(P-IG-13) Breakpoint Regions of an RHD-CE(4-9)-D Allele Expressing a C Antigen and a Rare JK Allele in a Pacific Islander Individual

Background/Case Studies: Incongruences between serologic and molecular results for blood groups are mostly caused by technical or clerical errors while performing serology but can also be attributed to rare or novel nucleotide variations.
Expression of CcEe antigens from an altered RHD gene and D antigens from an altered RHCE gene have previously been observed. Among >700 RHD alleles, 3 alleles have been shown to express CcEe antigens and, among 67 hybrid alleles of the RHD gene, at least 2 alleles have evolved to include RHCE exons 4-9, one of which likely expresses a C antigen. However, no breakpoint region had been described for any of the RHD-CE(4-9)-D hybrid alleles.
Several studies have noted indigenous populations of Pacific island nations to harbor a high prevalence of rare blood group alleles. We investigated a self-identified Pacific Islander who presented with an incongruence between serologic and molecular results for his C and Jkb antigens at our routine blood group testing for donors.

Study

Design/Methods: Blood samples were obtained from the index case with written informed consent. Another 8 individuals, whose blood samples were collected at our service between July 2011 and April 2023, presented with genotype-phenotype discrepancies of the Kidd blood group system. In a quality improvement project, we compared standard serology and molecular tests, including commercial kits and in-house sequencing for SLC14A1 gene.

Results/Findings: We identified the 2 breakpoint regions of an RHD-CE(4-9)-D hybrid allele in the index case, associated with a C antigen expression despite normal RHCE*ce alleles. Nucleotide sequencing of his SLC14A1 gene documented the JK*02N.01 null allele causing the Jkb-negative phenotype. In the other 8 samples, 5 previously known SLC14A1 nucleotide substitutions were identified. The JK*02N.17 null allele was unexpectedly determined to be Jkb-positive.

Conclusions: Hybrid alleles that are seemingly identical based on their mRNA need to be distinguished in clinical blood transfusion practice, because the precise location of the breakpoint region affects the expression and stability of mRNA transcripts which may still result in any D phenotype, such as RhD-negative, DEL, weak D or RhD-positive. Commercial red cell genotyping kits still have limitations in correctly predicting the antigens expressed by hybrid RH and rare JK alleles. A quality improvement project prompted us to establish a standard operating procedure for SLC14A1 genotyping, allowing to routinely resolve discrepancies between serology and red cell genotyping kits in the Kidd blood group system. We corroborated the molecular structure of the, erroneously labeled non-functional, JK*02N.17 allele expressing a Jkb-positive phenotype.

Importance of research: Our study highlights the importance of identifying the precise location of breakpoint regions to distinguish RHD alleles with identical coding sequences, which can still express distinct Rh phenotypes. We corroborated the molecular structure of a JK allele, previously labeled non-functional, which in fact expresses a Jkb-positive phenotype.