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 Vps33bfl/fl-ERT2 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.
BACKGROUND: Integrins are heterodimeric (alpha/beta) membrane proteins that play fundamental roles in many biological processes, for example, cell adhesion and spreading, which are important for platelet function and hemostasis. The molecular mechanism that regulates integrin activation is not comp
letely understood. METHODS AND RESULTS: Here, we show that VPS33B, a member of the Sec1/Munc18 family, binds directly to the integrin beta subunit. Overexpression of VPS33B in Chinese hamster ovary cells potentiated alphaIIbbeta3 outside-in signaling but not inside-out signaling. Platelets, from megakaryocyte- and platelet-specific VPS33B conditional knockout mice, had normal morphology, yet their spreading on fibrinogen was impaired and they failed to support clot retraction. Platelet aggregation and ATP secretion in response to low-dose agonists were reduced in the VPS33B knockout mice. alphaIIbbeta3-mediated endocytosis of fibrinogen was also defective. Tail bleeding times and times to occlusion in an FeCl3-induced thrombosis model were prolonged in the VPS33B knockout mice. Furthermore, VPS33B acted upstream of the RhoA-ROCK-MLC and Rac1-dependent pathways that lead to clot retraction and cell spreading, respectively. CONCLUSIONS: Our work demonstrates that vesicular trafficking complexes, containing VPS33B, are a novel class of modifiers of integrin function. Our data also provide insights into the molecular mechanism and treatment of arthrogryposis, renal dysfunction, and cholestasis syndrome.
ARC syndrome (OMIM 208085) is an autosomal recessive multisystem disorder characterized by neurogenic arthrogryposis multiplex congenita, renal tubular dysfunction and neonatal cholestasis with bile duct hypoplasia and low gamma glutamyl transpeptidase (gGT) activity. Platelet dysfunction is common.
Affected infants do not thrive and usually die in the first year of life. To elucidate the molecular basis of ARC, we mapped the disease to a 7-cM interval on 15q26.1 and then identified germline mutations in the gene VPS33B in 14 kindreds with ARC. VPS33B encodes a homolog of the class C yeast vacuolar protein sorting gene, Vps33, that contains a Sec1-like domain important in the regulation of vesicle-to-target SNARE complex formation and subsequent membrane fusion.
The typical phenotype of arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome involves three cardinal symptoms as the name describes, harboring biallelic mutations on VPS33B or VIPAS39. Except for ARC syndrome, low gamma-glutamyltransferase (GGT) ch
olestasis often implies hereditary hepatopathy of different severity; however, some remain undiagnosed. Several monogenic defects typically with multiorgan manifestations may only present liver dysfunction at times, such as DGUOK defect and AGL defect. Previously, four VPS33B mutated cases were reported without arthrogryposis, or with less severe symptoms and longer lifespan, indicating the possibility of incomplete ARC phenotype of isolated hepatopathy. So we retrospectively reviewed all patients with confirmed VPS33B/VIPARS39 defect in our center and identified three presenting isolated low-GGT cholestasis with intractable pruritus. Distinguished from others with typical ARC phenotype, these patients did not suffer the other two typical characteristics, survived much longer, and shared a novel missense VPS33B variation c.1726T>C, p.Cys576Arg, causing declined protein expression and abolished interaction with VIPAS39 in-vitro. Serum bile acid profiles of our VPS33B/VIPAS39 mutated patients revealed similar changes to primary defect of bile salt export pump, among which those with isolated cholestasis phenotype had a higher level of total secondary bile acids than that with typical ARC phenotype, indicating the partial residual function of VPS33B.
Bem D, etal., Blood. 2015 Jul 9;126(2):133-43. doi: 10.1182/blood-2014-12-614677. Epub 2015 May 6.
Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is caused by deficiencies in the trafficking proteins VPS33B or VIPAR, and is associated with a bleeding diathesis and a marked reduction in platelet alpha-granules. We generated a tamoxifen-indu
cible mouse model of VPS33B deficiency, Vps33b(fl/fl)-ER(T2), and studied the platelet phenotype and alpha-granule biogenesis. Ultrastructural analysis of Vps33b(fl/fl)-ER(T2) platelets identified a marked reduction in alpha-granule count and the presence of small granule-like structures in agreement with the platelet phenotype observed in ARC patients. A reduction of approximately 65% to 75% was observed in the alpha-granule proteins von Willebrand factor and P-selectin. Although platelet aggregation responses were not affected, a defect in delta-granule secretion was observed. Under arteriolar shear conditions, Vps33b(fl/fl)-ER(T2) platelets were unable to form stable aggregates, and tail-bleeding measurement revealed a bleeding diathesis. Analysis of bone marrow-derived megakaryocytes (MKs) by conventional and immuno-electron microscopy from Vps33b(fl/fl)-ER(T2) mice revealed a reduction in mature type-II multivesicular bodies (MVB II) and an accumulation of large vacuoles. Proteins that are normally stored in alpha-granules were underrepresented in MVB II and proplatelet extensions. These results demonstrate that abnormal protein trafficking and impairment in MVB maturation in MKs underlie the alpha-granule deficiency in Vps33b(fl/fl)-ER(T2) mouse and ARC patients.
