Autosomal Recessive Keratoderma-Ichthyosis-Deafness (ARKID) Syndrome Is Caused by VPS33B Mutations Affecting Rab Protein Interaction and Collagen Modification

Abstract

In this paper, we report three patients with severe palmoplantar keratoderma associated with ichthyosis and sensorineural deafness. Biallelic mutations were found in VPS33B, encoding VPS33B, a Sec1/Munc18 family protein that interacts with Rab11a and Rab25 proteins and is involved in trafficking of the collagen-modifying enzyme LH3. Two patients were homozygous for the missense variant p.Gly131Glu, whereas one patient was compound heterozygous for p.Gly131Glu and the splice site mutation c.240-1G>C, previously reported in patients with arthrogryposis renal dysfunction and cholestasis syndrome. We demonstrated the pathogenicity of variant p.Gly131Glu by assessing the interactions of the mutant VPS33B construct and its ability to traffic LH3. Compared with wild-type VPS33B, the p.Gly131Glu mutant VPS33B had reduced coimmunoprecipitation and colocalization with Rab11a and Rab25 and did not rescue LH3 trafficking. Confirming the cell-based experiments, we found deficient LH3-specific collagen lysine modifications in patients' urine and skin fibroblasts. Additionally, the epidermal ultrastructure of the p.Gly131Glu patients mirrored defects in tamoxifen-inducible VPS33B-deficient Vps33b^fl/fl-ER^T2 mice. Both patients and murine models revealed an impaired epidermal structure, ascribed to aberrant secretion of lamellar bodies, which are essential for epidermal barrier formation. Our results demonstrate that p.Gly131Glu mutant VPS33B causes an autosomal recessive keratoderma-ichthyosis-deafness syndrome.

Figure 1

Clinical features of the three individuals with ARKID syndrome. (a) Patient 1: 13-year-old boy of medium height and build, presenting with diffuse PPK, sensorineural deafness, requiring hearing aids, and generalized fine scaling. (b) Patient 2: marked palmoplantar hyperkeratosis, generalized ichthyosis, contractures of fingers, and acromicria in a 43-year-old obese woman with sensorineural hearing loss. (c) Patient 3: 49-year-old man of normal weight showing pronounced PPK with the absence of the small toes after autoamputation, generalized fine ichthyosis, perimamillar hyperkeratosis, and sensorineural deafness. ARKID, autosomal recessive keratoderma-ichthyosis-deafness; PPK, palmoplantar keratoderma.

Figure 2

VPS33B(p.Gly131Glu) does not affect the VPS33B-VIPAR interaction. (a, b) Representative mIMCD3 cells coexpressing RFP-VIPAR and (a) wt YFP-VPS33B or (b) YFP-VPS33B(p.Gly131Glu). Scale bars = 10 μm. (c) Quantification of colocalization between VPS33B constructs and VIPAR. Data are mean ± standard deviation, unpaired t-test, ns = nonsignificant. (d) Anti-HA co-IP on HEK-293 cell lysates coexpressing HA-VPS33B or HA-VPS33B(p.Gly131Glu) with Myc-VIPAR. (e) Mapping of p.Gly131Glu mutation on structural models of the VPS33B-VIPAR complex. Left: VPS33B in orange and alpha solenoid region of VIPAR (residues 140–450) in blue; the disordered N-terminus of VIPAR is omitted. VPS33B residue Gly131 is a green sphere. Right: surface potential of VPS33B, from −5 k_bTe_c⁻¹ (red) to +5 k_bTe_c⁻¹ (blue), VIPAR is dark gray. co-IP, co-immunoprecipitation; HA, hemagglutinin; HEK, human embryonic kidney; IP, immunoprecipitation; mIMCD3, murine inner medullary collecting duct 3; RFP, red fluorescent protein; WB, western blot; wt, wild type; YFP, yellow fluorescent protein.

Figure 3

VPS33B(p.Gly131Glu) cannot rescue defective LH3 delivery in VPS33B-deficient mIMCD3 cells. (a–b) Representative, (a) Control, or (b) VPS33B shRNA treated mIMCD3 cells expressing LH3-mCherry, stained for collagen IV (c–d) Representative VPS33B shRNA-treated mIMCD3 cells expressing (c) YFP-VPS33B or (d) YFP-VPS33B(p.Gly131Glu) and CFP-VIPAR, LH3-mCherry and stained for collagen IV. (e) Quantification of LH3-mCherry/collagen IV colocalization. One-way ANOVA, **P ≤ 0.01, ****P ≤ 0.0001. (f, g) LC-MS-MS analysis for relative quantification of hydroxylysines (Lys-OH), galactosyl-hydroxylysines (Lys-O-Gal), and glucosylgalactosyl-hydroxylysines (Lys-O-GalGlc) from (f) urine of patient 1 and father, patient 2 and mother, age-matched controls, and patients with ARC, (g) collagen I from control, patient 2, and ARC patient skin fibroblasts, statistics not performed as only single patient samples were analyzed. Scale bars = 10 μm. Data are mean ± standard deviation. ANOVA, analysis of variance; ARC, arthrogryposis renal dysfunction and cholestasis; CFP, cyan fluorescent protein; LC-MS-MS, liquid chromatography tandem mass spectrometry; mIMCD3, murine inner medullary collecting duct 3; wt, wild type; YFP, yellow fluorescent protein.

Figure 4

VPS33B(p.Gly131Glu) alters VPS33B interaction with Rab25 and Rab11a. (a, b) Representative mIMCD3 cells coexpressing wt YFP-VPS33B (a) or YFP-VPS33B(p.Gly131Glu) (b), RFP-VIPAR and CFP-Rab25. Scale bars = 10 μm. (c) Quantification of the colocalization between VPS33B constructs and Rab25 in transfected mIMCD3 cells. Data are mean ± standard deviation, unpaired t-test, **** P ≤ 0.0001. (d, e) Representative mIMCD3 cells coexpressing wt YFP-VPS33B (d) or YFP-VPS33B(p.Gly131Glu) (e), RFP-VIPAR and Myc-Rab11a, stained with an α-Myc antibody. Scale bars = 10 μm. (f) Quantification of the colocalization between VPS33B constructs and Rab11a in transfected mIMCD3 cells. Data are mean ± standard deviation, unpaired t-test, **** P ≤ 0.0001. (g) α-HA co-IP on lysates of HEK293 cells coexpressing HA-VPS33B or HA-VPS33B(p.Gly131Glu) with Myc-VIPAR and Rab25-GFP. (h) a-HA co-IP on lysates of HEK293 cells coexpressing HA-VPS33B or HA-VPS33B(p.Gly131Glu) with Myc-VIPAR and Rab11a-GFP. CFP, cyan fluorescent protein; co-IP, co-immunoprecipitation; HA, hemagglutinin; HEK, human embryonic kidney; GFP, green fluorescent protein; IP, immunoprecipitation; mIMCD3, murine inner medullary collecting duct 3; RFP, red fluorescent protein; wt, wild type; YFP, yellow fluorescent protein.

Figure 5

Similar epidermal defects in ARKID and Vps33b^fl/fl-ER^T2 mice. (a) Control and (b) ARKID palmar skin, H&E, scale bars = 1 mm. (c–e) ARKID skin TEM (c) LBs (*) and (d) lipid bilayers (arrows) within corneocytes, (e) inhomogeneous LB secretion (double arrows) and aberrant LBs, scale bars = 0.5 μm. (f, g) Dermoepidermal junction in (f) control and (g) ARKID abdominal skin, anchoring fibrils (arrows), scale bars = 0.5 μm. (h) wt and (i) Vps33b^fl/fl-ER^T2 dorsal epidermis, H&E, scale bars = 50 μm. (j) wt and (k) Vps33b^fl/fl-ER^T2 epidermis TEM: normal (white arrows) versus entombed bilayers (black arrows), aberrant LBs (*), scale bars = 0.2 μm. (l) aberrant LBs at the SG-SC interface (double arrows), scale bar = 2 μm. ARKID, autosomal recessive keratoderma-ichthyosis-deafness; BM, basement membrane; h, hemidesmosomes; H&E, hematoxylin and eosin; LB, lamellar body; SB, stratum basale; SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum; TEM, transmission electron microscopy; wt, wild type.