Analysis of beta-thalassemia mutations and beta-locus control region hypersensitive sites 2, 3 and 4 in southern Thailand.

beta-Thalassemia mutations in 221 chromosomes of unrelated southern Thai patients were analyzed. Using dot blot hybridization of PCR amplified DNA with 15 allele specific oligonucleotide probes for beta-thalassemia mutations 196/221 (89%) of the alleles were characterized. Ten mutations were identified, of which six [codon 41/42 (TTCTTT-TT), IVS1 nt5(G-C), codon 19 (AAC-AGC), codon 17 (AAG-TAG), IVS1 nt1(G-T), -28 TATA (A-G)], accounted for 85%. Among the 25 uncharacterized alleles, 15 were analyzed by automated fluorescent DNA sequencing of the whole beta-globin gene with normal results in 7 alleles. Four mutations, previously described were detected in 8 alleles. They were a G-A at IVS1 nt1 in one heterozygote, a G-T at IVS1 nt1 in one heterozygote, codon 15 (TGG-TAG) in two heterozygotes and poly A(AATAAA-AATAGA) in two homozygotes. The polyadenylation mutations, previously demonstrated in the Malaysian population have been first detected in Thailand. It is remarkable that the IVS1 nt1 (G-A) mutation, previously reported in the Mediterranean population has been found only in the south of Thailand. This mutation was probably imported from Portugal. In former times the Portuguese had settled in Phuket in southern Thailand. In order to find a causative mutation in the rest of 7 true unknowns we performed direct DNA sequencing of the core fragments of the beta-Locus Control Region Hypersensitive Sites (LCR HS) 2,3 and 4 in these 7 samples. DNA sequencing of HS2 and HS3 fragments showed normal results. The heterozygote A/G was present in the palindromic sequence of the LCR HS4 (TGGGGACCCCA) in 6 beta-thalassemia samples. The same heterozygote A/G was found in 5/12 normal subjects. The allele frequency of A (0.79) is obviously higher than that of G (0.21). This could be due to the stability of the palindromic structure. When an A is in the middle of the palindromic sequence, the hairpin structure is formed. In contrast the hairpin structure disappears when a G is in the middle of the palindromic sequence. This structure is not further symmetric and may not be so stable as the hairpin structure. beta-Thalassemia mutations in southern Thailand are very heterogeneous and their distribution is different from other parts of the country.

Alpha-thalassemia incidence in southern Thailand by restriction endonuclease analysis of globin DNA from placental blood at Songklanagarind Hospital.

The incidence of alpha-thalassemia has been studied previously based on the levels of Hb Barts' in cord blood. This method is an inadequate indicator of alpha-thalassemia. Thus in this study we use DNA analysis to get more accurate data. Hb Barts' was detected in placental blood samples from 15.5% of 375 infants born at Songklanagarind Hospital. The white blood cell DNA of 300 samples was studied for alpha-globin gene deletions by hybridization of DNA fragments digested by the restriction endonuclease Eco RI with specific 32P-labled zeta-globin gene probe. The incidence of alpha-thal 2 and alpha-thal 1 traits were 12.0% and 4.3%, with the gene frequencies 0.0650 and 0.0217 for -alpha/and --/, respectively. The incidence of HB CS trait was 5.8%, with the gene frequency of 0.0292 for alpha cs alpha/. We also found that the incidence of the triplicated zeta and triplicated alpha were 14.7 and 1.0%, with the gene frequencies of 0.0733 and 0.0050 for zeta zeta zeta/and alpha alpha alpha/, respectively. The DNA lesion of alpha-thalassemia in the south is similar to the study of Tanphaichitr et al (1988) in central Thailand. Knowledge of alpha-globin gene deletion would be useful for prenatal diagnosis of Bart's hydrops to prevent toxemia of pregnancy in the south of Thailand.

The problem of thalassemia in Thailand.

About one per cent of the Thai population are affected with thalassemic diseases. In each year there are almost 50,000 pregnancies at risk of having an affected fetus, one fourth of which result in thalassemic newborns. Both alpha- and beta -thalassemia, including hemoglobins E and Constant Spring, are common in Thailand. Their distribution varies from region to region and among different ethnic groups. About 30-40% of the population are carriers of at least one of the abnormal genes. Thalassemias and hemoglobinopathies are common and heterogeneous in Thailand. They combine to give more than 60 thalassemic syndromes with varying clinical severity. Abnormalities can be detected in every organ system. Studies in detail into each clinical problem will lead to better management. Hematological and molecular studies on different types of thalassemia in Thailand have made it possible to give prenatal diagnosis service to those pregnancies at risk of having a thalassemic child. Sporadic services have been given in three centers. Systematic prevention and control program is being planned by the cooperation of both the public and private sectors.

Molecular heterogeneity of beta-thalassemia in Thailand.

beta-Globin genes in 294 chromosomes of beta-thalassemia homozygotes and patients of beta-thalassemia/HbE in the northeast, the middle and the south of Thailand were analyzed by the PCR related techniques: dot blot hybridization, direct restriction assay, direct cloning and direct sequencing of the amplified DNA fragments. Twelve different mutations were detected at various frequencies. They are an A-G at-28, codon 19 (AAC-AGC), a G-T at IVS-1 nt1,a G-C at IVS-1 nt5, a C-T at IVS-2 nt654, a G addition in codons 8/9, a C deletion in codon 41, a 4 bp deletion in codons 41/42, an A addition in codons 71/72, an AAG-TAG in codon 17, a CAG-TAG in codon 26, a TAC-TAA in codon 35 and a 8 bp deletion in codons 123-125. We also developed allele specific-polymerase chain reaction to facilitate non-radioactive detection of the mutation. Origins and spread of mutations are speculated based on the results of determination of haplotypes and frameworks that are linked to the thalassemia alleles.