TMEM16A deficiency: a potentially fatal neonatal disease resulting from impaired chloride currents.

INTRODUCTION: TMEM16A is a calcium-activated chloride channel expressed in various secretory epithelia. Two siblings presented in early infancy with reduced intestinal peristalsis and recurrent episodes of haemorrhagic diarrhoea. In one of them, the episodes were characterised by hepatic pneumatosis with gas bubbles in the portal vein similar to necrotising enterocolitis of the newborn. METHODS: Exome sequencing identified a homozygous truncating pathogenic variant in ANO1. Expression analysis was performed using reverse transcription PCR, western blot and immunohistochemistry. Electrophysiological and cell biological studies were employed to characterise the effects on ion transport both in patient respiratory epithelial cells and in transfected HEK293 cells. RESULTS: The identified variant led to TMEM16A dysfunction, which resulted in abolished calcium-activated Cl- currents. Secondarily, CFTR function is affected due to the close interplay between both channels without inducing cystic fibrosis (CF). CONCLUSION: TMEM16A deficiency is a potentially fatal disorder caused by abolished calcium-activated Cl- currents in secretory epithelia. Secondary impairment of CFTR function did not cause a CF phenotyp, which may have implications for CF treatment.

Impaired antibacterial autophagy links granulomatous intestinal inflammation in Niemann-Pick disease type C1 and XIAP deficiency with NOD2 variants in Crohn's disease.

OBJECTIVE: Patients with Niemann-Pick disease type C1 (NPC1), a lysosomal lipid storage disorder that causes neurodegeneration and liver damage, can present with IBD, but neither the significance nor the functional mechanism of this association is clear. We studied bacterial handling and antibacterial autophagy in patients with NPC1. DESIGN: We characterised intestinal inflammation in 14 patients with NPC1 who developed IBD. We investigated bacterial handling and cytokine production of NPC1 monocytes or macrophages in vitro and compared NPC1-associated functional defects to those caused by IBD-associated nucleotide-binding oligomerization domain-containing protein 2 (NOD2) variants or mutations in X-linked inhibitor of apoptosis (XIAP). RESULTS: Patients with the lysosomal lipid storage disorder NPC1 have increased susceptibility to early-onset fistulising colitis with granuloma formation, reminiscent of Crohn's disease (CD). Mutations in NPC1 cause impaired autophagy due to defective autophagosome function that abolishes NOD2-mediated bacterial handling in vitro similar to variants in NOD2 or XIAP deficiency. In contrast to genetic NOD2 and XIAP variants, NPC1 mutations do not impair NOD2-receptor-interacting kinase 2 (RIPK2)-XIAP-dependent cytokine production. Pharmacological activation of autophagy can rescue bacterial clearance in macrophages in vitro by increasing the autophagic flux and bypassing defects in NPC1. CONCLUSIONS: NPC1 confers increased risk of early-onset severe CD. Our data support the concept that genetic defects at different checkpoints of selective autophagy cause a shared outcome of CD-like immunopathology linking monogenic and polygenic forms of IBD. Muramyl dipeptide-driven cytokine responses and antibacterial autophagy induction are parallel and independent signalling cascades downstream of the NOD2-RIPK2-XIAP complex.

VPS33B regulates protein sorting into and maturation of α-granule progenitor organelles in mouse megakaryocytes.

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 α-granules. We generated a tamoxifen-inducible mouse model of VPS33B deficiency, Vps33b(fl/fl)-ER(T2), and studied the platelet phenotype and α-granule biogenesis. Ultrastructural analysis of Vps33b(fl/fl)-ER(T2) platelets identified a marked reduction in α-granule count and the presence of small granule-like structures in agreement with the platelet phenotype observed in ARC patients. A reduction of ∼65% to 75% was observed in the α-granule proteins von Willebrand factor and P-selectin. Although platelet aggregation responses were not affected, a defect in δ-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 α-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 α-granule deficiency in Vps33b(fl/fl)-ER(T2) mouse and ARC patients.

Anti-TNF therapy for inflammatory bowel disease in patients with neurodegenerative Niemann-Pick disease Type C.

Background:  Blockade of tumour necrosis factor (anti-TNF) is effective in patients with Crohn's Disease but has been associated with infection risk and neurological complications such as demyelination. Niemann-Pick disease Type C1 (NPC1) is a lysosomal storage disorder presenting in childhood with neurological deterioration, liver damage and respiratory infections. Some NPC1 patients develop severe Crohn's disease. Our objective was to investigate the safety and effectiveness of anti-TNF in NPC1 patients with Crohn's disease. Methods: Retrospective data on phenotype and therapy response were collected in 2019-2020 for the time period 2014 to 2020 from patients in the UK, France, Germany and Canada with genetically confirmed NPC1 defects and intestinal inflammation. We investigated TNF secretion in peripheral blood mononuclear cells treated with NPC1 inhibitor in response to bacterial stimuli . Results: NPC1 inhibitor treated peripheral blood mononuclear cells (PBMCs) show significantly increased TNF production after lipopolysaccharide or bacterial challenge providing a rationale for anti-TNF therapy. We identified 4 NPC1 patients with Crohn's disease (CD)-like intestinal inflammation treated using anti-TNF therapy (mean age of onset 8.1 years, mean treatment length 27.75 months, overall treatment period 9.25 patient years). Anti-TNF therapy was associated with reduced gastrointestinal symptoms with no apparent adverse neurological events. Therapy improved intestinal inflammation in 4 patients. Conclusions: Anti-TNF therapy appears safe in patients with NPC1 and is an effective treatment strategy for the management of intestinal inflammation in these patients.