Stig Berglund

Thin-layer isoelectric focusing for haemoglobin screening and its application to haemoglobin Malmö.

Abstract A thin-layer technique for isoelectric focusing of proteins in polyacrylamide gel was developed and applied to various haemoglobins. Small differences in charge not detectable by conventional electrophoretic techniques were revealed. Thus Hb Malmo ( α 2 β 2 97 His → Gln ) could be separated from Hb-A, and Hb-F I from Hb-F II . The method may preferably replace conventional electrophoretic techniques in clinical work.

A novel mutation in the β-globin gene causing β-thalassaemia in a Swedish family

To the Editor: P-Thalassaemia is very rarely found in the indigenous population of Scandinavia. Scarce cases due to mutations commonly found in the Mediterranean area are seen, and a few novel mutations have been demonstrated (1,2). Experiences from Japan and Great Britain show that a rather high proportion of the indigenous thalassaemic cases in these temperate countries are classified as thalassaemia intermedia or dominant thalassaemia (3), i.e. the carriers present findings not only of classical pthalassaemia minor but also suffer from varying degrees of haemolysis. The underlying mutations in these cases are usually localized to exon 3 of the P-globin gene (4, 5), thus interfering with the Cterminal structure of the P-globin peptide chain. The more severe phenotype seen with these mutations is explained by the finding that variant P-globin chains are in fact synthesized in several of these cases. These variant P-globin chains retain their haem-binding capacity and the haem-globin complex will be relatively resistant to proteolytic degradation but prone to form intracellular aggregates and thereby lead to dyserythropoietic anaemia. In this report we describe a family where several members turned out to have a novel thalassaemic mutation in exon 2 of the p-globin gene. The index case was a 74-yr-old man with anaemia, who at admission to hospital presented signs of both iron deficiency and haemolysis (Table 1). Because of the pronounced microcytosis the possibility of coinciding thalassaemia was considered. An increased HbA, level was found, although the sample was obtained 6 d after transfusion of 3 units of red blood cells. Isoelectric focusing revealed no haemoglobin variant. Testing of haemoglobin stability in isopropanol-containing buffer gave a normal result. The increased HbA, level thus supported the diagnosis of P-thalassaemia minor. By this time it was also realized that some of the proband’s relatives had been previously investigated because of microcytic anaemia. The alnon-a-globin chain synthesis ratio had been studied in vitro in a sample from his sister and found to be 1.9, i.e. indicating heterozygous p-thalassaemia. The proband’s sister, daughter and grandson all demonstrated typical thalassaemic features with increased HbA, levels but normal HbF. Except for the proband none of the family members had any signs of haemolysis (Table 1). p-Globin cluster haplotyping was performed by ampllfying the regions surrounding 6 different polymorphic restriction enzyme sites (6). The thalassaemic condition was linked to an allele of the haplotype I or V (7). Nucleotide sequencing of double-stranded amplified material obtained from the proband revealed heterozygosity for a deletion (-T) in codon 54 of the second exon of the P-globin gene (Fig. 1). The same mutation was also found in the other thalassaemic family members. This mutation leads to an out of frame reading, introducing an inphase termination at codon 59. If ever translated, the resulting P-globin chain would contain only 58 (instead of normally 146) amino acid residues, and participation in the formation of normal alp-globin dimers would not be possible. However, there is also evidence in the literature (8,9) that mutations in this part of the P-globin gene impedes the stability of the mRNA by a hitherto unknown mechanism. Reduced mRNA survival will explain a typical Po-thalassaemia phenotype but not a dominant thalassaemia. As suggested from the family history the haemolysis found in the proband must then have a cause separate from his thalassaemic mutation. Further investigation of the proband revealed that he suffered from a malignant lymphoma with pronounced splenomegaly as well as a prostatic cancer invading the wall of the urinary bladder. He died 1 month after admission to hospital and at autopsy the spleen, weighing more than 1 kg, was filled with malignant lymphomatous tissue. Phagocytosis of erythroblasts in the abundant histiocytic cells could well explain the haemolysis occurring in this thalassaemic patient.

Hb Trollhättan [β20(B2)Val& RR; Glu] — a new haemoglobin variant with increased oxygen affinity causing erythrocytosis

Abstract: Erythrocytosis is sometimes caused by the presence of haemoglobin (Hb) variants with increased oxygen affinity. Here we describe a β‐globin variant found by isoelectric focusing (IEF) of Hb from a 23‐year‐old Swedish male with moderate erythrocytosis. Amplification of DNA corresponding to the β‐globin gene and subsequent nucleotide sequencing revealed heterozygosity for a GTG& RR; GAG mutation at codon 20, thus suggesting a Val& RR; Glu substitution, which was confirmed at the protein level. This mutation occurs at the same position as Hb Olympia [β20(B2)Val& RR; Met], another variant associated with erythrocytosis due to increased oxygen affinity. The novel variant, Hb Trollhättan [β20(B2)Val& RR; Glu], which was also associated with increased oxygen affinity, was shown to be present in three generations of the patients family.

Two different mutations in codon 97 of the β-globin gene cause Hb Malmö in Sweden

An abnormal hemoglobin with increased oxygen affinity, Hb Malmö [α2β297(FG4)His→Gln], was found to cause erythrocytosis in two apparently unrelated Swedish families. Direct nucleotide sequencing of amplified DNA demonstrated a CAC→CAA substitution in one family and a CAC→CAG substitution in the other. Both mutations resulted in a His→Gln substitution in codon 97. This finding prompted us to examine the possible point mutations underlying the different hemoglobin variants reported in the literature.