RESUMEN
INTRODUCTION: Phenotype studies still occupy a key position in the diagnosis of hemoglobin (Hb) disorders. MATERIAL AND METHODS: In addition to the conventional methods for diagnosis of Hb disorders which are mostly based on differences in charge of the Hb molecules, some progresses have been brought by studying other properties of the globin chains. Among those, difference in hydrophobicity that may be investigated by reversed-phase HPLC (RP-HPLC) discriminates between variants displaying identical charges. RESULTS: In this study, we show how an update of this method allows to recognize an α-chain variant from a γ-chain variant, a problem frequently during neonatal screening. We illustrate that RP-HPLC may also unravel unclear phenotypes which are modified by the presence of an additional variant not detected by the conventional methods, and help to characterize rare mutants. Also we show that it allows a clear distinction between variants with identical electrophoretical charges as exemplified by Hb Lepore Boston-Washington and Lepore Baltimore. CONCLUSIONS: In view of our results, RP-HPLC is a technique that needs to be used as a second step in the general strategy for a correct characterization of Hb variants.
Asunto(s)
Cromatografía de Fase Inversa , Subunidades de Hemoglobina/química , Hemoglobinopatías/diagnóstico , Fenotipo , Alelos , Sustitución de Aminoácidos , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa/métodos , Subunidades de Hemoglobina/genética , Hemoglobinopatías/genética , Hemoglobinas Anormales/química , Hemoglobinas Anormales/genética , Humanos , Recién Nacido , MutaciónRESUMEN
Hemoglobin (Hb) Esch, is an alpha1 variant, expressed at less than 5%, resulting from the duplication of the 12 nucleotides corresponding to CD65 through 68. The effect of this insertion is the repetition of the sequence Ala-Leu-Thr-Asn, which corresponds to the last turn of helix E. In this variant the presence of a one-turn elongated helix E causes instability and increased ligand affinity. Hb Esch was characterized by DNA sequencing and confirmed by electrospray mass spectrometry. Functional studies were performed by flash photolysis measurements on a fraction isolated by flatbed isoelectric focusing, which was enriched in the abnormal hemoglobin. Similar to other alpha chain variants due to short insertion (or deletion), Hb Esch probably results from a slipped mispairing mechanism. The stability of such modified proteins depends upon the region which is added or deleted and usually is more stable when involving a flexible loop or complete helix turn(s) near by.
Asunto(s)
Hemoglobinas Anormales/genética , Hemoglobinas/genética , Mutagénesis Insercional , Fragmentos de Péptidos/genética , Adulto , Secuencia de Aminoácidos , Duplicación de Gen , Variación Genética , Hemoglobinas Anormales/biosíntesis , Humanos , Masculino , Portugal , Talasemia alfa/diagnóstico , Talasemia alfa/genéticaRESUMEN
Hemoglobinopathies have become a significant national health problem in France. The biologists have a pivotal role in the genetic diagnoses. Although sickle cell disease (SCD) is the most frequent abnormality found: not less than 200 new cases are observed each year at birth, many other globin gene variations are found in the various ethnic groups. Since 1995 a neonatal sickle cell screening program has been established for at risk newborns. This programme is supported by the "Association française de dépistage et prévention des handicaps de l'enfant" (AFDPHE). The characterization of hemoglobin genetic variations requires a comprehensive set of laboratory techniques for which we specify here main clinical and technical recommendations.
Asunto(s)
Hemoglobinas/análisis , Análisis Químico de la Sangre/métodos , Análisis Químico de la Sangre/normas , Recolección de Muestras de Sangre , Hemoglobinopatías/sangre , HumanosRESUMEN
Hemoglobin Lyon-Bron was found in two members of a family of German ascent presenting with a moderate normocytic anemia. In this alpha 2 globin variant, the N-terminal valine of the chain was replaced by an alanine. Electrospray mass spectrometry of the alpha chain showed that, as normally, the initiator methionine was cleaved during globin processing but that the N alpha-terminal group was totally acetylated. This resulted in structural modifications of a region crucial for oxygen binding. As a consequence, hemoglobin Lyon-Bron displayed both a reduced chloride effect and a decreased oxygen affinity, this last point explaining the apparent anemia.
Asunto(s)
Globinas/genética , Hemoglobinas Anormales/genética , Oxígeno/metabolismo , Acetilación , Adolescente , Sustitución de Aminoácidos , Sitios de Unión/genética , Cloro/metabolismo , Salud de la Familia , Femenino , Variación Genética , Globinas/química , Hemoglobinas Anormales/química , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Masculino , Persona de Mediana Edad , Oxihemoglobinas , Espectrometría de Masa por Ionización de Electrospray , VolumetríaAsunto(s)
Globinas/genética , Hemoglobinas Anormales/análisis , Adulto , Sustitución de Aminoácidos , Electroforesis , Salud de la Familia , Variación Genética , Hemoglobinas Anormales/química , Hemoglobinas Anormales/genética , Humanos , India , Focalización Isoeléctrica , Masculino , Mapeo PeptídicoAsunto(s)
Hemoglobina Falciforme/genética , Hemoglobinas Anormales/genética , Eliminación de Secuencia/genética , Talasemia alfa/genética , Adulto , Sustitución de Aminoácidos , Camerún , Análisis Mutacional de ADN , Femenino , Globinas/análisis , Globinas/genética , Heterocigoto , Humanos , Focalización Isoeléctrica , Mutación , Mapeo Peptídico , Embarazo , Complicaciones Hematológicas del Embarazo/diagnósticoRESUMEN
Laboratory methods allowing the detection and characterization of hemoglobin variants are reviewed. Protein chemistry techniques such as isoelectrofocusing, electrophoreses under various experimental conditions, cation exchange and reversed phase high performance liquid chromatography, are the most frequently used for the detection of variants. When associated with a few additional data they may lead to a presumptive diagnosis. DNA studies are also developed in many laboratories. Final identification of a variant may be achieved either by molecular biology techniques or by protein sequence analysis in which mass spectrometry now occupies a key position.
Asunto(s)
Técnicas de Laboratorio Clínico , Hemoglobinas Anormales/análisis , Técnicas de Química Analítica/métodos , Técnicas de Laboratorio Clínico/instrumentación , Variación Genética , Hemoglobinas Anormales/genética , Humanos , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína/métodosRESUMEN
Building on the pioneering efforts of Professor Huisman, several different databases of hemoglobin variants have been developed, each with progressively increased capacity for sophisticated queries and prompt updating. These resources are reviewed in the context of a larger plan for providing related resources on hemoglobins, benign and pathological variation in these proteins and the genes that encode them, and the regulation of the globin genes.
Asunto(s)
Bases de Datos de Ácidos Nucleicos , Hemoglobinas/genética , Expresión Génica , Variación Genética , Humanos , Internet , Alineación de SecuenciaRESUMEN
The diagnosis of any sickle cell disease syndrome is based on the unambiguous identification of HbS. Electrophoretic tests are usually the first to be performed. A much better resolution is obtained with isoelectricfocusing than with the more conventional cellulose acetate electrophoresis at alkaline pH. In some laboratories the first test is cation exchange HPLC. The diagnosis of HbS should never be accepted if not confirmed by a second test, more specific of this Hb such as the solubility test or electrophoresis on agar in citrate buffer. The laboratory should also evaluate other factors interacting with HbS, such as HbF level, sickle cell restriction haplotype, association with alpha-thalassemias. It should also evaluate other cellular factors and, in case of symptomatic heterozygous patients, help to understand of the underlying mechanisms.
Asunto(s)
Anemia de Células Falciformes/diagnóstico , Hemoglobina Falciforme/análisis , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/complicaciones , Cromatografía Líquida de Alta Presión , Electroforesis/métodos , Recuento de Eritrocitos , Heterocigoto , Humanos , Concentración de Iones de Hidrógeno , Talasemia/complicaciones , Talasemia/diagnósticoAsunto(s)
Anemia Hemolítica Congénita/sangre , Globinas/genética , Hemoglobinas Anormales/análisis , Complicaciones Hematológicas del Embarazo/sangre , Adulto , Sustitución de Aminoácidos , Fenómenos Químicos , Química Física , Cromatografía Líquida de Alta Presión , Etnicidad/genética , Femenino , Francia , Hemoglobinas Anormales/genética , Humanos , Focalización Isoeléctrica , Espectrometría de Masas , Marruecos/etnología , Mutación Missense , EmbarazoRESUMEN
Hb Sitia [beta128(H6)Ala-->Val] was found in a Greek female with slightly reduced red blood cell indices. The abnormal hemoglobin was indistinguishable from Hb A by electrophoresis but eluted after Hb A on cation exchange high performance liquid chromatography. DNA sequence analysis revealed a GCT-->GTT mutation at codon 128, which is predicted to encode an Ala-->Val substitution. This was confirmed by mass spectrometry analyses of the beta-globin chain. Since alanine at beta128(H6) interacts with several amino acids of the alpha1beta1 contact, its replacement by a larger residue results in a mild instability of the molecule and slight modifications of the oxygen binding properties.
Asunto(s)
Sustitución de Aminoácidos , Globinas/genética , Hemoglobinopatías/genética , Hemoglobinas Anormales/aislamiento & purificación , Mutación Missense , Adulto , Cromatografía por Intercambio Iónico , Codón/genética , Análisis Mutacional de ADN , Eritropoyetina/sangre , Femenino , Globinas/biosíntesis , Globinas/aislamiento & purificación , Grecia , Cuerpos de Heinz/ultraestructura , Hemoglobinopatías/sangre , Hemoglobinas Anormales/química , Hemoglobinas Anormales/genética , Humanos , Cinética , Espectrometría de Masas , Oxígeno/metabolismo , Unión Proteica , Conformación Proteica , ARN Mensajero/sangre , Receptores de Transferrina/sangre , Transcripción GenéticaRESUMEN
Hb Mont Saint-Aignan [beta128(H6)Ala-->Pro] is a mildly unstable variant, associated with hemolytic anemia, marked microcytosis and increased alpha/beta biosynthetic ratio (1.55 versus 1.1 +/- 0.1 in the control). The abnormal chain was isolated by selective precipitation with isopropanol and the structural modification determined by protein chemistry methods (reversed phase high performance liquid chromatography and mass spectrometry). Possible mechanisms underlying the beta(+)-thalassemia-like expression of this variant are discussed.
Asunto(s)
Sustitución de Aminoácidos , Anemia Hemolítica Congénita/genética , Globinas/genética , Hemoglobinopatías/genética , Hemoglobinas Anormales/aislamiento & purificación , Mutación Missense , Adulto , Secuencia de Aminoácidos , Anemia Hemolítica Congénita/sangre , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Codón/genética , Femenino , Globinas/biosíntesis , Hemoglobinopatías/sangre , Hemoglobinas Anormales/química , Hemoglobinas Anormales/genética , Humanos , Espectrometría de Masas , Persona de Mediana Edad , Datos de Secuencia Molecular , Oxígeno/metabolismo , Embarazo , Complicaciones Hematológicas del Embarazo/sangreAsunto(s)
Sustitución de Aminoácidos , Globinas/genética , Hemoglobinopatías/genética , Hemoglobinas Anormales/genética , Hemoglobinas Anormales/aislamiento & purificación , Mutación Missense , Camerún/etnología , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Francia , Hemoglobinopatías/sangre , Hemoglobinas Anormales/química , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Espectrometría de Masa por Ionización de ElectrosprayAsunto(s)
Hemoglobina Fetal/genética , Hemoglobinas Anormales/genética , Sustitución de Aminoácidos , Cromatografía Líquida de Alta Presión , Francia/etnología , Variación Genética , Globinas/genética , Humanos , Recién Nacido , Peso Molecular , Tamizaje Neonatal , Portugal/etnología , Espectrometría de Masa por Ionización de ElectrosprayAsunto(s)
Hemoglobinas Anormales/genética , Adulto , Sustitución de Aminoácidos , Análisis Mutacional de ADN , Variación Genética , Hemoglobinas Anormales/química , Hemoglobinas Anormales/metabolismo , Humanos , Focalización Isoeléctrica , Masculino , Oxígeno/metabolismo , Estructura Cuaternaria de Proteína , Subunidades de ProteínaRESUMEN
Hb Bushey, found in a Chinese baby and his father, is a new variant with a point mutation leading to the substitution Phe-->Leu at position beta122. Hb Casablanca, found in a family from Morocco, is a further example of a hemoglobin variant that carries two abnormalities in the same chain; the first is identical to that of Hb Bushey and the second to that of Hb J-Antakya [beta65 (E9)Lys-->Met]. Structural abnormalities of both Hbs were determined by protein chemistry methods including electrospray and tandem mass spectrometry. Their stability and oxygen binding properties were found to be identical to those of Hb A. Various mechanisms that may lead to two point mutations in the same chain are reviewed briefly.
Asunto(s)
Hemoglobinas Anormales/química , Hemoglobinas Anormales/genética , 2,3-Difosfoglicerato/farmacología , Adulto , Sustitución de Aminoácidos , Pueblo Asiatico/genética , Cromatografía Líquida de Alta Presión , Salud de la Familia , Femenino , Cromatografía de Gases y Espectrometría de Masas , Variación Genética , Globinas/química , Globinas/genética , Hemoglobinas Anormales/efectos de los fármacos , Humanos , Recién Nacido , Masculino , Marruecos/etnología , Oxígeno/metabolismo , Mutación Puntual , Reino UnidoRESUMEN
Two new fetal hemoglobin variants affecting the Ggamma chain are reported. Hb F-Clamart was found during investigation of a French newborn who presented with a mild microcytemia. The second variant was found during neonatal screening for hemoglobinopathies of 30,000 babies from a population-at-risk living in the Paris region. It was named Hb F-Ouled Rabah because its structural modification and ethnic distribution is similar to that of Hb D-Ouled Rabah [beta19(B1)Asn-->Lys]. Hb F-Ouled Rabah is clinically silent and occurs at a frequency of ca. 0.1% in newborns originating from Maghreb. Structural characterization of both variants was done by protein chemistry methods, including amino acids analysis and mass spectrometry.
Asunto(s)
Hemoglobina Fetal/química , Hemoglobina Fetal/genética , Globinas/química , Globinas/genética , Hemoglobinas Anormales/química , Hemoglobinas Anormales/genética , África del Norte/etnología , Argelia/etnología , Sustitución de Aminoácidos , Cromatografía Líquida de Alta Presión , Salud de la Familia , Francia/epidemiología , Francia/etnología , Variación Genética , Heterocigoto , Humanos , Recién Nacido , Peso Molecular , Marruecos/etnología , Tamizaje Neonatal , Túnez/etnología , Talasemia alfa/genéticaRESUMEN
The relative proportions of the two gamma chain species (G gamma and A gamma) have been reinvestigated in newborns, during the physiological switch from foetal to adult haemoglobin, and in adults with some persistent expression of HbF. In newborns, with about 80% HbF, the G gamma percentage was close to 70% while in adult RBC, with less than 0.5% HbF, the G gamma chain was almost non-detectable and may reflect the completion of the foetal to adult switch. Conversely, in adult patients with HbF above 0.6%, usually accompanying some degree of marrow stimulation, the relative ratio of G gamma varied between 40 and 60%, independently of HbF level. This ratio corresponds to what has been described in the literature as being the adult type of HbF. In all the cases where we found higher levels of G gamma, the results could be explained by the presence of a specific genetic background such as the Senegalese haplotype in sickle cell disease.
