Clinical Course of Two Children with Unstable Hemoglobins: The Effect of Hydroxyurea Therapy.

Case reports on the effect of hydroxyurea (HU) therapy for unstable hemoglobins (Hbs) are sparse; only three adult cases have been reported. We report for the first time on the effect of HU therapy in children carrying unstable Hbs. The first case concerns a female child with a familial history of chronic hemolytic anemia. She was diagnosed with Hb Volga (HBB: c.83C>A) at the age of 7 months. At age 6, treatment options were reconsidered due to increasing fatigue and decreasing Hb concentration. The second case also concerns a female child with chronic hemolytic anemia and icterus since the age of 5. She was diagnosed with Hb Köln (HBB: c.295G>A) at the age of 9. At age 10, treatment options were reconsidered due to decreased general condition and poor school performance. Both children were started on HU therapy. The child with Hb Volga showed reduced clinical symptoms and increased average Hb concentrations. She has been on HU therapy for over 7 years at preparation of this manuscript. The child with Hb Köln showed decreasing Hb concentrations upon start of therapy; clinical symptoms did not improve. Therapy was discontinued after 3½ months. The Hb Volga case report suggests that HU therapy could improve clinical symptoms in some patients with unstable Hbs. Based on these and previously published cases, it was speculated that response can be predicted by the percentage of Hb F and reticulocyte counts.

Two new variants with the same substitution at position beta122: Hb Bushey [beta122(GH5)Phe-->Leu] and Hb Casablanca [beta65(E9)lys-->Met; beta122(GH5)Phe-->Leu].

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.

Conformational fluctuations in deoxy hemoglobin revealed as a major contributor to anionic modulation of function through studies of the oxygenation and oxidation of hemoglobins A0 and Deer Lodge beta2(NA2)His --> Arg.

Organisms rely on regulation at the molecular level, such as the allosteric regulation of hemoglobin (Hb) function by anions, to meet challenges presented by changing environmental and physiological conditions. A comparison of the effects of anions on oxygenation, oxidation, and sulfhydryl reactivity of Hb leads us to suggest that a large and significant part of the shift in oxygen affinity brought about by anion binding occurs as a result of increased conformational rigidity of the T state of deoxy Hb. As conformational rigidity increases, it becomes increasingly difficult for subunits in the deoxygenated T-state tetramer to assume higher oxygen affinity forms (T', T", T"'...) with less steric hindrance. The oxygen affinity reflects the average of the rapidly equilibrating conformations within the T state and is correspondingly decreased when anion levels are increased. The initial stage of the oxidation of Hb is relatively insensitive to steric alterations and thus reflects, primarily, the electronic aspects of the quaternary (T, T', T", T"'...) <--> equilibrium. We show that the reactivity of the sterically obscured sulfhydryl of beta93 Cys in deoxy Hb is much greater in chloride-free buffers than in buffers with added chloride. Anion-induced decreases in the extent and frequency of conformational fluctuations of subunits within the T-quaternary state thus reduce sulfhydryl reactivity as well as oxygen affinity. This parallel in anionic control of function allowed us to test, and disprove, the possibility that uncompensated positive charges in the central cavity of Hb Deer Lodge increase the frequency and extent of conformational fluctuations in its deoxy structure. This Hb variant exhibits increased anion sensitivity, increased oxygen affinity, and increased ease of oxidation, but without increased reactivity of its sulfhydryl groups, indicating that active-site alterations in deoxy Hb Deer Lodge are primarily electronic and not associated with increased conformational fluctuations within its T state. The restoration of normal properties in Hb Deer Lodge by addition of anions reinforces our conclusion that anionic control can be exerted through both steric and electronic alterations. The anionic control of fluctuations within the T state of Hb illustrates an important principle of macromolecular structure-function relationships: that functional regulation can be achieved by alterations in conformational rigidity.

Clinical and hematological response to hydroxyurea in a patient with Hb Lepore/beta-thalassemia.

The possibility of increasing Hb F in vivo using drugs like 5-azacytidine, hydroxyurea, and butyrate has been established. However, in many cases this does not entail an increase in total hemoglobin. We report on a patient with Hb Lepore/beta-thalassemia being treated with hydroxyurea (30 mg/Kg/day) because of the presence of erythroid extramedullary masses with severe neurological abnormalities. During therapy the patient showed a remarkable improvement in neurological signs due to the reduction in extra-medullary masses, a significant increase in both total hemoglobin (from 5.8 to 9.7 g/dl) and Hb F (from 4.9 g/dl to 9.1 g/dl). The marked improvement in hemoglobin level in our patient with Hb Lepore/beta-thalassemia suggests gamma-globin gene activation due to the DNA structure determined by the crossover event.

Regulation of oxygen affinity by quaternary enhancement: does hemoglobin Ypsilanti represent an allosteric intermediate?

Recent crystallographic studies on the mutant human hemoglobin Ypsilanti (beta 99 Asp-->Tyr) have revealed a previously unknown quaternary structure called "quaternary Y" and suggested that the new structure may represent an important intermediate in the cooperative oxygenation pathway of normal hemoglobin. Here we measure the oxygenation and subunit assembly properties of hemoglobin Ypsilanti and five additional beta 99 mutants (Asp beta 99-->Val, Gly, Asn, Ala, His) to test for consistency between their energetics and those of the intermediate species of normal hemoglobin. Overall regulation of oxygen affinity in hemoglobin Ypsilanti is found to originate entirely from 2.6 kcal of quaternary enhancement, such that the tetramer oxygenation affinity is 85-fold higher than for binding to the dissociated dimers. Equal partitioning of this regulatory energy among the four tetrameric binding steps (0.65 kcal per oxygen) leads to a noncooperative isotherm with extremely high affinity (pmedian = .14 torr). Temperature and pH studies of dimer-tetramer assembly and sulfhydryl reaction kinetics suggest that oxygenation-dependent structural changes in hemoglobin Ypsilanti are small. These properties are quite different from the recently characterized allosteric intermediate, which has two ligands bound on the same side of the alpha 1 beta 2 interface (see ref. 1 for review). The combined results do, however, support the view that quaternary Y may represent the intermediate cooperativity state of normal hemoglobin that binds the last oxygen.