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Alpha thalassaemia myelodysplastic syndrome (ATMDS) is an unusual complication of chronic myeloid malignancy that is associated with a striking red cell phenotype. It represents an acquired form of alpha-thalassaemia that most commonly arises in the context of myelodysplasia. It has recently been shown that this condition occurs in association with somatic mutations of a known X-encoded trans-acting regulator of alpha globin gene (HBA) expression, ATRX. There is an unexplained, strong male preponderance of individuals with the ATMDS phenotype with a >5:1 male-female ratio and furthermore, all the somatic ATRX mutations described to date have been in males. Here we report the identification, in a single centre, of two females with ATMDS and mutations in the ATRX gene, proving that ATMDS associated with such mutations may occur, albeit rarely, in females. It seemed possible that females might be less likely to develop ATMDS if the inactivated copy of the ATRX gene (ATRX) became progressively re-activated throughout life. This study ruled out this hypothesis by investigating the pattern of ATRX inactivation in a cross-sectional analysis of normal females at ages ranging from newborn to 90 years.

Patients with clonal myeloid disorders, especially myelodysplastic syndromes (MDS), may acquire alpha-thalassaemia. To estimate the prevalence of this erythrocyte phenotype, we examined brilliant cresyl blue-stained blood smears from 201 patients with neoplastic myeloid disorders and 282 controls (195 non-clonal anaemia, 62 with medical illnesses without anaemia and 25 healthy persons). Haemoglobin H inclusions were detected in 8/100 patients with MDS (8%) and 2/81 (2.5%) patients with myeloproliferative disorders, but in none of the acute leukaemia patients or controls. We conclude that the emergence of thalassaemic clones may be relatively common in the disordered marrow milieu of MDS.

We have identified and characterized a Scottish individual with alpha thalassaemia, resulting from a de novo 48 kilobase (kb) deletion from the telomeric flanking region of the alpha globin cluster which occurred as a result of recombination between two misaligned repetitive elements that normally lie approximately 83 kb and 131 kb from the 16p telomere. The deletion removes two previously described putative regulatory elements (HS-40 and HS-33) but leaves two other elements (HS-10 and HS-8) intact. Analysis of this deletion, together with eight other published deletions of the telomeric region, showed that they all severely downregulated alpha globin expression. Together they defined a 20.4-kb region of the human alpha cluster, which contains all of the positive cis-acting elements required to regulate alpha globin expression. Comparative analysis of this region with the corresponding segment of the mouse alpha globin cluster demonstrated conserved non-coding sequences corresponding to the putative regulatory elements HS-40 and HS-33. Although the role of HS-40 as an enhancer of alpha globin expression is fully established, these observations suggest that the role of HS-33 and other sequences in this region should be more fully investigated in the context of the natural human and mouse alpha globin loci.

To date, more than 35 single or oligonucleotide mutations of the alpha genes that cause alpha thalassaemia have been described. Their interactions give rise to widely variable clinical manifestations, from a mild hypochromic, microcytic anaemia to a lethal intrauterine anaemia associated with hydrops fetalis. Understanding the molecular genetics enables accurate genotyping, genetic counselling and prenatal testing for the most severe forms of alpha thalassaemia. Here we show for the first time that the interaction between two relatively common forms of alpha thalassaemia (--MED/(alpha)TSaudi(alpha)) may be associated with a clinically severe form of alpha thalassaemia, Hb H hydrops fetalis.

We have identified three individuals of Greek or Greek Cypriot origin with an atypical form of HbH disease characterized by a severe hypochromic microcytic anaemia associated with relatively small amounts of HbH in the peripheral blood. Molecular analysis has shown that each is a compound heterozygote for a previously described mutation affecting the poly A addition signal (AATAAA-->AATAAG) and a previously undescribed mutation involving a T-->C transition in codon 29 of the alpha 2 gene causing a leucine-->proline substitution. Although this mutation would be expected to produce an unstable haemoglobin and hence a haemolytic anaemia, simple heterozygotes for the alpha 29Leu-->Pro mutation have the phenotype of alpha-thalassaemia trait.

A rapid and inexpensive polymerase chain reaction (PCR) based strategy is described which detects the three common, severe alpha thalassaemia determinants observed in southeast Asia (--SEA) and the Mediterranean (--MED and -(alpha)20.5). Oligonucleotide primers have been chosen which allow specific identification of both normal (alpha alpha) and abnormal (--) chromosomes using identical conditions in either the same or parallel PCR reactions. This strategy should be useful in the development of screening programmes to identify carriers of alpha thalassaemia (--/alpha alpha) and prenatal diagnosis of the Hb Bart's hydrops fetalis syndrome (--/--) for those populations in which this represents a major cause of perinatal death.

The gene frequencies of abnormal haemoglobins have been determined in a group of 100,000 Jamaican newborns screened over a period of 8 1/2 years. The population is predominantly of West African origin and the survey represents approximately one quarter of all island deliveries within the period of the study. The common beta globin chain abnormalities beta s and beta c occurred with gene frequencies of 0.055 and 0.019 respectively; beta thalassaemia was relatively rare. In contrast, alpha thalassaemia was quite common, occurring with a gene frequency of 0.183. In addition to these common abnormalities, the frequencies of 256 rare abnormal haemoglobins are described. This survey thus represents a complete and accurate documentation of the alpha and beta globin variants that occur in the Jamaican 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.

A prospective study of 2191 Negro infants in Jamaica showed that approximately 7% of them had detectable levels of Hb Bart's (gamma 4) in the neonatal period. The red cell indices, globin chain biosynthesis and restriction endonuclease mapping of DNA from these infants were used to determine the significance of Hb Bart's at birth. The results indicate that the genotypes alpha alpha/alpha alpha, -- alpha/alpha alpha and -- alpha/ -- alpha are associated with 0%, 0.1-2%, and greater than 2% Hb Bart's respectively. Although trace amounts of Hb Bart's may be associated with the genotype -- alpha/alpha alpha this is not always the case and therefore haemoglobin analysis in the neonatal period cannot be used to diagnose this genotype with any certainty.

We have studied the interaction of the alpha alpha alpha/alpha alpha gene arrangement with various beta globin genotypes (AA, AS, AC, SS and SC). Whereas this interaction has no detectable clinical or haematological effects in subjects with AA, SS or SC genotypes it is associated with a significantly increased level of Hb S or Hb C in heterozygotes for these variants. These findings indicate that the additional alpha globin gene in the alpha alpha alpha gene arrangement is functional.

Three identical alpha + thalassemia genes, one of which always carried the Hb J Tongariki mutation, have been observed in Vanuatuans. Despite the fact that at least two of them have arisen by different types of crossover event, the expression of all three haplotypes is identical.

A new alpha thalassemia defect has been detected in the South African population. Restriction mapping of the alpha globin gene cluster in affected individuals has established that the defect is associated with the removal of 22.8-23.7 kb of DNA, including the psi zeta 1, psi alpha 1, psi alpha 2, alpha 2 and alpha 1 globin genes. The 5' endpoint of the deletion has been localized between the zeta 2 and psi zeta 1 globin genes, and the 3' endpoint lies 4-5 kb 3' to the alpha 1 globin gene. We have called the deletion - -SA in order to distinguish it from alpha zero thalassaemia defects described in other populations.

The effects of alpha thalassaemia on sickle cell-beta zero thalassaemia have been studied by comparing haematological and clinical features in four subjects homozygous for alpha thalassaemia 2 (2-gene group), 27 heterozygotes (3-gene group), and 55 with a normal alpha globin gene complement (4-gene group). Alpha thalassaemia was associated with significantly higher haemoglobin levels and lower reticulocyte counts independent of the presence of splenomegaly. Contrary to expectation, alpha thalassaemia was associated with small but significant increases in mean cell volume and mean corpuscular haemoglobin concentration. Splenomegaly at age 5 years and episodes of acute splenic sequestration were significantly more frequent in the 4-gene group. There were no significant differences in painful crises, acute chest syndrome, or other clinical features.

Haemoglobin Constant Spring (Hb CS) is a variant with an elongated alpha-chain associated with an alpha + thalassaemia phenotype. The amount of alpha mRNA relative to beta mRNA in reticulocytes was reduced in carriers of Hb CS by an amount equivalent to the reduction observed in carriers of alpha + thalassaemia. In a patient with Hb CS-H disease there was greater alpha/beta mRNA ratio in bone marrow nuclear RNA than in the peripheral blood. Furthermore, all the alpha mRNA in the patient's peripheral blood was derived from the alpha 1 (alpha A) gene. The data suggest that alpha CS mRNA is unstable and degraded in the cytoplasm. This instability may be due to destabilization of a specific sequence in the 3' non-coding region during translation.

The possibility of using alpha/beta globin messenger RNA (mRNA) ratios to distinguish between the carrier states for different forms of alpha thalassaemia has been explored. Alpha/beta globin mRNA ratios were determined in the red cells of a series of normal individuals and in members of four Cypriot families, one Thai and one Chinese family in which at least one person has haemoglobin H disease. It was found that there was a clear distinction in the ratios between normals, alpha thalassaemia 1 carriers, alpha thalassaemia 2 carriers, and those with haemoglobin H disease. This method should be a valuable addition to haematological analysis, haemoglobin synthesis and restriction mapping of DNA for the further elucidation of the genetics of alpha thalassaemia.

A study of rheological determinants (plasma viscosity, whole-blood viscosity, and erythrocyte deformability) was made in 24 matched pairs of patients with homozygous sickle cell disease, with and without homozygous alpha-thalassaemia 2. Patients with coexisting alpha-thalassaemia showed a significant increase in erythrocyte deformability measured as filtration of washed erythrocytes through 5 micron diameter pores and also as viscosity of whole blood at high shear rate (230s-1) and standard haematocrit (0.45). This rheological advantage may explain the beneficial effect of alpha-thalassaemia 2 on haematological parameters and clinical events in homozygous sickle cell disease.

Restriction endonuclease analysis has been performed on the alpha and beta globin gene clusters of 57 Cypriots homozygous for beta thalassaemia, 30 with the transfusion dependent form of the condition (thalassaemia major) and 27 who are less severely affected (thalassaemia intermedia). There was a significant difference in the incidence of alpha thalassaemia between the two groups: 14/27 of the patients with thalassaemia intermedia also had deletion forms of alpha thalassaemia, while only 4/30 of the patients with thalassaemia major were similarly affected. Thus in Cypriot patients who are homozygous for beta thalassaemia the co-inheritance of alpha thalassaemia is an important factor in determining the clinical course.

We have studied seven Jamaican Negro families in whom the genes for alpha thalassaemia and the sickle cell mutation (betas) were independently segregated. Using a combination of techniques we identified two alpha thalassaemia phenotypes which resemble the severe (alpha thalassaemia 1) and mild (alpha thalassaemia 2) determinants previously described in Orientals. This study has enabled us to clearly correlate the phenotype of alpha thalassaemia with the genotype in this population. Furthermore, since in each family alpha thalassaemia was present in association with the gene for the sickle cell mutation we have determined the proportion of Hb S in the peripheral blood of individuals with the alpha alpha/alpha alpha, -alpha/alpha alpha and -alpha/-alpha genotype who are also heterozygous for the betas mutation. Genetic analysis in these families shows that in each case subjects with the alpha thalassaemia 1 phenotype are homozygous for the alpha thalassaemia 2 defect (-alpha/-alpha). We have found no instances of the genotype --/alpha alpha in this population which may explain the rarity of the severe alpha thalassaemia syndromes in Jamaica. Restriction mapping data in the alpha thalassaemia 2 homozygotes from this population shows that the (-alpha/) haplotype results from a deletion of one of the linked pair of alpha globin genes and that this has probably arisen by an unequal crossover between non-homologous alpha genes.

A Chinese family has been studied in which two siblings have haemoglobin Q-H disease. Using a combination of haematological and haemoglobin analysis, globin chain synthesis, analysis of alpha/beta globin messenger RNA ratios and restriction endonuclease mapping, it has been shown that each of these siblings has received one chromosome on which both alpha chain genes have been deleted and another on which there is only a single alpha chain locus which carries the alpha Q mutation. Their genotype is thus --/-alpha Q. Despite the fact that the haemoglobin Q mutation in this family is carried on a chromosome with a single alpha chain locus, heterozygous carriers for the variant have only 25% or less haemoglobin Q. Our observations indicate that the molecular basis for haemoglobin Q-alpha thalassaemia is similar to that for the common form of haemoglobin H disease in Orientals. Furthermore, they provide clear evidence that the level of an alpha chain variant in heterozygous carriers is not a reliable reflection of the number of alpha globin genes.

We have fully characterized two alpha thalassaemia mutants that occur in Southeast Asia, - -THAI and - -FIL. Each mutant is due to a deletion that removes the entire zeta-alpha-globin gene complex. Localization of the 5' breakpoints described here, allows the identification of unique fragments that are specific for each of the two mutations. This information can be used to assess the frequency of these mutants in Southeast Asia and will be of value in prenatal testing for alpha thalassaemia in this area.

Ten patients with sickle cell (SS) disease from a Jamaican family were found to have unusually high levels of haemoglobin F for this population. Each of them has inherited one sickle cell gene on a chromosome characterized by an arrangement of restriction fragment length polymorphisms (haplotype) which is very rare in the Jamaican population. Genetic analysis of the family suggests that there is a determinant linked to the beta-globin gene cluster, characterized by this haplotype, which is responsible for increased haemoglobin F production in response to anaemia. Interestingly this particular haplotype appears to be common in patients with SS disease in eastern Saudi Arabia in whom a high level of haemoglobin F is the rule rather than the exception. Hence it is possible that this haplotype (++-++) acts as a genetic marker for elevated levels of haemoglobin F in sickle cell disease.