Heterozygous missense mutation of Unc119 at glycine 22 in an ICL patient 1 (P1). (A) Western blot analysis of CD4 T-cell lysates. CD4 T cells from subjects as in Figure 1 were lysed and blotted with anti-Unc119, anti-Lck, and anti-actin Abs. Each blot represents one of 3 independent experiments. (B) Sequence of cDNA clones. RNA from PBMCs obtained from P1 was reverse transcribed. Unc119 cDNA was amplified by PCR, cloned, and sequenced. Wild-type (WT) and mutant clones were identified. The G to T transversion in codon 22 resulted in a glycine (Gly) to valine (Val) substitution. (C) Sequence of the genomic DNA PCR products. Genomic DNA was isolated from P1 PBMCs (left panel) and from the P1 buccal swab sample (right panel). The Unc119 exons were amplified and the PCR products were sequenced. The figure shows select sequence from the exon 1 PCR product. In the sequence, a single peak of G overlapped with that of T at codon 22. The sequencer read these overlapping peaks as an unidentified base K. (D) Schematic representation of the Unc119 protein structure. The position of the G22V mutation in P1 is marked with an arrow. GMP-PDE δ domain, the cyclic guanosine monophosphate phosphodiesterase δ domain