A. L. Reese

Separation of Human Hemoglobins by Deae-Cellulose Chromatography using Glycine-Kcn-Nacl Developers

This chromatographic procedure uses DEAE-cellulose as ion exchanger and glycine-KCN-NaC1 solutions as developers. Blood samples from several adults and newborn infants with alpha, beta, delta, or gamma chains variants have been analysed. The hemoglobins are eluted as compact and symmetrical zones, and the separation of many hemoglobin types is greatly improved. The procedure is relatively fast, simple, and inexpensive.

β-Thalassemia in Turkey

A review is presented of the various β-thalassemia alleles observed in nearly 191 patients with β-thalassemia major and their 182 heterozygous relatives. Determination was by gene amplification and dot-blot hybridization with synthetic probes, specific for 27 different mutations. Eighteen mutations have been observed; six of these acount for nearly 83% of all thalassemia abnormalities (Table I). A new mutation, i.e. a G ↣ C mutation at the acceptor splice site of IVS-I, was found in one teenager who was homozygous for this disease. the high consanguinity among the families was considered the main reason for the high number of patients with a homozygosity for the IVS-I-110 (G ↣ A) mutation. Combinations of different mutations were present in many patients; some were mildly affected because of the specific mutation present on one chromosome. Combinations of classical β-thalassemia and an abnormal hemoglobin mainly concerned Hb S. Hbs Knossos and Lepore were rare occurrences. A comparison of hematological da...

Molecular characterization of β‐thalassaemia in 174 Greek patients with thalassaemia major

The mutations producing β‐thalassaemia in 174 Greek patients with thalassaemia major were investigated by dot‐blot hybridization of oligonucleotide probes to genomic DNA amplified by the polymerase chain reaction procedure, by direct sequencing of amplified DNA, and by gene mapping. β‐thalassaemia in Greeks was found to be very heterogeneous at the molecular level as 17 different mutations were observed; 86.6% of the β‐thalassaemic genes, however, could be identified with five probes: IVS‐I‐110 (G→A) (42.5%), codon 39 (C→T) (17%), IVS‐I‐1 (G→A) (13.2%), IVS‐I‐6 (T°C) (7.2%) and IVS‐II‐745 (C→G) (6→9%). Several mutations which had not previously been reported in the Greek population and which occurred at an incidence of 2% or lower were observed in this study. The information obtained will facilitate the prenatal diagnosis of β‐thalassaemia in Greece.

Heterogeneity in the molecular basis of three types of hereditary persistence of fetal hemoglobin and the relative synthesis of the Gγ and Aγ types of γ chain

Restriction endonuclease analyses of DNA from one Black GγAγ-HPFH homozygote and four Black and one Indian GγAγ-HPFH heterozygotes have identified three different HPFH types which are the result of large deletions including the δ and β genes. Two of the types are comparable to those characterized previously, but the third, which is present in the Indian heterozygote, shows a distinct difference in the size of the deletion. The 5′ end point of the deletion in this type III GγAγ-HPFH extends 0.5–1.0 kb beyond the 5′ end point of one of the Black types of HPFH (type I). Each of the three types is associated with a distinct ratio between the Gγ and the Aγ chains, an observation supported by family data. The highest ratio is found in the heterozygote with the Indian type III GγAγ-HPFH, with 69.3% Gγ chains, while the averages for the other types were 50.7% Gγ (type I) and 32.3% Gγ (type II).

Types of Thalassemia Among Patients Attending a Large University Clinic in Kuala Lumpur, Malaysia

We have identified the beta-thalassemia mutations in 59 patients with thalassemia major and 47 patients with Hb E-beta-thalassemia, and the deletional and nondeletional alpha-thalassemia determinants in 23 out of 24 patients with Hb H disease. All persons were attending the Haematology Clinic at the National University of Malaysia in Kuala Lumpur (Malaysia). Most patients (76) were of Malay descent, while 52 patients were Chinese, and two came from elsewhere. The most frequently occurring beta-thalassemia alleles among the Malay patients were IVS-I-5 (G----C) and G----A at codon 26 (Hb E), while a few others were present at lower frequencies. The Chinese patients carried the mutation characteristic for Chinese [mainly codons 41/42 (-TTCT) and IVS-II-654 (C----T)]; Malay mutations were not observed among Chinese and Chinese mutations were virtually absent in the Malay patients. The large group of patients with Hb E-beta-thalassemia and different beta-thalassemia alleles offered the opportunity of comparing hematological data; information obtained for patients with Hb E-beta-thalassemia living in other countries was included in this comparison. Twenty-three patients with Hb H disease carried the Southeast Asian (SEA) alpha-thalassemia-1 deletion; 13 had the alpha CS alpha (Constant Spring) nondeletional alpha-thalassemia-2 determinant, while the deletional alpha-thalassemia-2 (-3.7 or -4.2 kb) was present in 10 subjects. The --/alpha CS alpha condition appeared to be the most severe with higher Hb H values. Both deletional and nondeletional types of alpha-thalassemia-2 were seen among Malay and Chinese patients.

β-Thalassemia in Yugoslavia

This study concerned the evaluation of β-thalassemia alleles in nearly 50 patients with β-thalassemia major and in 130 -thalassemia heterozygotes using gene amplification and dot-blot hybridization with synthetic probes. Fourteen different mutations were observed (Table I); of these, three (IVS-I-110; IVS-I-6; IVS-I-1) account for some 75% of all β-thalassemia alleles. Newly discovered variants, i.e. T ↣ C in the initiation codon and AATAAA ↣ AATGAA in the poly A site were observed in a few patients. the poly A mutation with classicall β-thalassemia alleles result in thalassemia intermedia. Hb Lepore is a rather common abnormality and combinations of this variant with β-thalassemia often result in severe disease; a search for β-thalassemia mutations among patients affected with this disease should include an analysis to detect this hemoglobin abnormality.

β-Thalassemia in Bulgaria

Analyses of DNA from 64 patients with thalassemia major using the hybridization technique of amplified DNA with radiolabeled synthetic oligonucleotide probes identified 13 different β-thalassemia mutations (Table I). the codon 39 (C ↣ T) and IVS-I-110 (G ↣ A) mutations occurred most frequently but seven additional mutations were observed which were present at frequencies of 3.9 to 10.2%. This broad spectrum of β-thalassemia alleles complicates the analyses for institutions involved in prenatal diagnosis. Promoter mutations were rare and the frequencies of two other mild mutations [IVS-I-6 (T ↣ C) and the poly A mutation] were relatively low indicating that β-thalassemia is a severe disease among Bulgarians. the high frequencies of 4.7-5.5% for the four frameshifts at codons 5, 6, 8, and 8/9 may be specific for this population.

Five haplotypes in Black β‐thalassaemia heterozygotes: three are associated with high and two with low Gγ values in fetal haemoglobin

Genotypes at seven different polymorphic restriction sites (5’to the ° gene, at the Gγ, at the Aγ, at the Ψβ, 3’to the Ψβ, at the β, and 3’to the β genes) were analysed by restriction endonuclease mapping of the DNA from 66 Black β‐thalassaemia heterozygotes from Georgia and several of their normal relatives. Five different haplotypes were observed. Three of these were associated with high Gγ values in the small amount of Hb F (0.8‐8.3%) present in the blood of these patients and two with low Gγ values. One haplotype [‐ + ‐ ++++] that occurred on two of every three β thalassaemia chromosomes was associated with high Gγ levels, and is the same as that found in some Black SS patients also having high Gγ values (Gilman & Huisman, 1984). Two others [‐ ++ ‐ + ‐ +] and [−+−−+++] were also associated with high Gγ, while two [−−−−+++] and [+−−−−++] were associated with low Gγ. Variation in haematological data, mainly MCV and MCH values, was found to be caused in part by the type of β‐thalassaemia (defined by its haplotype) and by the presence of an additional α‐thalassaemia‐2 heterozygosity or homozygosity.

An Improved Chromatographic Procedure for Quantitation of Human Fetal Hemoglobin

The DEAE-cellulose chromatographic procedure which uses glycine-KCN-NaCl solutions as Developers and a NaCl gradient to elute the hemoglobin zones was found to be useful for the quantitation of Hb F in samples containing Hb A, provided the level of Hb F exceeds 2 percent. The method has been evaluted through a study of artificial mixtures and of blood samples from patients with various disorders. The data have been compared with results obtained by four other procedures that are used for the quantitation of Hb F.

(Aγδβ)°-Thalassaemia in Blacks is due to a deletion of 34 kbp of DNA

DNA from members of 10 Black families with conditions considered to be Gγδβ)°‐thalassaemia or Gγδβ)°‐HPFH were studied by using restriction enzyme analysis. One or more affected members from each family were shown to have the same deletion of 34 kbp of DNA in the human β‐globin gene cluster. A clone spanning the deletion was isolated from the DNA of one such person and studied in detail. The deletion removed part of the Aγ and all of the Ψβ, δ and β‐globin genes and is different from the four previously identified deletions which caused a condition presently known as (Aγδβ)°‐thalassaemia.