(P-IG-2) N Antigen Discrepancy Between Serologic and DNA Testing Explained by the Hybrid GYP(B-A-B) Allele Encoding the GP.Mur Phenotype

The MNS blood group system is known for its many variant alleles arising from duplication, deletion and conversion events in the glycophorin (GYP) genes. The M and N antigens are encoded by changes in GYPA exon 2: c.59C >T (p.Ser20Leu), 71G >A and 72T >G (p.Gly24Glu). The M antigen resides solely on GPA whereas N is expressed on both GPA (GPA^N) and on GPB (GPB^N, or usually denoted ‘N’ [MNS30]). Commercial anti-N reagents are formulated to detect N on GPA and not ‘N’. We investigated a sample (S1) that initially typed 3+ N+, yet N- by DNA, and two frozen RBC samples (S2,S3) from GYP*Mur heterozygotes with a N+ serologic type.

Study

Design/Methods:

Serology was performed on 3 samples (S1-S3) by standard tube method using commercial anti-N (Immucor Gamma-clone, Bio-Rad Seraclone, Quotient ALBAclone) and anti-M (Immcuor Gama-clone). Genomic DNA was isolated from WBCs. Samples were analyzed on PreciseType HEA (Immucor). Long-range amplification of GYPA exons 2-7 and GYPB exons 2-6 followed by sequencing of exon 2 and (pseudo)exons 2-4, respectively, was done on S1 DNA. Frozen stored RBCs from S1 were retested and RBCs from S2-S3 were typed for N.

Results/Findings:

On initial testing fresh RBCs from S1 typed M+(3+) and N+(3+^S) with Immucor reagents. By HEA, S1-S3 were c.59C/C predicting M+N-. Fresh RBCs from S1 were typed with 2 additional anti-N and reacted 1+^S with Quotient and 1+ with Bio-Rad. For S1, GYPA sequencing showed c.59C/C, c.71G/G and c.72T/T, indicative of GYPA*M/M and consistent with HEA. GYPB sequencing showed c.200C/G, c.209C/A, c.220A/G, c.223A/G, c.227A/C, and c.229+1T/G, consistent with GYP*Mur, as well as c.143C, indicative of GYPB*s. Frozen RBCs from S1 retyped 2+ with Immucor, 1+ with Quotient and were nonreactive with Bio-Rad anti-N. RBCs from S2-S3 typed 2+-3+ with Immucor, 3+ with Quotient and were nonreactive with Bio-Rad anti-N.

Conclusions: We report three samples that typed N+ by serology and by DNA had no GYPB*N allele but rather a hybrid GYP(B-A-B ) hybrid, GYP*Mur. The variable 1+-3+ reactivity with anti-N is likely due to cross reactivity with the elevated ‘N’ on GP.Mur. Indeed some anti-N package inserts allude to this possibility, but reactivity strength is described as only weak. Here we observed robust 3+ reactivity with Immucor for all three samples and for 2 of 3 with Quotient anti-N. GP.Mur while rare in most populations, can be as high as 10% in some Asian populations, suggesting this discrepancy may not be uncommon and emphasizes the usefulness of DNA testing to obtain an accurate N antigen type. 

Importance of research: This highlights the value of both serological and DNA based testing to aid in resolving antigen typing result discordances.