Figure 1.
Pedigree and AP4E1 mutation in P1 and P2.
A). Pedigree of the family. The segregation of the AP4E1 p.R1105X mutation is also indicated. B). Electrophoregram showing the homozygous mutation of P1 with respect to the control sequence. C). Illumina sequencing reads displayed for patient P1. D). Schematic diagram of the structure of AP-4ε, with the delimitation and numbering of the corresponding exons. The other known mutations of the AP4E1 gene and the mutation described here (in red) are also indicated.
Table 1.
Summary of clinical neurological presentations.
Table 2.
Summary of whole-exome sequencing results.
Figure 2.
mRNA and protein levels for the subunits of the AP-4 complex.
A). RT-qPCR to assess mRNA levels for the components of the AP-4 complex in EBV-B cells from P1. B). RT-PCR to assess the splicing of AP4E1 mRNA. C). Western blot: whole-cell homogenates from EBV-B cells from P1 and a healthy control were subjected to western blotting for clathrin heavy chain (CHC; loading control), AP-4ε, AP-4β or AP-4 μ. The loss of AP-4ε results in a concomitant decrease in the levels of AP-4β and AP-4 μ (specific bands are indicated by an arrow). These experiments were carried out at least twice.
Figure 3.
Immunoprecipitation and immunofluorescence of the AP-4 complex in P1.
A). EBV-B cells and SV40 fibroblasts from P1 and a healthy control were subjected to immunoprecipitation under native conditions with antibodies against AP-4ε or AP-4β, and the immunoprecipitates were then western blotted with antibodies against AP-4ε or AP-4β. Equal amounts of protein were used for immunoprecipitation from patient and control cells. A small amount of AP-4β is coassembled with AP-4ε, and the AP-4ε assembled with AP-4β in P1 appears to be slightly smaller (AP-4ε*) than the AP-4ε in control cells. B). Fibroblasts from P1 and a healthy control were double-labeled for AP-4ε and the AP-4-associated protein tepsin. Note the specific loss of AP-4ε and tepsin from the cells of P1. Bar 20 µm. These experiments were carried out at least three times.
Figure 4.
Functional study of the IFN-γ/IL-12 axis and oxidative burst in P1.
A). Whole-blood IL-12/IFN-γ pathway screening. P1 and P2 had normal responses to IFN-γ and IL-12. B). EMSA detecting GAF DNA-binding activity after IFN-γ stimulation in the EBV-B cells from two healthy controls, P1 and individuals with complete recessive IFN-γR2 deficiency, used as a negative control. Cells were stimulated for 15 minutes with the indicated dose of IFN-γ. C). Oxidative burst. The production of superoxide in the EBV-B cells from healthy controls, P1 and patients with chronic granulomatous disease, used as negative controls, was determined following stimulation with PMA. These experiments were carried out at least twice.