Duffy blood group phenotype-genotype correlations using high-resolution melting analysis PCR and microarray reveal complex cases including a new null FYA allele: the role for sequencing in genotyping algorithms

• Published in: Vox sanguinis 2015-10, Vol.109 (3), p.296-303
• Main Authors: Lopez, G. H., Morrison, J., Condon, J. A., Wilson, B., Martin, J. R., Liew, Y.-W., Flower, R. L., Hyland, C. A.
• Format: Article
• Language: English
• Subjects: Algorithms; Alleles; Base Sequence; Duffy blood group genotyping; Duffy Blood-Group System - genetics; Duffy null FY01N.07; Genetic Association Studies; high-resolution melting analysis; Humans; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Phase Transition; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; RBC antigens and antibodies; Receptors, Cell Surface - genetics; Sequence Analysis, DNA; serological testing
• ISSN: 0042-9007; 1423-0410
• DOI: 10.1111/vox.12273

Background and objectives Duffy blood group phenotypes can be predicted by genotyping for single nucleotide polymorphisms (SNPs) responsible for the Fya/Fyb polymorphism, for weak Fyb antigen, and for the red cell null Fy(a−b−) phenotype. This study correlates Duffy phenotype predictions with serotyping to assess the most reliable procedure for typing. Materials and methods Samples, n = 155 (135 donors and 20 patients), were genotyped by high‐resolution melt PCR and by microarray. Samples were in three serology groups: 1) Duffy patterns expected n = 79, 2) weak and equivocal Fy(b) patterns n = 29 and 3) Fy(a−b−) n = 47 (one with anti‐Fy3 antibody). Results Discrepancies were observed for five samples. For two, SNP genotyping predicted weak Fyb expression discrepant with Fy(b−) (Group 1 and 3). For three, SNP genotyping predicted Fya, discrepant with Fy(a−b−) (Group 3). DNA sequencing identified silencing mutations in these FY*A alleles. One was a novel FY*A 719delG. One, the sample with the anti‐Fy3, was homozygous for a 14‐bp deletion (FY*01N.02); a true null. Conclusion Both the high‐resolution melting analysis and SNP microarray assays were concordant and showed genotyping, as well as phenotyping, is essential to ensure 100% accuracy for Duffy blood group assignments. Sequencing is important to resolve phenotype/genotype conflicts which here identified alleles, one novel, that carry silencing mutations. The risk of alloimmunisation may be dependent on this zygosity status.