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

ABSTRACT

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.