Haploinsufficiency for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma

Abstract

Palmoplantar keratodermas (PPKs) are a group of disorders that are diagnostically and therapeutically problematic in dermatogenetics. Punctate PPKs are characterized by circumscribed hyperkeratotic lesions on the palms and soles with considerable heterogeneity. In 18 families with autosomal dominant punctate PPK, we report heterozygous loss-of-function mutations in AAGAB, encoding α- and γ-adaptin-binding protein p34, located at a previously linked locus at 15q22. α- and γ-adaptin-binding protein p34, a cytosolic protein with a Rab-like GTPase domain, was shown to bind both clathrin adaptor protein complexes, indicating a role in membrane trafficking. Ultrastructurally, lesional epidermis showed abnormalities in intracellular vesicle biology. Immunohistochemistry showed hyperproliferation within the punctate lesions. Knockdown of AAGAB in keratinocytes led to increased cell division, which was linked to greatly elevated epidermal growth factor receptor (EGFR) protein expression and tyrosine phosphorylation. We hypothesize that p34 deficiency may impair endocytic recycling of growth factor receptors such as EGFR, leading to increased signaling and cellular proliferation.

Figure 1. Clinical and histological features of punctate palmoplantar keratoderma

(a) The palms of the proband in PPKP1 Family 1 show numerous small, hard, slightly indented hyperkeratotic lesions (arrow; see inset), which typically appear around age 10 and increase in number throughout life. (b) On the soles, lesions tend to coalesce at pressure points and so the presentation can resemble a focal form of keratoderma. (c&d) In some cases, the phenotype is much more severe, as seen here in the proband from Family 15. (e) This H&E stained section shows a punch sample of skin incorporating a well defined central epidermal depression associated with hypergranulosis and a prominent layer of overlying orthokeratosis. Scale bar = 500 μm. (f) The proliferation marker Ki67 shows an increase in the numbers of keratinocytes in cell cycle within the floor of the epidermal depression (arrowheads) compared to perilesional epidermis (arrow). Scale bar = 100 μm.

Figure 2. Identification of mutations in AAGAB in PPKP1 families

Protein domain organization of the p34 protein encoded by AAGAB, showing positions of all 8 alterations identified. Further details of the mutations are listed in Table 4. Examples of sequence traces are shown in Supplementary Fig. 3.

Figure 3. AAGAB is expressed in skin and keratinocytes and its depletion leads to increased cell numbers over time

(a) QRT-PCR showed that AAGAB mRNA is expressed in skin and is present at broadly comparable levels in HeLa cells, primary epidermal keratinocytes and the epidermal keratinocyte cell line HaCaT. Conventional RT-PCR showed expression in palmoplantar epidermis comparable to abdominal skin (data not shown). (b) Western blotting shows that two independent siRNAs (designated 1239 and 2164) potently knocked down p34 expression in HaCaT cells. Non-specific control siRNA = NSC4 (inverted lacZ sequence). (c) AAGAB knockdown by either siRNA leads to an approximate 2-fold increase in HaCaT cell division at 96 hours post-transfection. Similar data were seen at early time-points (not shown). Error bars show standard deviation. Non-specific control siRNA = NSC4 (inverted lacZ sequence).

Figure 4. p34 associates with AP-1 and AP-2 in the cytosol

(a) Native immunoprecipitation (IP) of HeLa cytosolic extracts was performed with antibodies against p34 (p34-1 and p34-2), followed by Western blotting (WB) with antibodies against p34, AP-1 or AP-2. Note the association of both AP-1 and AP-2 adaptor complexes with p34. (b) HeLa cells were transiently transfected with either C-terminally (p34GFP) or N-terminally (GFPp34) GFP-tagged p34 constructs. Both constructs show a mainly cytosolic localization and this is consistent with the staining pattern (albeit faint) observed using an antibody against p34 in either transfected or untransfected cells (#). (c) HeLa lysates were subjected to fractionation into cytosol, membrane, and clathrin-coated vesicle (CCV) fractions, and then Western blotted for clathrin heavy chain (CHC), AP-1 and p34. Note that p34 is found principally in the cytosolic fraction and is virtually undetectable in membrane or CCV fractions in which AP-1 and CHC are enriched. (* = cross reacting band) (d) HeLa cells were treated with siRNA to knock down expression of p34 (kd), and western blotted for clathrin heavy chain (CHC) and p34 (* = cross reacting band). Note the efficient knockdown of p34 (>95%). (e) Localization of AP-1 and AP-2 visualized by immunofluorescence following knock down of p34 (kd). Note that the AP-2-containing membrane-associated vesicles or AP-1-trans-Golgi network appear unperturbed. Bar = 20 μm.

Figure 5. Transmission electron microscopy of lesional plantar skin reveals vesicle abnormalities within basal keratinocytes

Transmission electron microscopy of a basal epidermal keratinocyte (E) within PPKP1 lesional epidermis reveals an abnormal abundance of membrane-bound vesicles abutting the plasma membrane (arrows), close to the cutaneous basement membrane (*). D = dermis. Further electron micrographs are shown in Supplementary Fig. 7.

Figure 6. Knockdown of p34 greatly increases EGFR protein expression

(a) Knockdown of p34 with siRNA 1239 leads to greatly increased EGFR protein compared to non-specific control siRNA NSC4. Part of the blot was stained for β-actin as a loading control and to normalize EGFR quantification. Using the LI-COR Odyssey system, the observed upregulation was found to be >10-fold (data not shown). +, p34 siRNA; −, NSC4 siRNA control. (b) Knockdown of p34 with siRNA 1239 also leads to greatly increased phosphorylation of tyrosine-992 on the EGFR protein compared to non-specific control siRNA NSC4. Using the LI-COR Odyssey system, the observed increase in phosphorylation of this residue was found to be >20-fold (data not shown).