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We used quantitative real-time polymerase chain reaction (qPCR) and high resolution melting (HRM) analyses for the detection of the common α-thalassemia (α-thal) genotypes in 40 northern Thai Hb H (β4) patients. The α(0)-thal [- -(SEA) (Southeast Asian) deletion] was detected by a multiplex gap real-time PCR. To determine the α(+)-thal, three primer pairs were designed. The A-primer pair was used to amplify the 3' terminal DNA sequences of the HBA2 gene, and the B-primer pair amplified the 5' flanking region of the HBA1 gene. The C-primer pair amplified the 3' terminal DNA sequences of the HBA1 gene and was used as an internal control. The -α(4.2) (leftward) and -α(3.7) (rightward) deletions were determined by monitoring the absence of PCR product(s). The Hb H patients who had a negative PCR result for the A-primer pair but positive for the B- and C-primer pairs carried the -α(4.2) deletion, while the -α(3.7) deletion carriers were negative for the A- and B-primer pairs. In the case of Hb H with Hb Constant Spring (Hb CS, α142, Term→Gln; HBA2: c.427T>C), all primer pairs were positive, HRM analysis of the PCR product of the A-primer pair was introduced to analyze the Hb CS gene. It can distinguish clearly between normal and Hb CS genotypes. The applied methods for the determination of the α(0)- and α(+)-thal genotypes revealed the results to be as accurate as conventional gap-PCR and the direct DNA sequencing methods but resulted in a much simpler and faster procedure.

We developed two sets of a multiplex amplification refractory mutation system (M-ARMS) assay to identify specific beta-thalassemia (beta-thal) mutations that are common in Thailand. The first one was for the detection of mutants with codon 17 (A>T), IV S-I-1 (G >T)), codons 41/42 (-TCT T) and codons 71/72 (+A), while the second one was for the -87 (C>A), -28 (A>G) and IVS-II-654 (C>T). Application of the proposed assay to 282 persons with beta-thal trait revealed a positive result in 276 cases (97.8%). There were 258 cases (91.5%) positive for the set 1 M-ARMS assay and 18 cases (6.4%) were positive for set 2. Six cases (2.2%) were negative for both sets 1 and 2, and were further characterized by DNA sequencing. The mutations detected by the set 1 M-ARMS assay were 113 cases (40.1%) of codons 41/42, 95 (33.7%) of codon 17, 41 (14.5%) of IVS-I-1 and nine cases (3.2%) of codons 71/72, while by set 2 there were 12 cases (4.2%) of -28, four cases(1.4%) of -87 and two cases (0.7%) of IVS-II-654. Mutations undetectable by M-ARMS assay were two cases of codons 27/28 (+C), one case of codon 35 (C>A), one of codon 43 (G>T), one of -31 (A>G) and one of IVS-I-5 (C>G). The M-ARMS assay proved to be a valuable tool for the analysis of beta-thal mutations. The method is robust, accurate, simple, speedy and cost-effective. The application of this assay will facilitate genetic counseling and prenatal diagnosis for severe thalassemia in high-risk pregnancies.