects on complete blood count parameters. HbA2 and HbF levels suggest that these mutations are not effective on the phenotype alone and there may be additional factors which should be clarified. We think that there may be BTC in cases with low RBC, Mentzer index ≥13 and RDW index ≥220, HbA2 &lt;3.5 and studying the mutation analysis of these patients will contribute significantly to the literature.