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Hemoglobin Pakse: Presence on Red Blood Cell Membrane and Detection by Polymerase Chain Reaction-Single-Strand Conformational Polymorphism

Nondeletional gene mutations giving rise to α-thalassemia can be found at polymorphic frequency in Southeast Asia. Although the most common is hemoglobin Constant Spring (Hb CS), caused by a termination codon mutation (UAA →CAA, Gln) in the α2-globin gene and resulting in reduced synthesis of the elongated α-globin variant, Hb Pakse (UAA→UAU,Tyr) also has been observed at a significant prevalence.Western blot analysis of ghost membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from an individual with α-thal 1/Hb Pakse revealed the existence of a higher molecular weight globin of 18 kd consistent with an αPakse-globin chain.The presence of αPakse-globin on membranes of Hb Pakse-containing red blood cells affords an explanation for the severity of anemia observed in such patients. However, because the 2 Hb variants cannot be distinguished by current biochemical techniques, we developed a convenient single-tube polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) protocol for the simultaneous diagnosis of Hb CS and Hb Pakse by amplifying a short fragment covering the termination codon of the α2-globin gene.This PCR-SSCP method required no internal control coamplification or use of restriction enzymes and has the potential of identifying all the other possible termination codon mutations in a single reaction with only 1 pair of primers.