Deletional HPFH Vs. Delta Beta Thalassemia: Closing in on a Possible Hb F Silencer Location

Abstract Background: The control of hemoglobin F (Hb F) expression has proven an elusive puzzle with several postulated but not mutually exclusive hypotheses. A silencing mechanism hypothesis, which predicts the existence of a regulatory element that suppresses HbF expression, has been supported by recent investigations into a 3.5 kb region upstream from the delta globin gene (HBD). To test this hypothesis, we have investigated our historical case files in a tertiary care high-throughput clinical laboratory and compared the patient phenotype (Hgb, MCV, MCH, RDW, Hb F %, and flow cytometry Hb F distribution) with the status of this 3.5 kb region. Methods: A query of clinical testing patient files from Mayo Clinic Metabolic Hematology Laboratory yielded 179 patients confirmed by Multiplex Ligation-dependent Probe Amplification (MLPA) to contain large deletions located within the epsilon through beta globin genes. Of these, 27 unrelated patients with breakpoints between the pseudobeta (HBBP1) and HBD genes were identified. Four additional MLPA probe pairs were placed in this region and the patient phenotypes were compared. Results: The interior two (of the four added) probes between HBBP1 and HBD stratified all 27 cases. The other two (flanking) probes were never discriminatory, thus refining the area of interest to 2.4 kb. Sixteen cases showed breakpoints within hg19 g.5260154-5259135 (5' region) and eleven cases showed breakpoints within hg19 g.5259136-5257692 (3' region) upstream of HBD. Patients who displayed a phenotype of HPFH (n = 9) contained breakpoints in the 5' region whereas those with a delta beta thalassemia (DBT) phenotype (n = 11) were associated with breakpoints in the 3' region. A subset (n = 7) with breakpoints in the 5' region had indefinite phenotypic features, but of these, all but one showed homocellular Hb F distribution. Conclusion: Our molecular and phenotypic correlation of 27 patients with large deletional breakpoints between HBBP1 and HBD supports the hypothesis of a silencing element located upstream of HBD and further narrows the area that segregates many HPFH and DBT patient phenotypes from 3.5 kb to 2.4 kb. Several potential silencing effectors with binding sites in this region include HDAC1, GATA1 and H3K27me3; interestingly, the BCL11A binding site may be outside of the regulatory area. Table 1. Phenotypic data of patients with deletions between HBBP1 and HBD. Case Age/Sex Hb F Hb A2 Hb X Hgb RBC MCV MCH MCHC RDW Hb F Flow (Y) (%) (%) (%) (g/dL) ( 10^12/L) (fL) (pg/cell) (hg/cell) (%) 3' Region 1 47 M 25.5 2.1 16.7 6.3 81.4 26.7 32.8 15.7 H 2 5 M 28.3 2.4 12.5 4.9 74.6 25.4 34 15.9 H 3 24 F 30.3 1.7 11.6 4.23 92.7 27.4 29.6 15.2 H 4 9 F 36.3 2.2 S = 62 13.1 5.2 69.9 NA NA 14.8 5 24 F 37.3 2.1 C = 61 12.9 5.1 72.9 25.5 35 14.3 6 3 M 39.7 2.1 12.4 4.4 88.7 28.1 31.7 16.3 7 27 F 40.2 3.4 S = 56 12.2 4.4 79.7 27.8 34.9 15 8 16 M 43.7 2.2 S = 54 15.9 6.8 71.5 23.5 32.9 14.1 9 7m M 100.0* 0.0 12.8 4.8 76 26.5 34.9 16.8 3' Region 10 28 F 20.9 2 10.11 4.4 70.9 22.7 32.1 19 H 11 64 M 21.4 2.3 10.51 4.1 81.1 25.8 31.8 18.1 12 2.5 M 24 2.1 10.21 5.3 59.4 19.3 32.5 20.9 H 13 58 F 24 2.1 9.51 4.4 72.2 21.5 29.8 17.7 H 14 14 F 25.4 1.5 10.81 4.9 69.4 22 31.8 17.6 H 15 52 F 26.9 1.8 8.42 3.6 72 23.5 32.7 19.4 T 16 34 M 38.7 1.7 S = 60 11.4 4.7 71.6 NA NA 21.6 5' Region 17 24 F 2.7 2.9 12.3 6.1 62.2 20 32.2 18.2 18 32 F 6.1 2.7 10.2 5.1 61.3 19.9 32.5 19.3 19 27 F 7.2 2.9 11 5.1 72.9 21.7 29.8 19.9 20 29 F 7.8 2.7 10.2 4.7 72.2 21.7 30 20.5 21 15 M 7.8 2.5 12.7 6.2 64.7 20.4 31.5 21.2 22 42 M 7.8 3.1 10.6 4.9 75.1 21.5 28.6 23.7 23 60 M 10.1 2.5 11 4.5 74.8 24.7 33 23.4 24 41 M 10.7 2.8 8.6 4.1 73.3 21.2 29 26.8 25 30 F 11.5 2.7 12.1 5.5 67.4 21.9 32.5 21.8 26 25 F 13.2 2.8 12.1 5.3 68.4 22.8 33.3 21.6 27 3 F 18.3 2.6 11.9 5.79 63.6 20.6 32.3 22.7 T Normal values <1 2-3.3 0 H = homocellular; T= heterocellular; I = indeterminate; *homozygous deletion -- = no deletion; + = deletion present (all alpha deletions were single -3.7 kb 'rightward' deletions) 1 = Suspected Fe deficiency; 2 = recent transfusion Disclosures No relevant conflicts of interest to declare.

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Hemoglobin F (HbF) inducers; History, Structure and Efficacies

Mini-Reviews in Medicinal Chemistry ◽

10.2174/1389557521666210521221615 ◽

2021 ◽

Vol 21 ◽

Author(s):

Zahra Hashemi ◽

Mohammad Ali Ebrahimzadeh

Keyword(s):

Clinical Trials ◽

Natural Products ◽

Biological Activities ◽

Globin Gene ◽

Beta Thalassemia ◽

Gene Promoters ◽

Hemoglobin F ◽

Chemical Structures

Abstract: Inherited beta-thalassemia is a major disease caused by irregular production of hemoglobin through reducing beta-globin chains. It has been observed that increasing fetal hemoglobin (HbF) production improves symptoms in the patients. Therefore, an increase in the level of HbF has been an operative approach for treating patients with beta-thalassemia. This review represents compounds with biological activities and pharmacological properties that can promote the HBF level and therefore used in the β-thalassemia patients' therapy. Various natural products with different mechanisms of action can be helpful in this medication cure. Clinical trials were efficient in improving the signs of patients. Association of in vivo, and in vitro studies of HbF induction and γ-globin mRNA growth displays that in vitro experiments could be an indicator of the in vivo response. The current study shows that; (a) HbF inducers can be grouped in several classes based on their chemical structures and mechanism of actions; b) According to several clinical trials, well-known drugs such as hydroxyurea and decitabine are useful HbF inducers; (c) The cellular biosensor K562 carrying genes under the control of the human γ-globin and β-globin gene promoters were applied during the researches; d) New natural products and lead compounds were found based on various studies as HbF inducers.

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A novel basis for delta beta-thalassemia in a Chinese family

Blood ◽

10.1182/blood.v68.5.1108.1108 ◽

1986 ◽

Vol 68 (5) ◽

pp. 1108-1113 ◽

Cited By ~ 5

Author(s):

GF Atweh ◽

DE Zhu ◽

BG Forget

Keyword(s):

Gene Cluster ◽

Globin Gene ◽

Beta Thalassemia ◽

Chinese Family ◽

Compound Heterozygous ◽

Beta Globin ◽

Hemoglobin F ◽

Beta Globin Gene ◽

Globin Gene Cluster ◽

Alpha Beta

Abstract We have studied a Chinese family in which beta-thalassemia and delta beta-thalassemia were found in simple and compound heterozygous states. The delta beta-thalassemia heterozygote (the mother) had 22.3% hemoglobin F, of which 40% was G gamma and 60% A gamma; globin chain studies showed an alpha/beta + gamma ratio of 1.36. The compound heterozygote for delta beta-thalassemia and beta-thalassemia (the child) had the clinical picture of thalassemia intermedia and an alpha/beta + gamma ratio of 4.44. Gene mapping studies were performed using DNA from the affected child. Seventy kilobases of DNA in the beta- globin gene cluster starting upstream from the epsilon-globin gene and ending downstream from the beta-globin gene were mapped, and no detectable deletions or rearrangements were detected. In addition, heterozygosity was detected at multiple polymorphic restriction sites in and 3′ to the beta-globin gene, which excludes the possibility of a deletion of the entire beta-globin gene cluster. This is the first example of a nondeletion delta beta-thalassemia associated with increased expression of both G gamma and A gamma genes.

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Elevated Systolic Blood Pressure and Low Fetal Hemoglobin Are Risk Factors for Silent Cerebral Infarcts in Children with Sickle Cell Anemia.

Blood ◽

10.1182/blood.v114.22.262.262 ◽

2009 ◽

Vol 114 (22) ◽

pp. 262-262

Author(s):

Sharada A. Sarnaik ◽

James F. Casella ◽

Bruce A Barton ◽

Michele Afif ◽

Gladstone Airewele ◽

...

Keyword(s):

Risk Factors ◽

Blood Pressure ◽

Systolic Blood Pressure ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Conflicts Of Interest ◽

Beta Thalassemia ◽

Cerebral Infarcts ◽

Hemoglobin F ◽

Elevated Systolic Blood Pressure

Abstract Abstract 262 Introduction: The most common cause of neurological injury in sickle cell anemia is silent cerebral infarcts (SCI). In the Silent Cerebral Infarct Multi-Center Clinical Trial (SIT Trial) cohort, we sought to identify risk factors associated with SCI. Patients and Methods: In this cross-sectional study, we evaluated the clinical history, baseline laboratory values and performed magnetic resonance imaging of the brain. For those children with SCI-like lesions, a pediatric neurologist examined the child and neuroradiology and neurology committees adjudicated the presence of SCI. Children between the ages of 5 and 15 years with hemoglobin SS or S-beta° thalassemia and no history of overt strokes or seizure were evaluated. Results: A total of 542 children were evaluated; 173 (31.9%) had SCI. The mean age of the children was 9.3 years, with 280 males (51.7%). In a multivariate logistic analysis, two covariates were significant: a single systolic blood pressure (SBP) obtained during a baseline well-visit, p = 0.015 and hemoglobin F (Hgb F) level obtained after three years of age, p = 0.038. Higher values of SBP and lower values of Hgb F increased the odds of SCI; Figure. Baseline values of white blood cell count, hemoglobin level, oxygen saturation, reticulocytes, pain, or ACS event rates were not associated with SCI. Conclusion: SBP and Hgb F level are two previously unidentified risk factors for SCI in children with sickle cell disease. Modulation of SBP and Hgb F levels might decrease the risk of SCI. Disclosures: No relevant conflicts of interest to declare.

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Unraveling the Erythroid Function of Tfr2 in Beta-Thalassemia

Blood ◽

10.1182/blood.v128.22.73.73 ◽

2016 ◽

Vol 128 (22) ◽

pp. 73-73

Author(s):

Antonella Nai ◽

Maria Rosa Lidonnici ◽

Irene Artuso ◽

Giacomo Mandelli ◽

Mariateresa Pettinato ◽

...

Keyword(s):

Conflicts Of Interest ◽

Continuous Production ◽

Globin Gene ◽

Intermediate Phenotype ◽

Beta Thalassemia ◽

Severe Anemia ◽

Erythroid Cells ◽

Ineffective Erythropoiesis ◽

Iron Deficient ◽

Time Point

Abstract Transferrin receptor 2 (TFR2), a liver activator of the iron hormone hepcidin mutated in hemochromatosis type 3 (Camaschella C. et al., Nat Genet 2000), is an erythropoietin (EPO) receptor partner in erythroid cells (Forejtnikovà H. et al., Blood 2010). We have recently shown that the loss of bone marrow (BM) Tfr2 increases erythroblast EPO sensitivity and mimics a condition of mild iron-deficiency in mice (Nai A. et al., Blood 2015). β-thalassemias are iron-loading anemias due to recessive mutations in the β-globin gene, characterized by severe ineffective erythropoiesis. Since several studies demonstrated that iron restriction ameliorates the β-thalassemia phenotype, we generated thalassemic mice (Hbbth3/+) with selective BM inactivation of Tfr2 (Tfr2BMKO/Hbbth3/+) through transplantation of BM cells (BMT) from Hbbth3/+ and Tfr2-/-/Hbbth3/+ mice into lethally irradiated wt recipients. Two months after BMT Tfr2BMKO/Hbbth3/+ mice have Hb levels higher than Hbbth3/+ (Hbbth3/+: 9.47±0.59 g/dL; Tfr2BMKO/Hbbth3/+: 11.22±0.47 g/dL; p<0.0001). In contrast, 4 months after BMT Tfr2BMKO/Hbbth3/+ mice have more severe anemia, ineffective and extramedullary erythropoiesis, increased iron in serum (TS) and liver (LIC), reduced spleen iron (SIC), high hepcidin levels, increased Epo-target gene (Epor, Bcl-xL, Erfe) expression in BM and high levels of serum Erfe. These results suggest that deleting Tfr2 in thalassemic erythroblasts increases their Epo sensitivity as occurs in normal ones. This effect is positive during BM repopulation, but transient: on a long-term a severe phenotype develops, similar to the one observed in thalassemia major models (Rivella S. et al., Blood 2003; Huo Y. et al., Blood 2009), likely induced by excessive EPO stimulation of ineffective immature erythropoiesis. We speculate thatin Tfr2BMKO/Hbbth3/+ mice the continuous production of Erfe, the hepcidin inhibitor released by the expanded marrow (Kautz L. et al., Nat Genet 2014), worsens iron-overload, further damaging the thalassemic erythropoiesis. In order to exclude any interference of BMT, we analyzed also Hbbth3/+, Tfr2+/-/Hbbth3/+ and Tfr2-/-/Hbbth3/+ mice, lacking germ-line Tfr2 at the heterozygous and homozygous state respectively. Preliminary data indicate that young Tfr2-/-/Hbbth3/+ mice have less severe anemia than Hbbth3/+, while Tfr2+/-/Hbbth3/+ animals have an intermediate phenotype, suggesting that Tfr2 haploinsufficiency may be sufficient to improve the ineffective erythropoiesis at short time point. Follow up analysis will test whether the phenotype of Tfr2-/-/Hbbth3/+ mice worsens with aging as occurs in transplanted animals. Interestingly we noticed that 2-month-old Tfr2-/-/Hbbth3/+ mice, beside higher Hb levels, have higher LIC than thalassemic littermates, with comparable SIC. This observation lead us to hypothesize that low SIC, rather than high LIC, contributes to the worsening of the phenotype in Tfr2BMKO/Hbbth3/+ mice 4 months after BMT, likely impairing the compensatory spleen erythropoiesis. To dissect causality between high LIC and low SIC we are challenging transplanted mice with iron-deficient diet to decrease LIC or with iron-dextran injection to increase SIC. Moreover, a group of animals sacrificed 2 months after BMT will help to elucidate the mechanism(s) of the early amelioration of anemia. Preliminary results confirm the Hb and red cells count increase in Tfr2BMKO/Hbbth3/+ as compared to Hbbth3/+mice 2 months after BMT without increased spleen size, thus suggesting a real erythropoiesis improvement at this time point. Erythroid cells at the different stages of maturation sorted from these animals will be subjected to RNAseq analysis in comparison to cells isolated from animals at the time of phenotype worsening. The results are expected to define the pathway/s altered by the loss of Tfr2 in thalassemic erythroid cells and those responsible for the amelioration of the phenotype with the aim of identifying potential novel target/s for therapy of β-thalassemia. Disclosures No relevant conflicts of interest to declare.

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Genetic Engineering in Hematopoietic Stem Cells for Gene Therapy of Hemoglobinopathies

Blood ◽

10.1182/blood-2019-121119 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. SCI-23-SCI-23

Author(s):

Giuliana Ferrari

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Globin Gene ◽

Genetically Modify ◽

Fetal Hemoglobin ◽

Beta Thalassemia ◽

Globin Genes ◽

Hematopoietic Stem

Beta-thalassemia and sickle cell disease (SCD) are congenital anemias caused by mutations in the beta-globin gene, resulting in either reduced/absent production of globin chains or abnormal hemoglobin structure. At present, the definitive cure is represented by allogeneic hematopoietic stem cell transplantation, with a probability to find a well-matched donor of <25%. Experimental gene therapy for hemoglobinopathies is based on transplantation of autologous hematopoietic stem cells genetically modified to express therapeutic hemoglobin levels. Approaches to genetically modify HSCs for treatment of hemoglobinopathies include: 1) the addition of globin genes by lentiviral vectors and 2) gene editing by nucleases to reactivate fetal hemoglobin either through inhibition of repressors or by reproducing mutations associated with high fetal hemoglobin levels. The outcomes of early clinical trials are showing the safety and potential efficacy, as well as the hurdles still limiting a general application.Current challenges and improved strategies will be presented and discussed. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Plerixafor

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Therapeutic approaches in patients with β-thalassemia

Iranian Journal of Pediatric Hematology & Oncology ◽

10.18502/ijpho.v12i1.8364 ◽

2022 ◽

Author(s):

Mohammad Eini ◽

Mohammad Shoae ◽

Ebrahim Miri-moghaddam

Keyword(s):

Iron Overload ◽

Clinical Symptoms ◽

Globin Gene ◽

Micro Rna ◽

Beta Thalassemia ◽

Chemical Components ◽

Therapeutic Approaches ◽

Hemoglobin F ◽

Erythroid Hyperplasia ◽

Gene Reactivation

Beta-thalassemia (β-thal) is a congenital hemoglobinopathy explained by a decreased level (β+) or absence (βο) of β-globin gene expression. Microcytic hypochromic anemia and various clinical symptoms comprising severe anemia to clinically nonsymptomatic features. Treatment with an ordered blood transfusion and iron chelator agents can decrease transfusion iron overload that causes normal maturation. These patients also are at high risk for secondary iron overload because of erythropheron (GF15–TWSG1) release from erythroblasts resulting in erythroid hyperplasia. Based on the previous studies, chemicals such as hydroxyurea and 5-azacytidine are useful in treating β-hemoglobinopathy, including β-thal and sickle cell disease (SCD). Regarding both side effects and lifelong treatment of these chemical components, researchers have recently regarded gene-based treatments. These techniques, such as micro RNA gene silencing, viral-mediated gene editing, and clustered regulatory interspaced short palindromic repeats (CRISPR)-CAS9 systems, are the most commonly used gene therapy methods. Nowadays, ɣ-globin (fetal globin) gene reactivation is one of the most popular treatments for β-thal. Researches showed that these gene modification methods for γ-globin gene reactivation are also useful in increasing hemoglobin F (HbF) and helping patients with β-thal. In this review study, new therapeutic approaches to manage this disorder are regarded.

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Beta Thalassemia In Different Tribal Population In West Bengal, India

Blood ◽

10.1182/blood.v116.21.5170.5170 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5170-5170

Author(s):

Deboshree Majumdar ◽

Jayasri Basak ◽

Abhijit Chakraborty ◽

Soma Mukhopadhyay ◽

Nabamita Pal ◽

...

Keyword(s):

West Bengal ◽

Conflicts Of Interest ◽

Globin Gene ◽

Genetic Defect ◽

Beta Thalassemia ◽

Dihydrogen Phosphate ◽

Common Mutation ◽

Beta Globin ◽

Beta Globin Gene ◽

North Bengal

Abstract Abstract 5170 Background: The Thalassemias are a group of anemia that results from a genetic defect whch reduces the rate of synthesis of normal globin chains. Beta Thalassemia results in severe microcytic, hypochromic, haemolytic anaemia among affected patients. It has emerged as one of the most common health problems among the tribal populations in West Bengal. A project entitled, “Status of Prevalence and Awarness Among Thalassaemia Population: Special reference to various tribes belonging in different Districts of West Bengal” has been sanctioned by the Department of Biotechnology, Govt. of India(BT/PR10336/SPD/24/293/2007). This study is aimed to observe the spectrum of various beta mutations among the tribes. In the screening schedule thalassemia awarness programme was conducted in various rural areas of West Bengal. We mainly confronted with the Sardar tribes in extreme south of West Bengal. In North Bengal there were Toto, Rabha, Oraon, Munda, Toppo, Baraik, Nagbanshi, Khalko, Kheria, Kerketta and Soren. Method: In the year 2009–2010, 1289 tribes were screened. Mass awarness programme were followed by collection of peripheral blood samples in EDTA vials and transported to the laboratory in ice packs. NESTROFT was done on spot using 0.36% Saline Buffer solution (Sodium chloride, Sodium dihydrogen phosphate, Disodium hydrogen Phosphate). Complete Blood Count was performed within 24 hours of collection. HPLC (High Performance Liquid Chromatography) was done to identify the beta samples. DNA was extracted from the blood of beta carrier and beta major and subjected to ARMS (Amplification Refractory Mutation System) PCR (Polymerase Chain Reaction) to detect the point mutations. Result: Conclusion: Molecular Characterization of Beta globin gene mutations among these tribes have confirmed the presence of the following mutations: IVS-1 nt5 (G>C), IVS-1 nt1(G>T), codon 15 (G>A), codon 26 (G>A), the mutation which leads to HbE, codon 19 (A>G), codon 17 (A>T), -28 (A>G). The most common mutation observed among Totos and Rabhas were codon 26 (G>A) of North Bengal. The prevalence of Beta carrier among them is negligible. The mutation IVS-1 nt5 (G>C) is prevalent among the Oraon, Nagbanshi and Sardar tribes of Bengal. The other mutations which are present among them are codon 15 (G>A) and codon 30 (G>C). There are two codon 17 (A>T) and one -28 (A>G) mutations which are present among Mundas. The rest of them are mutation IVS-1 nt5 (G>C). In few cases presence of sickle cell anemia was observed. Our results have added to the existing data on the common beta globin gene defects which are prevalent among the general population of West Bengal, India. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2018-99-113903 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3640-3640

Author(s):

Deborah Rund ◽

Dvora Filon ◽

Natalie Bloch Isenberg ◽

Neta Goldschmidt

Keyword(s):

Sequence Analysis ◽

Clinical Significance ◽

Base Pair ◽

Conflicts Of Interest ◽

Hematological Parameters ◽

Globin Gene ◽

Beta Thalassemia ◽

Exon 2 ◽

Functional Variants ◽

Functional Variant

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

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HQK-1001 Is Well Tolerated and Augments Hemoglobin F and Hemoglobin Levels In Patients with Beta Thalassemia Intermedia

Blood ◽

10.1182/blood.v116.21.4280.4280 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4280-4280

Author(s):

Suthat Fuchareon ◽

Adlette C. Inati ◽

Noppadol Siritanaratkul ◽

Suzanne Koussa ◽

Ali Taher ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Globin Gene ◽

Beta Thalassemia ◽

Thalassemia Intermedia ◽

Employment Equity ◽

Advisory Committees ◽

Hemoglobin F ◽

Pharmacodynamic Effects ◽

Equity Ownership

Abstract Abstract 4280 Beta thalassemia intermedia syndromes are serious conditions for which there is no satisfactory therapy to correct the underlying globin chain imbalance. Some agents that induce fetal globin gene expression have ameliorated anemia in thalassemia patients by reducing the imbalance in alpha: non-alpha globin synthesis, but none have been broadly accepted or are currently approved by regulatory authorities. HQK-1001 is an oral agent that targets the fetal globin gene promoter, thereby increasing fetal hemoglobin (HbF) expression. It has been well tolerated in single dose and multiple dose escalation clinical studies in healthy volunteers. We now report the results of a randomized, double blind, placebo-controlled, multiple ascending dose Phase I/II trial in 21 adult patients with beta thalassemia intermedia (BTI), including 14 with HbE/ß0 thalassemia and 7 with ß+/ß0 thalassemia (including 12 different beta globin gene mutations). Study medication was taken as a single daily dose for 8 weeks. Four ascending dose levels (10, 20, 30, and 40 mg/kg/day) were sequentially evaluated in 4 dose level cohorts after the preceding dose and schedule were determined safe by an independent and unblinded Safety Monitoring Committee. HQK-1001 was well-tolerated. Adverse events in treated subjects included headache, upper respiratory infection and nausea, but the rates of such events were not markedly different than those observed in the placebo-treated subjects. The 20 mg/kg dose was associated with a 10% mean increase above baseline in HbF, (p< 0.001). Total hemoglobin (Hgb) increased by a mean of 1.1 gram/dL in 3 of 6 treated BTI patients with Mediterranean mutations. F-cells increased over the study period with maximal increases often observed 2 weeks following therapy. Doses higher than 20 mg/kg were not associated with the same magnitude of pharmacodynamic effects. These observations indicate that HQK-1001 is well-tolerated at doses associated with favorable pharmacodynamic effects on Hgb and HbF. These findings with brief treatment provide a rationale for conducting larger and longer studies in BTI patients. Disclosures: Fuchareon: HemaQuest Pharmaceuticals, Inc: Honoraria, Research Funding. Inati:HemaQuest Pharmaceuticals, Inc: Honoraria, Research Funding. Boosalis:HemaQuest Pharmaceuticals, Inc: Equity Ownership, Research Funding. Thein:HemaQuest Pharmaceuticals, Inc: Research Funding. Wallis:HemaQuest Pharmaceuticals: Consultancy, Equity Ownership. Bobbitt:HemaQuest Pharmaceuticals, Inc: Employment, Equity Ownership, Patents & Royalties. Thomson:HemaQuest Pharmaceuticals: Employment, Equity Ownership. Johnson:HemaQuest Pharmaceuticals: Employment, Equity Ownership. Berenson:HemaQuest Pharmaceuticals, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Perrine:HemaQuest Pharmaceuticals, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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Homozygous Expression of a Novel Senegalese-Type Partial Deletion of the Beta and Delta Genes Causes a Delta0 Beta+ Thalassemia.

Blood ◽

10.1182/blood.v120.21.2128.2128 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2128-2128

Author(s):

Hernan Sabio ◽

Natalia Dixon ◽

Ferdane Kutlar ◽

Niren Patel ◽

Hanfang Zhang ◽

...

Keyword(s):

Gene Expression ◽

Dna Analysis ◽

Conflicts Of Interest ◽

Clinical Phenotype ◽

Globin Gene ◽

African Ancestry ◽

Beta Thalassemia ◽

Globin Genes ◽

Chain Imbalance ◽

Globin Gene Expression

Abstract Abstract 2128 Clinical phenotype in β-thalassemia syndromes is determined by the degree of chain imbalance. An increase in γ-globin production will compensate for the absent or deficient β-globin synthesis and will result in the amelioration of the chain imbalance, and hence an improvement in clinical features. The known genotypes of δβ-thalassemia are associated with an increase in Hb F production, which results in the amelioration of the clinical presentation. Most δβ-thalassemias result from deletions that remove the δ- and β-globin genes, (δβ)0 with a compensatory increase in γ-globin (Hb F) expression. We report an unusual case of homozygous δ0β+ thalassemia that provides interesting insights into increased γ-globin expression and the regulation of β-globin gene expression. An 8-year old boy of African ancestry presented with lifelong jaundice and pallor. He also experienced episodes of worsening symptoms. He exhibited frontal bossing, pale mucosa, scleral icterus, and moderate splenomegaly. He was known to have G6PD deficiency and was suspected of having additional erythrocyte pathology. The CBC revealed a Hb of 8.7, Hct 26.4, MCV 64.7, WBC 10,700, platelets 283,000, reticulocytes 2.2%, and total bilirubin 5.3. Hemoglobin analysis by HPLC and IEF revealed HbA 13.4%, Hb F 86.6%, and no additional components. Alpha thalassemia −3.7kb deletion was not detected. Globin chain analysis revealed α, β, Gγ and AγI chains. DNA analysis revealed a novel Senegalese-type deletion of the beta and delta genes, resulting in a delta0 beta+ thalassemia. The subject's parents who were both from the same small village in Niger had normal hematology values. Their hemoglobin analyses revealed Hb A 94. 8%, Hb A2 2.0%, Hb F 3.2% and Hb A 93.5%, Hb A2 2.1%, Hb F 4.5% in the father and mother, respectively. They were both heterozygous for the delta-beta deletion identified in their son. DNA analysis revealed a breakpoint in the delta gene at nucleotides 54755–54760 and a breakpoint in the beta gene at nucleotides 62153– 62158 [GenBank Ref ID: HUMHBB] with a 5 nucleotide “CAACA” bp region overlapping area. The subject, who is homozygous for the identified deletions, has a clinical phenotype of thalassemia intermedia. He has not yet required red cell transfusions. This is the first instance of a Senegalese-type deletion occurring in the homozygous state. The genotype provides insights into regulation of globin gene expression. While the ∼7 Kb deletion in the δβ-intergenic region may be responsible for the increased expression of the γ-globin gene similar to Hb Lepore deletions, the continued low level expression of the β-globin gene is most probably the result of the juxtaposition of the inefficient δ-globin promoter brought in the vicinity of the β-globin gene. Disclosures: No relevant conflicts of interest to declare.

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