Phenotypic and molecular diversity of haemoglobin H disease: a Greek experience

Haemoglobin H (Hb H) disease is the severest form of alpha-thalassaemia compatible with post-natal life and occurs when alpha-thalassaemia mutations interact to reduce alpha-globin synthesis to levels approximately equivalent to the output of a single alpha-globin gene. Hb H disease has variable clinical expression, mainly related to underlying genotypes. The spectrum of alpha-thalassaemia determinants in Greece appears greater than in any other population studied and, in 75 Greek Hb H disease patients, we found 12 alpha-thalassaemia mutations interacting to produce 15 Hb H disease genotypes. Evaluation of haematological, biochemical and clinical findings, and correlation with genotypes, defined genetic predictors of disease severity and factors involved in disease progression. In accordance with previous reports, patients with non-deletion alpha-thalassaemia mutations had more severe clinical expression. Additionally, we found that all patients with the most severe phenotypes had alpha-thalassaemic globin variants. Phenotypic severity was not simply related to the degree of alpha-globin deficiency: high Hb H levels were found to exacerbate anaemia by negatively influencing tissue oxygenation, and both Hb H and alpha-thalassaemic haemoglobin variants appear to reduce red cell survival within the bone marrow and circulation. Together with the long-term follow-up in many patients, this report provides comprehensive information for management of Hb H disease and appropriate family counselling.     

Different patterns of intraerythrocytic inclusion body distribution in the two types of haemoglobin H disease. An ultrastructural study

Electron microscopic examination of intraerythrocytic inclusion bodies induced by methylene blue was carried out in 7 non-splenectomized patients with haemoglobin (Hb) H disease. In classical Hb H disease with the genotype of alpha-thalassaemia 1/alpha-thalassaemia 2 the inclusion bodies were mostly membrane-associated. In contrast, in Hb H disease of alpha-thalassaemia 1/Hb Constant Spring genotype the majority of inclusion bodies were in a floating stage. Possible mechanisms for the unexpected difference are discussed.     

Screening and genetic diagnosis of haemoglobin disorders

The inherited haemoglobinopathies are large group of disorders that include the thalassaemias and sickle cell disease. Carrier detection methods must be able to detect alpha-, beta- and deltabeta-thalassaemias, HPFH disorders and haemoglobin variants. Carrier diagnosis involves the accurate measurement of MCH, MCV, Hb A(2) and Hb F values in combination with an understanding of the haematological characteristics of the different types of thalassaemia genes and their interactions. The majority of the common thalassaemia mutations and abnormal haemoglobins can be identified by PCR-based techniques. The main applications of molecular analysis for carrier diagnosis are: the analysis of alpha-thalassaemia mutations by gap-PCR to discriminate between heterozygous alpha-thalassaemia and homozygous alpha-thalassaemia; the identification of beta-thalassaemia mutations for patients requiring prenatal diagnosis and for the prediction of the severity of the clinical phenotype of homozygous beta-thalassaemia; to discriminate between deltabeta-thalassaemia and HPFH deletions by gap-PCR.     

Haemoglobin GPhiladelphia and its interaction with haemoglobin S and alpha-thalassaemia in Nigerians

The diagnosis of Hb SS/GPhiladelphia disease was made in four young Nigerians from separate families. Their Hb electrophoretic patterns on cellulose acetate membrane at alkaline pH were similar to those obtained in sickle-cell haemoglobin C (HbSC) disease, but their clinical features and haematological data were consistent with the diagnosis of homozygous sickle-cell disease. Family studies also revealed that they had inherited an additional alpha-chain mutant haemoglobin. In one of the families, fingerprints of the globin peptides and amino acid analysis confirmed that the mutant haemoglobin was Hb GPhiladelphia (alpha 2 68 Asn----Lys beta 2 A). The results of the whole blood solubility test for sickle-haemoglobin provided firm support for the diagnosis of homozygous sickle-cell disease and distinguished clearly Hb SS/GPhiladelphia disease from Hb SC disease and Hb AS from Hb AGPhiladelphia heterozygotes. Restriction endonuclease mapping of the globin genes of the propositus and some relatives of one of the families revealed also that they were carriers of the alpha-thalassaemia-2 gene (deletion-type). The globin gene-analysis data indicate also that the alpha GPhiladelphia and alpha-thalassaemia genes are linked closely in Nigerians.     

Erythrocyte volume and haemoglobin concentration in haemoglobin H disease: discrimination between the two genotypes.

Erythrocyte volume and haemoglobin concentration in individual red cells from 62 patients with Hb H disease: 37 with H genotype (alpha-thalassaemia 1/alpha-thalassaemia 2) and 25 H/CS genotype (alpha-thalassaemia 1/Hb Constant Spring) were measured using the H*1 haematology analyser. All 25 cases with H/CS genotype, the more severe genotype, had microcytes (red cells with a volume smaller than 60 fl) less than 35% and hypochromic red cells (red cells with haemoglobin concentration less than 28 g/dl) more than 35%. A discriminant function, the ratio between the percentage of hypochromic red cells and the percentage of microcytes (Hypo/Micro), was proposed. Most of the H/CS patients (76%) had Hypo/Micro greater than 2.5 whereas those of H patients (82%) were below 2.5. Red cell volume histograms were also characteristically different between the two genotypes: the H/CS had a peak between 60 and 90 fl while the H genotype showed a peak at or very close to 60 fl, indicating a greater degree of microcytosis. Increased hypochromia with a slight decrease in cell size of H/CS red cells suggests that the poor degree of haemoglobinization has no linkage or very little role in disturbing the synthesis of membrane proteins and their assembly to the plasma membrane.     

Hemoglobin constant spring defined by specific oligonucleotide hybridization and hemoglobin D Punjab (beta 121----Gln) in a Batak Indonesian family

A Batak Indonesian from North Sumatra with hemoglobin (Hb) D Punjab (alpha 2 beta 2 121----Gln) and hemoglobin Constant Spring (Hb CoSp) is described. The 24-year-old man did not have clinical symptoms, and his hematological indices were normal. However, he had a persistent slight elevation of fetal hemoglobin level. His mother and his brother were heterozygous for Hb D Punjab; his father had Hb CoSp trait. A sister did not have any abnormal hemoglobin. To show the exact molecular defect leading to the synthesis of Hb CoSp in this family, genomic DNA from the father was analyzed by hybridization with synthetic oligonucleotides. Genomic DNA was digested with Sst I and Hind III producing a 1.05-kb fragment from the 3' end segment of the alpha 2-globin gene, including the termination codon. Two nonadecamers were synthesized to serve as probes: one, entirely homologous to the normal 3' end of alpha 2A-globin gene sequence, including the termination codon TAA, the other different from it by a replacement of the T in the termination codon TAA with C, changing it to CAA, the codon for the amino acid glutamine. DNA from normal controls gave a positive signal with the normal alpha 2TAA oligonucleotide probe but negative with the alpha 2 CAA probe. The father of propositus who had Hb CoSp trait gave a positive signal with the normal alpha 2TAA oligonucleotide probe as well as with the alpha 2CAA oligonucleotide probe, showing him to be heterozygous for the alpha 2CAA-globin gene. This result shows that the Hb CoSp in the Batak family is indeed due to a replacement of T by C in the TAA termination codon of the alpha 2-globin gene changing it to CAA the condon for glutamine. This explains the resulting readthrough of the untranslated sequence of the mRNA.     

Red cell metabolism and severe neonatal jaundice in West Malaysia.

A study was carried out of 332 babies suffering from severe neonatal jaundice who were admitted to the General Hospital, Kuala Lumpar, Malaysia. Of the 332 neonates, 51 were premature and 281 were full-term babies, 178 (110 Chinese, 58 Malay, 9 Indian and 1 European-Pakistani) had bilirubin levels of 20 mg% or higher, requiring exchange blood transfusion. Of the Chinese neonates, 23 (20.9%) had G6PD deficiency, 9 (8.2%) had Hb Bart's and 2 (1.8%) had an abnormal haemoglobin, one Hb Q and one fetal variant. Among the Malay infants, 10 (17.2%) had G6PD deficiency, 7 (12.1%) had Hb Bart's and 10 (17.2%) had abnormal haemoglobins (four had Hb E trait, one had Hb K and Bart's in addition to Hb E, three had Hb CoSp with Hb Bart's, one had Hb Q and one Hb Tak). One of the nine Indian neonates had G6PD deficiency and one had Hb S trait. The one European-Pakistani baby was a carrier of Hb D Punjab. In addition to G6PD deficiency, abnormal haemoglobins seem to have contributed to the high incidence of severe neonatal jaundice in Malaysia. The mean activities of GP, GR and GR after stimulation with FAD were higher, while the mean activity of PK and mean level of reduced glutathione were lower than in normal cord bloods. The percent increase of GR after FAD stimulation was significantly lower; fewer in this group had increases above 20% than in normal cord blood. The possible significance of the findings is discussed.     

Interaction between beta-thalassaemia and Hb G Philadelphia associated with alpha-thalassaemia.

A man who did not produce and beta-chains did not suffer from a severe beta-thalassaemia. He was heterozygous for Hb G Philadelphia. It has been suggested that this haemoglobin variant was associated with alpha-thalassaemia and that interaction between alpha-thalassaemia and beta-thalassaemia decreased the imbalance of alpha/beta-globin biosynthesis and thereby the severity of the beta-thalassaemic disorder. Association of Hb G Philadelphia and alpha-thalassaemia in this man and his family is now demonstrated using bone marrow and reticulocytes of the propositus and one of his sons and reticulocytes only of another son.     

Gene mapping of Malaysian alpha thalassemias with alpha and zeta globin gene probes

Restriction enzyme analysis of the alpha and zeta globin genes was carried out in four cases of Hb Bart's hydrops fetalis, in three patients with Hb H disease without Hb CoSp, in three patients with Hb H disease with Hb CoSp, in 47 individuals with alpha thalassemia trait, and in 47 normal individuals. All four cases of Hb Bart's hydrops fetalis resulted from deletions of alpha 1 and alpha 2 globin genes which did not extend to the psi zeta 1 and zeta 2 globin genes. The same type of deletion was observed in alpha thal1 carriers, but two newborns (one Malay and one of Chinese extraction) had a nondeletion type of alpha thal1 which was confirmed by quantitative alpha globin gene analysis. In addition, two other newborns diagnosed as alpha thal1 trait carriers (one Malay, one Chinese) were shown to have a deletion of both alpha globin genes by quantitative alpha globin gene analysis, but further testing with zeta globin gene probe failed to reveal an abnormal fragment length characteristic of an alpha globin gene deletion. We believe that this last condition is due to a large deletion which includes all alpha globin genes and all zeta globin genes on the same chromosome. On another front, Bgl II restriction analysis of all four Hb Bart's hydrops fetalis cases and the alpha thal1 trait carriers showed a 10.5-kb Bgl II restriction fragment, in the hydrops fetalis as a single band, while in the carriers this 10.5-kb fragment was accompanied by the usual normal 12.5-kb and 11.3-kb fragments. We report that this 10.5-kb fragment, previously thought to be specific for the Southeast Asian alpha thal1 gene deletion, is also common in normal individuals. Nevertheless, digestion with other enzymes can clearly differentiate the alpha thal1 and normal genotypes. We distinguish the findings in the alpha thalassemias from the extensive DNA polymorphism in the region of the alpha and zeta globin genes.     

Heterogeneity of beta thalassaemia in Thailand

Beta thalassaemia in Thailand is heterogeneous. Clinical, genetical, haematological and globin chain biosynthetic studies were performed in seven beta-thalassaemia families. The results showed different gene combinations. These were alpha-thalassaemia/homozygous beta 0-thalassaemia, questionable double heterozygosity between a beta-thalassaemia and a silent beta-thalassaemia genes with low Hb F of unexplained cause, silent beta-thalassaemia/beta+ - or beta 0-thalassaemia, high Hb A2 high Hb F-beta-thalassaemia - a new mutant, mild beta+-thalassaemia/Hb E, and beta 0-thalassaemia/heterocellular HPFH or delta beta-thalassaemia associated with alpha-thalassaemia. Most of variability of clinical and haematological findings in these families is due to heterogeneity of the beta-thalassaemia and related genes.     

Abnormal haemoglobins and hereditary ovalocytosis in the Ulu Jempul District of Kuala Pilah, West Malaysia.

A survey of abnormal haemoglobins and hereditary ovalocytosis was carried out among 629 Malays of Minangkabau descent in the Ulu Jempul District of Kuala Pilah, in the state of Negri Sembilan in West Malaysia.. Several abnormal haemoglobins were found with the following frequencies: Hb E 5.25%, Hb CoSp 2.38%, Hb A2 indonesia 0.80%, a fast moving Hb with a Mobility between A and Bart's 0.64% and Hb Q 0.16%. Hereditary ovalocytosis was found in 13.2% of these people. None of the persons with hereditary ovalocytosis had any evidence of haemolysis.     

Alpha thalassaemia in an Italian population

The incidence of alpha-thalassaemia in an Italian population has been determined by a survey of random cord bloods for the presence of Hb Bart's. 144 out of 4730 (3%) had detectable amounts of Hb Bart's. Furthermore, alpha-globin gene analysis of 100 random cord bloods showed that five out of 100 had the common type of alpha-thalassaemia caused by a single alpha-globin gene deletion (-alpha). The molecular basis of alpha-thalassaemia was also determined in a selected group of 34 newborns with detectable levels of Hb Bart's. 25 of these cases had the -alpha 3.7 deletion type of alpha-thalassaemia and nine had nondeletion types of alpha-thalassaemia in four of which the molecular defect was detectable directly by restriction enzyme analysis.     

Haemoglobin Barts in newborn Tanzanians.

Using both starch gel and cellulose-acetate electrophoresis is screening procedures, haemoglobin (Hb) Barts was detected in 11.08% of 325 cord blood samples from newborn Tanzanians. Red cell studies in these and in normals and a search for inclusion bodies of Hb H did not suggest alpha-thalassaemia. The mothers of these babies do not show any evidence of alpha-thalassaemia. It is suggested that the presence of Hb Barts in samples of cord blood is not due to the presence of alpha-thalassaemia in the Tanzanian population.     

Studies on sickle cell heterozygotes in Saudi Arabia--interaction with alpha-thalassaemia

This study was conducted in the Eastern Province of Saudi Arabia where both thalassaemia and Hb S genes occur at a high frequency. In 171 Saudi Hb S heterozygotes, the range for Hb S was found to be 17-45% with a mean of 31.0%. Frequency distribution histograms showed a trimodal distribution. Peak A, B and C had 18-28, 28-35 and 35-45% Hb S values, respectively. Determination of alpha/beta ratio showed that these findings result from interaction of Hb S with the alpha-thalassaemia gene. Peak C with mean for Hb S of 40% consisted of a majority of individuals with no thalassaemia gene, and the alpha/beta ratio was 0.98% +/- 0.06. Peak B individuals were heterozygous for alpha-thalassaemia 2 with an alpha/beta ratio of 0.80% +/- 0.06 and peak A individuals were homozygous to alpha-thalassaemia 2 with an alpha/beta ratio of 0.58 +/- 0.08. The presence of alpha-thalassaemia 2 in homozygotes results in hypochromia and microcytosis, with little effect on haematological parameters.     

Antioxidant deficit and enhanced susceptibility to oxidative damage in individuals with different forms of alpha-thalassaemia

alpha-Thalassaemia is a common red cell disorder in Taiwan, affecting 6-8% of Taiwanese. Previous studies have shown that reactive oxygen species are generated in increased amounts in thalassaemic red cells. This implies the possible alteration of redox status in thalassaemic patients, which may adversely affect their health. In the present study, the redox status of patients with alpha-thalassaemia trait and haemoglobin H (Hb H) disease was investigated. Lipid peroxidation, as measured by the level of plasma thiobarbituric acid reactive substances (TBARS), was increased in alpha-thalassaemic patients, with the highest level of TBARS in Hb H disease patient. The plasma levels of vitamin A, C, and E were significantly lower in alpha-thalassaemic patients than in controls. The overall antioxidant capacity in plasma was inversely correlated with the severity of alpha-globin gene defect: the more severe the form of alpha-thalassaemia, the lower the overall antioxidant capacity in plasma. Erythrocytes isolated from alpha-thalassaemia patients had lower levels of vitamin E, glutathione, catalase and superoxide dismutase. In addition, these alpha-thalassaemic red cells were more susceptible to hydrogen peroxide-induced lipid peroxidation and decrease in deformability. All these data suggest that the alpha-thalassaemic patients suffer from increased oxidative stress and antioxidant deficit, which may complicate the pathophysiology of alpha-thalassaemia.     

Heterogeneity and variation of clinical and haematological expression of haemoglobin S in Saudi Arabs.

Sickle cell haemoglobin (Hb S) occurs at a high frequency in the Eastern (EP), South-Western (SWP) and North-Western (NWP) Provinces of Saudi Arabia and the presentation of the Hb S is believed to exhibit clinical diversity in the different regions. Three areas of Saudi Arabia were screened to determine the frequency of Hb S and alpha- and beta-thalassaemias and glucose-6-phosphate dehydrogenase deficiency genes. Furthermore, in an attempt to investigate and compare the clinical and haematological presentation of sickle cell disease (SCD) in the different regions of Saudi Arabia, the individuals identified as Hb S homozygotes were investigated further. The patients were further classified on the basis of whether there was associated alpha- or beta-thalassaemia. A severity index (SI) was calculated for each patient and the clinical presentations and laboratory findings were compared. The results showed significantly variable severity of SCD in patients from different regions. The patients from the EP generally had a mild clinical presentation, while in the SWP and NWP majority of the patients suffered from a severe disease as judged by the SI. No correlation could be established between Hb F level and SI, though the WBC level correlated positively with the SI. The lowest SI values were encountered in patients with associated alpha-thalassaemia who also had the lowest WBC count and MCV and the highest RBC count and packed cell volume.     

Hb H hydrops foetalis syndrome: a case report and review of literature

Haemoglobin H (Hb H) disease is caused by deletion or inactivation of three alpha-globin genes, leaving only one intact and active alpha-globin gene. People with Hb H disease usually have moderate anaemia, but are generally thought to be asymptomatic. Some Hb H disease patients require transfusions, and there are reports of fetuses with Hb H disease who have severe anaemia in utero resulting in fatal hydrops foetalis syndrome. We now report a case of Hb H hydrops foetalis syndrome, caused by the inheritance of a hitherto novel alpha-globin gene point mutation (codon 35 TCC-->CCC or Serine-->Proline) and an alpha-thalassaemia deletion of the Filipino type removing all zeta-alpha-globin genes on the other chromosome 16. The infant was delivered prematurely because of pericardial effusion and fetal distress, and was found to have severe anaemia and congenital anomalies. A review of the relevant literature on this syndrome is presented, and serves to underscore the phenotypic variations of Hb H disease and the need for surveillance for this condition among newborns and genetic counselling in communities with a high proportion of at-risk populations.     

Laboratory diagnosis of a compound heterozygosity for Hb Hekinan [alpha27(B8) Glu-Asp] and a deletional alpha-thalassaemia 2 in Thailand

We report the haematological and molecular characterization of a previously undescribed condition of compound heterozygosity for haemoglobin (Hb) Hekinan [alpha27(B8) Glu-Asp] and a deletional alpha-thalassaemia 2 detected in a Thai individual. Hb analysis demonstrated that although this Hb variant co-migrates with Hb A on cellulose acetate electrophoresis and cation-exchange high-performance liquid chromatography (HPLC), the HPLC procedure using a weak cation-exchange material with polyaspartic acid could clearly differentiate the two Hb. The variant could then be confirmed using the polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis of the amplified alpha1-globin gene.     

Antioxidant system and serum trace elements in alpha-thalassaemia and Hb Lepore trait

Erythrocyte antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase were found to be significantly high in subjects with alpha-thalassaemia and Hb Lepore trait, as a consequence of the increased oxidant stress which is known to exist in these conditions. Among the serum trace elements present in these enzymes, selenium was increased in subjects with Hb Lepore trait and significantly low in those with alpha-thalassaemia trait, while selenium erythrocyte content was significantly increased in alpha-thalassaemic subjects.     

Homozygous State for Hb Constant Spring (Slow-moving Hb X Components)

A 12-yr-old Malay boy who was studied because his youngest brother and both hisparents had the slow-moving Hb Xcomponents (earlier reported to lead to HbH disease when combined with -thalassemia) was found to be homozygous forthe same slow-moving components. He hadsplenomegaly and a just palpable liver, mildanemia with microcytosis, hypochromia,slight morphologic changes of the red bloodcells, and slight reticulocytosis. Of eightchildren in the family, six had the trait forthe abnormality, one was normal, and one,the propositus, was homozygous. Structuralstudies of the isolated abnormal hemoglobinshowed it to be identical to Hb ConstantSpring (Hb CoSp), an -chain variant with172 residues instead of the usual 141, theadditional 31 being attached to the C-terminal end. In addition to the abnormal variant for which the propositus washomozygous, he also had normal Hb A andnormal Hb A₂ with normal -chains. If thetheory that Hb CoSp is due to a structuralmutation affecting the terminator codon iscorrect, this case provides evidence for a duplication of the gene for -chain production.Results of study of several erythrocyteenzymes are also reported.

Submitted on March 15, 1973 Revised on July 30, 1973 Accepted on August 2, 1973