Multiplex allele-specific PCR assay for differential diagnosis of Hb S, Hb D-Punjab and Hb Tak

Abstract

Background: Apart from hemoglobin (Hb) E, Hb D-Punjab [β121(GH4)Glu–Gln] and Hb Tak [β147Term-Thr] are the two most common β-chain variants among the Asian population. These two Hb variants have similar alkaline electrophoretic mobilities and HPLC profiles as those of the Hb S [β6(A3)Glu–Val]. Differential diagnosis of these clinically relevant hemoglobinopathies is therefore problematic. Direct detection of the β-globin gene mutations would be another diagnostic alternative. Methods: A simultaneous DNA diagnosis of the three Hb variants was developed based on the multiplex allele-specific polymerase chain reaction (PCR) approach. The method was validated on 10 carriers of Hb D-Punjab, 5 carriers of Hb Tak, 2 carriers of Hb S and 50 normal individuals of Thai origin. Results: The three abnormal Hbs could be correctly diagnosed with the simultaneous PCR approach, and a complete concordance with results using other established methods was obtained. Conclusions: The multiplex allele-specific PCR approach developed should prove useful in complementing routine Hb analysis for differential diagnosis of these three common Hb variants and should facilitate a program of hemoglobinopathy screening in the region.