Real‐time quantitative PCR analysis for α‐thalassemia‐1 of Southeast Asian type deletion in Taiwan

Abstract

Since homozygosity of the alpha-thalassemia-1 of Southeast Asian (SEA) type deletion results in hydrops fetalis, a novel protocol based on the real-time quantitating polymerase chain reaction (PCR) technique has been developed to quantify the intact and aberrant alpha-globin genes in adults. The ratio of the normal/SEA-bearing alpha-globin genes was expressed in cycle threshold (C(T)) values. Theoretically, a relative ratio of one to one was anticipated in individuals carrying the SEA type deletion. Twenty-five heterozygous and 20 normal cases were analyzed retrospectively with this protocol. Data showed that the CT values for the intact alpha-globin gene allele and the allele bearing the SEA type deletion in carriers were 28.74+/-1.49 and 26.46+/-2.05, respectively. Therefore, the ratio of normal/SEA type deletion-bearing alpha-globin genes in the carriers was 1.09+/-0.043. No ambiguous results were observed from other less common genotypes associated with alpha-thalassemia, such as the Philippine type deletion. Based on the results, we concluded that this protocol could provide a rapid method to mass screen carriers with alpha-thalassemia-1 of SEA type deletion in this region.

Molecular diagnosis of inherited disorders: lessons from hemoglobinopathies

“…viennalab.com), the BeTha Gene TM 1 Kit for Mediterranean b-thalassemia mutations [Ugozzoli et al, 1998], the BeTha TM Gene 2 Kit for Asian b-thalassemia mutations, and the Alpha Gene 1 Kit for the most common HBA2/HBA1 gene mutations (all in microtiter plate formats; Biorad Laboratories, Hercules, CA; www.biorad.com) and the b-hemoglobinopathy mutation assay (six b-globin variants and 42 b-thalassemia mutations; Roche Molecular Diagnostics, Pleasanton, CA; http://us.diagnostics.roche.com) [Jarvis et al, 2004], which has yet to be offered commercially. Finally, real-time PCR has recently emerged for rapid genotyping in the HBAC [Sun et al, 2001] and HBBC [Moreno et al, 2002, Vrettou et al, 2003] without the need for post-PCR sample manipulation. The method is based on the use of fluorescently labeled hybridization probes that are specific for each mutation.…”

Diagnosis of α+-Thalassemias by Determining the Ratio of the Two α-Globin Gene Copies by Oligonucleotide Hybridization and Melting Curve Analysis

“…Quantitative PCR has been proposed to avoid these problems (9 ). Because of limited sensitivity, however, real-time PCR approaches have been successful only for the diagnosis of ␣ 0 -thalassemias (9 -11 ).…”

“…MCA does not require the analysis of a single-copy gene for reference because HBA1 and HBA2 serve as reference to each other. Because MCA does not use allele-specific PCR, it will not be affected by small amounts of contaminating DNA (7)(8)(9).…”

“…In addition, gapPCRs require the use of deletion-type-specific primers, which limits their sensitivities to distinct variants. Moreover, as for any PCR-based, allele-specific method, they carry the risk that alleles may be amplified from minimal contaminants, such as maternal DNA in prenatal diagnostics or fetal DNA circulating in maternal blood (7)(8)(9).…”

“…We modified the method so that it became faster and less expensive and then used it to identify 3 polymorphisms related to homocysteine and B Clinical Chemistry 51, No. 9,2005 …”