IMMUNOHEMATOLOGY Molecular analysis of the York antigen of the Knops blood group system Barbera Veldhuisen, Peter C. Ligthart, Gestur Vidarsson, Ingrid Roels, Claudia C. Folman, C. Ellen van der Schoot, and Masja de Haas BACKGROUND: Antigens of the Knops blood group system are present on complement component (3b/4b) receptor 1 (CR1/CD35), which is a transmembrane gly- coprotein encoded by the CR1 gene. Eight of the nine known antigens of this system are linked to polymor- phisms in Exon 29. The molecular background of one antigen, York (Yk a ), has not yet been described. STUDY DESIGN AND METHODS: We aimed to iden- tify a polymorphism associated with the absence of Yk a to enable molecular typing. Yk a -negative individuals were identified by serologic typing. Their CR1 gene was partially sequenced and compared to that of Yk a - positive individuals. Loss of Yk a antigen was investi- gated by expressing the SCR22/23 domain of both wild- type and mutated CR1 as a GPI-linked protein on HEK293 cells. RESULTS: We observed that absence of the Yk a antigen is caused by a mutation in Exon 26 of the CR1 gene. This 4223C>T mutation results in a 1408T>M change at the protein level. Ten of 117 donors (8.5%) were homozygous TT, confirming the Caucasian fre- quency of 8% Yk a -negative individuals. Serologically, these TT donors showed a Yk a -negative phenotype, while CC/CT individuals were Yk a -positive. While the Yk a antigen was present on HEK293 cells expressing wild-type constructs, cells expressing the 4223C>T variant were Yk a negative. CONCLUSION: We identified a 4223C>T sequence variation in the CR1 gene causing absence of the Yk a antigen of the Knops blood group system. With this finding, all polymorphisms of the known Knops blood group antigens have been revealed, enabling molecular testing to contribute to red blood cell alloantibody identi- fication procedures. A ntigens of the Knops blood group system are part of the component (3b/4b) receptor 1 protein (CR1) encoded by the CR1 gene on chromosome 1q32. 1,2 The Knops blood group system is the 22nd system recognized by the International Society of Blood Transfusion. 3 The first antibody forma- tion against an antigen of the Knops system was reported in 1970 after a blood transfusion of a Caucasian patient lacking the Kn a antigen. 4 To date, eight different Knops antigens are described, which arise from amino acid changes due to single-nucleotide polymorphisms (SNPs) in the CR1 gene. Polymorphisms in Exon 29 of this gene are responsible for the antithetical antigens Kn a and Kn b (KN1/KN2), 5 Sl1 and Sl2 (KN3/KN6), 6 McC a and McC b (KN4/KN7) 6 and the antigens Sl3 (KN8) 7 and KCAM (KN9) 8 (Table 1). A sixth SNP present in Exon 29 has not been associated with a specific blood group antigen. 9 Although antibody formation against the York (Yk a ) antigen (KN5) was already detected in 1965, 10 it was much later assigned to the Knops system. Yk a is a high-frequency antigen present in approximately 98% of the African and 92% of the Caucasian population. 10 The absence of Yk a has not been reported as caused by changes in Exon 29 of CR1. 11 Expression of CR1 (CD35) has been shown on red blood ABBREVIATIONS: AHG = anti-human immunoglobulin G; HTLA = high-titer, low-avidity; LHR(s) = long homologous region; MAIEA = monoclonal antibody-specific immobilization of erythrocyte antigen; SCR = short consensus repeat(s); SNP(s) = single-nucleotide polymorphism(s). From the Sanquin Research, Amsterdam and Landsteiner Labo- ratory, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands. Address reprint requests to: Barbera Veldhuisen, Depart- ment of Experimental Immunohematology, Sanquin Research, Plesmanlaan 125, 1066 CX Amsterdam, the Netherlands; e-mail: b.veldhuisen@sanquin.nl. Received for publication July 16, 2010; revision received November 9, 2010, and accepted November 9, 2010. doi: 10.1111/j.1537-2995.2010.02999.x TRANSFUSION 2011;51:1389-1396. Volume 51, July 2011 TRANSFUSION 1389