RESUMO
Unstable gamma globin variants can cause transient neonatal hemolytic anemia. We have identified a novel variant in a newborn who presented with jaundice and anemia requiring phototherapy and red blood cell transfusion. The patient was found to be heterozygous for the mutation HGB2:c.290T>C, p.Leu97Pro, which we have termed hemoglobin (Hb) Wareham. This substitution is expected to generate an unstable hemoglobin with increased oxygen affinity based on the homologous mutation previously described in the beta globin gene, which is termed as Hb Debrousse. The patient fully recovered by 9 months of age as expected with the transition from fetal to adult hemoglobin.
Assuntos
Anemia Hemolítica , Hemoglobinas Anormais , gama-Globinas , Humanos , Recém-Nascido , Anemia Hemolítica/genética , Globinas beta/genética , gama-Globinas/genética , Hemoglobinas Anormais/genética , Heterozigoto , Mutação , LactenteRESUMO
HbVar (http://globin.bx.psu.edu/hbvar) is a widely-used locus-specific database (LSDB) launched 20 years ago by a multi-center academic effort to provide timely information on the numerous genomic variants leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Here, we report several advances for the database. We made clinically relevant updates of HbVar, implemented as additional querying options in the HbVar query page, allowing the user to explore the clinical phenotype of compound heterozygous patients. We also made significant improvements to the HbVar front page, making comparative data querying, analysis and output more user-friendly. We continued to expand and enrich the regular data content, involving 1820 variants, 230 of which are new entries. We also increased the querying potential and expanded the usefulness of HbVar database in the clinical setting. These several additions, expansions and updates should improve the utility of HbVar both for the globin research community and in a clinical setting.
Assuntos
Bases de Dados Genéticas , Hemoglobinas/genética , Mutação , Software , Talassemia/genética , Expressão Gênica , Loci Gênicos , Genoma Humano , Genômica/métodos , Genótipo , Hemoglobinas/química , Hemoglobinas/metabolismo , Heterozigoto , Humanos , Internet , Fenótipo , Talassemia/classificação , Talassemia/patologiaRESUMO
Sickle cell disease (SCD) is a genetic disorder that affects millions around the world. Enhancement of fetal γ-globin levels and fetal haemoglobin (HbF) production in SCD patients leads to diminished severity of many clinical features of the disease. We recently identified the transcriptional co-activator PGC-1α as a new protein involved in the regulation of the globin genes. Here, we report that upregulation of PGC-1α by infection with a lentivirus expressing PGC-1α or by the small-molecule PGC-1α agonist ZLN005 in human primary erythroid progenitor CD34+ cells induces both fetal γ-globin mRNA and protein expression as well as the percentage of HbF-positive cell (F cells) without significantly affecting cell proliferation and differentiation. We further found that the combination of ZLN005 and hydroxyurea (hydroxycarbamide) exhibited an additive effect on the expression of γ-globin and the generation of F cells from cultured CD34+ cells. In addition, ZLN005 induced robust expression of the murine embryonic ßh1-globin gene and to a lesser extent, human γ-globin gene expression in sickle mice. These findings suggest that activation of PGC-1α by ZLN005 might provide a new path for modulating HbF levels with potential therapeutic benefit in ß-hemoglobinopathies.
Assuntos
Anemia Falciforme , Hemoglobinopatias , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Animais , Hemoglobina Fetal/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos , gama-Globinas/genéticaRESUMO
The thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from ß-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of ß-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent ß-thalassemia genotypes with currently available gene-editing technology.
Assuntos
Edição de Genes , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas , Sítios de Splice de RNA , Splicing de RNA , Globinas beta , Talassemia beta , Sistemas CRISPR-Cas , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Mutação Puntual , Globinas beta/biossíntese , Globinas beta/genética , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/terapiaRESUMO
We report a novel frameshift ß-thalassemia (ß-thal) mutation due to a two-nucleotide deletion at codon 118 of the ß-globin gene (HBB: c.356_357delTT) in a 4-year-old Iraqi Kurd female presenting as transfusion-dependent ß-thal. This frameshift mutation, unlike many others involving the third exon, behaved as a recessive ß0 defect and not as dominant ß-thal mutation.
Assuntos
Globinas beta , Talassemia beta , Pré-Escolar , Códon , Feminino , Mutação da Fase de Leitura , Humanos , Iraque , Globinas beta/genética , Talassemia beta/diagnóstico , Talassemia beta/etnologia , Talassemia beta/genéticaRESUMO
Induced pluripotent stem cells (iPSCs) stand to revolutionize the way we study human development, model disease, and eventually, treat patients. However, these cell sources produce progeny that retain embryonic and/or fetal characteristics. The failure to mature to definitive, adult-type cells is a major barrier for iPSC-based disease modeling and drug discovery. To directly address these concerns, we have developed a chemically defined, serum and feeder-free-directed differentiation platform to generate hematopoietic stem-progenitor cells (HSPCs) and resultant adult-type progeny from iPSCs. This system allows for strict control of signaling pathways over time through growth factor and/or small molecule modulation. Through direct comparison with our previously described protocol for the production of primitive wave hematopoietic cells, we demonstrate that induced HSPCs are enhanced for erythroid and myeloid colony forming potential, and strikingly, resultant erythroid-lineage cells display enhanced expression of adult ß globin indicating definitive pathway patterning. Using this system, we demonstrate the stage-specific roles of two key signaling pathways, Notch and the aryl hydrocarbon receptor (AHR), in the derivation of definitive hematopoietic cells. We illustrate the stage-specific necessity of Notch signaling in the emergence of hematopoietic progenitors and downstream definitive, adult-type erythroblasts. We also show that genetic or small molecule inhibition of the AHR results in the increased production of CD34+ CD45+ HSPCs while conversely, activation of the same receptor results in a block of hematopoietic cell emergence. Results presented here should have broad implications for hematopoietic stem cell transplantation and future clinical translation of iPSC-derived blood cells. Stem Cells 2018;36:1004-1019.
Assuntos
Hematopoese/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Notch/genética , Diferenciação Celular , Humanos , Transdução de SinaisRESUMO
The HBS1L-MYB intergenic region (chr6q23) regulates erythroid cell proliferation, maturation, and fetal hemoglobin (HbF) expression. An enhancer element within this locus, highlighted by a 3-bp deletion polymorphism (rs66650371), is known to interact with the promoter of the neighboring gene, MYB, to increase its expression, thereby regulating HbF production. RNA polymerase II binding and a 50-bp transcript from this enhancer region reported in ENCODE datasets suggested the presence of a long noncoding RNA (lncRNA). We characterized a novel 1283bp transcript (HMI-LNCRNA; chr6:135,096,362-135,097,644; hg38) that was transcribed from the enhancer region of MYB. Within erythroid cells, HMI-LNCRNA was almost exclusively present in nucleus, and was much less abundant than the mRNA for MYB. HMI-LNCRNA expression was significantly higher in erythroblasts derived from cultured adult peripheral blood CD34+ cells which expressed more HBB, compared to erythroblasts from cultured cord blood CD34+ cells which expressed much more HBG. Down-regulation of HMI-LNCRNA in HUDEP-2 cells, which expressed mostly HBB, significantly upregulated HBG expression both at the mRNA (200-fold) and protein levels, and promoted erythroid maturation. No change was found in the expression of BCL11A and other key transcription factors known to modulate HBG expression. HMI-LNCRNA plays an important role in regulating HBG expression, and its downregulation can result in a significant increase in HbF. HMI-LNCRNA might be a potential therapeutic target for HbF induction treatment in sickle cell disease and ß-thalassemia.
Assuntos
Cromossomos Humanos Par 6 , DNA Intergênico/genética , Hemoglobina Fetal/genética , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Genes myb , RNA Longo não Codificante , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Eritroblastos/metabolismo , Células Eritroides/metabolismo , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/metabolismo , Humanos , Locos de Características QuantitativasRESUMO
Alpha thalassemia due to nondeletional mutations usually leads to more severe disease than that caused by deletional mutations. Devastating outcomes such as hydrops fetalis can occur with two nondeletional mutations, therefore warranting DNA-based workup for suspected carriers with subtle hematological abnormalities for family counseling purposes. We describe three cases with hemoglobin (Hb) Adana, a nondeletional alpha chain mutation, compounded with an alpha globin gene deletion resulting in thalassemia intermedia. We review the literature, draw genotype-phenotype correlations from published cases of Hb Adana, and propose that this correlation can be used by clinicians to help direct diagnostic studies and urge hematologists to thoroughly workup high-risk patients.
Assuntos
Hemoglobinas Anormais/genética , alfa-Globinas/genética , Talassemia alfa/genética , Pré-Escolar , Códon/genética , Feminino , Deleção de Genes , Estudos de Associação Genética , Aconselhamento Genético , Humanos , Hidropisia Fetal/etiologia , Hidropisia Fetal/genética , Masculino , Mutação Puntual , Deleção de SequênciaRESUMO
ß thalassemia is characterized by a deficient production of functional ß-globin chains and a relative excess of α-globin chains. An extremely diverse clinical spectrum-asymptomatic to transfusion-dependent-is primarily due to homozygosity or compound heterozygosity for the very large number of ß-thalassemia-causing mutations, along with interacting mutations that affect the α-globin and γ-globin genes and their expression. We report a case of a 16-month-old boy who was initially diagnosed with iron deficiency anemia until he was later found to be homozygous for a severe ß-thalassemia genotype with a mild hematologic phenotype. This was likely as a result of his ability to produce high levels of fetal hemoglobin.
Assuntos
Talassemia beta , Anemia/genética , Hemoglobina Fetal/biossíntese , Genótipo , Homozigoto , Humanos , Lactente , Masculino , Mutação , Fenótipo , Talassemia beta/complicações , Talassemia beta/genéticaRESUMO
BACKGROUND: Sickle cell anemia causes severe complications and premature death. Five common ß-globin gene cluster haplotypes are each associated with characteristic fetal hemoglobin (HbF) levels. As HbF is the major modulator of disease severity, classifying patients according to haplotype is useful. The first method of haplotype classification used restriction fragment length polymorphisms (RFLPs) to detect single nucleotide polymorphisms (SNPs) in the ß-globin gene cluster. This is labor intensive, and error prone. METHODS: We used genome-wide SNP data imputed to the 1000 Genomes reference panel to obtain phased data distinguishing parental alleles. RESULTS: We successfully haplotyped 813 sickle cell anemia patients previously classified by RFLPs with a concordance >98%. Four SNPs (rs3834466, rs28440105, rs10128556, and rs968857) marking four different restriction enzyme sites unequivocally defined most haplotypes. We were able to assign a haplotype to 86% of samples that were either partially or misclassified using RFLPs. CONCLUSION: Phased data using only four SNPs allowed unequivocal assignment of a haplotype that was not always possible using a larger number of RFLPs. Given the availability of genome-wide SNP data, our method is rapid and does not require high computational resources.
Assuntos
Anemia Falciforme/genética , Haplótipos , Polimorfismo de Nucleotídeo Único , Globinas beta/genética , Adolescente , Adulto , Anemia Falciforme/patologia , Criança , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco Pluripotentes/metabolismo , Adulto JovemRESUMO
Hemoglobin S/Black (A γδß)0 -thalassemia is a rare sickle cell disease (SCD) variant. On the basis of limited descriptions in the literature, the disease is reported as a mild microcytic anemia with an uncomplicated course. We report the clinical and laboratory data of nine patients whose diagnoses were confirmed by DNA-based techniques. Despite having mild anemia and high fetal hemoglobin level postinfancy, these patients developed many of the classic complications of SCD, including vaso-occlusive crisis, acute chest syndrome, avascular necrosis, and cholelithiasis. On the basis of these findings, we recommend that patients with this rare disorder receive specialized hematology care according to SCD guidelines.
Assuntos
Anemia Falciforme/patologia , Negro ou Afro-Americano/genética , Hemoglobina Fetal/genética , Hemoglobina Falciforme/genética , Talassemia/patologia , Adolescente , Adulto , Anemia Falciforme/genética , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Prognóstico , Índice de Gravidade de Doença , Talassemia/genética , Adulto JovemRESUMO
Hemoglobin (Hb) is the protein responsible for oxygen transportation. It is a tetrameric protein comprising two α- and two ß-globin subunits. In the literature, a large number of mutations in the α- and ß-globin genes have been documented. Among these mutations, Hb Presbyterian (HBB: c.327 C>G), is a naturally occurring mutant exerting low oxygen affinity. The C to G exchange (AAC>AAG) at codon 108 of the ß-globin gene results in the substitution of asparagine by lysine. Here, we document the identification of HBB: c.327 C>G in a 6-year-old female patient and her father from Nicaragua and Cuba, respectively. The presence of the abnormal Hb was confirmed by cellulose acetate electrophoresis, high performance liquid chromatography (HPLC) and genomic DNA sequencing. The ß-globin gene sequences for both, father and daughter, disclosed the heterozygous mutation at codon 108 to be Hb Presbyterian or HBB: c.327 C>G. The mutant Hb was previously reported in four families from North America, Germany, Japan and Spain, respectively. This is the fifth family carrying HBB: c.327 C>G described to date and the first report from Latin America.
Assuntos
Hemoglobinopatias/diagnóstico , Hemoglobinopatias/genética , Hemoglobinas Anormais/genética , Mutação , Globinas beta/genética , Adulto , Alelos , Criança , Códon , Análise Mutacional de DNA , Feminino , Genótipo , Hemoglobinopatias/sangue , Humanos , Masculino , Nicarágua , FenótipoRESUMO
Two 21-year old dizygotic twin men of Iraqi descent were homozygous for HBB codon 8, deletion of two nucleotides (-AA) frame-shift ß(0) -thalassaemia mutation (FSC8; HBB:c25_26delAA). Both were clinically well, had splenomegaly, and were never transfused. They had mild microcytic anaemia (Hb 120-130 g/l) and 98% of their haemoglobin was fetal haemoglobin (HbF). Both were carriers of Hph α-thalassaemia mutation. On the three major HbF quantitative trait loci (QTL), the twins were homozygous for G>A HBG2 Xmn1 site at single nucleotide polymorphism (SNP) rs7482144, homozygous for 3-bp deletion HBS1L-MYB intergenic polymorphism (HMIP) at rs66650371, and heterozygous for the A>C BCL11A intron 2 polymorphism at rs766432. These findings were compared with those found in 22 other FSC8 homozygote patients: four presented with thalassaemia intermedia phenotype, and 18 were transfusion dependent. The inheritance of homozygosity for HMIP 3-bp deletion at rs66650371 and heterozygosity for Hph α-thalassaemia mutation was found in the twins and not found in any of the other 22 patients. Further studies are needed to uncover likely additional genetic variants that could contribute to the exceptionally high HbF levels and mild phenotype in these twins.
Assuntos
Doenças em Gêmeos/genética , Mutação da Fase de Leitura , Talassemia beta/genética , Proteínas de Transporte/genética , Feminino , Hemoglobina Fetal/análise , Hemoglobina Fetal/genética , Genes myb , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Proteínas Repressoras , Gêmeos Dizigóticos/genética , Adulto JovemRESUMO
Pharmacologic augmentation of γ-globin expression sufficient to reduce anemia and clinical severity in patients with diverse hemoglobinopathies has been challenging. In studies here, representative molecules from four chemical classes, representing several distinct primary mechanisms of action, were investigated for effects on γ-globin transcriptional repressors, including components of the NuRD complex (LSD1 and HDACs 2-3), and the downstream repressor BCL11A, in erythroid progenitors from hemoglobinopathy patients. Two HDAC inhibitors (MS-275 and SB939), a short-chain fatty acid derivative (sodium dimethylbutyrate [SDMB]), and an agent identified in high-throughput screening, Benserazide, were studied. These therapeutics induced γ-globin mRNA in progenitors above same subject controls up to 20-fold, and increased F-reticulocytes up to 20%. Cellular protein levels of BCL11A, LSD-1, and KLF1 were suppressed by the compounds. Chromatin immunoprecipitation assays demonstrated a 3.6-fold reduction in LSD1 and HDAC3 occupancy in the γ-globin gene promoter with Benserazide exposure, 3-fold reduction in LSD-1 and HDAC2 occupancy in the γ-globin gene promoter with SDMB exposure, while markers of gene activation (histone H3K9 acetylation and H3K4 demethylation), were enriched 5.7-fold. These findings identify clinical-stage oral therapeutics which inhibit or displace major co-repressors of γ-globin gene transcription and may suggest a rationale for combination therapy to produce enhanced efficacy.
Assuntos
Benserazida/farmacologia , Butiratos/farmacologia , Células Precursoras Eritroides/efeitos dos fármacos , Hemoglobinopatias/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Ativação Transcricional/efeitos dos fármacos , gama-Globinas/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Células Precursoras Eritroides/metabolismo , Hemoglobinopatias/genética , Humanos , RNA Mensageiro/genéticaRESUMO
Fetal hemoglobin (HbF) modulates the phenotype of sickle cell anemia by inhibiting deoxy sickle hemoglobin (HbS) polymerization. The blood concentration of HbF, or the number of cells with detectable HbF (F-cells), does not measure the amount of HbF/F-cell. Even patients with high HbF can have severe disease because HbF is unevenly distributed among F-cells, and some cells might have insufficient concentrations to inhibit HbS polymerization. With mean HbF levels of 5%, 10%, 20%, and 30%, the distribution of HbF/F-cell can greatly vary, even if the mean is constant. For example, with 20% HbF, as few as 1% and as many as 24% of cells can have polymer-inhibiting, or protective, levels of HbF of â¼10 pg; with lower HbF, few or no protected cells can be present. Only when the total HbF concentration is near 30% is it possible for the number of protected cells to approach 70%. Rather than the total number of F-cells or the concentration of HbF in the hemolysate, HbF/F-cell and the proportion of F-cells that have enough HbF to thwart HbS polymerization is the most critical predictor of the likelihood of severe sickle cell disease.
Assuntos
Anemia Falciforme/imunologia , Anemia Falciforme/terapia , Hemoglobina Fetal/imunologia , Adulto , Anemia Falciforme/sangue , Eritrócitos/citologia , Deleção de Genes , Haplótipos , Hemoglobina Falciforme/imunologia , Heterozigoto , Humanos , Hidroxiureia/uso terapêutico , Modelos Teóricos , Família Multigênica , Fenótipo , Polímeros/químicaAssuntos
Anemia Falciforme/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Células Eritroides/metabolismo , Hemoglobina Fetal/biossíntese , Histona Desmetilases/antagonistas & inibidores , Células-Tronco Pluripotentes Induzidas/metabolismo , Anemia Falciforme/metabolismo , Anemia Falciforme/patologia , Animais , Inibidores Enzimáticos/química , Células Eritroides/patologia , Histona Desmetilases/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , CamundongosRESUMO
Fetal hemoglobin (HbF) levels are higher in the Arab-Indian (AI) ß-globin gene haplotype of sickle cell anemia compared with African-origin haplotypes. To study genetic elements that effect HbF expression in the AI haplotype we completed whole genome sequencing in 14 Saudi AI haplotype sickle hemoglobin homozygotes-seven selected for low HbF (8.2% ± 1.3%) and seven selected for high HbF (23.5% ± 2.6%). An intronic single nucleotide polymorphism (SNP) in ANTXR1, an anthrax toxin receptor (chromosome 2p13), was associated with HbF. These results were replicated in two independent Saudi AI haplotype cohorts of 120 and 139 patients, but not in 76 Saudi Benin haplotype, 894 African origin haplotype and 44 AI haplotype patients of Indian origin, suggesting that this association is effective only in the Saudi AI haplotype background. ANTXR1 variants explained 10% of the HbF variability compared with 8% for BCL11A. These two genes had independent, additive effects on HbF and together explained about 15% of HbF variability in Saudi AI sickle cell anemia patients. ANTXR1 was expressed at mRNA and protein levels in erythroid progenitors derived from induced pluripotent stem cells (iPSCs) and CD34+ cells. As CD34+ cells matured and their HbF decreased ANTXR1 expression increased; as iPSCs differentiated and their HbF increased, ANTXR1 expression decreased. Along with elements in cis to the HbF genes, ANTXR1 contributes to the variation in HbF in Saudi AI haplotype sickle cell anemia and is the first gene in trans to HBB that is associated with HbF only in carriers of the Saudi AI haplotype. Am. J. Hematol. 91:1118-1122, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Anemia Falciforme/genética , Hemoglobina Fetal/genética , Haplótipos , Adolescente , Adulto , Árabes/genética , Proteínas de Transporte/genética , Criança , Pré-Escolar , Feminino , Expressão Gênica , Humanos , Masculino , Proteínas dos Microfilamentos , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Polimorfismo de Nucleotídeo Único , Receptores de Superfície Celular/genética , Proteínas Repressoras , População Branca/genética , Adulto Jovem , Globinas beta/genéticaRESUMO
We report an infant with a compound heterozygosity for Hb C (HBB: c.19G > A) and Hb Osu Christiansborg (HBB: c.157G > A) and a phenotype of mild microcytic anemia with target cell morphology but without overt hemolysis.
Assuntos
Anemia Hipocrômica/genética , Hemoglobina C/genética , Hemoglobinas Anormais/genética , Heterozigoto , Humanos , Lactente , FenótipoRESUMO
Hereditary persistence of fetal hemoglobin (HPFH) can be caused by point mutations in the γ-globin gene promoters. We report three rare cases: a child compound heterozygous for Hb S (HBB: c.20A > T) and HPFH with a novel point mutation in the (A)γ-globin gene promoter who had 42.0% Hb S, 17.0% Hb A and 38.0% Hb F; a man with Hb SC (HBB: c.19G > A) disease and a point mutation in the (G)γ-globin gene promoter who had 54.0% Hb S, 18.0% Hb C and 25.0% Hb F; a child heterozygous for Hb S and HPFH due to mutations in both the (A)γ- and (G)γ-globin gene promoters in cis [(G)γ(A)γ(ß(+)) HPFH], with 67.0% Hb A, 6.5% Hb S and 25.0% Hb F.