Figure 3.

Genetic analysis of DOCK8. (a) T cell blast cDNA chromatograms show wildtype sequence in a healthy control, double peaks in patient II.2 and altered sequence in patient II.3. Both patients’ gDNA is homozygous for alteration c.4626 + 76 A > G; vertical black lines: 3′ junction of exon 36; black letters: wildtype; red letters: altered sequence. (b) Schematic model of affected region in DOCK8 gDNA and transcripts showing exon extension (dotted line) due to the novel splice site (*) introduced at c.4626 + 76 A > G; filled boxes: exons; horizontal line: intronic region. (c) Quantification of wildtype and altered transcripts in T cell blasts by ddPCR indicating percentages of wildtype (wt) of total DOCK8 transcripts in patient II.2, patient II.3 and healthy controls (HC). (d) ddPCR analysis of healthy controls (homozygous wt) and healthy carriers of a c.3120 + 1 G > T DOCK8 alteration resulting in exon 25 skipping (heterozygous). (e) Sashimi plot of RNA sequencing data based on GTEx samples^21,22 showing exon 32 skipping as a rare event; read counts accumulated over all samples. (f) Schematic model of wildtype and mutated minigene vectors. Sequence tags (PT1/PT2) flanked the minigene sequence to differentiate minigene transcripts from endogenous DOCK8 transcripts; filled boxes: exons; dotted line: exon extension; horizontal line: intronic regions; *: novel splice site. (g) The altered or physiologic transcription products of the minigene vectors were differentiated by size. Agarose gel with canonical splice site usage (378 nucleotide transcript) in cDNA of control PBMCs transfected with wildtype (Mini wt) and usage of the novel splice site (453 nucleotide transcript) in cDNA of PBMCs transfected with the mutated minigene vector (Mini mut); GFP- and mock-transfected as negative controls.