PMC8085392_01
Identification of compound heterozygous DOCK8 variants in the index patient. (A) Pedigree of the family. Solid symbols indicate affected patient; half solid symbols, heterozygous persons; circles, female family members; square, male family members. (B) Electropherograms showing the novel c.T824A mutation in DOCK8 patient's epithelial cells and T-cell blasts. (C) Reversion of the deletion in patient's T cells blasts detected by multiplex ligation-dependent probe amplification. PBMC, peripheral blood mononuclear cells; PT, patient.
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The index patient was a 8-year-old female patient born from non-consanguineous parents ( Figure 1A ). NGS revealed a heterozygous NM_203447:c.T824A variant in exon 7 of the DOCK8 gene, resulting in a premature stop codon (p.L275*), which was confirmed by Sanger sequencing ( Figure 1B ). Single nucleotide polymorphism (SNP) array and MLPA revealed the maternally inherited deletion of exons 1 to 3 ( Figure 1C ). Sanger sequencing analysis performed on epithelial cells as well as on peripheral T-cells confirmed the presence of the heterozygous status for the T824A variant in all the lineages, excluding the possibility of somatic reversion for this variant, while a somatic reversion of the deletion was detected by MLPA analysis in patient's T-cells blasts ( Figures 1B, C ).
Sclerosing cholangitis in the index patient. (A) Upper abdomen magnetic resonance showing diffuse and marked enlargement of intra- and extrahepatic bile ducts. (B) Liver biopsy showing one dilated duct with "onion-skin" type of periductal fibrosis.
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Marked dilation of intrahepatic bile ducts (more pronounced at the right system) and dilation of the common bile duct (up to 7 mm and without any mass forming lesion in the lumen) were then confirmed by means of abdominal ultrasound and magnetic resonance imaging ( Figure 2A ). Figure 2B ). Fecal calprotectin was mildly increased (185 mug/mg; normal up to 50 mug/mg).
Functional characterization of the DOCK8 variants. (A) Evaluation of DOCK8 levels in IL2-PHA expanded T-cell of the index patient, family members and two healthy controls (HC1 and HC2) by means of flow cytometry (upper panel; one representative experiment of 2 independent experiments) and western blot (lower panel; one representative experiment of 2 independent experiments). (B) Flow cytometric analysis of DOCK8 expression in peripheral T-cells and monocytes of the patient and a healthy control, showing hypomorphic DOCK8 expression in CD3+CD4+ and CD3+CD8+. (representative experiment) (C) Reduction of STAT5 phosphorylation in patient's peripheral CD4+ and CD8+ T-cells at baseline and following stimulation with IL2 or CD3/CD28 monoclonal antibodies. (one representative experiment performed in duplicates) (D) Representative images of Actin (Red), LFA-1 (green) and DAPI (Blue) staining on unstimulated or CD3/CD28 stimulated CD8+ cells from the index patient and a healthy donor (representative experiment). Scale bar: 20um. In the right panel, box plots show the quantification of actin signal in analyzed samples. (**p < 0.01; ***p < 0.001). HC, healthy control; PT, patient; Stim, stimulated; Unstim, unstimulated. NS, not significant.
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T-cells, CD3+CD4+ and CD3+CD8+ while DOCK8 expression was absent in monocytes. These results collectively suggested hypomorphic residual function in T-cells ( Figures 3A, B ). T-cells (both CD3+CD4+ and CD3+CD8+) was reduced either after anti-CD3/CD28 or IL-2 stimulation ( Figure 3C ). To examine the role of the novel variants in the IS, CD8+ were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies to mimic the interaction between CD8+ cells and dendritic cells. Patient's cells exhibited a significantly reduced actin polymerization in response to stimuli, whose distribution pattern was similar to the one displayed by LFA-1 ( Figure 3D ).