15 research outputs found
Collagen α5 and α2(IV) chain coexpression: Analysis of skin biopsies of Alport patients
Alport syndrome is a collagen type IV disease caused by mutations in the COL4A5 gene with the X-linked form being most prevalent. The resultant α5(IV) collagen chain is a component of the glomerular and skin basement membranes (SBMs). Immunofluorescent determination of the α5(IV) chain in skin biopsies is the procedure of choice to identify patients. In 30% of patients, however, the mutant protein is still found in the SBM resulting in a normal staining pattern. In order to minimize or eliminate false results, we compared the distribution of the α2(IV) chain (another SBM component) and the α5(IV) chain by standard double label immunofluorescence (IF) and by confocal laser scanning microcopy. The study was performed on 55 skin biopsies of patients suspected of Alports and five normal control specimens. In normal skin, IF showed the classical linear pattern for both collagens along the basement membrane. Additionally, decreased α5(IV) was found in the bottom of the dermal papillary basement membrane. Confocal analysis confirmed the results and show α5(IV) focal interruptions. In suspected patients, both techniques showed the same rate of abnormal α5(IV) expression: segmental in women and absent in men. Our results show a physiological variation of α5(IV) location with focal interruptions and decreased expression in the bottom of the dermal basement membrane. Comparison of α5(IV) with α2(IV) expression is simple and eliminates technical artifacts
Mutations in TRAF3IP1/IFT54 reveal a new role for IFT proteins in microtubule stabilization
Ciliopathies are a large group of clinically and genetically heterogeneous disorders caused by defects in primary cilia. Here we identified mutations in TRAF3IP1 (TNF Receptor-Associated Factor Interacting Protein 1) in eight patients from five families with nephronophthisis (NPH) and retinal degeneration, two of the most common manifestations of ciliopathies. TRAF3IP1 encodes IFT54, a subunit of the IFT-B complex required for ciliogenesis. The identified mutations result in mild ciliary defects in patients but also reveal an unexpected role of IFT54 as a negative regulator of microtubule stability via MAP4 (microtubule-associated protein 4). Microtubule defects are associated with altered epithelialization/polarity in renal cells and with pronephric cysts and microphthalmia in zebrafish embryos. Our findings highlight the regulation of cytoplasmic microtubule dynamics as a role of the IFT54 protein beyond the cilium, contributing to the development of NPH-related ciliopathies
X-linked susceptibility to mycobacteria is caused by mutations in NEMO impairing CD40-dependent IL-12 production
Germline mutations in five autosomal genes involved in interleukin (IL)-12–dependent, interferon (IFN)-γ–mediated immunity cause Mendelian susceptibility to mycobacterial diseases (MSMD). The molecular basis of X-linked recessive (XR)–MSMD remains unknown. We report here mutations in the leucine zipper (LZ) domain of the NF-κB essential modulator (NEMO) gene in three unrelated kindreds with XR-MSMD. The mutant proteins were produced in normal amounts in blood and fibroblastic cells. However, the patients' monocytes presented an intrinsic defect in T cell–dependent IL-12 production, resulting in defective IFN-γ secretion by T cells. IL-12 production was also impaired as the result of a specific defect in NEMO- and NF-κB/c-Rel–mediated CD40 signaling after the stimulation of monocytes and dendritic cells by CD40L-expressing T cells and fibroblasts, respectively. However, the CD40-dependent up-regulation of costimulatory molecules of dendritic cells and the proliferation and immunoglobulin class switch of B cells were normal. Moreover, the patients' blood and fibroblastic cells responded to other NF-κB activators, such as tumor necrosis factor-α, IL-1β, and lipopolysaccharide. These two mutations in the NEMO LZ domain provide the first genetic etiology of XR-MSMD. They also demonstrate the importance of the T cell– and CD40L-triggered, CD40-, and NEMO/NF-κB/c-Rel–mediated induction of IL-12 by monocyte-derived cells for protective immunity to mycobacteria in humans
IFT81, encoding an IFT-B core protein, as a very rare cause of a ciliopathy phenotype
Background: Bidirectional intraflagellar transport (IFT) consists of two major protein complexes, IFT-A and IFT-B. In contrast to the IFT-B complex, all components of IFT-A have recently been linked to human ciliopathies when defective. We therefore hypothesised that mutations in additional IFT-B encoding genes can be found in patients with multisystemic ciliopathies. Methods: We screened 1628 individuals with reno-ocular ciliopathies by targeted next-generation sequencing of ciliary candidate genes, including all IFT-B encoding genes. Results: Consequently, we identified a homozygous mutation in IFT81 affecting an obligatory donor splice site in an individual with nephronophthisis and polydactyly. Further, we detected a loss-of-stop mutation with extension of the deduced protein by 10 amino acids in an individual with neuronal ceroid lipofuscinosis-1. This proband presented with retinal dystrophy and brain lesions including cerebellar atrophy, a phenotype to which the IFT81 variant might contribute. Cultured fibroblasts of this latter affected individual showed a significant decrease in ciliated cell abundance compared with controls and increased expression of the transcription factor GLI2 suggesting deranged sonic hedgehog signalling. Conclusions: This work describes identification of mutations of IFT81 in individuals with symptoms consistent with the clinical spectrum of ciliopathies. It might represent the rare case of a core IFT-B complex protein found associated with human disease. Our data further suggest that defects in the IFT-B core are an exceedingly rare finding, probably due to its indispensable role for ciliary assembly in development
Abnormal apical-to-basal transport of dietary ovalbumin by secretory IgA stimulates a mucosal Th1 response.
In celiac disease, enhanced permeability to gliadin peptides can result from their apico-basal transport by secretory immunoglobulin A1 (SIgA1) binding to the CD71 receptor ectopically expressed at the gut epithelial surface. Herein, we have established a mouse model in which there is apico-basal transport of the model antigen ovalbumin (OVA) by specific SIgA1 and have analyzed local T-cell activation. Transgenic DO11.10 mice were grafted with a hybridoma-secreting OVA-specific humanized IgA1, which could bind mouse CD71 and which were released in the intestinal lumen as SIgA. CD71 expression was induced at the gut apical surface by treating the mice with tyrphostin A8. Following gavage of the mice with OVA, OVA-specific CD4(+) T cells isolated from the mesenteric lymph nodes displayed higher expression of the activation marker CD69 and produced more interferon gamma in mice bearing the hybridoma-secreting OVA-specific IgA1, than in ungrafted mice or in mice grafted with an irrelevant hybridoma. These results indicate that the protective role of SIgA1 might be jeopardized in human pathological conditions associated with ectopic expression of CD71 at the gut surface
Abnormal apical-to-basal transport of dietary ovalbumin by secretory IgA stimulates a mucosal Th1 response.
International audience: In celiac disease, enhanced permeability to gliadin peptides can result from their apico-basal transport by secretory immunoglobulin A1 (SIgA1) binding to the CD71 receptor ectopically expressed at the gut epithelial surface. Herein, we have established a mouse model in which there is apico-basal transport of the model antigen ovalbumin (OVA) by specific SIgA1 and have analyzed local T-cell activation. Transgenic DO11.10 mice were grafted with a hybridoma-secreting OVA-specific humanized IgA1, which could bind mouse CD71 and which were released in the intestinal lumen as SIgA. CD71 expression was induced at the gut apical surface by treating the mice with tyrphostin A8. Following gavage of the mice with OVA, OVA-specific CD4(+) T cells isolated from the mesenteric lymph nodes displayed higher expression of the activation marker CD69 and produced more interferon gamma in mice bearing the hybridoma-secreting OVA-specific IgA1, than in ungrafted mice or in mice grafted with an irrelevant hybridoma. These results indicate that the protective role of SIgA1 might be jeopardized in human pathological conditions associated with ectopic expression of CD71 at the gut surface.Mucosal Immunology advance online publication 10 July 2013; doi:10.1038/mi.2013.49
Human IFT52 mutations uncover a novel role for the protein in microtubule dynamics and centrosome cohesion
Mutations in genes encoding components of the intraflagellar transport (IFT) complexes have previously been associated with a spectrum of diseases collectively termed ciliopathies. Ciliopathies relate to defects in the formation or function of the cilium, a sensory or motile organelle present on the surface of most cell types. IFT52 is a key component of the IFT-B complex and ensures the interaction of the two subcomplexes, IFT-B1 and IFT-B2. Here, we report novel IFT52 biallelic mutations in cases with a short-rib thoracic dysplasia (SRTD) or a congenital anomaly of kidney and urinary tract (CAKUT). Combining in vitro and in vivo studies in zebrafish, we showed that SRTD-associated missense mutation impairs IFT-B complex assembly and IFT-B2 ciliary localization, resulting in decreased cilia length. In comparison, CAKUT-associated missense mutation has a mild pathogenicity, thus explaining the lack of skeletal defects in CAKUT case. In parallel, we demonstrated that the previously reported homozygous nonsense IFT52 mutation associated with Sensenbrenner syndrome [Girisha et al. (2016) A homozygous nonsense variant in IFT52 is associated with a human skeletal ciliopathy. Clin. Genet., 90, 536-539] leads to exon skipping and results in a partially functional protein. Finally, our work uncovered a novel role for IFT52 in microtubule network regulation. We showed that IFT52 interacts and partially co-localized with centrin at the distal end of centrioles where it is involved in its recruitment and/or maintenance. Alteration of this function likely contributes to centriole splitting observed in Ift52(-/-) cells. Altogether, our findings allow a better comprehensive genotype-phenotype correlation among IFT52-related cases and revealed a novel, extra-ciliary role for IFT52, i.e. disruption may contribute to pathophysiological mechanisms
X linked susceptibility to Mycobapteria is caused by mutation in NEMO impairing CD40-dependent IL-12production.
X-linked susceptibility to mycobacteria is caused by mutations in NEMO impairing CD40-dependent IL-12 production
Germline mutations in five autosomal genes involved in interleukin
(IL)-12-dependent, interferon (IFN)-gamma-mediated immunity cause
Mendelian susceptibility to mycobacterial diseases (MSMD). The molecular
basis of X-linked recessive (XR)-MSMD remains unknown. We report here
mutations in the leucine zipper (LZ) domain of the NF-kappa B essential
modulator (NEMO) gene in three unrelated kindreds with XR-MSMD. The
mutant proteins were produced in normal amounts in blood and
fibroblastic cells. However, the patients’ monocytes presented an
intrinsic defect in T cell-dependent IL-12 production, resulting in
defective IFN-gamma secretion by T cells. IL-12 production was also
impaired as the result of a specific defect in NEMO- and NF-kappa
B/c-Rel-mediated CD40 signaling after the stimulation of monocytes and
dendritic cells by CD40L-expressing T cells and fibroblasts,
respectively. However, the CD40-dependent up-regulation of costimulatory
molecules of dendritic cells and the proliferation and immunoglobulin
class switch of B cells were normal. Moreover, the patients’ blood and
fibroblastic cells responded to other NF-kappa B activators, such as
tumor necrosis factor-alpha, IL-beta, and lipopolysaccharide. These two
mutations in the NEMO LZ domain provide the first genetic etiology of
XR-MSMD. They also demonstrate the importance of the T cell- and
CD40L-triggered, CD40-, and NEMO/NF-kappa B/c-Rel-mediated induction of
IL-12 by monocyte-derived cells for protective immunity to mycobacteria
in humans