Anna Straatman-Iwanowska

Mutations in VIPAR cause an arthrogryposis, renal dysfunction and cholestasis syndrome phenotype with defects in epithelial polarization

Arthrogryposis, renal dysfunction and cholestasis syndrome (ARC) is a multisystem disorder associated with abnormalities in polarized liver and kidney cells. Mutations in VPS33B account for most cases of ARC. We identified mutations in VIPAR (also called C14ORF133) in individuals with ARC without VPS33B defects. We show that VIPAR forms a functional complex with VPS33B that interacts with RAB11A. Knockdown of vipar in zebrafish resulted in biliary excretion and E-cadherin defects similar to those in individuals with ARC. Vipar- and Vps33b-deficient mouse inner medullary collecting duct (mIMDC-3) cells expressed membrane proteins abnormally and had structural and functional tight junction defects. Abnormal Ceacam5 expression was due to mis-sorting toward lysosomal degradation, but reduced E-cadherin levels were associated with transcriptional downregulation. The VPS33B-VIPAR complex thus has diverse functions in the pathways regulating apical-basolateral polarity in the liver and kidney.

Mutations in SLC29A3, Encoding an Equilibrative Nucleoside Transporter ENT3, Cause a Familial Histiocytosis Syndrome (Faisalabad Histiocytosis) and Familial Rosai-Dorfman Disease

The histiocytoses are a heterogeneous group of disorders characterised by an excessive number of histiocytes. In most cases the pathophysiology is unclear and treatment is nonspecific. Faisalabad histiocytosis (FHC) (MIM 602782) has been classed as an autosomal recessively inherited form of histiocytosis with similarities to Rosai-Dorfman disease (RDD) (also known as sinus histiocytosis with massive lymphadenopathy (SHML)). To elucidate the molecular basis of FHC, we performed autozygosity mapping studies in a large consanguineous family and identified a novel locus at chromosome 10q22.1. Mutation analysis of candidate genes within the target interval identified biallelic germline mutations in SLC29A3 in the FHC kindred and in two families reported to have familial RDD. Analysis of SLC29A3 expression during mouse embryogenesis revealed widespread expression by e14.5 with prominent expression in the central nervous system, eye, inner ear, and epithelial tissues including the gastrointestinal tract. SLC29A3 encodes an intracellular equilibrative nucleoside transporter (hENT3) with affinity for adenosine. Recently germline mutations in SLC29A3 were also described in two rare autosomal recessive disorders with overlapping phenotypes: (a) H syndrome (MIM 612391) that is characterised by cutaneous hyperpigmentation and hypertrichosis, hepatomegaly, heart anomalies, hearing loss, and hypogonadism; and (b) PHID (pigmented hypertrichosis with insulin-dependent diabetes mellitus) syndrome. Our findings suggest that a variety of clinical diagnoses (H and PHID syndromes, FHC, and familial RDD) can be included in a new diagnostic category of SLC29A3 spectrum disorder.

Mutation in the TCRα subunit constant gene (TRAC) leads to a human immunodeficiency disorder characterized by a lack of TCRαβ+ T cells

Inherited immunodeficiency disorders can be caused by mutations in any one of a large number of genes involved in the function of immune cells. Here, we describe two families with an autosomal recessive inherited immunodeficiency disorder characterized by increased susceptibility to infection and autoimmunity. Genetic linkage studies mapped the disorder to chromosomal region 14q11.2, and a homozygous guanine-to-adenine substitution was identified at the last base of exon 3 immediately following the translational termination codon in the TCRα subunit constant gene (TRAC). RT-PCR analysis in the two affected individuals revealed impaired splicing of the mRNA, as exon 3 was lost from the TRAC transcript. The mutant TCRα chain protein was predicted to lack part of the connecting peptide domain and all of the transmembrane and cytoplasmic domains, which have a critical role in the regulation of the assembly and/or intracellular transport of TCR complexes. We found that T cells from affected individuals were profoundly impaired for surface expression of the TCRαβ complex. We believe this to be the first report of a disease-causing human TRAC mutation. Although the absence of TCRαβ+ T cells in the affected individuals was associated with immune dysregulation and autoimmunity, they had a surprising level of protection against infection.

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

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. Hum Mutat 33:1656–1664, 2012.

Molecular Investigations to Improve Diagnostic Accuracy in Patients With ARC Syndrome

Arthrogryposis, Renal dysfunction and Cholestasis (ARC) syndrome is a multi‐system autosomal recessive disorder caused by germline mutations in VPS33B. The detection of germline VPS33B mutations removes the need for diagnostic organ biopsies (these carry a>50% risk of life‐threatening haemorrhage due to platelet dysfunction); however, VPS33B mutations are not detectable in∼25% of patients. In order further to define the molecular basis of ARC we performed mutation analysis and mRNA and protein studies in patients with a clinical diagnosis of ARC. Here we report novel mutations in VPS33B in patients from Eastern Europe and South East Asia. One of the mutations was present in 7 unrelated Korean patients. Reduced expression of VPS33B and cellular phenotype was detected in fibroblasts from patients clinically diagnosed with ARC with and without known VPS33B mutations. One mutation‐negative patient was found to have normal mRNA and protein levels. This patients clinical condition improved and he is alive at the age of 2.5 years. Thus we show that all patients with a classical clinical course of ARC had decreased expression of VPS33B whereas normal VPS33B expression was associated with good prognosis despite initial diagnosis of ARC.

Regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis

Post-translational modifications are necessary for collagen precursor molecules (procollagens) to acquire final shape and function. However, the mechanism and contribution of collagen modifications that occur outside the endoplasmic reticulum and Golgi are not understood. We discovered that VIPAR, with its partner proteins, regulate sorting of lysyl hydroxylase 3 (LH3, also known as PLOD3) into newly identified post-Golgi collagen IV carriers and that VIPAR-dependent sorting is essential for modification of lysines in multiple collagen types. Identification of structural and functional collagen abnormalities in cells and tissues from patients and murine models of the autosomal recessive multisystem disorder Arthrogryposis, Renal dysfunction and Cholestasis syndrome caused by VIPAR and VPS33B deficiencies confirmed our findings. Thus, regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis and for the development and function of multiple organs and tissues.

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

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.

Vps33b is crucial for structural and functional hepatocyte polarity

Graphical abstract

Glomerular involvement in the arthrogryposis, renal dysfunction and cholestasis syndrome

Background Arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome is a multisystem autosomal-recessive disorder caused by defects in the VPS33B and VIPAR genes, involved in localization of apical membrane proteins. Affected children usually die by 1 year of age, often secondary to infective complications. The classic renal manifestation previously described in ARC syndrome is proximal–tubular dysfunction. The aim of this study is to gain further insight into the renal manifestations of this syndrome. Methods Clinical review of three cases of ARC syndrome presenting to a tertiary centre. Together with measurement of VPS33B and VIPAR protein expression in the human glomerulus. Results The cases demonstrated severe failure to thrive and in addition to commonly described features profound proteinuria and albuminuria, together with hypoalbuminaemia, suggesting glomerular involvement of this syndrome. Western blotting of conditionally immortalized human glomerular cells and ex vivo immunofluorescent analysis of the human glomerulus revealed that VPS33B and VIPAR were highly expressed in glomerular endothelium, and podocytes, but not in the mesangium. Conclusions ARC syndrome affects the glomerulus as well as the proximal tubule in the kidney. Our molecular studies suggest that both cell types that constitute the glomerular filtration barrier are affected in this condition, providing an explanation for the albuminuria that we have observed in our cases.

Nephrotic range albuminuria as a feature of arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome: three new cases

Aims Arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome is an autosomal recessive disorder caused by defects in two proteins (VPS33B and VIPAR), involved in regulation of intracellular protein trafficking. ARC patients usually die by 2 years of age, often secondary to infective complications. It was previously thought that the defect affects the proximal tubule in the kidney, as well as the liver and the musculoskeletal system. We have recently managed three cases with varying clinical presentations and found that in addition to commonly described features, they all demonstrated marked albuminuria suggesting the glomerulus is also involved in this syndrome. Methods Case-note review of the three cases of ARC syndrome and glomerular mapping of the VPS33B expression using western blotting in conditionally immortalised cells of the glomerulus (Podocytes, Glomerular Endothelial cells and Mesangial cells). Results All cases had severe failure to thrive, arthrogryposis, renal tubular acidosis, nephrotic range albuminuria, conjugated hyperbilirubinaemia with normal γ GT, gastro-oesophageal reflux with difficulty establishing well tolerated feed regimens, metabolic bone disease with fractures and recurrent septic episodes. The primary case had a confirmed VPS33B mutation and died of overwhelming sepsis at the age of 4 months. The subsequent two cases have a clinical diagnosis of ARC, but negative initial VPS33B mutation analysis. These children are still alive at five and 6 months of age respectively. We are currently analysing the expression of VPS33B in the cells of the glomerulus to identify the mechanism of action of this mutation in this site. The initial experiments showed that VPS33B was highly expressed in human glomerular endothelium but much less in podocyte and mesangial cell lines. Review of the literature suggests ARC syndrome has an expanding clinical spectrum and may be more common than previously thought. Nephrotic range albuminuria as a feature of ARC syndrome has only been previously described in a small number of cases. Conclusion The cases demonstrate severe failure to thrive and nephrotic range albuminuria as common features of ARC syndrome. Nephrotic range albuminuria may contribute to the infective complications of this condition.