Figure 1.

Strategy for identification of the homozygous ITK mutation in a family with epidermodysplasia verruciformis (EV). A, Clinical presentation of EV manifesting with flat warts (arrows) on the hands of patient III-5. B, Histopathology of a flat wart in acral skin from patient III-3. Note epidermal thickening with hyperkeratosis, acanthosis, and hypergranulosis (hematoxylin and eosin staining, ×10 magnification); note koilocytes with large cytoplasmic halo (inset, ×40). C, Viral typing by real time quantitative polymerase chain reaction from a skin biopsy revealed the presence of human papillomavirus types 5 and 8, while Epstein-Barr virus was present in plasma (viral load, 10 000 IU/mL) of both patients III-5 and III-3 (data from III-3 is shown). D, Fluorescence-activated cell sorting of CD3⁺ T cells in patients III-3 and III-5 (upper and lower right panels, respectively) for T-cell receptor (TCR)–Vα24⁺ and TCR-Vβ11⁺. Reduced percentage of cells with these markers in patients with the ITK mutation (right panels: <0.02%), in comparison to 2 healthy controls (left panels: 0.36% and 0.42%), indicated lack of natural killer T cells. E, Pedigree of the family. Segregation analysis of the mutation is shown, with + sign representing the wild-type allele. F, Genome-wide homozygosity mapping was performed in patients III-3 and III-5, as well as in the healthy brother III-4 and the mother II-2. Regions of homozygosity (ROH) are represented by the blue vertical lines. The only ROH shared between the patients and not present in the unaffected individual was of 10.2 Mb on chromosome 5, containing the ITK locus. Locations of genes associated with EV are shown with black dotted lines. G, Whole exome sequencing and filtering of variants in patients III-3 and III-5, and the mother, II-2. Filtering criteria used to arrive at the pathogenic mutation in ITK are shown. H, Sanger sequencing confirmed that the c.362_364del mutation in ITK is homozygous in patient III-3 (lower panel); control DNA showed wild-type alleles only (upper panel). I, Structural analysis of the wild-type (I-1) and mutant (I-2) ITK protein motif in 3D structure. Interactions of Zn²⁺ ion with amino acids in the Cys₃His₁ motif are represented by orange dashed lines. Oxygen, hydrogen, nitrogen, sulphur, and carbon atoms are in red, white, blue, yellow, and cyan, respectively (stick-and-ball structures in I-1 and I-2). Backbone root-mean-square deviation (I-3), pairwise distance between Cys132–Zn²⁺, Cys133–Zn²⁺, Cys143–Zn²⁺, and His121–Zn²⁺ (I-4). Wilcoxon signed-rank test significance: *P < 2.2x10(-16) . Interaction energies of Zn²⁺ –Btk motif during the molecular dynamics trajectories in wild-type (blue line) and mutant (red line) structures (I-5). Abbreviations: EBV, Epstein-Barr virus; FITC, fluorescein isothiocyanate; HPV, human papillomavirus; ITK, interleukin 2–inducible T-cell kinase; MAF, minor allele frequency; MD, molecular dynamics; PE, phycoerythrin; RMSD, root-mean-square deviation; ROH, region of homozygosity; TCR, T-cell receptor.