OR28 Advances in molecular typing of abo/rhd and ccr5Δ32 mutation

Abstract

Aim ABO/RhD blood group matching is considered an important factor in hematopoietic stem cell transplantation (HSCT) since ABO incompatibility may affect the engraftment and/or ABO expressed on recipient endothelial cells may represent targets for graft versus host disease. The CCR5 deletion (CCR5Δ32) is associated with HIV resistance and provides a therapeutic strategy for eradicating HIV infection in HIV+ patients undergoing HSCT. Currently, HLA typing of NMDP donors is performed on DNA isolated from buccal swabs. The goal of this study was to develop molecular typing methods for ABO/RhD and CCR5Δ32 for HSCT registry donors. Methods 96 reference buccal swab samples were obtained from NMDP. The SSP typing for ABO/RhD and CCR5Δ32 were performed using a multiplex PCR (One Lambda) and the sizes of the amplicons were detected on 3730xl DNA Analyzer and interpreted with Gene Mapper 5. ABO typing was performed by NGS using in-house primers on Illumina MiSeq system and the results were analyzed using Galaxy (UCLA). Results For ABO blood group typing, both SSP and NGS typing methods yielded 100% concordance with reference in all major blood groups (A = 29.2%, B = 20.8%, AB = 20.8%, O = 29.2% respectively) and differentiated A1 and A2 subtypes. NGS typing identified a total of 4 A alleles, 1 B allele, and 10 O alleles with 7 novel mutations. Two mutations occurred in B101 (A930G) and O02 (G1105A). We discerned 7 O alleles without the nt261G deletion which included 5 O03 and 2 O49 alleles. O03 and O49 alleles have G802A compared to A101 which presumably inactivates the enzyme by altering its sugar-binging sites. In addition, the G1096A novel mutation was detected in all 5 O03 alleles. 100% concordance was achieved for RhD typing with 23 (24%) RhD- homozygous samples detected. In this cohort, no CCR5Δ32 double deletion was detected, but 13 CCR5Δ32/CCR5 heterozygous were found (13.5%). The CCR5Δ32 typing was confirmed by in-house SSP with 100% concordance. Conclusion We demonstrate the ability to perform accurate molecular tying of blood group ABO/RhD and CCR5Δ32. Successful molecular typing of registry donors for ABO/RhD and CCR5Δ32 may reduce donor search time and time to transplant. In addition, the ability to identify novel/rare alleles can fill in gaps in knowledge of the existing blood antigen registry and further improve the HSCT outcome.