Mutations in the PIGG gene are responsible for the Emm-negative blood group. (A-B) Sanger sequencing of PIGG mutations found in Emm-negative proband 1 and proband 2, respectively. (C) Schematic representation of the deletion in the PIGG gene and Sanger sequencing of Emm-negative proband 3. (D) Cell surface expression of the Emm antigen and CD59 in control RBCs (Ctrl RBCs) and Emm-negative RBCs (Emm- RBCs). The gray profile corresponds to RBCs incubated with only the secondary antibody. (E) Cell surface expression of the Emm antigen and CD59 in K562 WT and K562 PIGG KO cells. The gray profile corresponds to K562 cells incubated with only the secondary antibody. (F) Surface level of Emm antigen in K562 PIGG KO cells stably transfected with WT PIGG cDNA or mutant PIGG cDNA (c.640C>T; p.H214Y). The gray profile corresponds to K562 cells incubated with only the secondary antibody. (G) Immunofluorescence analysis of Emm antigen shows the plasma membrane localization of this antigen. Nonpermeabilized WT and PIGG KO K562 cells were stained by using an anti-Emm (green) antibody and 4′,6-diamidino-2-phenylindole (blue).