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1.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(1): 33-36, 2020 Jan 10.
Artigo em Chinês | MEDLINE | ID: mdl-31922592

RESUMO

OBJECTIVE: To explore hematological and genotypic characteristics of patients with hemoglobin E (Hb E) disorders from Yunnan Province. METHODS: One hundred individuals with Hb E disorders indicated by high performance liquid chromatography (HPLC) were subjected to genetic testing through multiple gap-PCR and reverse dot-blotting analysis. RESULTS: All patients were found to harbor a mutation to the 26th codon of the ß -globin chain (HBB: c.79G>A). Ninety patients were heterozygotes, and 10 co-inherited c.79G>A and an α -thalassemia mutation (7 α α /-α3.7, 2 α α /--SEA and 1 -α 3.7/-α3.7). Hematological characteristics of the heterozygotes were: Hb A2 (26.02±3.64)%, Hb F(1.35±1.25)%, MCV(78.83±4.68) fl, MCH(26±1.54) pg, MCHC (329.65±10.73) g/L, HGB (141.08±16.53) g/L, while that of the co-inherited cases was decided by the type of α -thalassemia mutation. CONCLUSION: Hb E can be effectively detected by HPLC. The type of α -thalassemia mutations will determine hematological features of co-inherited cases. Hb E disorders may be missed by relying only on routine blood test upon prenatal screening.


Assuntos
Hemoglobina E , Talassemia alfa , China , Feminino , Genótipo , Hemoglobina E/genética , Humanos , Mutação , Gravidez , Talassemia alfa/genética , Globinas beta/genética
2.
Ann Hematol ; 99(1): 23-29, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31776727

RESUMO

Hemoglobin (Hb) F has a modulatory effect on the clinical phenotype of ß-thalassemia disease. High expression of Hb F in Hb E-related disorders has been noted, but the mechanism is not well understood. We have examined the association of a novel SNP rs11759328 on ARHGAP 18 gene and other known modulators with a variability of Hb F in Hb E-related disorders. Genotyping of SNP rs11759328 (G/A) was performed based on high-resolution melting analysis. The rs11759328 (A allele) was shown to be significantly associated with Hb F levels (p < 0.05) in heterozygous and homozygous Hb E. High levels of Hb F in both heterozygous and homozygous Hb E were also found to be associated with SNPs in the study of other modifying genes including KLF 1 mutation, rs7482144 (Gγ-XmnI), rs4895441, rs9399137 of (HBS1L-MYB), and rs4671393 (BCL11A). Multivariate analysis showed that KLF1 mutation and SNP rs11759328 (GA) (ARHGAP18) modulated Hb F expression in heterozygous Hb E. For homozygous Hb E, this was found to be related to five modifying factors, i.e., KLF1 mutation, rs4895441 (GG), rs9399137 (CC), rs4671393 (AA), and rs4671393 (GA). These results indicate that a novel SNP rs11759328 is a genetically modifying factor associated with increased Hb F in Hb E disorder.


Assuntos
Hemoglobina Fetal/biossíntese , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica , Hemoglobinúria/genética , Mutação , Polimorfismo de Nucleotídeo Único , Hemoglobina Fetal/genética , Proteínas Ativadoras de GTPase/metabolismo , Hemoglobina E/genética , Hemoglobina E/metabolismo , Hemoglobinúria/sangue , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Tailândia
3.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(5): 1580-1584, 2019 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-31607315

RESUMO

OBJECTIVE: To analyze the hematological characteristics of HbE homozygotes. METHODS: Complete blood cells count and hemoglobin electrophoresis were used for phenotypic analysis of 78 cases with HbE homozygotes from Yunnan province, China. The PCR-fluorescence hybridization was used to detect the common gene mutation of thalassemia. The hematological indexes, including MCV, MCH, Hb, HbA2, HbF and HbE were statistically analyzed between groups with different sex, ages and compound α thalassemia status. RESULTS: In HbE homozygotes (HbEE), 89.5% (17/19) children presented mild to moderate microcytic hypochromic anemia, and 10.5% of them presented moderate anemia. 39.6% (19/48) of women with HbEE developed mild anemia ,while 11 cases of male with HbE homozygotes were asymptomatic. The levels of MCV and MCH in HbE homozygotes increased by co-inheritance of α thalassemia mutation. CONCLUSION: The clinical phenotype of HbE homozygote shows highly heterogeneous, which is relates with age, sex and co-inheriting α-globin genotypes. In Hb EE women and children are more likely to develop mild to moderate anemia. The microcytic hypochromic anemia degree is relieved when HbEE combined with α- thalassemia.


Assuntos
Hemoglobina E/genética , Criança , China , Feminino , Genótipo , Homozigoto , Humanos , Masculino , Fenótipo , Talassemia alfa
4.
Ann Hematol ; 98(8): 1827-1834, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31190133

RESUMO

In this study, we aimed to investigate the pattern and association of genetic mutations occurring within the alpha hemoglobin-stabilizing protein (AHSP) gene among HbE beta thalassemia patients with varying phenotypic expressions. Fifty-four diagnosed cases of HbE beta thalassemia (transfusion dependent and independent) were included in the study. Among them, 38 patients with similar genotypes (IVS 1-5, alpha gene deletion and triplication, Xmn polymorphism) were selected for further analysis. AHSP gene sequencing was done for these 38 samples to study associated mutations in AHSP gene. HbE beta thalassemia patients with similar genotypes but different phenotypic expressions were found to have mutations in the AHSP gene. There were five mutations found most prevalent among the samples analyzed for AHSP gene sequencing. Among these, two mutations were from intron 1 region of AHSP and three mutations were found in exon 3. The most prevalent mutation was found at the Oct binding site at intron 1 of AHSP. The mutations in exon 3 were more prevalent among the TDT groups. A mutation in exon 3 changing the amino acid (33rd) from serine to phenylalanine was found to be associated with only TDT group. This study documents that among the HbE beta thalassemia patients with varying severity, an exon mutation in AHSP is significantly prevalent only among the TDT group. Further understanding of the mechanism will shed light upon the impact of AHSP in modifying the disease severity in thalassemia.


Assuntos
Proteínas Sanguíneas/genética , Deleção de Genes , Duplicação Gênica , Hemoglobina E/genética , Chaperonas Moleculares/genética , Talassemia beta/genética , Adolescente , Adulto , Sequência de Bases , Proteínas Sanguíneas/metabolismo , Transfusão de Sangue/estatística & dados numéricos , Criança , Pré-Escolar , Análise Mutacional de DNA , Eritrócitos/metabolismo , Eritrócitos/patologia , Éxons , Feminino , Expressão Gênica , Genótipo , Hemoglobina E/metabolismo , Humanos , Íntrons , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Fenótipo , Estrutura Secundária de Proteína , Índice de Gravidade de Doença , Talassemia beta/metabolismo , Talassemia beta/patologia , Talassemia beta/terapia
5.
Hematology ; 24(1): 459-466, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31124399

RESUMO

Background: The clinical consequences and significance of many unstable hemoglobins interacting with other hemoglobinopathies remain unrecognized. Here we first explore molecular and hematological characterizations of previously undescribed compound heterozygosity states for unstable hemoglobin Rush (Hb Rush, Beta 101 Glu > Gln, HBB:c.304G > C) with Hb E and different forms of thalassemia. Methods: Hematological assays, globin gene mutation assays and ß-globin gene cluster haplotype were conducted in 11 patients from 8 unrelated Chinese ethnic families with unexplained hemoglobin separation fraction in hemoglobin gel electrophoresis. Results: Hb Rush in various combinations with Hb E, ß0-thalassemias and α+-thalassemia were identified. Hb Rush simple heterozygote was generally associated with mild hemolytic anemia, and the compound heterozygotes of Hb Rush and the other ß-globin variants led to thalassemia intermedia phenotypes with moderate anemia. Hemoglobin electrophoreses showed that the co-presence of Hb Rush with either Hb E or ß0-thalassemias increased proportion of Hb Rush due to relative decrease of other globin chain synthesis. Beta-globin gene cluster haplotype analysis suggested a common origin of the Hb Rush variant in the Chinese families of different ethnic ancestry. Conclusions: Unstable Hb Rush interacting with ß-thalassemia result in thalassemia intermedia phenotypes, which demonstrated the clinical significance of Hb Rush and new insights into complex mechanism of clinical heterogeneity of thalassemia.


Assuntos
Hemoglobina E/genética , Hemoglobinas Anormais/genética , Talassemia beta/genética , Adolescente , Adulto , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
7.
J Clin Pathol ; 72(4): 322-324, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30630871

RESUMO

Interaction of ßE-globin gene with an in trans ß-thalassaemia gene leads to thalassaemia syndrome, known as haemoglobin (Hb) E-ß-thalassaemia disease, with variable clinical and haematological severity. Here, we reported for the first time the Hb E-ß-thalassaemia syndrome caused alternatively by an in cis interaction of ßE and a novel IVSI#7;A>G mutation, namely the Hb E-Udon Thani (HBB:c.[79G>A;92+7 A>G]). The syndrome was found in an adult Thai man (32) who was generally healthy but had an unexplained hypochromic microcytosis. Hb analysis identified heterozygous Hb E with very low Hb E expression (3.1%) and elevated Hb A2 (5.7%). Final diagnosis was made on DNA analysis, which confirmed a double mutation in a single ß-globin gene of the patient. A multiplex allele-specific PCR assay was developed for use in the screening of this novel form of Hb E-ß-thalassaemia in the population.


Assuntos
Hemoglobina E/genética , Mutação , Talassemia beta/genética , Adulto , Análise Mutacional de DNA/métodos , Predisposição Genética para Doença , Hemoglobina E/metabolismo , Heterozigoto , Humanos , Masculino , Reação em Cadeia da Polimerase Multiplex , Fenótipo , Síndrome , Talassemia beta/sangue , Talassemia beta/diagnóstico
8.
Ann Lab Med ; 39(2): 209-213, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30430785

RESUMO

Hb mutations can alter the structure, behavior, stability, or quantity of the globin chain produced. Some Hb variants shorten the erythrocyte life span, resulting in physiologically lower hemoglobin A1c (HbA1c) levels. The hemoglobin E (HbE) phenotype involves a single-nucleotide polymorphism that reduces ß-globin chain synthesis. We compared the HbA1c levels of subjects with normal Hb (HbAA; N=131) and HbE (N=148) phenotypes, examining potential hematological and biochemical factors contributing to differences in HbA1c levels. All had normal fasting plasma glucose (<5.6 mmol/L), AST, ALT, and creatinine levels. Mean±SD HbA1c levels differed between HbAA and HbE subjects: 5.5±0.3% and 5.3±0.3% (P<0.001) according to an immunoassay, and 5.5±0.3% and 5.3±0.3% (P<0.001) according to cation-exchange HPLC, respectively. In multiple logistic regression, only mean corpuscular volume (P<0.001) contributed to the difference in HbA1c levels between groups. Although a 0.2% difference in HbA1c is relatively small and unlikely to alter clinical decisions, epidemiologically, this can lead to misclassification of a significant proportion of the population, especially since the threshold of non-diabetes HbA1c (≤5.6%) falls very close to the HbA1c median of the general population.


Assuntos
Hemoglobina A Glicada/análise , Hemoglobina E/genética , Glicemia/análise , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Creatinina/sangue , Estudos Transversais , Hemoglobina A/genética , Humanos , Imunoensaio , Modelos Logísticos , Fenótipo
9.
Ann Hematol ; 98(2): 289-299, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30413899

RESUMO

Thalassaemia are the most common inherited autosomal recessive single gene disorders characterized by chronic hereditary haemolytic anaemia due to the absence or reduced synthesis of one or more of the globin chains. Haemoglobin E-ß thalassaemia is the genotype responsible for approximately one half of all severe beta-thalassaemia worldwide. This study proposes to evaluate the effect of various molecular parameters on the response of hydroxyurea. Hydroxyurea was started at an initial dose of 10 mg/kg of body weight/day on 110 transfusion-dependent HbE-ß thalassaemia patients. HbF level was measured by HPLC analysis. ß-Thalassaemia mutations, XmnI and five other SNPs, and α-globin gene deletions and triplications were detected by ARMS-PCR, RFLP-PCR and Gap-PCR, respectively. Based on the factors for evaluating hydroxyurea-response, 42 patients were responders as they showed an increment of Hb from a mean baseline value of 6.45 g/dl (± 0.70) to 7.78 g/dl (± 0.72) post-therapy. Based on increase in HbF above the median value (14.72%) post-therapy, 78 patients were found to be responders. All the 78 responders showed mean decrease in transfusion of 74.26% (± 8.32) with a maximum decrease of 98.43%. There was a significant correlation between decrease in transfusion and increase in HbF level for all 78 responders. XmnI polymorphism showed the strongest association (p < 0.0001) with increase in HbF levels and Hb levels. Patients with α-globin gene deletions were better responders. It was concluded that hydroxyurea treatment is effective in transfusion-dependent HbE-ß thalassaemia patients and the response is best in patients having both XmnI polymorphism and α-deletion.


Assuntos
Transfusão de Sangue , Hemoglobina Fetal/biossíntese , Hemoglobina E/metabolismo , Hidroxiureia/administração & dosagem , Mutação , Polimorfismo de Nucleotídeo Único , Talassemia beta , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Hemoglobina Fetal/genética , Hemoglobina E/genética , Humanos , Masculino , Talassemia beta/sangue , Talassemia beta/genética , Talassemia beta/terapia
11.
Lab Med ; 50(1): 47-53, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30016461

RESUMO

Objective: To determine the number and intensity of phosphatidylserine (PS) expression of the red blood cells (RBCs), fragmented RBCs, and RBC-derived microparticles (RMPs) in patients with ß-thalassemia/hemoglobin (Hb)E. Methods: We used flow cytometry to determine the number and levels of PS expression. Results: The number of PS-exposing RBCs was statistically significantly higher (P <.001) than that of PS-exposing fragmented RBCs or RMPs. In contrast, the intensity of PS expression was significantly higher (P <.001) in RMPs than in RBCs or fragmented RBCs. Our study showed a trend of association between RBC distribution width (RDW) and both the number of fragmented RBCs and RMPs and their intensity of PS expression. Conclusion: In ß-thalassemia/HbE, PS-exposing RBCs, fragmented RBCs, and RMPs all differed in their numbers and their intensity of PS expression. The effects of these differences among PS-exposing populations on the pathophysiology of the disease require further investigation.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Eritrócitos/metabolismo , Talassemia beta/sangue , Adolescente , Adulto , Estudos de Casos e Controles , Micropartículas Derivadas de Células/patologia , Criança , Pré-Escolar , Eritrócitos/patologia , Feminino , Hemoglobina E/genética , Humanos , Lactente , Masculino , Fosfatidilserinas/genética , Fosfatidilserinas/metabolismo
13.
J Infect ; 77(5): 435-439, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29964138

RESUMO

OBJECTIVES: Hemoglobin E (HbE, ß26 Glu-Lys) is the most prevalent hemoglobinopathy in Southeast Asia. This study aimed to determine whether HbE protects against clinical Plasmodium vivax malaria in Southeast Asia. METHODS: In a case-control study performed in villages along the China-Myanmar border, we determined the prevalence of HbE in 257 villagers who had acute P. vivax infections and in 157 control healthy villagers. RESULTS: HbE in P. vivax patients (17.4%) was significantly less prevalent than in the healthy villager population (36.3%). Moreover, there was a complete lack of HbEE homozygotes in the vivax patients as compared to 9.5% prevalence in the healthy villagers. Using the HbAA group as the reference, both the HbEA heterozygotes and HbEE homozygotes had significantly lower odds of presenting with acute P. vivax infections. Furthermore, HbEA heterozygotes also had significantly lower P. vivax asexual parasite densities. HbEA did not affect the proportion of P. vivax patients with gametocytemia nor the gametocyte densities. CONCLUSIONS: HbE offers significant protection against the occurrence and parasite density of acute P. vivax infections and provides a renewed perspective on P. vivax malaria as a potentially strong driving force behind the high frequencies of HbE in the Kachin population.


Assuntos
Resistência à Doença/genética , Hemoglobina E/genética , Malária Vivax/etnologia , Adolescente , Adulto , Idoso , Ásia Sudeste/epidemiologia , Estudos de Casos e Controles , Criança , Pré-Escolar , China/epidemiologia , Feminino , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Mianmar/epidemiologia , Plasmodium vivax , Prevalência , Adulto Jovem
14.
Clin Chem Lab Med ; 56(9): 1507-1513, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-29668441

RESUMO

BACKGROUND: The objective of the study was to describe a formula based on hemoglobin (Hb)A2 and HbF levels for differentiation of homozygous HbE and HbE-ß-thalassemia. METHODS: A total of 1256 subjects suspected for homozygous HbE or HbE-ß0-thalassemia were recruited at the ongoing thalassemia screening program at Khon Kaen University, Thailand. Hb analysis was done using capillary electrophoresis. Genotyping was based on DNA analysis. An arbitrary formula based on HbA2 and HbF was developed statistically for differentiation of the two conditions. Validation was carried out prospectively on another 139 subjects encountered at routine laboratory. RESULTS: Among 1256 subjects, Hb and DNA analyses identified cases with homozygous HbE (n=1076, 85.7%), HbE-ß0-thalassemia (n=140, 11.1%), HbE-δß0-thalassemia (n=30, 2.4%) and unknown HbE-related disorder (n=10, 0.8%). An inverse correlation between the amounts of HbA2 and HbF in HbE-ß0-thalassemia was observed. With differences in the amounts of HbA2 and HbF between the groups, an arbitrary score (7.3 HbA2+HbF) was developed where score above 60 indicated HbE-ß0-thalassemia. Application of this score on another 139 subjects showed accurate prediction of HbE-ß0-thalassemia with 100% sensitivity, 96.5% specificity, 85.7% positive predictive value and 100% negative predictive value. Successful application onto couples at risk was demonstrated. CONCLUSIONS: An established score should prove useful in the differentiation of homozygous HbE and HbE-ß0-thalassemia in routine setting and lead to a significant reduction in number of referring cases for molecular testing.


Assuntos
Hemoglobina E/análise , Talassemia beta/diagnóstico , Área Sob a Curva , Análise Mutacional de DNA , Diagnóstico Diferencial , Eletroforese Capilar , Hemoglobina Fetal/análise , Genótipo , Hemoglobina A2/análise , Hemoglobina E/genética , Homozigoto , Humanos , Curva ROC , Sensibilidade e Especificidade , Talassemia beta/genética
15.
Biochem Biophys Res Commun ; 499(1): 86-92, 2018 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-29550480

RESUMO

Repair of a splicing defect of ß-globin pre-mRNA harboring hemoglobin E (HbE) mutation was successfully accomplished in erythroid cells from patients with ß-thalassemia/HbE disorder by a synthetic splice-switching oligonucleotide (SSO). However, its application is limited by short-term effectiveness and requirement of lifelong periodic administration of SSO, especially for chronic diseases like thalassemias. Here, we engineered lentiviral vectors that stably express U7 small nuclear RNA (U7 snRNA) carrying the splice-switching sequence of the SSO that restores correct splicing of ßE-globin pre-mRNA and achieves a long-term therapeutic effect. Using a two-step tiling approach, we systematically screened U7 snRNAs carrying splice-switching SSO sequences targeted to the cryptic 5' splice site created by HbE mutation. We tested this approach and identified the most responsive element for mediating splicing correction in engineered U7 snRNAs in HeLa-ßE cell model cell line. Remarkably, the U7 snRNA lentiviral vector (U7 ßE4+1) targeted to this region effectively restored the correctly-spliced ßE-globin mRNA for at least 5 months. Moreover, the effects of the U7 ßE4+1 snRNA lentiviral vector were also evident as upregulation of the correctly-spliced ßE-globin mRNA in erythroid progenitor cells from ß-thalassemia/HbE patients treated with the vector, which led to improvements of pathologies in erythroid progenitor cells from thalassemia patients. These results suggest that the splicing correction of ßE-globin pre-mRNA by the engineered U7 snRNA lentiviral vector provides a promising, long-term treatment for ß-thalassemia/HbE.


Assuntos
Células Precursoras Eritroides/metabolismo , Engenharia Genética/métodos , Terapia Genética/métodos , Precursores de RNA/genética , Processamento de RNA , RNA Nuclear Pequeno/genética , Globinas beta/genética , Sequência de Bases , Células Precursoras Eritroides/patologia , Éxons , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HeLa , Hemoglobina E/genética , Hemoglobina E/metabolismo , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Mutação , Cultura Primária de Células , Precursores de RNA/metabolismo , Sítios de Splice de RNA , RNA Nuclear Pequeno/metabolismo , Globinas beta/metabolismo , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/patologia , Talassemia beta/terapia
16.
Mymensingh Med J ; 27(1): 205-208, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29459615

RESUMO

Here we present a Bangladeshi family where out of four family members two (mother and son) inherited both haemoglobin (HbS) S and haemoglobin (HbE) E gene confirming the diagnosis of double heterozygous state for HbS and HbE, presented in the Armed Forces Institute of Pathology (AFIP), Dhaka Cantonment, Dhaka, Bangladesh on the month of July 2016. Among other two members, one (daughter) inherited HbS and other (father) HbE. Double heterozygous state for HbS and HbE is a rare condition in this subcontinent especially in Bangladesh. HbS is rare but HbE is prevalent in Bangladesh. Co-inheritance of both HbS and HbE is therefore uncommon in this country in comparison to HbE/ß - thalassaemia. Though the double heterozygous state for HbS and HbE is rare and the patients are usually asymptomatic but their documentation is important for genetic counseling and to determine the reproductive risk of the family.


Assuntos
Hemoglobina E , Hemoglobina Falciforme , Bangladesh , Documentação , Feminino , Aconselhamento Genético , Hemoglobina E/genética , Hemoglobina Falciforme/genética , Humanos
17.
Stem Cell Res Ther ; 9(1): 46, 2018 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-29482624

RESUMO

BACKGROUND: Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and ß-thalassemia (HbE/ß-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia. METHODS: We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression. RESULTS: The hemoglobin E mutation of HbE/ß-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. CONCLUSIONS: Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/ß-thalassemia in the future.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Hemoglobina E , Células-Tronco Pluripotentes Induzidas/metabolismo , Talassemia beta , Autoenxertos , Feminino , Hemoglobina E/genética , Hemoglobina E/metabolismo , Humanos , Masculino , Mutação , Transplante de Células-Tronco , Talassemia beta/genética , Talassemia beta/metabolismo , Talassemia beta/terapia
18.
Hemoglobin ; 42(1): 54-57, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29484903

RESUMO

Hb Q-Thailand [α74(EF3)Asp→His (α1), GAC>CAC, HBA1: c.223G>C] is an abnormal hemoglobin (Hb) frequently found in Thailand and Southeast Asian countries. The association of the αQ-Thailand allele with other globin gene disorders has important implications in diagnosis. Here, we report how to diagnose the coinheritance of Hb Q-Thailand with ß-thalassemia (ß-thal)/Hb E disease in four Thai samples from high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) testing results. Understanding of the HPLC chromatogram and CE electropherogram patterns of this complex mutation is important for interpretation of testing results and providing genetic counseling.


Assuntos
Hemoglobina E/genética , Hemoglobinopatias/genética , Hemoglobinas Anormais/genética , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Hemoglobinopatias/diagnóstico , Tailândia
19.
BMC Genet ; 19(1): 1, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29295702

RESUMO

BACKGROUND: Bangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92 + 1 - c.92 + 130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh. RESULTS: Our HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G > A, c.92 + 5G > C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result. CONCLUSIONS: Targeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and electrophortic indices in order to avoid false positive and false negative results.


Assuntos
Triagem de Portadores Genéticos/métodos , Hibridização de Ácido Nucleico/métodos , Globinas beta/genética , Talassemia beta/diagnóstico , Adolescente , Bangladesh , Criança , Pré-Escolar , Triagem de Portadores Genéticos/economia , Hemoglobina E/genética , Humanos , Lactente , Mutação , Talassemia beta/genética
20.
Hematology ; 23(7): 423-428, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29157161

RESUMO

OBJECTIVES: The inherited genetic disorder beta0-thalassemia/Hb E disease is associated with the over-suppression of the master regulator of iron homeostasis, the peptide hormone hepcidin. How developing erythroid cells mediate the suppression of hepcidin remains controversial, although a number of inhibitors have been proposed. METHODS: To investigate the ability of erythroid cells to suppress hepcidin expression in liver cells, conditioned media from the culture of in vitro differentiating erythroblasts (from normal controls and beta0-thalassemia/Hb E patients) was used to treat HepG2 cells, and the effects on hepcidin expression were assayed by real-time quantitative PCR and confocal microscopy. RESULTS: Early activation followed by later suppression of hepcidin expression was seen posttreatment. Markedly, however, no significant differences were observed between suppression of hepcidin as mediated by media from the culture of erythroblasts from normal controls and beta0-thalassemia/Hb E patients Discussion: Previous studies investigating the suppression of hepcidin expression in beta0-thalassemia/Hb E disease have used patient-derived serum samples, which are complex fluids with contributions from multiple cell types. This study has developed a simple in vitro system that allows investigation of how a single cell type mediates hepcidin expression. The results support proposals that over-suppression of hepcidin seen in beta-thalassemia/Hb E patients is a consequence of the increased mass of erythropoiesis and not defects in the signaling process per se. CONCLUSION: The in vitro cell system developed here allows further investigation into the processes mediating erythroid cell suppression of liver hepcidin expression in both normal and pathological states.


Assuntos
Eritroblastos/metabolismo , Regulação da Expressão Gênica , Hemoglobina E/genética , Hepcidinas/genética , Talassemia beta/sangue , Talassemia beta/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células Hep G2 , Humanos
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