High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies.

Glycophorins are the most abundant sialoglycoproteins on the surface of human erythrocyte membranes. Genetic variation in glycophorin region of human chromosome 4 (containing , , and genes) is of interest because the gene products serve as receptors for pathogens of major public health interest, including , , Influenza virus, El Tor Hemolysin, and . A large structural rearrangement and hybrid glycophorin variant, known as , which was identified in East African populations, has been linked with a 40% reduction in risk for severe malaria. Apart from , other large structural variants exist, with the most common being deletion of the whole gene and its surrounding region, resulting in multiple different deletion forms. In West Africa particularly, these deletions are estimated to account for between 5 and 15% of the variation in different populations, mostly attributed to the forms known as DEL1 and DEL2. Due to the lack of specific variant assays, little is known of the distribution of these variants. Here, we report a modification of a previous DEL1 assay and the development of a novel DEL2 assay as high-throughput PCR-RFLP assays, as well as the identification of the crossover/breakpoint for DEL2. Using 393 samples from three study sites in Ghana as well as samples from HapMap and 1000 G projects for validation, we show that our assays are sensitive and reliable for genotyping DEL1 and DEL2. To the best of our knowledge, this is the first report of such high-throughput genotyping assays by PCR-RFLP for identifying specific deletion types in populations. These assays will enable better identification of GYPB deletions for large genetic association studies and functional experiments to understand the role of this gene cluster region in susceptibility to malaria and other diseases.