Search

During a recent survey of hemoglobin (Hb) abnormalities among the population of Northern Sardinia, we observed an electrophoretically slow-moving variant in two brothers. The abnormal Hb could be detected by cellulose acetate electrophoresis at pH 8.9 (it moved like Hb F), by citrate agar electrophoresis at pH 6.1 (it moved like Hb C), and by isoelectricfocusing (IEF) where it moved between Hb F and Hb G-Philadelphia (Fig. 1) (see Refs. 1 and 2 for methodologies). Quantitation by densitometric scanning of the IEF gels gave the following percentages for the two brothers: 24.5 and 23.6% Hb X, and 0.6 and 0.6% Hb X2. In one person Hb X was quantitated by DEAE Sephadex chromatography (5); its value was 21.4%. The aX chain also readily separated from the normal α chain by polyacrylamide gel electrophoresis (PAGE) (3) (Fig. 2) and by reverse phase high performance liquid chromatography (HPLC) (4); the values for the two brothers were 21.9 and 21.7%, respectively (Fig. 3). Hematological data showed a distinct erythrocytosis with slight microcytosis in both brothers (Table I).

Fetal hemoglobin analysis and globin gene mapping have identified one type of βo-thalassemia and four different γ globin gene arrangements among newborn babies from the northern part of Sardinia. The βo-thalassemia with a nonsense mutation at codon 39 was found on two chromosomes, each with a distinct pattern of polymorphic restriction sites; one had the AγT (Aγ75 Ile → Thr) mutation, while the second did not. Four closely related haplotypes were identified for chromosomes with the AγT mutation. The γ-thalassemia heterozygosity with the —GAγ— hybrid gene fell into two categories. One apparently originated through crossing-over between mismatched chromosomes characterized by the most common haplotype, while the other had polymorphisms resembling those of a less frequently occurring chromosome. Chromosomes with the —Gγ—AGγ—Aγ— triplication had polymorphic sites to be expected for this condition, being complimentary to the —GAγ— thalassemias. Of the two additional γ globin gene variations the —Gγ—Gγ— arrangement was associated with the chromosome with the most commonly occurring haplotype, while the chromosome with the —Aγ—Aγ— arrangement had a haplotype characteristic for that with the AγT mutation, which identified an —Aγ—AγT— arrangement. The incidental discovery of a silent β-chain mutant, Hb Hamilton, with the Val → Ile substitution at position β11, in five newborns was also reported.

DNA amplification combined with hybridization with 32P-labeled synthetic oligonucleotide probes has been used to identify base substitutions in the 5' promoter region of the A gamma globin gene in members of eleven families from China, Sardinia, Canada, and the United States who had a heterozygosity for the A gamma-beta+-hereditary persistence of fetal hemoglobin (HPFH), and in members of six black families with a possible G gamma-beta+-HPFH heterozygosity. All three known A gamma types were observed, ie, the British type (-198, T----C), the Chinese type (-196, C----T), and the Green type (-117, G----A); the latter has been found in a black family. Of the six families with G gamma-beta+-HPFH, three had C----G at -202 and none T----C -175. Conditions for hybridization of amplified DNA with the specific probes are provided and the usefulness of the technique is discussed. The increase in numbers of A gamma(G gamma)-beta+-HPFH heterozygotes with specific base substitutions greatly enhances the probability of a direct correlation between these substitutions and the increase in the production of a specific gamma chain.

DNA amplification combined with hybridization with 32P-labeled synthetic oligonucleotide probes has been used to identify base substitutions in the 5’ promoter region of the Aγ globin gene in members of eleven families from China, Sardinia, Canada, and the United States who had a heterozygosity for the Aγ-β+-hereditary persistence of fetal hemoglobin (HPFH), and in members of six black families with a possible Gγ-β+-HPFH heterozygosity. All three known Aγ types were observed, ie, the British type (— 198, T → C), the Chinese type (-196, C → T), and the Greek type ( —117, G → A); the latter has been found in a black family. Of the six families with Gγ-β+-HPFH, three had C → G at —202 and none T → C — 175. Conditions for hybridization of amplified DNA with the specific probes are provided and the usefulness of the technique is discussed. The increase in numbers of Aγ(Gγ)-β+-HPFH heterozygotes with specific base substitutions greatly enhances the probability of a direct correlation between these substitutions and the increase in the production of a specific γ chain.