Alpha-thalassemia of Southeast Asian deletion (-- SEA/) is very common in Southeast Asia. Homozygosity of this genotype is the major cause of Hb Bart's hydrops fetalis in Taiwan. With polymerase chain reaction using three oligonucleotide primers bridging the common deletion breakpoint, a DNA fragment of 194 basepairs (bp) was amplified in chromosomes with the -- SEA determinant and a DNA fragment of 287 bp was amplified in chromosomes without this deletion. In our pilot study including 8 normal subjects, 20 obligate carriers, and 11 homozygotes of the deletion, all the genotypes were determined and then confirmed by Southern blotting and DNA hybridization with phi zeta globin gene probe. For prenatal diagnosis, 55 at-risk pregnancies were collected. Chorionic villus sampling was done in 51 cases and early amniocentesis was done in 4 cases. Fourteen cases (25.5%) were diagnosed as normal, 25 (45.5%) as heterozygotes, and 16 (29%) as homozygotes of -- SEA. All of the diagnoses were also confirmed as aforementioned. With polymerase chain reaction, the determination of the -- SEA deletion is straightforward and is much quicker and easier than with conventional Southern blotting and DNA hybridization. In areas with a high prevalence of -- SEA deletion, this method provides a rapid tool for carrier detection and prenatal diagnosis.

Polymerase chain reaction-based techniques were used to study the molecular defects of 480 unrelated beta-thalassemia heterozygotes in Taiwan. Analysis of artificially created restriction sites and gap-polymerase chain reaction were performed to detect four common mutations, i.e. IVS-II-654 (C-->T), codons 41/42 (-TCTT), codon 17 (A-->T), -28 (A-->G), and a deletional form of delta beta-thalassemia in the Chinese population. In cases with negative or ambiguous results with the aforementioned methods, direct DNA cycle sequencing using either S35-dATP or a fluorescent dye terminator, was carried out to determine the defects. A total of 14 different mutations have been found in this series. The IVS-II-654 mutation was the most common (39.6%), followed by the codons 41/42 mutation (37.9%). The four common genotypes accounted for 92.3% of defects. Two new mutations were detected: codon 31 (-C) and codons 40/41 (+T). Both defects resulted in a frameshift and a premature terminator, the former at codon 60, the latter at codon 43. Although we have studied our cases extensively, the molecular defects in seven alleles are still unknown.

Thailand deletion of alpha-Thalassemia (thal) 1 involves the zeta2-, phi zeta1-, alpha2-, alpha1-, and theta1-globin genes. In Southeast Asians and Taiwanese, this mutation is the second most common long-segment deletion of two alpha-globin genes, after the Southeast Asian deletion. To define the Thailand deletion breakpoints, we used polymerase chain reaction (PCR) to amplify the normal-sequence DNA fragments across the breakpoints. The amplified products were sequenced directly or after cloning into pGem-3Z or pCR2.1 vectors. Comparison of the normal and mutant sequences revealed that the 5' breakpoint lies between nucleotides 1,269 and 1,290 upstream of the initiator codon adenine of the zeta2-globin gene, and the 3' breakpoint lies between nucleotides 29,387 and 29,408 downstream of it. A total of 30,677 nucleotides were deleted. Both breakpoints mentioned above lie within the Alu repetitive sequences and an extensive sequence homology is present around the two breakpoints. These findings suggest that homologous recombination is the mechanism by which the deletion occurs. Based on our data, we used three oligonucleotide primers to amplify the regions across the deletion and its corresponding normal sequence. The feasibility of PCR diagnosis was confirmed in 20 carriers with this deletion.

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