Macrothrombocytopenia and dense granule deficiency associated with FLI1 variants: ultrastructural and pathogenic features.

Congenital macrothrombocytopenia is a family of rare diseases, of which a significant fraction remains to be genetically characterized. To analyze cases of unexplained thrombocytopenia, 27 individuals from a patient cohort of the Bleeding and Thrombosis Exploration Center of the University Hospital of Marseille were recruited for a high-throughput gene sequencing study. This strategy led to the identification of two novel FLI1 variants (c.1010G>A and c.1033A>G) responsible for macrothrombocytopenia. The FLI1 variant carriers' platelets exhibited a defect in aggregation induced by low-dose adenosine diphosphate (ADP), collagen and thrombin receptor-activating peptide (TRAP), a defect in adenosine triphosphate (ATP) secretion, a reduced mepacrine uptake and release and a reduced CD63 expression upon TRAP stimulation. Precise ultrastructural analysis of platelet content was performed using transmission electron microscopy and focused ion beam scanning electron microscopy. Remarkably, dense granules were nearly absent in the carriers' platelets, presumably due to a biogenesis defect. Additionally, 25-29% of the platelets displayed giant α-granules, while a smaller proportion displayed vacuoles (7-9%) and autophagosome-like structures (0-3%). In vitro study of megakaryocytes derived from circulating CD34+ cells of the carriers revealed a maturation defect and reduced proplatelet formation potential. The study of the FLI1 variants revealed a significant reduction in protein nuclear accumulation and transcriptional activity properties. Intraplatelet flow cytometry efficiently detected the biomarker MYH10 in FLI1 variant carriers. Overall, this study provides new insights into the phenotype, pathophysiology and diagnosis of FLI1 variant-associated thrombocytopenia.

Functional polymorphisms in the regulatory regions of the VNN1 gene are associated with susceptibility to inflammatory bowel diseases.

BACKGROUND: Vanin-1 is an epithelial pantetheinase, which regulates intestinal inflammation in mouse. We investigated whether human VNN1 levels could be associated to the susceptibility to inflammatory bowel diseases (IBD) and explored the participation of PPARg to these processes. METHODS: We studied VNN1 expression in colon biopsies from IBD patients. We investigated polymorphisms in the regulatory regions of the VNN1 gene and examined their genetic association with the disease. Functional relevance of these single-nucleotide polymorphisms (SNPs) was assayed, and we tested PPARg in nuclear complexes associated with specific VNN1 polymorphic sequences. In mouse, we examined Vanin-1 expression in gut and feces during dextran sulfate sodium-induced colitis and assayed the effect of PPARg on Vanin-1 regulation. RESULTS: VNN1 is expressed by enterocytes and is upregulated in IBD. Three SNPs are statistically associated to IBD. The regions containing these SNPs specifically bind nuclear complexes and are correlated with the VNN1 transcript abundance in colon in an allele-dependent manner. One rare SNP is associated to severe ulcerative colitis with strong VNN1 and dropped PPARg levels. PPARg is involved in nuclear complexes that bound to VNN1 regulatory sites. Similarly, Vanin-1 is tightly regulated in the mouse gut in normal and colitis conditions and PPARg regulates its expression. CONCLUSIONS: VNN1 is a marker for IBD. Polymorphic positions in the VNN1 locus are direct targets for nuclear factors that might regulate the level of VNN1 in colon, and this could be linked to IBD susceptibility. It is hoped that modulating locally VNN1 expression or activity can be exploited to develop future therapeutic strategies against IBD.

Epithelial vanin-1 controls inflammation-driven carcinogenesis in the colitis-associated colon cancer model.

BACKGROUND: Vanin-1 is an epithelial pantetheinase that provides cysteamine to tissue and regulates response to stress. Vanin-1 is expressed by enterocytes, and its absence limits intestinal epithelial cell production of proinflammatory signals. A link between chronic active inflammation and cancer is illustrated in patients with ulcerative colitis, who have an augmented risk of developing colorectal cancer. Indeed, sustained inflammation provides advantageous growth conditions to tumors. We examined whether epithelial cells affect tumorigenesis through vanin-1-dependent modulation of colonic inflammation. METHODS: To vanin-1(-/-) mice, we applied the colitis-associated cancer (CAC) protocol, which combines injection of azoxymethane (AOM) with repeated administrations of dextran sodium sulfate (DSS). We numbered tumors and quantified macrophage infiltration and molecular markers of cell death and proliferation. We also tested DSS-induced colitis. We scored survival, tissue damages, proinflammatory cytokine production, and tissue regeneration. Finally, we explored activation pathways by biochemical analysis on purified colonic epithelial cells (CECs) and in situ immunofluorescence. RESULTS: Vanin-1(-/-) mice displayed a drastically reduced incidence of colorectal cancer in the CAC protocol and manifested mild clinical signs of DSS-induced colitis. The early impact of vanin-1 deficiency on tumor induction was directly correlated to the amount of inflammation and subsequent epithelial proliferation rather than cell death rate; all this was linked to the modulation of NF-kappaB pathway activation in CECs. CONCLUSIONS: These results emphasize the importance of the intestinal epithelium in the control of mucosal inflammation acting as a cofactor in carcinogenesis. This might lead to novel anti-inflammatory strategies useful in cancer therapy.