Haemoglobin Radcliffe (α2β299(G1)Ala): A High Oxygen-Affinity Variant Causing Familial Polycythaemia

Three members of an Oxfordshire family have polycythacmia. In each case their whole-blood oxygen affinity is increased. This is due to a previously undescribed haemoglobin variant which has been named haemoglobin Radcliffe (α₂β₂^99(G1)Ala). In addition to having a high oxygen affinity haemoglobin Radcliffe shows virtually no haem-haem interaction and a diminished Bohr effect. It is synthesized at the same rate and is as stable as haemoglobin A. X-ray analysis indicates that crystals of deoxyhaemoglobin Radcliffe are isomorphous with those of deoxyhaemoglobin A. Solutions of haemoglobin Radcliffe were also studied by high-resolution proton nuclear magnetic resonance spectroscopy. The structure/function relationships of haemoglobin Radcliffe are discussed in the light of these studies.     

Haemoglobin F Jamaica (α2γ261 Lys→Glu; 136 Ala)

S ummary . Two siblings of mixed Negro, East Indian and Caucasian parentage showed an abnormal haemoglobin component in infancy which had electrophoretic characteristics similar to Hb F Roma (Silvestroni & Bianco, 1963). The amino-acid substitution responsible for the peculiar electrophoretic mobility was found to be in the gamma polypeptide chain (γ— 61(E5) Lys→Glu). The finding of an alanyl residue at position 136 of the γ-chain of the abnormal component in the first born of these infants was repeated in the abnormal γ-chain of the second infant. No functional abnormalities were found to be associated with this abnormal haemoglobin, which is designated Hb F Jamaica.     

Haemoglobin F Texas I (α2γ25Glu→Lys): a Variant of Haemoglobin F

A survey of 3000 cord bloods in Great Britain yielded one electrophoretically abnormal variant of Haemoglobin F in an infant of West Indian parentage. This haemoglobin resembled Haemoglobin F Texas. Haemoglobin F Texas might either be α₂γ₂^5Glu→^Lys or α₂γ₂^6Glu→^Lys. The present variant F has its substitution in the fifth residue of the γ-chain, and is designated Haemoglobin F Texas I.     

Haemoglobin α2β223Val → Ile produced in Escherichia coli facilitates Hb S polymerization

The doubly substituted variant Hb S-Antilles (beta 6 Glu----Val, beta 23 Val----Ile) produces sickling in heterozygous carriers. The Csat value for pure deoxyHb S-Antilles is nearly half that of deoxyHb S. Dilute solutions of pure Hb S-Antilles have a lower oxygen affinity than those of Hb A or Hb S. The mutant Hb alpha 2 beta 2 23 Val----Ile was synthesized in E. coli. It exhibits a decreased oxygen affinity compared to Hb A and does not polymerize in 1.8 M phosphate buffer. Mixtures of equal amounts of Hb S + Hb beta 23 Val----Ile have a decreased Csat value compared to mixtures of Hb S + Hb A. The beta 23 Val in Hb S contributes to the axial contact joining molecules in each single filament. Substituting Ile for Val at this site increases the strength of this contact through hydrophobic interactions, allowing increased stability of the lateral contact between filaments in pair, which is the specific unit structure of polymers in deoxyHb S.     

Haemoglobin F Port Royal (α2Gγ2, 125Glu → Ala)

S ummary . A ^Gγ-chain variant, Hb F Port Royal, with an electrophoretic mobility intermediate between Hb S and Hb C, was found in a Jamaican-Negro infant, and made up 14–15% of the total Hb F. A glycinamidation procedure was employed to aid in determining the amino-acid residue substitution of γ^{125Glu → Ala}, and the presence of glycinc in position 136.     

Haemoglobin Vanderbilt (α2β289Ser→Arg): a New Haemoglobin with High Oxygen Affinity and Compensatory Erythrocytosis

Haemolysates of family members from three generations, all of whom had polycythaemia, were analysed by polyacrylamide gel electrophoresis at pH 8.8. Two closely spaced major bands were observed, one of which corresponded to Hb A and the other to a new mutant designated Hb Vanderbilt. Whole blood from a heterozygote for Hb Vanderbilt was analysed for oxygen affinity which was found to be much higher than that of normal subjects. Haemoglobin Vanderbilt was separated from Hb A using anion exchange chromatography. Cation exchange chromatography yielded a variant β chain from which a mutant peptide was identified with a structure corresponding to residues β83–89 with a Ser→Arg replacement at position 89. The oxygen affinity of 'stripped' haemolysates from the heterozygote was found to be much less sensitive to added organic phosphates than haemolysates from normal subjects. In whole blood, the decreased sensitivity to 2,3-diphosphoglycerate results in an increased oxygen affinity, thus explaining the clinical observations of tissue hypoxia and compensatory polycythaemia.     

Haemoglobin F Texas II (α2γ26 Glu→Lys), the Second of the Haemoglobin F Texas Variants

S ummary A survey of 13 ,000 cord bloods in Great Britain yielded an electrophoretically abnormal variant of Haemoglobin F which resembled Haemoglobin F Texas. Haemoglobin F Texas might be either α₂γ₂^5G1u→Lys or α₂γ₂^6Glu→Lys. The first has been described and was named Haemoglobin F Texas I. The present variant has the same substitution in the sixth residue of the γ chain and is designated as Haenioglobin F Texas II.     

Homozygous deletional α+ thalassaemia associated with unequal expression of the two remaining α1 genes (α1A and α1Q)

S ummary . A Cambodian family presenting several haemoglobinopathies, Hb E, Hb Q and α⁺ thalassaemia, has been investigated. DNA analysis showed that the thalassaemia syndrome corresponds to a leftward type (4.2 kb) deletional from of α⁺ thalassaemia. Genotypes found in the family are: propositus -α^A/-α^Q, β^A/β^E, mother and older sister α^Aα^A/ -α^Q, β^A/β^E; father α^Aα^A/-α^A, β^A/β^A. The propositus consistently presents an α^Q/α^A chain ratio of 60/40 although both chains are products of α₁ loci. The relatively higher expression of the α^Q chain is not observed in the mother and therefore makes it unlikely to reflect anything other than differential expression of the maternal -α^Q/ and paternal -α^A/ haplotypes. This observation raises the possibility that both haplotypes are not strictly identical and that the region of the cross-over event is important for α gene expression.     

Haemoglobin D Iran: β222 Glutamic Acid→Glutamine (B4)

A new haemoglobin D (Iran) has been described. The substitution was in the beta chain at residue 22 (B₄), which is the most frequently mutated area in human haemoglobin. No clinical symptoms were associated with this haemoglobin.     

Hb Utrecht [α2 129(H12)Leu → Pro], a new unstable α2-chain variant associated with a mild α-thalassaemic phenotype

We describe a new α₂-globin gene point mutation found in six individuals of a three-generation Dutch family. The mutant, which is associated with a mild α-thalassaemic phenotype, is not detectable at the protein level. The α₂ cd129 (CTG → CCG) transition was found by molecular analysis using denaturing gradient gel electrophoresis (DGGE) and single-strand conformation analysis (SSCA) followed by direct sequencing of the α₂-globin gene. Southern analysis revealed a triplication of the ζ-gene in cis with the mutant α-globin gene.     

The Role of α1 Protease Inhibitor (α1 Antitrypsin) in the Regulation of Immunologic and Inflammatory Reactions

Summary: Twenty-six different alleles have been identified for α₁ protease inhibitor (α₁ antitrypsin), each designated by a letter of the alphabet. In any individual two alleles co-dominantly determine the characteristics of α₁ protease inhibitor (Pi), including mobility on electrophoresis, serum concentration and acute phase response. Recent evidence has linked some mildly deficient phenotypes of Pi with a variety of chronic immunologic and inflammatory disorders, such as rheumatoid arthritis, juvenile chronic arthritis, systemic lupus erythematosus, ankylosing spondylitis, uveitis, asthma and fibrosing alveolitis, in addition to the well recognised association of severe deficiency with emphysema and chronic liver disease. This disease susceptibility in phenotypes associated with reduced serum levels may be due to alteration in lymphocyte responses, complement activation and leukocyte migration. Pi can also influence the autolytic effects of leukocytic enzymes on tissues and may inhibit some aspects of coagulation and fibrinolysis. Therefore patients with deficient Pi phenotypes are likely to have exaggerated immunologic and inflammatory responses.     

Renal Metabolism of β2-Microglobulin

beta 2-microglobulin (beta 2M) is a protein of 11,800 daltons which occurs in the plasma of normal individuals at concentrations of approximately 2 microgram/ml. It is presumed to be relatively freely filterable. More than 99% of the filtered beta 2M is taken up by an active reabsorptive mechanism and catabolized by the renal tubule. The data presented here demonstrate that renal extraction is only slightly diminished by complete ureteral obstruction. The renal extraction of beta 2M is greater than can be accounted for by filtration alone. These data indicate that some uptake of beta 2M occurs from the peritubular capillary circulation. The loading of animals with beta 2M is associated with a marked tubular proteinuria suggesting that this protein may play a part in inducing tubular injury.     

Human GABA, Receptor αl and α3 Subunits Genes and Alcoholism

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain. GABA effects are largely mediated by binding to the postsynaptic GABA_A receptor, causing the opening of an integral chloride-ion channel. The GABA_A antagonists picrotoxin and bicuculline reduce some ethanol-induced behaviors, such as motor impairment, sedation, and hypnosis. The role of this receptor in alcoholism is further supported by effective alleviation of alcohol withdrawal symptoms by GABA_A agonists. To determine the role of the GABA, receptor (GABR) genes in the development of alcoholism, we have used α₁ and α ₃ simple sequence repeat polymorphisms in a sample of unrelated alcoholics, alcoholic probands with both parents, and psychiatrically normal controls. For the GABRα₁ gene, the differences between allele frequencies, when all alleles were compared together, were not significant between total alcoholics, subtypes of alcoholics, and normal controls. However, for GABRα₃, the differences between total alcoholics and normal controls were significant when all alleles were compared together. The differences between subtypes of alcoholics and normal controls were not significant. The results of haplotype relative risk analysis for both genes, GABRα₁ and GABRα₃, were also negative. It is possible that the sample size in the haplotype relative risk is too small to have power to detect the differences in transmitted versus nontransmitted alleles. There is a need for a replication study in a large family sample, that will allow haplotype relative risk or affected sib-pair analysis.     

β2 glycoprotein-I antigen is increased in primary hyperlipidaemia

Summary. In order to determine whether elevated levels of β₂ glycoprotein-I (β₂GPI) are associated with increased plasma lipids, we measured plasma β₂ GPI antigen levels in 47 patients with primary hyperlipidaemia (20 severe hypercholesterolaemia, nine severe hypertriglyceridaemia, and 18 mixed hyperlipidaemia) and 34 normal healthy subjects. Mean β₂GPI levels were significantly increased in each patient group (302.3, 272.9 and 299.1 mg/1. respectively) compared to controls (199.6 mg/1) (p<0.01). Significant correlations were demonstrated between β₂GPI levels and triglyceride and total cholesterol levels in the control group (r = 0.387, r =0.559: P<0.5), but were not observed in all patient groups. These results indicate that β₂GPI is increased in hyperlipidaemia and that its distribution between plasma lipid fractions is perturbed. Plasma lipid levels should therfore be considered when interpreting results of β₂GPI antigen assays.     

β2-glycoprotein 1 (β2GP1) enhances cardiolipin binding activity but is not the antigen for antiphospholipid antibodies

Some investigators have reported that a serum protein, beta 2-glycoprotein 1 (beta 2GP1), either alone or in combination with negatively charged phospholipid, may be the antigen for anticardiolipin (aCL) antibodies. To examine these reports further, ELISA tests, inhibition experiments, Ouchterlony and Western blot techniques were used to examine anticardiolipin binding to beta 2GP1. Sera from patients with the antiphospholipid syndrome (APS) and syphilis were studied, as well as whole IgG immunoglobulin and affinity purified (a.p.) IgG aCL antibodies. Results showed no binding of aCL antibodies to beta 2GP1 in the absence of cardiolipin. beta 2GP1 caused enhanced binding of aCL antibodies to cardiolipin, but this enhancement was not observed in inhibition experiments. Binding to cardiolipin occurred in the absence of beta 2GP1. Enhancement of cardiolipin binding activity by beta 2GP1 was observed for APS, but not for syphilis. We conclude that beta 2GP1 is not the antigen for aCL antibodies, nor is it likely that the antibody recognizes shared beta 2GP1-cardiolipin epitopes. Instead, this protein may make cardiolipin more available for aCL binding on solid surfaces by some yet undefined mechanism. This effect may not extend to aqueous suspensions.     

α4β1 integrin-mediated adhesion of CD34+ cells from patients with chronic myeloid leukaemia: influence of IL-3

Interactions between integrins on haemopoietic progenitor cells and their stromal ligands have an important role in the control of haemopoiesis. Growth factors can modulate these interactions (so-called 'inside-out' signalling) resulting in changes in ligand binding activity. We have studied alpha4beta1 integrin-mediated adhesion to the H120 fragment of fibronectin (which contains the strongest alpha4beta1 binding site) in CD34+ cells from patients with chronic myeloid leukaemia (CML) and have determined the effect of IL-3 on the level of adhesion. Compared to normal CD34+ cells isolated from cord blood and peripheral blood progenitor harvests (mean of 61.4 +/- 14.9% of cells attached) the CML CD34+ cells showed reduced levels of adhesion (mean of 41.9 +/- 14.7%, P < 0.05). The effect of 10 ng/ml of IL-3 resulting in reduced adhesion of normal CD34+ cells at 30 min was absent in 6/7 patients with CML. Abnormalities of adhesion to fibronectin may thus be related to IL-3 pathways affected by BCR-ABL. These findings will have implications for understanding the dysregulation of growth and adhesion in CML.     

Allotypes of Serum α2-Macroglobulin β-Lipoprotein and Immunoglobulins in the Domesticated Sheep (Ovis Aries)

Abstract. Antisera specific for allotypic markers carried by the serum α₂-macro-globulin, β-lipoprotein and IgGl and IgG2 were produced during cross-immunization experiments with sheep (Ovis aries). Two markers controlled by autosomal co-dominant genes, Ap ¹ and Ap ², were found on the α₂-macroglobulin; one marker controlled by an autosomal dominant Lp ¹ was found on the β-lipoprotein, two markers controlled by the autosomal codominants, Igl ¹ and Igl ², were found on the IgGl and one marker, controlled by Ig2 ¹ and linked to Igl ¹, was found on the IgG2. The immunoglobulin markers all occurred on the Fc fragments of the immunoglobulin molecules. There was no linkage between the Ap and Lp loci or between these and the immunoglobulin loci. A preliminary survey of the incidences of the markers in various flocks of sheep showed that the incidence of Lp ¹ and Igl ¹ was very much higher in Merinos than in the British breeds of sheep.