Molecular characterization of LAT mutation. (A) Genomic DNA sequence around the c.268_269delGG mutation site (arrows) of a patient (top), a parent (middle), and an unrelated healthy control (ctrl; bottom). (B) Schematic view of wild-type LAT showing the extracellular region (ER), the transmembrane domain (TM), and the intracellular region (IR) with the four tyrosine residues phosphorylated downstream of TCR signaling. Below is the mutated form of the protein. The presumptive position of the frame shift starting at 89 aa and the presumed truncation after 100 aa is shown in red. (C) Relative mRNA expression of LAT with hypoxanthine phosphoribosyltransferase as the housekeeping gene is shown in CD4 CD45R0 T cells of patient 2 and three controls normalized to Jurkat cells. (D) FACS plot for LAT expression gated on CD4 CD45R0 T cells and graph of the mean fluorescence intensity (MFI) of LAT in CD4 and CD8 T cells in patient 2, the heterozygous sister, and three healthy controls. (E) Anti-FLAG staining and ZsGreen1 expression are shown in J.CaM2.5 cells expressing the recombinant wild-type (LAT^wt) or the mutated LAT protein (LAT^mut). (F) Immunodetection of FLAG-tagged LAT^wt or LAT^mut in J.CaM2.5 cells. The immunoblot is representative of three independent experiments.