RESUMO
The alpha V/41 polymorphism of erythroid alpha-spectrin has been characterized initially by an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction (Alloisio N., L. Morlé, J. Maréchal, A.-F. Roux, M.-T. Ducluzeau, D. Guetarni, B. Pothier, F. Baklouti, A. Ghanem, R. Kastally, et al. 1991. J. Clin. Invest. 87:2169-2177). Until now, it has been found associated invariably with a low expression level of the corresponding alpha chain. Among 61 chromosomes investigated in French and North African individuals or kindreds, we observed 19 chromosomes with the alpha V/41 polymorphism. With no single exception, the latter displayed a point mutation in exon 40 (Leu-->Val; CTA-->GTA) at position alpha 1857. According to the triple helical model of spectrin structure, this change accounts for the peptide maps' abnormalities. Sequencing the entire alpha V domain cDNA disclosed, in addition, a partial skipping of exon 46. At the gene level, a substitution (C-->T) was evidenced at nucleotide -12 of intron 45. This mutation appeared linked to the exon 40 mutation in 17 chromosomes, again with no single exception, among 53 examined chromosomes. We hypothesized that the lack of exon 46 would hamper the nucleation process and eventually account for the low expression feature. The present doubly mutated allele was renamed allele alpha LELY (low expression, Lyon).
Assuntos
Éxons , Íntrons , Mutação , Mutação Puntual , Polimorfismo Genético , Espectrina/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , DNA/genética , DNA/isolamento & purificação , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Mapeamento de Peptídeos , Reação em Cadeia da Polimerase/métodosRESUMO
Spectrin alpha I/74 elliptocytosis results from abnormalities involving the "head" region of spectrin dimer. Increased susceptibility to trypsin enhances cleavage of the alpha spectrin chain, yielding an increased amount of the alpha I 74-kD fragment at the expense of the alpha I 80-kD parent fragment. Recently we showed that the mutations causing the Sp alpha I/74 abnormality may lie in the alpha- or the beta-chain, and that spectrin Culoz and spectrin Lyon were two (alpha I/74) alpha-variants, respectively. We now show that the spectrin Culoz alpha I domain undergoes prominent tryptic cleavage after Lys 42, whereas cleavage prevails after Arg 39 in spectrin Lyon. Applying the polymerase chain reaction (PCR) technique to exon 2 of the spectrin alpha I domain, we have established that the mutation responsible for spectrin Culoz is alpha I 40 Gly----Val; GGT----GTT. Applying the PCR technique to the cDNA derived from reticulocyte mRNA, we have shown that the mutation responsible for spectrin Lyon is alpha I 43 Leu----Phe; CTT----TTT. Studies of normal controls and of family members using dot blot hybridization with allele-specific oligonucleotide probes confirmed these results. Variants such as spectrin Culoz and spectrin Lyon should provide insight into a region that participates in spectrin dimer self-association and whose susceptibility to proteolysis must reflect subtle conformational changes.
Assuntos
Eliptocitose Hereditária/genética , Espectrina/genética , Sequência de Aminoácidos , Sequência de Bases , Genes , Humanos , Dados de Sequência Molecular , Mutação , Sondas de Oligonucleotídeos , Fragmentos de Peptídeos/análise , Reação em Cadeia da Polimerase , RNA Mensageiro/genéticaRESUMO
Protein 4.1 has been defined as a major component of the subcortical skeleton of erythrocytes. It binds the spectrin--actin scaffold through a 10-kD internal domain. This binding requires an essential 21-amino acid sequence motif, Motif I, which is retained by alternative splicing at the late stage of erythroid differentiation. We here analyze the molecular basis of heterozygous 4.1(-) hereditary elliptocytosis, associated with protein 4.1 partial deficiency, in nine related French families. cDNA sequencing revealed a single codon deletion (AAA) resulting in a lysine residue deletion within the 10-kD binding domain, 3' of Motif I. The mutated allele was designated allele 4.1 Aravis. In order to assess the functional effect of the codon deletion, recombinant 10-kD constructs were made and various binding assays were performed using spectrin, purified spectrin-actin complex, or red cell membranes. These experiments demonstrated that the deletion of the Lys residue clearly prevents the binding capacity. Similar results were obtained with a construct containing the Lys residue but lacking Motif I. These data strongly suggest that the binding site to the spectrin-actin complex must contain the Lys 447 (or 448), and therefore resides not only on Motif I but extends 3' of this essential motif.
Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto , Eliptocitose Hereditária/genética , Membrana Eritrocítica/metabolismo , Proteínas de Membrana/metabolismo , Neuropeptídeos , Espectrina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Análise Mutacional de DNA , Eliptocitose Hereditária/sangue , Feminino , França , Humanos , Lisina/fisiologia , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Dados de Sequência Molecular , Linhagem , Conformação Proteica , Proteínas Recombinantes de Fusão , Deleção de Sequência/genéticaRESUMO
Spectrin alpha-chain mutants associated with hereditary elliptocytosis are highly variable in their level of expression. It has been assumed that the degree of elliptocytosis can be increased when the spectrin alpha chain, encoded by the alpha gene in trans to the variant, is expressed at a low level. We now provide strong evidence for the existence of low-level expression of spectrin alpha chains. This condition is referred to as the alpha V/41 polymorphism. It has been observed in 15 different families or individuals of French, North African, and African ancestry in which seven distinct elliptocytogenic alpha-spectrin variants were co-inherited. Whenever the alpha V/41 polymorphism was present, the severity of the biochemical, morphological, and, sometimes, the clinical phenotype of elliptocytosis was increased. The alpha V/41 polymorphism was also frequently encountered among 36 unrelated control subjects in the heterozygous or homozygous states, and was entirely asymptomatic in both cases. The main biochemical feature was an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction. Alteration of the facing beta IV domain of spectrin was demonstrated by in vitro spectrin dimer reconstitution experiments. It appears that the alpha V/41 polymorphism is often required for alpha-spectrin elliptocytogenic variants to become manifest in the heterozygous state. Thus, alpha-spectrin-related elliptocytosis may be viewed as a bifactorial condition.
Assuntos
Eliptocitose Hereditária/genética , Espectrina/genética , Eletroforese em Gel Bidimensional , Humanos , Peso Molecular , Linhagem , Polimorfismo Genético , Espectrina/químicaRESUMO
Sialoglycoprotein beta, a minor sialoglycoprotein of the red cell membrane, was studied in homozygous and heterozygous 4.1(-) hereditary elliptocytosis, a variety of hereditary elliptocytosis characterized by total or partial absence of protein 4.1. Erythrocytes were treated with the periodic acid-NaB3H4 procedure. Following polyacrylamide gel electrophoresis in the presence of SDS, labelled sialoglycoproteins were revealed by fluorography. (i) In the ghosts from the 4.1(-) homozygote, sialoglycoprotein beta was sharply decreased. It is not sure whether the residual material is sialoglycoprotein beta itself, or a distinct sialoglycoprotein migrating in the same place. In long exposure fluorograms, sialoglycoprotein gamma (a sialoglycoprotein related to sialoglycoprotein beta) also turned out to be reduced. In the homozygote's Triton-shells, sialoglycoprotein beta and gamma appeared completely absent. (ii) In the 4.1(-) heterozygote, sialoglycoprotein beta appeared slightly reduced, whereas sialoglycoprotein gamma appeared normal. Both of these proteins were extracted in seemingly normal amounts in the Triton-shells. These observations bring further support to the view that there is an interaction between skeletal membrane protein 4.1 and sialoglycoprotein beta, that is additional to other interactions between the former protein and the lipid bilayer and/or other transmembrane proteins.
Assuntos
Proteínas do Citoesqueleto , Eliptocitose Hereditária/sangue , Membrana Eritrocítica/metabolismo , Glicoforinas/sangue , Heterozigoto , Homozigoto , Proteínas de Membrana/sangue , Neuropeptídeos , Sialoglicoproteínas/sangue , Adolescente , Autorradiografia , Proteínas Sanguíneas/deficiência , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Masculino , Coloração e RotulagemRESUMO
Mutations in the GJB2 gene encoding connexin26 (CX26) account for up to 50% of cases of autosomal recessive hearing loss. In contrast, only one GJB2 mutation has been reported to date in an autosomal dominant form of isolated prelingual hearing loss. We report here a novel heterozygous 605G-->T mutation in GJB2 in all affected members of a large family with late childhood onset of autosomal dominant isolated hearing loss. The resulting C202F substitution, which lies in the fourth (M4) transmembrane domain of CX26, may impair connexin oligomerisation. Finally, our study suggests that GJB2 should be screened for heterozygous mutations in patients with autosomal dominant isolated hearing impairment, whatever the severity of the disease.
Assuntos
Conexinas/genética , Surdez/genética , Genes Dominantes , Mutação , Adolescente , Adulto , Criança , Conexina 26 , Feminino , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
A gene responsible for autosomal dominant non-syndromic hearing impairment in two families (DFNA8 and DFNA12) has recently been identified as TECTA encoding alpha-tectorin, a major component of the tectorial membrane. In these families, missense mutations within the zona pellucida domain of alpha-tectorin were associated with stable severe mid-frequency hearing loss. The present study reports linkage to DFNA12 in a new family with autosomal dominant high frequency hearing loss progressing from mild to moderate severity. The candidate region refined to 3.8 cM still contained the TECTA gene. A missense mutation (C1619S) was identified in the zonadhesin-like domain. This mutation abolishes the first of the vicinal cysteines (1619Cys-Gly-Leu- 1622Cys) present in the D4 von Willebrand factor (vWf) type D repeat. These results further support the involvement of TECTA mutations in autosomal dominant hearing impairment, and suggest that vicinal cysteines are involved in tectorial membrane matrix assembly.
Assuntos
Proteínas da Matriz Extracelular/genética , Genes Dominantes , Perda Auditiva Neurossensorial , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Mutação , Feminino , Proteínas Ligadas por GPI , Ligação Genética , Humanos , Masculino , LinhagemRESUMO
Congenital cataracts are an important cause of visual impairment in children. Approximately one-third of congenital cataracts are hereditary. The disease, when inherited as an isolated abnormality, is phenotypically and genetically heterogeneous. Autosomal dominant forms with high penetrance appear to be the most common. To date, thirteen genes have been implicated in cataractogenesis. The identification of the genetic mutations causing congenital cataracts will provide a better understanding of cataractogenesis in childhood and provide further insights into normal lens development.
Assuntos
Catarata/congênito , Catarata/genética , Cristalinas/genética , Aquaporinas , Catarata/classificação , Mapeamento Cromossômico , Conexinas/genética , Proteínas do Olho/genética , Ferritinas/genética , Genes Dominantes , Genes Recessivos , Humanos , Recém-Nascido , Cristalino/embriologia , Glicoproteínas de Membrana/genética , Mutação , Fatores de Transcrição/genéticaRESUMO
Genetically-transferred hearing impairments account for more than 50% of cases of pediatric sensorineural hearing defects. Multiple clinical aspects are involved in genetic hearing impairment, including the involvement of other organs, genetic inheritance, and the degree and age at onset of hearing loss. Diagnosis relies on family history, on the systematic investigation of the symptomatology including an associated syndrome, and audiometry testing in parents and siblings. Analysis of the connexin 26 gene is also indicated, as it is frequently involved in this disorder. Further genetic analysis in affected families will aid in detecting other as yet unidentified genes responsible for hearing impairment.
Assuntos
Transtornos da Audição/diagnóstico , Transtornos da Audição/genética , Idade de Início , Audiometria , Criança , Genes Dominantes , Genes Recessivos , Aconselhamento Genético , Testes Genéticos/métodos , Genótipo , Transtornos da Audição/epidemiologia , Transtornos da Audição/prevenção & controle , Perda Auditiva Neurossensorial/genética , Humanos , Anamnese , LinhagemAssuntos
Citoesqueleto/ultraestrutura , Membrana Eritrocítica/ultraestrutura , Eritrócitos Anormais/ultraestrutura , Acantócitos/ultraestrutura , Sequência de Aminoácidos , Anemia Diseritropoética Congênita/genética , Anemia Diseritropoética Congênita/patologia , Sequência de Bases , Antígenos de Grupos Sanguíneos/genética , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/fisiologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Eliptocitose Hereditária/genética , Eliptocitose Hereditária/patologia , Genes , Hemoglobinopatias/genética , Hemoglobinopatias/patologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Fenótipo , Espectrina/genética , Espectrina/fisiologia , Esferocitose Hereditária/genética , Esferocitose Hereditária/patologiaAssuntos
Proteínas Sanguíneas/análise , Eliptocitose Hereditária/genética , Neuropeptídeos , Actinas/análise , Proteínas Sanguíneas/deficiência , Proteínas do Citoesqueleto/análise , Eliptocitose Hereditária/sangue , Deformação Eritrocítica , Eritrócitos/análise , Heterozigoto , Homozigoto , Humanos , Proteínas de Membrana/análise , Espectrina/análiseRESUMO
The genetic disorders of the red cell skeleton encompass hereditary spherocytosis, hereditary elliptocytosis and an array of ill-defined haemolytic anaemias. Protein chemistry and molecular genetics have illuminated the supramolecular arrangement of the skeleton, the sequence and three-dimensional structure of its protein components, the exon-intron organization of the corresponding genes, the complex splicing undergone by their transcripts. Basically, hereditary spherocytosis is often due to a defect of ankyrin, hereditary elliptocytosis usually results from alterations of spectrin or protein 4.1. Other conditions are related to changes in the anion transporter or protein 4.2. The heterogeneity of the genomic changes, their ultimate consequences at the protein level open windows on fundamental problems concerning alternative splicing of mRNAs and structure-function relationships in proteins.
Assuntos
Anemia Hemolítica/genética , Eliptocitose Hereditária/genética , Eritrócitos/ultraestrutura , Esferocitose Hereditária/genética , Anemia Hemolítica/sangue , Proteínas do Citoesqueleto/ultraestrutura , Eliptocitose Hereditária/sangue , Membrana Eritrocítica/ultraestrutura , Humanos , Proteínas de Membrana/ultraestrutura , Mutação , Esferocitose Hereditária/sangueRESUMO
We searched an interaction between (i) pentoxifylline and/or propentofylline, and (ii) the red cell membrane with special emphasis on the membrane skeleton. It appeared (i) that propentofylline has no permanent binding site on the membrane, (ii) that propentofylline and/or pentoxifylline do not detectably alter spectrin conformation (no change of spectrin dimer self-association or of spectrin limited digestion in the presence of trypsin), and (iii) that these compounds have no effect on membrane protein phosphorylation, particularly the cAMP-dependent and TPA-dependent phosphorylation of protein 4.1.
Assuntos
Membrana Eritrocítica/metabolismo , Proteínas de Membrana/metabolismo , Pentoxifilina/farmacologia , Teobromina/análogos & derivados , Xantinas/farmacologia , Proteínas do Citoesqueleto/metabolismo , Interações Medicamentosas , Membrana Eritrocítica/efeitos dos fármacos , Fosforilação , Espectrina/metabolismoRESUMO
Hereditary elliptocytosis (HE), its aggravated form hereditary pyropoikilocytosis (HPP), and hereditary spherocytosis (HS) designate a set of congenital hemolytic syndromes. The responsible mutations lie in several genes encoding proteins of the red cell membrane. In particular, they involve the SPTA1 and SPTB genes that encode erythroid spectrin alpha- and beta-chains, respectively. In situ, spectrin is a alpha 2 beta 2 fibrillar tetramer resulting from the head-to-head self-association of two alpha beta dimers. In HE, the 24 known alpha-chain mutations lie in the self-association site or its vicinity, whereas the 17 beta-chain mutations occur in the self-association site itself (record of November 30, 1995). Allele alpha LELY (LELY: Low Expression LYon) is found in ethnic groups remote from one another with a uniform frequency (20-30% of all alpha-alleles). It allows an expanded expression of any HE alpha-allele located in trans and results in severe HE or in HPP. In HS, a number of spectrin mutations have been recorded recently. Allele alpha LEPRA (LEPRA: Low Expression PRAgue) would occur in a recurrent fashion.
Assuntos
Eliptocitose Hereditária/genética , Mutação , Esferocitose Hereditária/genética , Eliptocitose Hereditária/diagnóstico , Membrana Eritrocítica/metabolismo , Humanos , Polimorfismo Conformacional de Fita Simples , Esferocitose Hereditária/diagnósticoRESUMO
We present a new abnormality of the red cell membrane observed in the course of a study on beta-thalassemia. In a child suffering from a beta 0-thalassemia major, the neutral membrane bound phosphatase, instead of having Michaelis-Menten kinetics, displayed a biphasic kinetics consistent with inhibition by substrate excess. This abnormality is discussed along with other erythrocyte membrane alterations that are known to occur in beta-thalassemia.
Assuntos
Membrana Eritrocítica/enzimologia , Eritrócitos/enzimologia , Monoéster Fosfórico Hidrolases/sangue , Talassemia/enzimologia , Criança , Humanos , CinéticaRESUMO
In a previous report (Blood 60:265, 1982), we described a family with an abnormal RBC membrane protein doublet, which we considered a shortened protein 4.1 on the basis of biochemical and genetic data. Using an anti-4.1 monoclonal antibody, we confirm here that the shortened protein derives from protein 4.1. One of the members of the family contemporaneously displayed the 4.1 (-) trait, eg, the heterozygous state of this variety of hereditary elliptocytosis that lacks protein 4.1. The 4.1a/4.1b ratio was low whenever the 4.1 trait was present, regardless of the type of protein 4.1 involved. The RBCs of the compound heterozygote, containing only the shortened species of protein 4.1, made it possible to analyze without interference the contact between shortened protein 4.1 and sialoglycoprotein beta, or glycoconnectin. Shortened protein 4.1 did not alter the amount of glycoconnectin in the ghosts nor did it change its extractability into the Triton shells. Limited proteolysis of shortened polypeptides 4.1a and 4.1b showed that they are sequence related. It is conflicting that the persons carrying the shortened protein 4.1 are devoid of specific clinical and morphological abnormalities, apart from those pertaining to the 4.1- trait, when the latter is present.
Assuntos
Proteínas Sanguíneas/genética , Proteínas do Citoesqueleto , Membrana Eritrocítica/análise , Proteínas de Membrana/sangue , Neuropeptídeos , Proteínas Quinases , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Proteínas Sanguíneas/imunologia , Fracionamento Químico , Conectina , Feminino , Variação Genética , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas Musculares/sangue , Linhagem , Peptídeos/análise , Sialoglicoproteínas/análiseRESUMO
We report on a case of congenital stomatocytosis in a French boy presenting with a haemolytic anaemia requiring splenectomy at the age of 6. The red cells included 15-20% stomatocytes and displayed a marked increase of volume. Their osmotic resistance and density were reduced; however, their deformability was unaltered in isotonicity. Erythrocyte Na+ was high (27 mEq/l) and K+ low (65 mEq/l). The newly described (K+, Cl-)-cotransporter normally triggered by hypo-osmotic stress, was activated to maximal capacity. Membrane band 7 was reduced by 72%. From anamnestic data, the condition appears to have been transmitted by the father. The mother proved to be strictly normal on clinical, morphological, osmotic and biochemical bases. We suggest that the partly missing band 7 may play an important role in the genesis of stomatocytosis.
Assuntos
Anemia Hemolítica Congênita/sangue , Proteínas Sanguíneas/análise , Proteínas de Transporte/sangue , Proteínas de Membrana , Simportadores , Criança , Eritrócitos Anormais , Humanos , Masculino , Cotransportadores de K e Cl-RESUMO
We have studied 20 caucasian individuals including 3 independent persons and 17 persons belonging to 6 unrelated families. These subjects are normal or elliptocytic. On biochemical grounds, families with hereditary elliptocytosis (HE) can be divided in two groups. (i) Three families in which HE is associated with a marked reduction (approximately 30%) of the band 4.1 percentage, upon scanning of SDS-polyacrylamide gels. This deficiency is associated with the absence of obvious clinical symptom and a dominant genetic transmission. (ii) Three HE families display a normal amount of band 4.1. Among two of them, HE is concomitant with moderate clinical manifestations and with a genetic transmission that seems to be morphologically recessive. In the last family, HE is transmitted as a dominant trait and is clinically silent. Finally, in the 3 isolated persons, HE is asymptomatic but the mode of genetic transmission could not be ascertained. One of them has a decreased amount of band 4.1 similar to that observed in the first group. To date, the band 4.1 deficiency is probably the best example of a relatively frequent specific molecular defect associated with a specific morphological abnormality. In one family with this type of HE, we have fortuitously discovered, at the heterozygous state, a variant of protein 4.1, shortened by about 8 500 daltons, involving both subcomponents 4.1a and 4.1b and morphologically silent.
Assuntos
Proteínas Sanguíneas/genética , Proteínas do Citoesqueleto , Eliptocitose Hereditária/sangue , Membrana Eritrocítica/análise , Neuropeptídeos , Proteínas Sanguíneas/isolamento & purificação , Eliptocitose Hereditária/genética , Humanos , Proteínas de Membrana/sangue , Proteínas de Membrana/isolamento & purificação , Valores de ReferênciaRESUMO
We studied the red cell membrane neutral phosphatase, which is part of the Na+K+ ATPase, in several types of oxidative hemolytic anemias. We used an artificial substrate, the p-nitrophenylphosphate. In controls and in patients heterozygous for various unstable hemoglobins (Hb Hope, Hb Köln, or Hb Hammersmith), the kinetics were of the Michaelis-Menten type. On the contrary, in nearly all patients with alpha- or beta-thalassemia, the kinetics displayed an abnormally biphasic character. The apparent Michaelis constant (KMapp) was significantly decreased. The biphasic character correlated with the imbalance of globin chain synthesis. The beta-mercaptoethanol markedly increased Vmax in controls, but had little effect on the biphasic kinetics. Omission of K+ abolished the biphasic kinetics. The abnormal kinetics failed to appear with another artificial substrate, the 4-methylumbelliferylphosphate, nor did it appear with ATP, the natural substrate. In vitro, H2O2 treatment of normal and thalassemic red cells was unable to induce or exaggerate, respectively, the biphasic kinetics, but generated alterations of a different nature. We suggest that the various kinetic alterations of the phosphatase in thalassemic syndromes originate from the imbalance of globin chain synthesis. However, the involvement of an oxidative process remains to be demonstrated.