Determining the genotype frequency of the Diego blood group system in Malta

Abstract

This study investigated the prevalence of key Diego (Di) blood group antigens (Dia , Dib , Wra , and Wrb ) in Maltese blood donors using a molecular genotyping approach, with the aim of improving rare blood group detection and enhancing transfusion compatibility. A polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) was developed to genotype four alleles: DI*A (Dia ), DI*B (Dib ), DI02.03* (Wra ), and DI02.04* (Wrb ). High-quality genomic DNA was extracted from donor blood samples, and allele-specific primers were used to amplify target regions of the solute carrier family 4 member 1 SLC4A1 gene. Selected samples were validated through direct DNA sequencing to confirm assay specificity. Additionally, a standardised questionnaire was administered to record donor parental ancestry. The PCR-RFLP method yielded consistent and reproducible results. All individuals tested negative for the rare DI*A and DI02.03* alleles, indicating an absence of the Dia and Wra antigens. The common DI*B and DI02.04* alleles were detected in all samples, suggesting a Di(a-b+) and Wr(a-b+) phenotype across the cohort. Ancestry data showed that the majority of participants reported full Maltese lineage, and allele frequencies were consistent with those expected in European populations. In conclusion, this study provides the first molecular characterisation of the Di Blood Group System in the Maltese population. Although no rare alleles were identified, the developed PCR-RFLP platform proved reliable for Di genotyping and is suitable for integration into routine blood donor screening. These findings establish a reference point for future studies and support the implementation of DNA-based methods to enhance transfusion safety in increasingly diverse populations.