Hemoglobin variants and methods used for their characterization during 7 years of screening at the Center for Disease Control

During 7 years of screening for hemoglobin variants, over 75 rare variants have been characterized. Of these, 18 were described for the first time. This report presents tabulated data regarding the structural and functional defects that were observed, information on the ethnic origin, and other special properties exhibited by these variants. The strategy and procedures for characterizing these variants are also summarized.     

Hemoglobin Dunn: alpha 6 (A4) aspartic acid replaced by asparagine.

Hemoglobin Dunn, alpha 6 (A4) Asp leads to Asn was found in a 39-year-old black woman and her daughter. The hematological data on the propositus were normal and there were no apparent clinical or pathological findings associated with this abnormal hemoglobin. The structural characterization of this variant is described.     

A case of hemoglobin Indianapolis [beta 112(G14) Cys----Arg] in an individual from Cordoba, Spain

Hemoglobin Indianapolis was first described by Adams et al (1,2) as a very unstable variant with a phenotype similar to severe beta-thalassemia. We have also characterized this variant, but there are several differences in the clinical expression of the variant described in our report and those described in the original case. We found Hb Indianapolis to be unstable, but not to the extent that it could not be detected by routine testing. The four family members heterozygous for the variant were not anemic, showed normal hematologic values, and did not exhibit any severe clinical disadvantages, although there was slight reticulocytosis. The variant could not be resolved from Hb A on cellulose acetate (pH 8.4), but isoelectric focusing showed a double band in the region of Hb A that is probably the variant and Hb A. However, the variant chain was clearly evident by globin chain analyses in acid and alkaline buffers. The condition of additional blood samples did not allow us to determine the oxygen dissociation properties of the variant or the rates of globin chain synthesis.     

Hb Rancho Mirage [beta 143(H21)His----Asp]; a variant in the 2,3-DPG binding site showing normal oxygen affinity at physiological pH.

Hb Rancho Mirage was detected in a 17-year-old male in association with a mild anemia. Hemoglobin electrophoresis revealed the variant had a mobility between Hbs A and J on cellulose acetate (pH 8.6) and a mobility like Hb F on citrate agar (pH 6.4). A substitution of His----Asp was found at position 143 in the beta chain, a residue that contributes to the anionic 2,3-DPG binding site in Hb. This variant exhibited normal oxygen affinity at physiologic pH and reduced affinity at alkaline pH. This suggested a subtle shift in the allosteric equilibrium due most likely to the introduction of a negative charge that stabilized the 2,3-DPG pocket. Both homotrophic (heme-heme) and heterotropic (2,3-DPG and protons) effects were reduced; this might be a consequence of an alteration in the carboxyl terminal region of the beta-subunits. Although a His----Asp substitution would be considered to cause reasonable disruption of the 2,3-DPG and C-terminal conformation of the beta- subunits, the properties of Hb Rancho Mirage suggest that, in fact, there appear to be no major perturbation of the critical C-terminal residues.     

Purification and characterization of a pilin specific for Brazilian purpuric fever-associated Haemophilus influenzae biogroup aegyptius (H. aegyptius) strains.

Brazilian purpuric fever (BPF) is a recently described fatal pediatric disease caused by systemic infection with Haemophilus influenzae biogroup aegyptius. Previous studies have shown that all H. influenzae biogroup aegyptius strains isolated from BPF cases and case contacts share several unique phenotypic and genotypic characteristics that differentiate them from other H. influenzae biogroup aegyptius strains isolated from conjunctivitis cases in Brazil. One key characteristic of this BPF clone is reactivity in a BPF-specific monoclonal antibody enzyme-linked immunosorbent assay. We have purified and partially characterized a pilin, referred to as the 25-kilodalton (kDa) protein. Aggregates of this protein contain a heat-labile epitope which is recognized by a monoclonal antibody used in the BPF-specific enzyme-linked immunosorbent assay. The protein has a molecular weight of approximately 25,000, is insoluble in most detergents, and fractionates with outer membrane vesicles after LiCl extraction. Biochemical analysis of the 25-kDa protein shows it to have an amino acid composition similar but not identical to that of the H. influenzae type b pilin. The sequence of 20 N-terminal amino acids of the 25-kDa protein shows almost complete homology with the N terminus of the H. influenzae type b pilin and the types 1 and P pilins of Escherichia coli. Transmission electron microscopic analysis of the purified protein shows the presence of filamentous structures similar in morphology to those of H. influenzae pili. Reactivity between the 25-kDa protein and the BPF-specific monoclonal antibody is demonstrated by Western blotting (immunoblotting) and colloidal gold-enhanced immunoelectron microscopy. Hemadsorption analysis shows that expression of this protein is associated with increases in piliated cells and enhanced binding of these cells to human erythrocytes. These studies indicate that expression of the 25-kDa protein is a characteristic unique to the BPF clone and suggest that this protein plays a role in the pathogenesis of BPF.