S. L. Thein

Association of thalassaemia intermedia with a beta-globin gene haplotype

We have identified 14 Asian patients with homozygous β° thalassaemia who had a mild clinical disorder related to an augmented production of haemoglobin F. None of their parents had an elevated level of Hb F. Restriction fragment length polymorphism analysis of the β‐globin cluster of these patients and a control group of Asian thalassaemia major patients showed that 6/14 of the thalassaemia intermedia patients were homozgyous for a particular 5′β‐globin haplotype (−+−++), in contrast to 1/42 of the thalassaemia major patients. Furthermore, the —+—++β haplotype is also associated with amelioration of disease severity in β thalassaemia in an Italian population. This β haplotype is linked to a DNA sequence variation 5′ (at position — 158) to the cγ globin gene which can be detected by the presence (+) of an Xmn I restriction enzyme site. The possible role of the Xmn I‐γ polymorphism in relation to this variant HPFH is discussed. We conclude that much of the observed clinical variability of β thalassaemia can now be explained by the inheritance of β thalassaemia chromosomes with different propensities for fetal haemoglobin production.

A genetic marker for elevated levels of haemoglobin F in homozygous sickle cell disease

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 β‐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.

Molecular genetics of antithrombin deficiency

Antithrombin is the major proteinase inhibitor of thrombin and other blood coagulation proteinases. Antithrombin has two functional domains, a heparin binding site and a reactive centre (that complexes and inactivates the proteinase). Its deficiency results in an increased risk of venous thromboembolism. Appreciable progress has been made in recent years in understanding the structure and function of this protein, the genetic cause of inherited deficiency and its clinical consequence. The structure of antithrombin is now considered in terms of the models derived from X-ray crystallography, which have provided explanations for the function of its heparin interaction site and of its reactive loop. The structural organization of the antithrombin gene has been defined and numerous mutations have been identifed that are responsible for antithrombin deficiency: these may reduce the level of the protein (Type I deficiency), alter the function of the protein (Type II deficiency, altering heparin binding or reactive sites), or even have multiple or ‘pleiotropic effects’ (Type II deficiency, altering both functional domains and the level of protein).

Thalassaemia intermedia: a new molecular basis

Summary. A 5‐year‐old child heterozygous for β thalassaemia has the clinical picture of thalassaemia intermedia. Restriction endonuclease mapping shows that the child is homozygous for a triplicated α gene complex. The greater degree of globin chain imbalance resulting from two additional α chain genes is the likely mechanism for this unusually severe clinical phenotype.

The molecular basis of HPFH in a British family identified by heteroduplex formation

Summary. Single‐base substitutions in the immediate 5′‐flanking region of the fetal Gγ and Aγ globin genes have been associated with non‐deletional forms of hereditary persistence of fetal haemoglobin (HPFH). Previously, the sole promoter mutation associated with HPFH in British individuals has been the T to C substitution at position – 198 relative to the Aγ globin gene CAP site.

Identification of nine novel mutations in type I antithrombin deficiency by heteroduplex screening

Summary. We have utilized DNA heteroduplex detection as a method for screening sequences of the antithrombin (AT) gene for the presence of mutations. Affected individuals from 41 kindreds with type Ia antithrombin deficiency were investigated. Heteroduplexes were detected in 12 cases; direct sequencing of the appropriate exons revealed nine cases with novel mutations, and two with previously described mutations. In addition, a new polymorphism in the 5’untranslated region was characterized. The defects included minor insertions and deletions which lead to the removal of intact codons or premature termination, and single base substitutions leading to premature termination or amino acid substitution. In all cases, the affected individuals were heterozygous for the defect and variant AT protein was not detected. In keeping with previous reports the defects associated with type Ia AT deficiency are extremely heterogeneous, the vast majority being point mutations. This study also demonstrates the efficiency of hydrolink gel electrophoresis as a method of screening for unknown mutations by heteroduplex detection.

Antithrombin Budapest 3 An antithrombin variant with reduced heparin affinity resulting from the substitution L99F

The molecular basis and functional properties of a variant antithrombin (AT) protein. AT Budapest 3, were studied. A single base substitution was identified in codon 99, TC→ TC, altering the normal leucine to phenylalanine. The proband presented with a history of venous thrombotic disease and was found to be homozygous for the mutation. The variant protein demonstrated reduced heparin affinity and reduced antiproteinase activity in the presence of either unfractionated heparin or the AT‐binding heparin pentasaccharide, when compared to normal AT. A small change in the isoelectric point was also identified. The substituted amino acid residue of AT Budapest 3 is located near to the proposed AT heparin binding site, and it is suggested that reduced heparin affinity of the variant protein may result from substitution‐induced distortion of positive charge geometry in the binding site and/or changes in its position relative to the rest of the inhibitor molecule.

Population and genetic studies suggest a single origin for the Indian deletion β° thalassaemia

Summary. In a study of β thalassaemia in the Asian Indian immigrant populations in the U.K., 23 out of 125 β‐Thalassaemic chromosomes (18%) were of the Indian deletion β° type (600 bp deletion involving the 3’ end). The individuals with β thalassaemia had originated from various parts of India and Pakistan. However, all those individuals with deletion β° thalassaemia were from Sind and the adjacent area of Gujarat. Analysis of restriction fragment length polymorphisms in the β globin gene cluster showed that all the 23 deletion β° thalassaemia chromosomes had an indentical haplotype. These findings suggest a single origin for the Indian deletion β° thalassaemia.

Thalassaemia intermedia: interaction of the triple α-globin gene arrangement and heterozygous β-thalassaemia

Five patients with heterozygous β‐thalassaemia with an unusually severe clinical picture, low haemoglobin levels occasionally requiring blood transfusion, splenomegaly and unusually prominent basophilic stippling were found to have co‐inherited a triple α‐globin gene arrangement on one chromosome (ααα/αα). It seems probable that the expression of a single extra α‐globin gene is sufficient in some patients with heterozygous β‐thalassaemia to give rise to a clinically significant degree of dyserythropoietic anaemia.

A novel δº mutation in cis with Hb Knossos: a study of different genetic interactions in three Egyptian families

Summary. We have defined the molecular basis of normal HbA2β‐thalassaemia associated with Hb Knossos. DNA sequence analysis of the δ globin gene in cis with βKnossos showed deletion of a single A in codon 59 leading to a premature termination at codon 60. This δº/βKnossos allele has been observed in three unrelated Egyptian families and associated with a single β haplotype (+ ‐ ‐ ‐ ‐ ++). One individual who was homozygous for the δº/βKnossos allele as well as heterozygous for a non‐deletional α thalassaemia, was completely clinically asymptomatic, while others have coinherited the δº/βKnossos allele with different β and α thalassaemia determinants. A study of the different genetic interactions giving rise to a spectrum of clinical phenotypes is reported.