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1.
Hemoglobin ; 43(1): 63-65, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31037981

RESUMO

Hb A'2 (or Hb B2) (HBD: c.49G>C) is the most frequent δ chain variant that has been described in Africa but not in Thailand. We report here a 10-month-old Thai infant with compound heterozygosity for ß0 codon 17 (A>T; HBB: c.52A>T) and ß+ IVS II-654 (C>T; HBB: c.316-197C>T). Under diagnosed ß-thalassemia (ß-thal) in her father, who carries Hb A'2 and a heterozygous ß0 codon 17 mutation, and the mother, who carries a heterozygous ß+ IVS II-654 mutation, was noted. Although Hb A'2 does not cause any problems, heterozygosity for Hb A'2 can lead to under diagnosis of ß-thal in Hb A'2 samples. This case highlights the importance of Hb A'2 in prenatal diagnosis (PND). Thus, molecular analysis for ß-thal mutations should be carried out when a small peak presents at the retention time (RT) of 4.71 min. on high performance liquid chromatography (HPLC) and the summation level of this peak and Hb A2 was equal or higher than 4.0%.


Assuntos
Hemoglobina A2/genética , Heterozigoto , Globinas beta/genética , Talassemia beta/diagnóstico , Talassemia beta/genética , Adulto , Cromatografia Líquida de Alta Pressão , Códon , Índices de Eritrócitos , Feminino , Genótipo , Hemoglobina A2/química , Hemoglobinas Anormais/química , Hemoglobinas Anormais/genética , Humanos , Lactente , Masculino , Mutação , Diagnóstico Pré-Natal , Globinas beta/química , Talassemia beta/sangue
2.
Hemoglobin ; 41(3): 213-215, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28795619

RESUMO

We report here the hematological and molecular features of a novel δ-globin chain variant found in a Southern Thai woman. Her complete blood count was as follows: red blood cell (RBC) count 5.90 × 1012/L, hemoglobin concentration (Hb) 12.6 g/dL, packed cell volume (PCV) 0.41 L/L, mean corpuscular volume (MCV) 69.5 fL, mean corpuscular Hb (MCH) 21.4 pg, mean corpuscular Hb concentration (MCHC) 30.7 g/dL and RBC distribution width (RDW) 13.1%. The blood smear demonstrated microcytic hypochromic RBCs suggestive of thalassemia trait. Hemoglobin analysis identified Hb A2 + Hb A2-Kiriwong (2.4%) and Hb F (0.1%) on high performance liquid chromatography (HPLC). To characterize the α-thalassemia (α-thal) genotype, common α-thal-1 and α-thal-2 alleles were characterized by multiplex gap-polymerase chain reaction (gap-PCR). The results revealed homozygous α-thal-2 (-α3.7/-α3.7) in this case. DNA sequencing showed the presence of a novel δ-globin gene mutation [δ77(EF1)His→Arg; HBD: c.233A>G] that we named Hb A2-Kiriwong for the village from where the proband lived. In summary, the presence of microcytic hypochromic RBCs in this case was likely the result of the homozygous -α3.7 (rightward) deletion and was not affected by this Hb A2 variant.


Assuntos
Hemoglobina A2/genética , Homozigoto , Mutação , Fenótipo , Talassemia alfa/sangue , Talassemia alfa/genética , Globinas delta/genética , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , Hemoglobina A2/química , Humanos , Tailândia , Talassemia alfa/diagnóstico
3.
Int J Lab Hematol ; 37(5): 577-82, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26372049

RESUMO

Automated high performance liquid chromatography and Capillary electrophoresis are used to quantitate the proportion of Hemoglobin A2 (HbA2 ) in blood samples order to enable screening and diagnosis of carriers of ß-thalassemia. Since there is only a very small difference in HbA2 levels between people who are carriers and people who are not carriers such analyses need to be both precise and accurate. This paper examines the different parameters of such equipment and discusses how they should be assessed.


Assuntos
Automação Laboratorial , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Hemoglobina A2/química , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Eletroforese Capilar/instrumentação , Eletroforese Capilar/métodos , Eletroforese Capilar/normas , Hemoglobina A2/genética , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Talassemia beta/sangue , Talassemia beta/diagnóstico , Talassemia beta/genética
4.
Hemoglobin ; 39(5): 340-5, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26193975

RESUMO

In this study, we describe the clinical features and provide experimental analyses of Hb Flurlingen (HBA2: c.177 C > G, p.His > Gln) that contrasted with Hb Boghé (HBA2: c.177 C > A, p.His > Gln). Despite the identical amino acid substitution in both variants, Hb Flurlingen shows the phenotype of α-thalassemia (α-thal), whereas Hb Boghé has no impact on α2-globin (HBA2) production. For in vitro transcription analysis, HBA2 expression constructs carrying the HBA2-WT (wild type), Hb Flurlingen and Hb Boghé sequences were generated and expressed in human bladder carcinoma 5637 cells for downstream analyses by quantitative real time-polymerase chain reaction (qReTi-PCR) and immunofluorochemistry (IFC). In silico analysis of secondary folding structures of the HBA2-WT, Hb Flurlingen and Hb Boghé mRNA sequences was performed using Mfold software. The gene transcription and translation analyses revealed that cells transfected with the Hb Flurlingen construct had significantly lower HBA2 transcription (-55.4%, p ≤ 0.01) and reduced protein synthesis when compared to the wild type group. In contrast, cells transfected with the Hb Boghé construct showed no significant changes in HBA2 transcription or translation activities when compared to the wild type group. The in silico prediction of possible effects of these mutations on the folding structures of the HBA2 transcripts showed a change of secondary folding pattern in the Hb Flurlingen transcript when compared to those of HBA2-WT and Hb Boghé. Our experimental findings support the clinical presentation of an α-thalassemic phenotype for Hb Flurlingen in contrast with Hb Boghé, despite identical amino acid substitutions. The results confirm the importance of experimental analysis in establishing the impact of novel base substitutions.


Assuntos
Substituição de Aminoácidos , Regulação da Expressão Gênica , Hemoglobina A2/genética , Hemoglobinas Anormais/genética , Mutação Puntual , Adolescente , Códon , Análise Mutacional de DNA , Índices de Eritrócitos , Ordem dos Genes , Vetores Genéticos/genética , Hemoglobina A2/química , Hemoglobina A2/metabolismo , Hemoglobinopatias/sangue , Hemoglobinopatias/diagnóstico , Hemoglobinopatias/genética , Humanos , Imuno-Histoquímica , Ferro/sangue , Masculino , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética , Transferrina/metabolismo
5.
Br J Haematol ; 170(6): 781-7, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26104837

RESUMO

HbA2 , a tetramer of α- and δ-globin chains, provides a diagnostic clue to the presence of ß-thalassaemia trait. This minor haemoglobin, which forms about 2-3% of the total, has no known physiological role, but has the interesting property of preventing polymerization of deoxy-sickle haemoglobin. If it were possible to increase the level of HbA2 sufficiently it could have a benefit in sickle cell disease similar to that of foetal haemoglobin. Moreover, HbA2 is present in all erythrocytes, an advantage not found with foetal haemoglobin, which is heterocellularly expressed. The molecular basis of HbA2 gene (HBD) expression is partially understood, and with new molecular tools, it might be possible to induce levels of HbA2 that could be clinically important. However, high concentrations of this positively charged haemoglobin might damage the erythrocyte membrane; also, the reciprocal relationship of δ- and γ-globin gene (HBD and HBG1/2, respectively) expression might negate any benefit of increasing transcription of the former.


Assuntos
Anemia Falciforme/etiologia , Hemoglobina A2/fisiologia , Anemia Falciforme/tratamento farmacológico , Animais , Evolução Molecular , Hemoglobina A2/química , Humanos , Biossíntese de Proteínas
6.
Int J Lab Hematol ; 37(3): 420-5, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25324031

RESUMO

INTRODUCTION: Capillary zone electrophoresis (CZE) at alkaline pH is one of the techniques used for hemoglobinopathy screening. In this study, an evaluation of the performance of a lower throughput CZE instrument, the Sebia Minicap Flex Piercing system, for this purpose is reported for the first time. METHODS: The analytical performance of the Sebia Minicap Flex Piercing system was evaluated. Furthermore, a method comparison between the Sebia Minicap Flex Piercing and two HPLC methods, that is, the Bio-Rad Variant Classic(™) and the Bio-Rad D-10(™) systems was performed by measuring samples with and without clinically relevant hemoglobin disorders. RESULTS: The analytical performance was acceptable for the determination of HbA, HbA2, HbS, and HbF, with an imprecision ≤2.0%. Method comparison showed a linear correlation for HbA2, HbF, and HbS measurements. Clinical concordance was acceptable when comparing CZE and HPLC. CONCLUSIONS: Lower throughput CZE using the Sebia Minicap Flex Piercing can be used for precise and accurate first line screening and follow-up of hemoglobinopathies.


Assuntos
Eletroforese Capilar/métodos , Hemoglobinopatias/diagnóstico , Cromatografia Líquida de Alta Pressão , Hemoglobina Fetal/química , Hemoglobina A2/química , Hemoglobina Falciforme/química , Hemoglobinopatias/sangue , Hemoglobinas/química , Humanos , Reprodutibilidade dos Testes
7.
Hemoglobin ; 38(4): 299-302, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24985928

RESUMO

Although δ-thalassemia (δ-thal) is not categorized as a severe disease, it is essential to know the molecular spectrum of the δ gene mutations frequently occurring in specific areas, particularly if these areas are characterized by a high rate of ß-thalassemia (ß-thal) such as Oman. This is because coinherited δ-globin gene defects can interfere with the basic diagnosis of a ß-thal carrier when this is based upon the measurement of the Hb A2 only. Because of that, we have investigated 33 patients with low Hb A2 levels, collected from different hospitals in Oman. Some cases had a second Hb A2 fraction, while others had only significantly lower Hb A2 levels. Among these patients, 20 did carry a δ-globin gene mutation, the rest were carriers of α thalassemia (α-thal) defects or could be iron depleted or both. In total, eight different known mutations and two novel δ variants were found. The characterization of the δ-globin gene mutation spectrum will improve carrier diagnostics and genetic counseling in the Omani population screened for ß-thal.


Assuntos
Hemoglobina A2/metabolismo , Mutação , Globinas delta/genética , Talassemia delta/sangue , Talassemia delta/genética , Cromatografia Líquida de Alta Pressão , Códon , Análise Mutacional de DNA , Feminino , Genótipo , Hemoglobina A2/química , Humanos , Masculino , Omã , Talassemia delta/diagnóstico
8.
Hemoglobin ; 38(1): 24-7, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24200152

RESUMO

Abstract The molecular basis of ß-thalassemia (ß-thal) syndromes have been well documented, while the spectrum of mutations causing δ-thalassemia (δ-thal) has not been well characterized. δ-Thalassemia has no clinical symptoms but its coinheritance with heterozygous ß-thal may cause misdiagnosis, especially in countries with a high prevalence of ß-thal where prevention programs have been implemented. The coinheritance of ß- and δ-globin mutations in India is not common. This association may interfere with correct diagnosis and genetic counseling of ß-thal in screening programs. Here we report two families showing borderline Hb A2 levels belonging to the Koli Community, indigenous to the Saurashtra Province of Gujarat, India. They were referred to us for thalassemia molecular screening as they had children clinically presenting before 2 years of age and requiring regular blood transfusions. Interestingly, both families carried a novel δ-globin gene mutation at codon 100 (C > T) linked to a polyadenylation (polyA) site [AATAAA > A(-AATAA)] 5 bp deletional ß-thal mutation, never before reported in the Indian population. This report highlights the importance of considering δ-globin gene analysis during ß-thal screening to avoid false-negative results in the detection of at-risk couples. It also highlights how incomplete diagnosis of a borderline or normal Hb A2 level may lead to the probable birth of a ß-thal major (ß-TM) child. This has important implications in prenatal diagnosis.


Assuntos
Hemoglobina A2/genética , Mutação , Talassemia beta/diagnóstico , Talassemia beta/genética , Globinas delta/genética , Adolescente , Adulto , Pré-Escolar , Índices de Eritrócitos , Feminino , Genótipo , Hemoglobina A2/química , Humanos , Lactente , Masculino , Adulto Jovem , alfa-Globinas/genética , Talassemia beta/sangue
9.
Hemoglobin ; 37(6): 593-8, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23952214

RESUMO

The high heterogeneity in regional profiles of ß-thalassemia (ß-thal) mutations highlights the need for population-specific carrier detection strategies. Our aim was to analyze the relationship between hematological values and ß(0) and ß(+) mutations in 154 Balearic ß-thal heterozygotes, in order to establish the most optimized mutation carrier detection strategy to be used to manage the disease in our population. The Hb A2 level was the best parameter for discriminating between both types of carriers. Taking into account the cut-off point value of 4.85% (Hb A2), obtained by a receiver-operating characteristic (ROC) curve analysis, we proposed an algorithm that would use a real-time polymerase chain reaction (RT-PCR) hybridization probe assay technique to detect one of the two most common mutations in the Balearic population, namely codon 39 (C>T) and IVS-I-110 (G>A), depending on the Hb A2 value of the patient.


Assuntos
Hemoglobina A2/genética , Talassemia beta/diagnóstico , Talassemia beta/genética , Algoritmos , Alelos , Índices de Eritrócitos , Genótipo , Hemoglobina A2/química , Humanos , Mutação , Fenótipo , Curva ROC , Espanha
10.
Hemoglobin ; 37(1): 80-4, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23227922

RESUMO

We report two new variants of the δ-globin gene: Hb A(2)-Saint-Etienne [δ14(A11)Leu→Pro] and Hb A(2)-Marseille [δ22(B4)Ala→Lys]. The first variant has a low rate of expression, the second results from a double nucleotide mutation on the same codon.


Assuntos
Hemoglobina A2/genética , Mutação , Globinas delta/genética , Sequência de Aminoácidos , Sequência de Bases , DNA/genética , Feminino , Hemoglobina A2/química , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Análise de Sequência de DNA , Talassemia beta/genética , Globinas delta/química
11.
Clin Chem Lab Med ; 50(9): 1625-9, 2012 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-22962223

RESUMO

BACKGROUND: Hemoglobin (Hb) A(2) is artifactually elevated in cases of heterozygous Hb Hope when measured by capillary electrophoresis (CE). However, there is no report of HbA(2) levels and capillary electrophoregrams for associations of heterozygote of Hb Hope with α-thalassemia nor ß-thalassemia. METHODS: Levels of HbA(0), HbA(2) and Hb Hope in 16 heterozygous Hb Hope, 3 Hb Hope/α-thalassemia-1 SEA type deletion and 2 Hb Hope/ß(0)-thalassemia were measured by CE. Electrophoregram and the levels of those were compared within these three groups. RESULTS: Artifactually elevated HbA(2) levels (≥4%) were found in both groups of heterozygous Hb Hope and Hb Hope/α-thalassemia-1 SEA type deletion. Manual corrections were performed by adjusting baselines, and results showed that means of HbA(2) in both groups decreased from 4.47% and 4.03% to 1.93% and 1.77%, respectively. The highest levels of HbA(2) and Hb Hope were observed in samples with Hb Hope/ß(0)-thalassemia. Moreover, HbA(0) was not observed in these cases. CONCLUSIONS: The elevation of HbA(2) in patients with heterozygous Hb Hope and with Hb Hope/α-thalassemia-1 SEA type deletion measured by CE leads to incorrect ß-thalassemia trait diagnosis. However, using CE electrophoregram together with levels of HbA(0), HbA(2) and Hb Hope would be a more accurate and precise method for diagnosis of Hb Hope/ß(0)-thalassemia.


Assuntos
Eletroforese Capilar , Hemoglobinas Anormais/química , Talassemia alfa/genética , Talassemia beta/genética , Alelos , Deleção de Genes , Hemoglobina A/química , Hemoglobina A2/química , Hemoglobinas Anormais/genética , Heterozigoto , Humanos , Talassemia alfa/metabolismo , Talassemia beta/metabolismo
12.
Biochemistry ; 50(34): 7350-60, 2011 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-21806075

RESUMO

This study is aimed at investigating the molecular basis of environmental adaptation of woolly mammoth hemoglobin (Hb) to the harsh thermal conditions of the Pleistocene ice ages. To this end, we have carried out a comparative biochemical-biophysical characterization of the structural and functional properties of recombinant hemoglobins (rHb) from woolly mammoth (rHb WM) and Asian elephant (rHb AE) in relation to human hemoglobins Hb A and Hb A(2) (a minor component of human blood). We have obtained oxygen equilibrium curves and calculated O(2) affinities, Bohr effects, and the apparent heat of oxygenation (ΔH) in the presence and absence of allosteric effectors [inorganic phosphate and inositol hexaphosphate (IHP)]. Here, we show that the four Hbs exhibit distinct structural properties and respond differently to allosteric effectors. In addition, the apparent heat of oxygenation (ΔH) for rHb WM is less negative than that of rHb AE, especially in phosphate buffer and the presence of IHP, suggesting that the oxygen affinity of mammoth blood was also less sensitive to temperature change. Finally, (1)H NMR spectroscopy data indicates that both α(1)(ß/δ)(1) and α(1)(ß/δ)(2) interfaces in rHb WM and rHb AE are perturbed, whereas only the α(1)δ(1) interface in Hb A(2) is perturbed compared to that in Hb A. The distinct structural and functional features of rHb WM presumably facilitated woolly mammoth survival in the Arctic environment.


Assuntos
Fenômenos Biofísicos , Elefantes , Hemoglobinas/química , Hemoglobinas/metabolismo , Mamutes , Ácidos Alcanossulfônicos/química , Sequência de Aminoácidos , Animais , Substitutos Sanguíneos/metabolismo , Tampões (Química) , Hemoglobina A2/química , Hemoglobina A2/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Morfolinas/química , Oxigênio/metabolismo , Fosfatos/química , Temperatura Ambiente
13.
Blood Cells Mol Dis ; 47(2): 120-4, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21664157

RESUMO

In order to update the molecular basis of ß-thalassemia and describe hematological features among different mutations and the concurrent of α- and ß-thalassemias, 849 unrelated ß-thalassemia heterozygotes recruited in northeast Thailand during a prevention and control program were studied. ß- and α-thalassemia mutations were investigated using the polymerase chain reaction (PCR)-based technologies and hematological parameters were recorded using standard methods. Seventeen different mutations including both ß(0)- and ß(+) -thalassemias were identified. Eight of these 17 ß-thalassemia alleles accounted for 97.4%, others were found at lower frequencies (<1.0%). Of the 849 cases, 626 were investigated for common α-thalassemia mutations and 155 (24.8%) were found to be co-inherited with different forms of α-thalassemia. Comparison of the hematological parameters among different ß-thalassemia mutations revealed an increasing trend of MCV and MCH in a group of heterozygous states for the 3.4kb deletion and the A-G substitution at nucleotide (NT) -28. Hb A(2) and Hb F levels in individuals with the 3.4kb deletion were significantly higher than those with other mutations. Interaction of each ß-thalassemia mutation with α-thalassemia did not affect the diagnostic ranges of Hb A(2) and Hb F, though the significantly increased MCV and MCH was noted. These findings underline the heterogeneity of ß-thalassemia and the importance of hematological and molecular analyses of both α-and ß-thalassemias in the diagnosis and genetic counseling of the couples at-risk of having babies with severe thalassemia diseases in the region.


Assuntos
Hemoglobina Fetal/análise , Globinas/genética , Hemoglobina A2/análise , Talassemia alfa/genética , Talassemia beta/genética , Alelos , Estudos de Coortes , Índices de Eritrócitos , Feminino , Hemoglobina Fetal/química , Aconselhamento Genético , Heterogeneidade Genética , Genótipo , Globinas/química , Hemoglobina A2/química , Heterozigoto , Humanos , Masculino , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Tailândia , Talassemia alfa/diagnóstico , Talassemia alfa/epidemiologia , Talassemia beta/diagnóstico , Talassemia beta/epidemiologia
14.
Hemoglobin ; 35(2): 97-102, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21417565

RESUMO

We describe a new nondeletional α-thalassemia (α-thal) determinant found in a Moroccan infant and in two members of his family. The new mutation generates an abnormal hemoglobin (Hb) as a consequence of a Pro→Ser amino acid substitution at codon 37 (old nomenclature) of the α2 gene. The new Hb variant is barely separable on high performance liquid chromatography (HPLC) but the expression of the α chain mutant measured on reversed phase chromatography is one-third of that expected from a stable α2 variant, which explains the mild α-thal phenotype observed in the carriers. As shown for other mutations described in our laboratory (i.e., Hb Gouda), this variant could also be common in the North African population, overlooked because of the mild phenotype and silent behavior on HPLC. Nevertheless, these silent variants could generate intermediate Hb H diseases in association with Mediterranean α(0)-thal deletion defect.


Assuntos
Substituição de Aminoácidos/genética , Hemoglobina A2/genética , Mutação Puntual/genética , Talassemia alfa/genética , Adulto , Sequência de Bases , Criança , Pré-Escolar , Códon , Feminino , Testes Hematológicos , Hemoglobina A2/química , Humanos , Masculino , Linhagem
15.
Electrophoresis ; 31(17): 2913-20, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20680969

RESUMO

To elucidate the protein-protein interactions of hemoglobin (Hb) variants A and A(2), HbA was first shown to bind with HbA(2) in live red blood cells (RBCs) by diagonal electrophoresis and then the interaction between HbA and HbA(2) outside the RBC was shown by cross electrophoresis. The starch-agarose gel electrophoresis of hemolysate, RBCs, freeze-thawed RBCs and the supernatant of freeze-thawed RBCs showed that the interaction between HbA and HbA(2) was affected by membrane integrity. To identify the proteins involved in the interaction, protein components located between HbA and HbA(2) in RBCs (RBC HbA-HbA(2)) and hemolysate (hemolysate HbA-HbA(2)) were isolated from the starch-agarose gel and separated by 5-12% SDS-PAGE. The results showed that there was a ≈22 kDa protein band located in the RBC HbA-HbA(2) but not in hemolysate HbA-HbA(2). Sequencing by LC/MS/MS showed that this band was a protein complex that included mainly thioredoxin peroxidase B, α-globin, δ-globin and ß-globin. Thus, using our unique in vivo whole blood cell electrophoresis release test, Hbs were proven for the first time to interact with other proteins in the live RBC.


Assuntos
Eletroforese/métodos , Eritrócitos , Hemoglobina A/metabolismo , Sequência de Aminoácidos , Extratos Celulares/química , Membrana Eritrocítica/metabolismo , Eritrócitos/química , Eritrócitos/metabolismo , Hemoglobina A/química , Hemoglobina A2/química , Hemoglobina A2/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Mapeamento de Interação de Proteínas , Espectrometria de Massas em Tandem
16.
Electrophoresis ; 30(17): 3041-3, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19685469

RESUMO

In this paper, we aimed to introduce a newly established red blood cells (RBCs) electrophoresis method hemoglobin release test (HRT) and tried to determine its significance. Human blood samples from beta-thalassemia patients and healthy controls were analyzed with HRT, which was carried out on starch-agarose mixed gel. First, the whole blood samples were electrophoresed for 2 h, then paused for 15 min and ran for 15 min by turns. This "pause-run-pause" experiment was performed for several turns and the total electrophoresis time lasted for about 6 h. The results showed that some other hemoglobin (Hb) components were released from the origin of each sample during the HRT, and the samples from beta-thalassemia patients released more Hb than the healthy controls. This finding demonstrates that Hb may exist differently associated in RBCs, and it may have an important theoretical and clinical significance in Hb and RBC research.


Assuntos
Eletroforese/métodos , Eritrócitos/química , Hemoglobinas/química , Proteômica/métodos , Contagem de Células Sanguíneas , Hemoglobina A2/química , Humanos , Talassemia beta/metabolismo
17.
Protein Sci ; 16(8): 1641-58, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17656582

RESUMO

The different types of naturally occurring, normal human hemoglobins vary in their tetramer-dimer subunit interface strengths (stabilities) by three orders of magnitude in the liganded (CO or oxy) state. The presence of embryonic zeta-subunits leads to an average 20-fold weakening of tetramer-dimer interfaces compared to corresponding hemoglobins containing adult alpha-subunits. The dimer-monomer interfaces of these hemoglobins differ by at least 500-fold in their strengths; such interfaces are weak if they contain zeta-subunits and exchange with added beta-subunits in the form of beta(4) (HbH) significantly faster than do those with alpha-subunits. Subunit exchange occurs at the level of the dimer, although tetramer formation reciprocally influences the amount of dimer available for exchange. Competition between subunit types occurs so that pairs of weak embryonic hemoglobins can exchange subunits to form the stronger fetal and adult hemoglobins. The dimer strengths increase in the order Hb Portland-2 (zeta(2)beta(2)) < Hb Portland-1 (zeta(2)gamma(2)) approximately equal Hb Gower-1 (zeta(2)epsilon(2)) < Hb Gower-2 (alpha(2)epsilon(2)) < HbF(1) < HbF (alpha(2)gamma(2)) < HbA(2) (alpha(2)delta(2)), i.e., from embryonic to fetal to adult types, representing maturation from weaker to stronger monomer-monomer subunit contacts. This increasing order recapitulates the developmental order in which globins are expressed (embryonic --> fetal --> adult), suggesting that the intrinsic binding properties of the subunits themselves regarding the strengths of interfaces they form with competing subunits play an important role in the dynamics of protein assemblies and networks.


Assuntos
Hemoglobina Fetal/química , Hemoglobinas Anormais/química , Hemoglobinas/química , Subunidades Proteicas/química , Animais , Dimerização , Embrião de Mamíferos/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Hemoglobina A2/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/metabolismo , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade
18.
Hemoglobin ; 31(1): 23-9, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17365002

RESUMO

We describe a new delta-globin variant, Hb A2-Pasteur-Tunis [delta59(E3)Lys-->Asn, AAG-->AAC]. This hemoglobin (Hb) displayed an electrophoretic mobility faster than normal Hb A2 and was expressed at 2.2 %. The molecular defect was characterized by DNA sequencing and confirmed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-designed protocol. Hb A2-Pasteur-Tunis was found in a carrier of a codon 39 (C-->T) beta0-thalassemia (thal), presenting with a normal Hb A2 level. Phenotype and genotype investigations revealed that the total Hb A2 level of the patient was that expected for a minor beta-thal (4.8%).


Assuntos
Anemia Hipocrômica/genética , Hemoglobina A2/isolamento & purificação , Hemoglobinas Anormais/isolamento & purificação , Talassemia beta/genética , Sequência de Aminoácidos , Sequência de Bases , Eletroforese das Proteínas Sanguíneas/métodos , Criança , Análise Mutacional de DNA , Feminino , Genótipo , Globinas/química , Globinas/genética , Hemoglobina A2/química , Hemoglobina A2/genética , Hemoglobinas Anormais/química , Hemoglobinas Anormais/genética , Humanos , Masculino , Fenótipo , Mutação Puntual , Polimorfismo de Fragmento de Restrição , Talassemia beta/diagnóstico
19.
Haematologica ; 91(12 Suppl): ECR56, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17194662

RESUMO

We describe the genotype/phenotype correlation in a 35 year old anemic female referred to our laboratory because a fast eluting minor fraction on HPLC, mild hemolysis and hematological parameters suggesting a Thalassemia trait, eventually in combination with iron depletion. Direct sequencing of the alpha globin genes revealed heterozygosity for HbJ-Meerut, a Glu-->Ala substitution at residue 120 not justifying the hematological parameters. No other point mutations were found on the alpha genes and Gap-PCR excluded the 6 common deletion defects. Direct sequencing of the beta-globin genes revealed the IVS-I-5 (G-->C) transversion in absence of the elevated HbA2 levels usually measured in carriers of this beta-Thalassemia mutation. The HbA2 tetramer in the presence of HbJ-Meerut divides in two parts. One alphaN2/delta2 migrating on the right spot on HPLC. The other alphaJ2/delta2 migrating under the HbA fraction. Classic alkaline electrophoresis and the modern capillary electrophoresis CE showed these two tetramers and the reduction of the elevated HbA2 level of the beta-Thalassemia trait by at least 20% due to HbA2 Meerut.


Assuntos
Globinas/genética , Hemoglobina A2/análise , Hemoglobina J/análise , Hemoglobinometria/métodos , Talassemia beta/diagnóstico , Adulto , Eletroforese das Proteínas Sanguíneas , Cromatografia Líquida de Alta Pressão , Reações Falso-Negativas , Feminino , Hemoglobina A2/química , Hemoglobina A2/isolamento & purificação , Hemoglobina J/química , Hemoglobina J/genética , Heterozigoto , Humanos , Índia/etnologia , Países Baixos , Fenótipo , Talassemia beta/sangue , Talassemia beta/genética
20.
Hematology ; 11(2): 109-12, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16753851

RESUMO

There are few studies investigating alpha globin gene triplications in beta-thalassemia in Asian Indians and its effect on phenotype, which was the primary aim of this study. Gap-PCR was performed in order to detect common alpha thalassemia determinants (-alpha(3.7), -alpha(4.2) and alpha alpha alpha(anti 3.7) triplication). Alpha-triplication was detected in 15.4% (10/65) of patients with thalassemia intermedia, 8.8% (4/45) of those with thalassemia minor and in 2.7% (2/74) of healthy controls. The severity of jaundice was higher in thalassemia intermedia cases with alpha-triplication and two of the alpha-triplication cases had a marked increase in serum bilirubin following intercurrent illness. Thus, alpha globin gene triplication is important genetic determinant underlying thalassemia intermedia in North Indians. Patients with alpha-triplication may develop prominent jaundice with marked increase in serum bilirubin following antecedent aggravating factors.


Assuntos
Amplificação de Genes/genética , Globinas/genética , Talassemia beta/genética , Adolescente , Adulto , Anemia Hemolítica/etiologia , Criança , Feminino , Hemoglobina Fetal/química , Genótipo , Hemoglobina A2/química , Humanos , Índia , Masculino , Talassemia beta/etnologia
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