The mutations producing beta-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. beta-thalassaemia in Greeks was found to be very heterogeneous at the molecular level as 17 different mutations were observed: 86.6% of the beta-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 beta-thalassaemia in Greece.

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.

Restriction endonuclease analyses of DNA from one Black G gamma A gamma-HPFH homozygote and four Black and one Indian G gamma A gamma-HPFH heterozygotes have identified three different HPFH types which are the result of large deletions including the delta and beta 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 G gamma A gamma-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 G gamma and the A gamma chains, an observation supported by family data. The highest ratio is found in the heterozygote with the Indian type III G gamma A gamma-HPFH, with 69.3% G gamma chains, while the averages for the other types were 50.7% G gamma (type I) and 32.3% G gamma (type II).

The gamma chain compositions of the fetal hemoglobins of 2453 newborn babies from East Asian countries (1350 babies), from Italy, Yugoslavia, Bulgaria, and Georgia (417 Caucasian babies), and 686 black babies from Georgia were determined by high pressure liquid chromatography. Unusual results for a limited number of babies were confirmed by chemical analyses, and were evaluated further by family studies. Statistical analyses indicated high gene frequencies for the A gamma T chain in Italian (f = 0.237), Yugoslavian and Bulgarian (f = 0.238), and white Georgia babies (f = 0.224), a lower frequency in Japan (f = 0.178), and India (f = 0.173), and particularly in mainland China (f = 0.079). The A gamma T gene frequency in normal (AA) Black babies was 0.102. When a beta S or beta C mutation was also present this frequency was greatly decreased, particularly in babies with the AC condition (f = 0.036). These results suggest the near absence of the A gamma T mutation on the chromosome also carrying the beta C determinant. Most babies had the expected G gamma values which vary between 60 and 80%, but several (mainly black) babies had higher values (between 80 and 90%), while one normal black baby had a G gamma value of (nearly) 100%. This condition may be a form of A gamma +1-thalassemia and has been discussed in detail elsewhere (Blood 58:491-500, 1981). Thirty-five clinically normal (mainly Chinese, Indian, and Japanese) babies had G gamma values of about 40%. Twenty-six babies had A gamma I values of about 60%, while the remaining nine babies had A gamma T and A gamma I chains in a ratio of either 1 to 2 or 1 to 1. Two additional newborns did not produce any G gamma chains, but had only A gamma I chains or A gamma T chains. Family studies failed to indicate a specific hematological abnormality. These unusual ratios between the G gamma and A gamma (either A gamma I or A gamma T) chains have led to speculations regarding possible genetic abnormalities present in these infants.

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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.

A review is presented of the various beta-thalassemia alleles observed in nearly 191 patients with beta-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 account for nearly 83% of all thalassemia abnormalities. 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 beta-thalassemia and an abnormal hemoglobin mainly concerned Hb S. Hbs Knossos and Lepore were rare occurrences. A comparison of hematological data for adults with heterozygosities for some of the common alleles confirmed the low Hb A2 values in IVS-I-6 (T----C) heterozygotes and the high Hb F values for codon 8 (-AA), IVS-II-1 (G----A), and IVS-I-1 (G----A) heterozygotes.

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