Molecular analysis of the perforin gene, perforin expression, and NK activity. (A) Molecular analysis of the perforin gene in the 3 patients who had the biallelic mutations. Study of the perforin gene was performed as reported by Stepp et al⁴  sequencing exon 2 and 3. The Blast program¹⁶  was used to compare the obtained sequences to the reported gene structure gene index ([GI] 190339). Red identifies thymidine (T); black identifies guanine (G); blue identifies cytosine (C); green identifies adenine (A); corresponding initials are shown above the curves. (B) Perforin (PRF) expression in CD8⁺ and CD56⁺ cells was detected by FACS analysis after membrane staining with CD8 FITC and CD56 FITC monoclonal antibodies (mAbs; Becton Dickinson [BD]) and intracytoplasmic staining with perforin phycoerythrin (PE) (BD). Patients 1 and 2 (PT.1 and PT.2) were tested. (C) In vitro NK activity tested against the K562 cell line as target and expressed as percent specific ⁵¹Cr release at the different effector-target ratios of 100:1, 30:1, and 10:1 of Pt 1 and Pt 2. Results are compared with that of a healthy control.