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BACKGROUND: The Colton blood group system currently comprises three antigens, Coa, Cob, and Co3. The latter is only absent in the extremely rare individuals of the Colton “null” phenotype, usually referred to as Co(a–b–), which lack the water channel AQP1 that carries the Colton antigens. The discovery of a Co(a–b–) individual with no AQP1 deficiency suggested another molecular basis for the Co(a–b–) phenotype.

STUDY DESIGN AND METHODS: Red blood cells were analyzed by stopped-flow light scattering and Western blotting and typed by hemagglutination and flow cytometry. Genotyping by sequencing and polymerase chain reaction–restriction fragment length polymorphism was applied. An expression system for Colton antigens was developed in mammalian cells.

RESULTS: Although Co(a–b–), the proband expressed fully functional AQP1 and had developed a novel Colton alloantibody. Sequencing of AQP1 revealed a homozygous nucleotide change (140A>G) encoding the single-amino-acid substitution Q47R. A second case was identified due to the presence of this novel Colton alloantibody. By generating an expression system for Colton antigens in K-562 cells, the Q47R substitution was shown to inhibit the expression of both Coa and Cob antigens. Other naturally occurring single-amino-acid substitutions, that is, A45T, P38L, and N192K, were also studied in this Colton antigen expression system.

CONCLUSIONS: The Co(a–b–) phenotype can be generated by a functional AQP1 allele, that is, AQP1 140G encoding AQP1 (Q47R) and allowing the development of a novel Colton alloantibody. This study also shows that the Cob antigen can be produced by at least two different substitutions at Amino Acid Position 45, that is, A45V and A45T.