title: Detection of Hb H disease genotypes common in northern Thailand by quantitative real-time polymerase chain reaction and high resolution melting analyses.
abstract: 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.
authors: Department of Biology, Faculty of Science, Naresuan University , Phitsanulok , Thailand.
authors: Teerapat
authors: T
authors: Seeratanachot
authors: Torpong
authors: T
authors: Sanguansermsri
authors: Dawan
authors: D
authors: Shimbhu
mesh_terms: D000328:Adult; D003062:Codon; D004252:DNA Mutational Analysis; D004909:Erythrocyte Indices; D005091:Exons; D005838:Genotype; D006447:Hemoglobin H / Q000235:genetics*; D006801:Humans; D008297:Male; D010641:Phenotype; D060888:Real-Time Polymerase Chain Reaction; D017085:alpha-Thalassemia / Q000175:diagnosis / Q000235:genetics; D055544:beta-Globins / Q000235:genetics
chemical_list: D003062:Codon; D055544:beta-Globins; D006447:Hemoglobin H