The alpha-chain-termination mutants and their relation to the alpha-thalassaemias.

The structure, synthesis, genetic transmission, clinical associations and distribution of the elongated alpha-chain haemoglobin variants has been described. The data indicate that the most likely molecular basis for these common abnormal haemoglobins is a single base substitution in the alpha-chain termination codon. Because these variants are produced inefficiently they give rise to the clinical picture of alpha-thalassaemia. When these findings are taken together with recent work regarding the molecular basis for other forms of alpha-thalassaemia it is possible to build up a fairly complete picture of the molecular pathology of the alpha-thalassaemias.     

Patterns of sickle cell, thalassaemia and glucose-6-phosphate dehydrogenase deficiency genes in north-western Saudi Arabia

This study was conducted on 429 blood samples collected from Saudi males and females from Al-Ula in the north-western province of Saudi Arabia in order to determine the frequency of the sickle cell gene, glucose-6-phosphate dehydrogenase (G6PD) deficiency gene, and alpha- and beta-thalassaemia genes, and to investigate the pattern of their interactions. The frequency of the sickle cell gene was 0.0785, while that of the beta-thalassaemia gene was 0.1195. Heterozygous alpha-thalassaemia 2 (- alpha/alpha alpha) was encountered at a frequency of 0.121, while homozygous alpha-thalassaemia 2 (- alpha/- alpha) occurred at a frequency of 0.0046. HbH disease and hydrops fetalis were not encountered. One case with triple alpha-gene arrangement, alpha alpha alpha anti-3.7, was identified. The G6PD deficiency gene frequency was 0.08 and 0.032 in males and females, respectively. Several cases with 2 abnormal genes were encountered. The haematological and biochemical data from the patients with sickle cell disease suggest that the disease in this population is more severe in comparison with cases reported from the eastern population.     

Interaction of deletional alpha-thalassaemia with sickle cell beta-thalassaemia and its influence on foetal haemoglobin expression

A rare association of three haemoglobin defects, viz: traits for deletion form of alpha-thalassaemia, beta-thalassaemia, and sickle cell gene, in a family of French West Indies origin, was studied both at phenotype and genotype levels. In this sickle cell beta-thalassaemia, interacting alpha-thalassaemia is shown to influence the foetal haemoglobin expression. A reverse relationship between the foetal haemoglobin level and the number of alpha genes was observed.     

Fetal haematology in rhesus isoimmunisation.

Haematological studies were carried out in pure fetal blood samples obtained fetoscopically in 29 rhesus isoimmunised pregnancies at 18-24 weeks'' gestation, and the values were compared with those obtained in 62 normal control pregnancies. Fetal reticulocytosis or erythroblastaemia was seen only in association with a haemoglobin concentration of 4 g/dl or less. Ten of the 14 fetuses with a haemoglobin concentration below 4 g/dl showed ultrasonographic evidence of hydrops.     

Association of red cell glucose-6-phosphate dehydrogenase with haemoglobinopathies

A total of 1,112 randomly selected Saudi Arabs, of both sexes, living in Jeddah and the surrounding areas were screened for the phenotypic distribution of red cell glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD). They were also investigated for haemoglobin and for thalassaemia. Phenotyping of the haemoglobins and the red cell enzymes was carried out by starch gel electrophoresis and the dye-decolouration screening test, while the investigation for thalassaemia was carried out by globin-chain biosynthesis, followed by column chromatography. The red cell Gd- alleles were significantly associated with the sickle-cell gene in both the males (chi 2(1): AS-28.80; SS-4.89) and females (chi 2(1): AS-10.99; SS-13.16). A similar association was also observed between G6PD deficiency and thalassaemias in males (chi 2(1): alpha-thalassaemia - 3.13; beta-thalassaemia - 11.06) and females (chi 2(1): alpha-thalassaemia - 6.63). However, no such association was detected between red cell 6PGD types and haemoglobin genes. The results suggest that the red cell G6PD deficiency, sickle-cell and thalassaemia genes might have evolved as a result of the same ecological factor, probably malaria.     

The molecular basis of alpha-thalassaemia in Thailand

The molecular basis of alpha-thalassaemia has been established in 48 Thai subjects with Hb H disease and 15 with the Hb Bart's hydrops fetalis syndrome. This study has shown that in this population there are at least 18 different types of chromosome carrying seven independent alpha-thalassaemia mutations one of which is a novel deletion removing the entire alpha-globin gene complex. Although there are a limited number of alpha-thalassaemia determinants in the Thai population, there is a remarkable degree of variation in the genetic markers which flank them. These markers may be of value in establishing the evolutionary history of the alpha-thalassaemias.     

Cardiac ultrasonography in structural abnormalities and arrhythmias. Recognition and treatment.

Fetal cardiac ultrasonography has become an important tool in the evaluation of fetuses at risk for cardiac anomalies. It can both guide prenatal treatment and assist the management and timing of delivery. We recommend that a fetal echocardiogram be done when there is a family history of congenital heart disease; maternal disease that may affect the fetus; a history of maternal drug use, either therapeutic or illegal; evidence of other fetal abnormalities; or evidence of fetal hydrops. The optimal timing of evaluation is 18 to 22 weeks'' gestation. An entire range of structural cardiac defects can be visualized prenatally, including atrioventricular septal defect, ventricular septal defect, cardiomyopathy, ventricular outlet obstruction, and complex cardiac defects. The outcome for a fetus with a recognized abnormality is unfavourable, with less than 50% surviving the neonatal period. Fetal cardiac arrhythmias are also a common occurrence, 15% in the series described here. Premature atrial or ventricular contractions are most commonly seen and usually require no treatment. Supraventricular tachycardia can result in hydrops and require in utero treatment to prevent fetal demise. Complete heart block, particularly in association with structural heart disease, has a poor prognosis for fetal survival.     

Thalassaemia in Cyprus

Frequencies of the thalassaemias in Cyprus were examined by a survey of hospital inpatients and haematological investigations of adult and newborn population samples. The data indicate that 15% of the Greek and Turkish Cypriots are carriers of beta-thalassaemia genes, while 10% of the population carry alpha-thalassaemia genes. These are the highest frequencies of thalassaemia genes found today in any Caucasian population.     

Structurally inherent antigenic sites. Localization of the antigenic sites of the alpha-chain of human haemoglobin in three host species by a comprehensive synthetic approach.

The antigenic structure of the alpha-chain of human haemoglobin was studied by a synthetic approach consisting of the synthesis of a series of consecutive overlapping peptides that together systematically represent the entire primary structure of the protein. This approach enabled the identification of a full profile of immunochemically active alpha-chain peptides and the localization of its major 'continuous' antigenic sites. Antibodies to haemoglobin raised in each of three different species (goat, rabbit and mouse) recognize similar sites on the alpha-chain. Further, the molecular locations of these sites coincide with alpha-chain regions extrapolated from antigenic sites of the conformationally similar myoglobin molecule. These findings support our earlier proposed concept of 'structurally inherent antigenic sites', namely that antigenicity is conferred on certain surface regions of proteins by virtue of their three-dimensional locations. Thus the antigenic sites of conformationally related proteins are likely to have similar molecular locations.     

Human red blood cell polymorphisms and malaria

Genetic factors are a major determinant of child survival in malaria endemic countries. Identifying which genes are involved and how they affect the malaria disease risk potentially offers a powerful mechanism through which to learn more about the host-parasite relationship. The past few years have seen significant progress towards achieving this goal for some of the best-known malaria resistance genes that determine the structure or function of red blood cells: Gerbich blood group antigen negativity; polymorphisms of the complement receptor genes (most notably CR1); Southeast Asian ovalocytosis; pyruvate kinase deficiency; haemoglobin E; the sickle cell trait; and alpha-thalassaemia are all examples. The challenge for the future must be to translate such advances into fresh approaches to the prevention and treatment of malaria.     

Amino acid sequence of the beta-chain of the tetrameric haemoglobin of the bivalve mollusc, Anadara trapezia

The amino acid sequence of the beta-chain of the principal haemoglobin from A. trapezia has been determined. The sequence was deduced from the sequences of tryptic peptides, which were fractionated using highperformance liquid chromatography and peptide mapping. Additional sequence data, particularly for the large tryptic peptides, was obtained from enzyme digests of both cyanogen bromide fragments and large citraconyltryptic peptides. The beta-chain has 151 residues which is longer than all the other sequenced haemoglobin chains except the alpha-chain of A. trapezia, which is 153 residues in length. The residues corresponding to those normally in the D helix are absent in this beta-chain. The additional residues are contributed by an extension of the N-terminal region, which was also found to be acetylated. Comparison of the beta-chain amino acid sequence with that of the alpha-chain of A. trapezia, the dimeric chain of A. trapezia, and the dimeric chain of A. broughtonii showed 53% identity in each case. In the E and F helices, the homology is particularly noticeable. There is 100% homology in the F helix of all four chains. The dimeric globin of A. trapezia also shows 100% homology with the beta-chain in the E helix, while the alpha-chain shows 75%. If the tertiary structure of the alpha- and beta-chains of A. trapezia haemoglobin is the same as that of horse haemoglobin, then there are many changes in the alpha 1 and beta 2 contact site residues.     

Strongyloidiasis in Allied ex-prisoners of war in south-east Asia

One hundred and sixty ex-servicemen who had been prisoners of war in south-east Asia during 1942-5 were investigated for infection with Strongyloides stercoralis. Larvae were found in 44 (27·5%) of the men, who had therefore been infected for 34-37 years. Direct microscopy of the faeces was the most successful diagnostic method, giving a positive result in 37 cases (84%); multiple examinations were often necessary. Faecal culture was positive in 30 cases (68%), but examination of duodenal fluid obtained with the string test gave a positive result in only 17 (39%). The mean blood eosinophil count and mean serum IgE concentration were higher in the infected men, though normal values were often found in individual cases. Clinical manifestations of isolated strongyloides infection were analysed by comparing the infected men with control groups of ex-prisoners in south-east Asia without proved strongyloidiasis and ex-prisoners in Europe. Twenty-nine infected men (66%) complained of non-specific urticaria, and 13 (30%) had pathognomonic larva currens. Gastrointestinal symptoms significantly more common in the infected group were diarrhoea, indigestion, lower abdominal pain, pruritus ani, and weight loss (p <0·05-p <0·0005).The study group was thought to be reasonably representative of Allied ex-servicemen imprisoned in south-east Asia during the second world war. Probably there are many thousands of infected persons in several countries. The worm has an unusual ability to multiply, and larvae may spread throughout the body in immunosuppressed subjects.     

Neuro-angiostrongyliasis: unresolved issues

Angiostrongylus cantonensis, the rat lungworm, probably evolved with its hosts, members of the genus Rattus and closely related species, in south-east Asia. Since its first discovery in rats in China and in a case of human infection in Taiwan, the parasite has been found to infect humans and other mammals across a wide and ever-increasing territory, which now encompasses much of south-east Asia, Melanesia, Polynesia and eastern Australia. It has also established a foothold in Africa, India, the Caribbean and south-eastern USA. This dispersal has been a direct result of human activity, and in some cases has been linked with the spread of the African giant land snail, Achatina fulica. However, this snail is not critical to the extension of the parasite's range, as numerous other indigenous molluscan species serve as adequate intermediate hosts; the importance of Achatina to the life cycle may have been over-emphasized. In Australia, the parasite is established along parts of the east coast, and the presence of an indigenous close relative, Angiostrongylus mackerrasae, suggests a long association of the parasite with its local rat hosts, a situation analogous to that of Angiostrongylus malaysiensis in south-east Asia. These three Angiostrongylus species share virtually the same life cycle, but only A. cantonensis has been confirmed to be a human pathogen.     

Haemoglobin binding with haptoglobin. Unequivocal demonstration that the beta-chains of human haemoglobin bind to haptoglobin.

Haptoglobin binding to haemoglobin and its isolated alpha- and beta-chains was studied by use of a highly sensitive solid-phase radiometric assay. As expected, adsorbents of haemoglobin bound 125I-labelled haptoglobin more efficiently than did adsorbents of the alpha-chain. However, unexpectedly, adsorbents of the beta-chain were found to be essentially identical with those of the alpha-chain in their ability to bind haptoglobin. These results demonstrate, unequivocally, the ability of beta-chains to bind to haptoglobin, and indicate that this assay is particularly convenient and useful for studying haptoglobin interactions with haemoglobin and its alpha- and beta-chains.     

Haemoglobin of the antarctic fish Pagothenia bernacchii. Amino acid sequence, oxygen equilibria and crystal structure of its carbonmonoxy derivative.

The Antarctic fish Pagothenia bernacchii has one major haemoglobin, Hb1 (over 95% of the total blood content). Hb1 has a strong alkaline Bohr effect and at low pH exhibits the reduced ligand affinity and co-operativity that comprise the Root effect. We have determined the complete amino acid sequence of P. bernacchii Hb1 and also the structure of its carbonmonoxy derivative by X-ray crystallography, to a resolution of 2.5 A. The crystallographic R-factor of the refined structure is 18%. The three-dimensional structure of this fish haemoglobin is similar to that of human haemoglobin A, with a root-mean-square difference in main-chain atom positions of 1.4 A after superimposition of the two structures, despite only 48% homology of their amino acid sequences (including insertion of a single residue in the CD region of the fish alpha-chain). Large structural differences occur only at the N and C termini of both the alpha- and beta-chains. Neither these nor other smaller structural differences provide any obvious explanation of the Root effect of this or other fish haemoglobins.     

Specific cyanylation and cleavage at cysteine-104 human hemoglobin alpha-chain. A novel approach to the problem of the alpha-chain tryptic core in the study of haemoglobin variants by "fingerprinting" methods.

1. A new approach to the analysis, by "fingerprinting", of the tryptic core region of human haemoglobin alpha-chain is described. 2. The alpha-chain is cyanylated at its single cysteine residue (alpha104) and then split, by exposure to mild alkali, at the N-peptide bond of the resulting beta-thiocyanoalanine residue. 3. The two cleavage fragments, alpha1-103 and alpha104-141, are separated by gel filtration, and the fragment alpha104-141, which contains all the residues of the alpha-chain tryptic core, is digested with pepsin. 4. Preparative "fingerprints" of these peptic peptides yield eight major peptides, which provide complete sequence information for the whole region alpha104-141. 5. The utility of the method is demonstrated by repeating the determination of the substitution in haemoglobin Hopkins-2, a known alpha-chain core variant in which histidine-alpha112 (G19) is replaced by an aspartic acid residue.     

The screening of Chinese cord blood for haemoglobinopathies

The frequency of haemoglobinopathies among the Hong Kong Chinese newborn was examined by screening 932 consecutive cord blood samples by electrophoresis. The findings indicate that 49 (5.2%) had electrophoretic abnormalities, of which 39 (4.2%) demonstrated the presence of Hb Bart's. 14 of these 39 babies were re-investigated after 1 year and all were proven to carry the alpha-thalassaemia gene. This is the first study in which Chinese babies with Hb Bart's in cord blood had the diagnosis of alpha-thalassaemia trait confirmed in later life. 3 out of 4 other babies had different haemoglobin variants, namely Hb alpha Q, Hb J and Hb New York. 1 out of the 6 babies who had elevated A2 levels at birth was found to carry the beta-thalassaemia trait. None had a serious haemoglobinopathy.     

Semi-Quantitative vs. Volumetric Determination of Endolymphatic Space in Menière’s Disease Using Endolymphatic Hydrops 3T-HR-MRI after Intravenous Gadolinium Injection

Magnetic resonance imaging enhances the clinical diagnosis of Menière''s disease. This is accomplished by in vivo detection of endolymphatic hydrops, which are graded using different semi-quantitative grading systems. We evaluated an established, semi-quantitative endolymphatic hydrops score and with a quantitative method for volumetric assessment of the endolymphatic size. 11 patients with Menière''s disease and 2 healthy subjects underwent high resolution endolymphatic hydrops 3 Tesla MRI with highly T2 weighted FLAIR and T2DRIVE sequences. The degree of endolymphatic hydrops was rated semi-quantitatively and compared to the results of 3D-volumetry. Moreover, the grade of endolymphatic hydrops was correlated with pure tone audiometry. Semi-quantitative grading and volumetric evaluation of the endolymphatic hydrops are in accordance (r = 0.92) and the grade of endolymphatic hydrops correlates with pure tone audiometry. Patients with a sickness duration of ≥ 30 months showed a significant higher total labyrinth fluid volume (p = 0.03). Fast, semi-quantitative evaluation of endolymphatic hydrops is highly reliable compared to quantitative/volumetric assessment. Endolymphatic space is significantly higher in patients with longer sickness duration.     

Haemoglobin synthesis in 28 obligatory cases for alpha-thalassemia traits.

In the Far East two types of alpha-thalassemia genes, namely alpha-thalassemia, (alpha-thal1), and alpha-thalassemia2 (alpha-thal2) exist. Definite diagnosis of the alpha-thal1 and alpha-thal2 traits is very difficult because their hematological findings are minimally abnormal or normal. This study attempts to characterize the heterozygotes by hemoglobin chain synthesis in reticulocytes from obligatory cases of the alpha-thal1 and alpha-thal2 traits. Twelve parents of babies with hemoglobin Bart's hydrops fetalis (obligatory alpha-thal1 trait) had the mean total radioactivity alpha/beta ratio of 0.76 +/- SD 0.04, while that of 7 normal controls was 1.06 +/- SD 0.04. The alpha/beta globin chain ratios of 16 cases, who were either parents or offspring of patients with hemoglobin H disease, were found to segregate into 2 groups, i.e. 0.78 +/- SD 0.03 (10 cases) and 0.9l1 and alpha-thal2 traits respectively. The hematological data of the first group showed definite hypochromic microcytic red cells, similar to those of the parents of the hydrops. The second group had significantly higher mean corpuscular hemoglobin than the first group, compatible with alpha-thal2 trait. Our globin chain synthesis study thus appears to be capable of discriminating normal, alpha-thal1 and alpha-thal2 traits.