Associations among genotype, clinical phenotype, and intracellular localization of trafficking proteins in ARC syndrome

Abstract

Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in vacuolar protein sorting 33 homologue B (VPS33B) and VPS33B interacting protein, apical-basolateral polarity regulator (VIPAR). Cardinal features of ARC include congenital joint contractures, renal tubular dysfunction, cholestasis, severe failure to thrive, ichthyosis, and a defect in platelet alpha-granule biogenesis. Most patients with ARC do not survive past the first year of life. We report two patients presenting with a mild ARC phenotype, now 5.5 and 3.5 years old. Both patients were compound heterozygotes with the novel VPS33B donor splice-site mutation c.1225+5G>C in common. Immunoblotting and complementary DNA analysis suggest expression of a shorter VPS33B transcript, and cell-based assays show that c.1225+5G>C VPS33B mutant retains some ability to interact with VIPAR (and thus partial wild-type function). This study provides the first evidence of genotype-phenotype correlation in ARC and suggests that VPS33B c.1225+5G>C mutation predicts a mild ARC phenotype. We have established an interactive online database for ARC (https://grenada.lumc.nl/LOVD2/ARC) comprising all known variants in VPS33B and VIPAR. Also included in the database are 15 novel pathogenic variants in VPS33B and five in VIPAR.

Figure 1

Patients with attenuated ARC syndrome. A–D: Patient AB. Aged (A) 3 years and (B) 5 years. Hyperkeratosis and lichenification of palm of right hand (C) and sole of left foot (D). E–I: Patient CD. Facies aged 3 (E); in plaster after corrective hip surgery (F). Hyperkeratosis, dorsum of right foot (G). Radiographs of right foot showing vertical talus (H) and of pelvis showing hip dislocation (I).

Figure 2

ARC–LOVD database content analysis. VPS33B Gene map of distribution of “pathogenic” and “probably pathogenic” variants in VPS33B. The boxes represent the exons (not to scale). All variants are described at the DNA level. Variants below the genogram represent intronic mutations, affecting splice sites. Variants above the genogram represent mutations in exons. Purple pointers indicate substitutions; → indicates a nonsense mutation, —• indicates a missense mutation. Orange pointers indicate deletions/duplications/indels; → indicates a deletion, —• indicates a duplication, and —♦ indicates an indel. VIPAR Gene map of distribution of “pathogenic” and “probably pathogenic” variants in VIPAR. The boxes represent the exons (not to scale). All variants are described at the DNA level. Variants above the genogram represent mutations in the exons. Purple pointers indicate substitutions; → indicates a nonsense mutation, —• indicates a missense mutation. The orange pointer → indicates a deletion.

Figure 3

VPS33B and VIPAR interaction. Confocal fluorescence photomicrographs of HEK293 cells cotransfected with wild-type and mutant constructs of YFP-tagged VPS33B and Myc-tagged VIPAR. Wild-type YFP-VPS33B and Myc-VIPAR colocalized (A). However, constructs modeled on severe phenotype mutant proteins (B) Myc-VIPAR(L213P) and (C) YFP-VPS33B(L30P) resulted in a disruption of VPS33B–VIPAR interaction. D: Transfection for YFP-VPS33B(c.1225G>C), modeling an attenuated phenotype, resulted in partial colocalization. Myc-VIPAR was immunostained with mouse monoclonal antibody anti-myc (Sigma) at a 1:400 concentration and anti-mouse ALEXA-568 conjugate secondary antibody (Invitrogen) at a concentration of 1:400. Nuclei are stained with TO-PRO-3. Scale bars, 10 μm.

Figure 4

Ultrastructual localizations of VPS33B, VPS33B(c.1225G>C), VIPAR, and VPS18 constructs. Transmission electron micrographs of ultrathin cryosections of HEK293 cells. A, B: Cells cotransfected with YFP-tagged VPS33B and Myc-tagged VIPAR were immunogold stained with anti-GFP/YFP (15 nanometer gold) and anti-Myc (10 nanometer gold) (A), or anti-GFP/YFP (15 nanometer gold) and anti-TfR (10 nanometer gold) (B). Colocalization was observed on endosome (E)-associated tubular–vesicular membranes typical of recycling endosomes, which was confirmed by the presence of TfR (arrows in B). (C) Cells co-overexpressing YFP-VPS33B(c.1225G>C) and Myc-VIPAR were immunogold labeled for anti-GFP/YFP (15 nanometer gold) and Myc (10 nanometer gold). The two proteins colocalized in cytosolic aggregates with partial staining of VPS33B(c.1225G>C) on nearby vesicles (arrows). D: Cells co-overexpressing YFP-tagged VIPAR (labeled with anti-GFP; 10 nanometer gold) and Myc-tagged VPS18 (labeled with anti-Myc, 15 nanometer gold) showed colocalization in cytosolic aggregates. E, endosome; N, nucleus; PM, plasma membrane. Scale bars, 200 nm.

Figure 5

VPS33B and VIPAR interaction with VPS18. Confocal fluorescence photomicrographs of HEK293 cells cotransfected with (A) YFP-VPS33B and Myc-VPS18 found these proteins do not colocalize. However, (B) mCherry–VIPAR and Myc-VPS18 do colocalize in clusters in the absence of YFP-VPS33B. If all three proteins were cotransfected, then YFP-VPS33B and mCherry-VIPAR formed clusters and Myc-VPS18 assumed independent cytoplasmic distribution (C). Myc-VPS18 was immunostained with mouse monoclonal antibody anti-myc (Sigma) and anti-mouse ALEXA-568 conjugate secondary antibody (Invitrogen), both at a concentration of 1:400. Scale bar, 10 μm.