Molecular Characterisation of the Diego and Wright Blood Group Systems of Blood Donors in Malta: A PCR-RFLP Study

Abstract

The Diego and Wright blood group systems are clinically important because antibodies directed against their antigens may cause haemolytic transfusion reactions (HTRs) and haemolytic disease of the foetus and newborn (HDFN). Despite their relevance in transfusion medicine, limited information is available regarding the prevalence of Diego and Wright blood group alleles in Malta. Molecular genotyping provides a reliable alternative to conventional serological methods, particularly for rare antigens for which  suitable antisera may be unavailable or difficult to obtain. This study aimed to establish a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the molecular detection of the Diego and Wright blood group alleles in blood donors in Malta and to determine the distribution of these alleles within the donor population. A total of 403 randomly selected blood donor samples were collected between February and July 2024. Genomic DNA was extracted and amplified using polymerase chain reaction (PCR). Restriction fragment length polymorphism analysis was performed using NaeI and Hpy188I restriction enzymes to differentiate the DI*A/DI*B and DI*02.03/DI*02.04 alleles, respectively. A subset of samples underwent Sanger sequencing to validate the genotyping results. Donor demographic information relating to parental ancestry was also collected to assess population diversity. The donor cohort was composed predominantly of individuals with Maltese ancestry, although 11.7% of participants reported at least one parent of foreign origin. PCR-RFLP analysis demonstrated the complete absence of the DI*A allele encoding the Dia antigen and the DI*02.03 allele encoding the Wra antigen. All successfully genotyped samples (403) carried the DI*B and DI*02.04 alleles, corresponding to the high-frequency Dib and Wrb antigens, respectively. Sequencing results showed complete concordance with PCR-RFLP findings and confirmed the presence of the DI*B and DI*02.04 alleles in all samples analysed. The study provides the first molecular data describing the distribution of Diego and Wright blood group alleles in blood donors in Malta. The exclusive detection of DI*B and DI*02.04 alleles is consistent with findings reported in other predominantly Caucasian and Middle Eastern populations. PCR-RFLP proved to be a reliable and cost-effective genotyping method and may serve as a useful tool for rare blood group screening and future implementation of molecular transfusion medicine strategies in Malta.