(P-IG-51) Two Novel Variants Identified Which Result in the Bombay Phenotype

Background/Case Studies: Investigations were performed on two samples requiring antibody identification. Sample A was referred to the immunohematology reference lab for a pregnant female presenting with preterm labor. The patient had a record of a positive antibody screen at the referring facility but no antibody identification was performed. Sample B was from a female blood donor with no history of pregnancy or transfusion. Anti-H was identified in both samples.

Study

Design/Methods: Antibody identifications were performed by tube method using N-HANCE (Immucor, Norcross, GA). Anti-H, anti-Le^a and anti-Le^b were used (Immucor, Norcross, GA) to determine the respective phenotypes. A select cell panel of H-negative red blood cells (RBCs) was tested by routine tube method to confirm the antibody specificity. Adsorption/elution studies were performed on Sample B using pooled polyclonal anti-A from healthy group O blood donors. Genomic DNA was extracted using the QIAcube (QIAGEN, Hilden Germany). An extended red cell genotype was obtained by next generation sequencing (NGS) on the MiniSeq (Illumina, San Diego, CA) using the HemoSelect version 3 assay (HAPLOGNX, San Diego, CA). Sanger sequencing for FUT1 and FUT2 was performed on both samples at New York Blood Center.

Results/Findings: Both samples were non-reactive with H lectin and anti-H was confirmed using H-negative RBCs from frozen droplets. Sample A’s initial FUT1 genotype prediction was indeterminate. A novel single nucleotide variant (SNV) of 760G >T was identified in both FUT1 alleles. Sanger sequencing confirmed the detected SNV and determined that 760G >T introduces a premature stop codon. Adsorption/elution studies on Sample B demonstrated weak expression of the A antigen. NGS of Sample B detected homozygous expression of a novel SNV 496G >T in FUT1. The FUT2 gene was reported as FUT2*01N.02 in both samples. These results confirmed the Le(a+b-) phenotypes observed in both samples and confirmed the Bombay phenotype.

Conclusions: The 760G >T mutation in Sample A disrupts the expression of the H antigen leading to the Bombay phenotype. Due to the A_el phenotype detected by adsorption/elution technique in Sample B, the novel SNV 496G >T in FUT1 appears to encode a weak H antigen. Both samples were obtained from sources with anti-H present in their plasma.