Genotyping of the common haptoglobin Hp 1/2 polymorphism based on PCR

Abstract

Background: A genetically defined molecular heterogeneity of haptoglobin, characterized by the major phenotypic forms Hp 1-1, Hp 2-1, and Hp 2-2, has been associated with distinct clinical manifestations. To enable the use of DNA samples for the study of this polymorphism, we established a haptoglobin genotyping method based on PCR. Methods: Taking advantage of the selectivity of PCR, we amplified DNA segments specifically representing haptoglobin alleles Hp 1 and Hp 2 from genomic DNA. The products were analyzed by agarose gel electrophoresis. Haptoglobin phenotyping of plasma samples was performed by polyacrylamide gel electrophoresis and peroxidase staining. Results: Exploiting the known size difference between Hp 1 and Hp 2, we amplified allele-specific DNA molecules with one pair of oligonucleotide primers. As an alternative, we used separate primer pairs to generate amplification products indicative of alleles Hp 1 and Hp 2. Because of the primer design, genotype determination was not compromised by sequence variations specifying haptoglobin allele subtypes S and F. For the same reason, the sequence similarity between the haptoglobin gene and the haptoglobin-related gene did not interfere with the accuracy of genotyping. Analysis with restriction enzymes demonstrated the authenticity of the allele-specific DNA products. Haptoglobin DNA genotyping and protein phenotyping, performed in parallel, yielded fully corresponding results. In a group of 249 individuals, the haptoglobin genotype distribution was as follows: 14.5% Hp 1-1, 48.2% Hp 2-1, and 37.3% Hp 2-2. Conclusion: The new method can be used for genotyping of a common haptoglobin polymorphism. © 2002 American Association for Clinical Chemistry.