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Biomedical subjects

H Wajcman

Publications and source records attributed to H Wajcman.

At least 37 records · Page 2Linked to original sources
PubMed1999

Detection of neutral and charged mutations in alpha- and beta-human globin chains by capillary zone electrophoresis in isoelectric, acidic buffers.

A simple and reliable method, for screening for point mutations in alpha- and beta-human globin chains, is reported here, utilizing capillary zone electrophoresis in isoelectric, acidic buffers. A solution of 50 mM iminodiacetic acid (pI 2.23) containing 7 M urea and 0.5% hydroxyethylcellulose (apparent pH 3.2) is used as background electrolyte for fast separation of heme-free, denatured globin (alpha and beta) chains. Due to the low conductivity of such buffers, high voltage gradients (600 V/cm) can be applied, thus reducing the separation time to only a few minutes. In presence of neutral to neutral amino acid substitutions, it is additionally shown that the inclusion of 3% surfactant (Tween 20) in the sample and background electrolyte induces the separation of the wild-type and mutant chains, probably by a mechanism of hydrophobic interaction of the more hydrophobic mutant with the detergent micelle, via a mechanism similar to "micellar electrokinetic chromatography". At this low operative pH, however, charged mutants, involving substitutions of acidic amino acids (Glu and Asp) are not detected, since these residues are extensively protonated. Curiously, however, they are still separated in presence of detergent, due to the large variation in hydrophobicity involved in such mutations. Of the 19 mutants analyzed, all but one were resolved: Hb St Nazaire (beta 103 Phe-->Ile). This is due to the fact that the delta G (in kcal/mol) in the substitution Phe-->Ile is zero, thus no separation can possibly take place between two chains exhibiting the same hydrophobicity parameter.

Buffers↗
PubMed1999

Quaternary structure sensitive tyrosine residues in human hemoglobin: UV resonance raman studies of mutants at alpha140, beta35, and beta145 tyrosine.

Recent studies noted the contribution of alpha42Tyr to the T-R-dependent UV resonance Raman (UVRR) spectral changes of HbA [Nagai, M., et al. (1996) J. Mol. Struct. 379, 65-75; Huang, S., et al. (1997) Biochemistry 36, 6197-6206], but the observed UVRR changes of the Tyr residue cannot be fully interpreted with alpha42Tyr alone. To identify the remaining contributions, the 235 nm-excited UVRR spectra of Tyr mutant Hbs at alpha140, beta35, and beta145 were investigated here. The Fe-His stretching mode demonstrated that all of these mutant Hbs take the T structure in the deoxy form under these experimental conditions. The UVRR change of the Trp residue of these mutants upon the T-R transition was the same as that in HbA, indicating that the T-R-dependent UVRR change of beta37Trp is not due to stacking with Tyr residues but is due to the formation or destruction of a hydrogen bond. The recombinant Hbs beta35Tyr --> Phe and beta35Tyr --> Thr both exhibited UVRR spectra identical with that of HbA, meaning that beta35Tyr is not responsible. In the spectra of des(beta146His,beta145Tyr)Hb with inositol hexaphosphate, the frequency shift of the Tyr RR bands was the same as that in HbA but the intensity enhancement in the CO form was small, suggesting that beta145Tyr contributes to a part of the intensity change, but scarcely relates to the frequency shift. In the spectra of Hb Rouen (alpha140Tyr --> His), the frequency shifts of bands at 1617 (Y8a) and 1177 (Y9a) cm-1 following ligation were half of those in HbA, while the intensity enhancement was not detected. This result means that alpha140Tyr is responsible for both the frequency shift and the intensity changes. It is suggested that the frequency shift of the Tyr RR bands upon the T --> R transition is due to changes in the hydrogen bonding state of alpha42- and alpha140Tyr and that the intensity enhancement is due to changes in the environment of the penultimate Tyr in both alpha and beta subunits (alpha140 and beta145). These alterations in the vibrational spectra clearly demonstrate which tyrosine residues are involved in the T-R transition as a result of modification of their local environments.

Amino Acid Substitution↗
PubMed1999

Hb Iraq-Halabja beta10 (A7) Ala-->Val (GCC-->GTC): a new beta-chain silent variant in a family with multiple Hb disorders.

A patient originating from Iraq was referred to our laboratory upon suspicion of a hemoglobinopathy. Routine hematological tests revealed a microcytic and slightly anemic phenotype with an elevated HbA2 suggestive of beta-thalassemia. Samples were obtained for several members of the family which upon examination revealed highly heterogeneous phenotypes that prompted us to investigate the case further. Sequencing of the beta-globin gene and alpha cluster mapping in the propositus and his brother showed a previously undescribed beta-globin variant:Hb Iraq-Halabja, beta10(A7) Ala-->Val (GCC-->GTC), associated with beta0-thalassemia IVS-2 nt1 G-->A and either alpha-thal-2-3.7 kb deletion (brother), or alpha-globin gene triplication anti-3.7 kb type (propositus). Detailed functional studies of the variant gave a normal oxygenation curve, a normal heterotopic action of 2,3 DPG, and normal heat stability and isopropanol precipitation tests. The variant shows a clear difference in migration properties compared to normal beta-chain only when run on PAGE urea Triton. As expected, alpha/beta-globin mRNA ratios were influenced by the concomitant presence of an alpha-globin gene pathology and the beta0 thalassemia and not by the presence of the beta-globin variant which apparently is clinically silent.

Adult↗
PubMed1999

Association of unstable hemoglobin variants and heterozygous beta-thalassemia: example of a new variant Hb Acharnes or [beta53(D4) Ala --> Thr].

We report here the functional and structural characterization of Hb Acharnes [beta53(D4) Ala --> Thr], an unstable and electrophoretically silent variant, that was found associated in trans with a beta(0)-thalassemic mutation (IVSI-1 G --> A), in a patient with thalassemia intermedia syndrome. This case is discussed in comparison with other sporadic cases that we have previously investigated, resulting from the co-inheritance of a beta(0)-thalassemic mutation (CD39 C --> T) with two other types of unstable hemoglobins, Hb Köln [beta98(FG5) Val --> Met], and Hb Arta [beta45(CD4) Phe --> Cys]. It may be concluded that, in these associated forms, both the degree of instability of the variant and the altered oxygen binding properties (affecting the degree of tissue hypoxia) are major determinants of their clinical expression.

Child↗
PubMed1999

Two hemoglobin variants with an alteration of the oxygen-linked chloride binding: Hb Antananarivo [alpha1(NA1)Val-->Gly] and Hb Barbizon [beta144(HC1)Lys-->Met].

We here report two new, clinically silent, hemoglobin variants in which the structural modification disturbs the oxygen-linked chloride binding. Hb Antananarivo [alpha1(NA1)Val-->Gly] was found during a systematic hematological study in a 24-year-old woman, who originates from Madagascar. Hb Barbizon [beta144 (HC1)Lys-Met] was found in several members of a French family. The oxygen binding properties of Hb Barbizon were similar to those of Hb Antananarivo showing, in vitro, a decreased chloride effect as compared to Hb A. In Hb Barbizon, the replacement of lysine beta144 by a methionine residue decreased from 4 to 2 the excess positive charges in the central cavity, thus leading to a reduction of about half of the chloride effect. For Hb Antananarivo, the mechanism is unclear since there is no difference in the number of positive charges in the central cavity but alterations are likely at the alpha1alpha2 interface.

Adult↗
PubMed1998

Genetic and hematological studies in a group of 114 adult patients with SC sickle cell disease.

The clinical and biological heterogeneity of sickle cell hemoglobin (Hb) C disease (SC disease) is similar to sickle cell anemia, but has a much milder course. The effect of genetic factors such as alpha thalassemia or beta-globin gene haplotype has been analyzed in a limited number of cases. In this work, we report about 114 adult SC patients, aged 15 to 65 years (M/F = 0.93). The frequency of deletional alpha thalassemia (alpha(-3.7)) was found to be about 35%. The coinheritance of an alpha-thalassemia trait with SC disease had no effect on the hemoglobin level but hemolysis was significantly reduced. In these patients, as described for homozygous Hb S individuals, the Hb F level was higher in females than in males and in individuals carrying the beta(s)-Senegal haplotype. This haplotype involves the presence of an Xmnl site 5' to Ggamma, which is considered responsible for an increased Ggamma/Agamma ratio. Our survey showed that some genetic factors may modulate hematological parameters in SC disease.

Adolescent↗
PubMed1998

Hemoglobins in Togolese newborns: Hb S, Hb C, Hb Bart's, and alpha-globin gene status.

The gene frequency of the most important hemoglobin (Hb) abnormalities is reported in a population of 171 Togolese newborns. Hb phenotypes, hematological parameters, and the more frequently described alpha-gene deletions were analyzed. Structural abnormalities of beta-globin were observed in 35.7% of the children with a gene frequency of 0.105 for beta(S) and 0.091 for beta(C). The frequency of the different alpha-globin genotypes was alpha alpha/ = 0.71, -alpha/ = 0.28, and alpha alpha alpha/ = 0.01. All of the individuals homozygous for the -alpha genotypes, and most of the heterozygous individuals, carried Hb Bart's. Within the alpha alpha/alpha alpha and the -alpha/alpha alpha groups, several individuals with or without Hb Bart's were found; they did not differ from the others by their red blood cell (RBC) parameters but by their levels of fetal hemoglobin (Hb F). The African alpha2 polymorphism marker, characterized by the replacement of G by TCGGCCC at position 7238 (EMBL HSHBA4, 1993) and of T 7174 by G, was found in 21 newborns. The mean value of Hb F was calculated for each genotype, the mean (G)gamma percentage was 69.4 +/- 4.0%, and the gene frequency of the AgammaT marker was 0.10; this marker was linked to the normal beta-globin cluster.

Blood Cell Count↗
PubMed1998

Hb Nancy and Hb Osler: two distinct genetic variants with identical clinical and hemoglobin phenotype.

Three hemoglobin variants (Hb Nancy, Osler and Fort Gordon), carrying the same Tyr-->Asp substitution at position beta 145 (HC2), have been independently described in 1975 in patients with marked polycythemia. The first one was found in a French caucasian family from Lorraine, and the two others in African Americans. Two unrelated individuals with Hb Osler have been recently reinvestigated at the DNA level and surprisingly, in their beta gene, codon 145 was found to be AAT which encodes for asparagine and not for aspartic acid, the aspartate at the protein level resulting, thus, from a very efficient posttranslational event. We reinvestigated a patient from the family of Hb Nancy and found that codon 145 was GAT, encoding for aspartate. This demonstrates that Hb Nancy is genetically distinct from Hb Osler despite an almost identical phenotype.

Asparagine↗
PubMed1998

Haemoglobin J-Biskra: a new mildly unstable alpha1 gene variant with a deletion of eight residues (alpha50-57, alpha51-58 or alpha52-59) including the distal histidine.

Single point mutation, which accounts for 92% of the 700 known variants, is the most frequent genetic defect responsible for abnormal haemoglobins. Small deletions (or insertions) involving from one to five residues are also observed, but in only approximately 5% of cases. The remaining variants produce fusion or extended haemoglobins. A deletion of eight residues, which included the distal histidine and its neighbours (alpha50-57, alpha51-58 or alpha52-59), was found in Hb J-Biskra. This new alpha-chain variant was mildly unstable in vitro only and there was no haematological or biochemical evidence of haemolysis in the affected family members. 24 nucleotides were missing in a region of the alpha1 gene showing an identical sequence of eight nucleotides at both ends. Several starting points could therefore lead to the same nucleotide and aminoacid remaining sequence. This deletion is the largest up to now reported in a haemoglobin molecule which is expressed at an almost normal level in the red blood cell. Comparison of the DNA sequences near to the deleted (or inserted) regions in the various haemoglobins carrying this type of abnormality almost always revealed the presence of a sequence that was hypothesized to slow down progression of the replication fork, and of repeats that may lead to possible secondary structures favouring slipped mispairing.

Amino Acid Sequence↗
PubMed1998

A new sickle cell disease phenotype associating Hb S trait, severe pyruvate kinase deficiency (PK Conakry), and an alpha2 globin gene variant (Hb Conakry).

A Guinean woman, heterozygous for haemoglobin (Hb) S, was studied because of episodes of marked anaemia, repeated typical metaphyseal painful crises and haemosiderosis. Her sickling syndrome resulted from the association of Hb S trait with a severe pyruvate kinase deficiency leading to a 2,3-DPG concentration of twice normal levels. Sequence of the PK-R gene revealed an undescribed mutation in the homozygous or hemizygous state within exon 5 (nucleotide 2670 C-->A), leading to the interchange of Ser 130 into Tyr (PK Conakry). In addition, the patient carried a new haemoglobin variant, Hb Conakry [alpha80(F1) Leu-->Val], which seemed to have a mild effect. The high intraerythrocytic 2,3-DPG concentration induced by the PK deficiency resulted in a decreased oxygen affinity which favoured sickling to a level almost similar to that of Hb S/C compound heterozygous patients. This was confirmed by oxygen binding measurements of Hb A/Hb S erythrocytes in which 2,3-DPG content was modified in vitro. Hysteresis between deoxy- and reoxygenation curves, as well as increase in the n(max) value, demonstrated that the extent of HbS polymerization in the propositus was almost the same as that of RBCs from a homozygous sickle cell patient or those of an A/S heterozygous patient with an artificial in vitro increase of 2,3-DPG concentration.

Adult↗
PubMed1998

Hb Godavari [alpha 95(G2)Pro-->Thr]: a neutral amino acid substitution in the alpha 1 beta 2 interface that modifies the electrophoretic mobility of hemoglobin.

Hb Godavari [alpha 95(G2)Pro-->Thr] was characterized independently in two families of different ethnic origin. The first case, found in the Netherlands, involved an Indian patient. The second one was identified a few months later in an African family from Mali, living in France. Hb Godavari is the fourth example of a substitution involving neutral residues at position alpha 95(G2). In all these variants the electrophoretic pattern suggested that the structural modification unmasks a charged residue buried in the alpha 1 beta 2 contact area. The oxygen affinity of this abnormal hemoglobin was approximately 10% higher than that of Hb A; in the absence of 2,3-diphosphoglycerate, its cooperativity was moderately decreased, suggesting a slightly unstable T state.

Adult↗
PubMed1998

HB Les Andelys [alpha83(F4)LEU-->PRO]: a new moderately unstable variant.

Hb Les Andelys [alpha83(F4)Leu-->Pro] is a mildly unstable variant that was found during glycated hemoglobin measurement in a French family. In this hemoglobin molecule the affected site, in the alpha chain, and the amino acid substitution are identical to those of Hb Santa Ana, an unstable beta chain variant. The structural abnormality was demonstrated by protein chemistry methods, involving, in addition to the classical techniques, a selective precipitation of the abnormal hemoglobin by isopropanol and a mass spectrometry analysis of the alphaT-9 peptide following carboxypeptidase digestion. DNA sequencing demonstrated that the mutation was CTG-->CCG at codon 83 of the alpha2 gene.

Amino Acid Sequence↗