Red cell membrane proteins were investigated in two unrelated children with congenital dyserythropoietic anemia (CDA) I and two siblings with CDA 11. The CDA I patients displayed globin chain synthesis imbalance, with reduction of the non a/cx ratio. One of the CDA I1 patients presented the reverse alteration. Whenever globin chain svnthesis was unbalanced. the membrane p-nitrophen~lphosphatask had an abnormally biphasic kinetics, consistent with substrate excess inhibition. as is observed in a-or P-thalassemic syndromes. One CDA 1 patient displayed a decrease of electrophoretic band 4.1 along with an ectopic phosphorylated protein at the level of band 4.2. In CDA I1 patients, band 3 was strikingly narrower than in controls. In CDA I1 and, to a lesser extent, in CDA I, the in vitro endogenous phosphorylation of band 2 + 2
Hb Knossos (beta 27 (B9) Ala----Ser) is a recently discovered hemoglobin variant endowed with beta-thalassemic properties (1,2) We present the first homozygous cases. The propositus, a 19-year-old man is originally from northeast Algeria, but is unrelated to other Algerians who have hemoglobin Knossos. He has a beta+-thalassemia intermedia syndrome, including microcytic, hypochromic anemia, enlargement of the spleen, and an increase in the number of reticulocytes. The reduction of beta-chain synthesis is pronounced (alpha/non alpha:2.76). Whole cells containing Hb Knossos have a dramatically low oxygen affinity (P50:38 mm Hg). The propositus also has homozygous delta degrees-thalassemia. The chromosome carrying these mutations is characterized by the DNA haplotype I.
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.
We report on a 54 years-old male patient from North-Eastern Algeria who combines two hemoglobin variants that are associated with thalassemia-like disorders: Hb Lepore and Knossos (beta 27 Ala----Ser) (1, 2). A beta-thalassemia intermedia picture gradually developed and finally required splenectomy at the age of 53. Total absence of Hb A2 indicated that the beta Knossos gene is most probably flanked with a delta(0)-thalassemia gene. No DNA deletion additional to the Lepore deletion was found. Hb F was elevated (12.3%) with 24% G gamma Hb F. In whole cells, Hb Knossos, representing 70% of total hemoglobin, displayed a decreased affinity for oxygen (P50 = 35 mm Hg), a fact presumably accounting for the relatively good tolerance of the condition.
In recent studies, we observed a decrease of KMapp, an abnormally biphasic kinetics of the red cell membrane neutral phosphatase and an increased binding of hemoglobin to the membrane in various forms of beta-thalassemia. Since the gene encoding the beta chain (beta E chain) of hemoglobin E (HbE) is endowed with some thalassemic characteristics, we studied the erythrocyte membrane in 25 individuals with Hb E trait or disease. The apparent Michaelis-Menten constant for p-nitrophenylphosphate (the artificial substrate used) was significantly decreased, as in beta-thalassemia. However, the kinetics was monophasic in all the heterozygotes and in four of the homozygotes. It was biphasic only in the three other homozygotes. Vmax was also significantly reduced, a fact that is masked, when not reversed in beta-thalassemia, owing to the rejuvenation of the red cell population. In 5 mM phosphate buffer (pH 8.00), the binding of Hb E to the erythrocyte ghosts was increased in the homozygotes. In the heterozygotes, Hb A binding was also increased, as is the case in beta-thalassemia. This latter fact suggests that the membrane binding site(s) of hemoglobin is (are) altered. We found a highly significant increase of Hb F in EE subjects. The present study extends to the red cell membrane the beta-thalassemic phenotype associated with the beta E gene.
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