Background & Aims Very early onset inflammatory bowel diseases (VEOIBD), including infant disorders, are a diverse group of diseases found in children less than 6 years of age. They have been associated with several gene variants. We aimed to identify genes that cause VEOIBD. Methods We performed whole-exome sequencing of DNA from 1 infants with severe enterocolitis and her parents. Candidate gene mutations were validated in 40 pediatric patients and functional studies were carried out using intestinal samples and human intestinal cell lines. Results We identified compound heterozygote mutations in the tetratricopeptide repeat domain 7 (TTC7A) gene in an infant from non-consanguineous parents with severe exfoliative apoptotic enterocolitis; we also detected the mutations in 2 unrelated families, each with 2 affected siblings. TTC7A interacts with EFR3 homolog B (EFR3B) to regulate phosphatidylinositol 4-kinase (PI4KA) at the plasma membrane. Functional studies demonstrated that TTC7A is expressed in human enterocytes. The mutations we identified in TTC7A result in either mislocalization or reduced expression of TTC7A. PI4KA was found to co-immunoprecipitate with TTC7A; the identified TTC7A mutations reduced this binding. Knockdown of TTC7A in human intestinal-like cell lines reduced their adhesion, increased apoptosis, and decreased production of phosphatidylinositol 4-phosphate. Conclusion In a genetic analysis, we identified loss of function mutations in TTC7A in 5 infants with VEOIBD. Functional studies demonstrated that the mutations cause defects in enterocytes and T cells that lead to severe apoptotic enterocolitis. Defects in the PI4KA–TTC7A–EFR3B pathway are involved in the pathogenesis of VEOIBD.
Hexavalent chromium (Cr(VI)) is a widespread environmental contaminant and a known human carcinogen, generally causing bronchial cancer. Recent studies have shown that the particulate forms of Cr(VI) are the potent carcinogens. Particulate Cr(VI) is known to induce a spectrum of DNA damage such as DNA single strand breaks, Cr-DNA adducts, DNA-protein crosslinks and chromosomal aberrations. However, particulate Cr(VI)-induced DNA double strand breaks (DSBs) have not been reported. Thus, the aim of this study was to determine if particulate Cr(VI)-induces DSBs in human bronchial cells. Using the single cell gel electrophoresis assay (comet assay), showed that lead chromate-induced concentration dependent increases in DSBs with 0.1, 0.5, 1 and 5 microg/cm2 lead chromate inducing a 20, 50, 67 and 109% relative increase in the tail integrated intensity ratio, respectively. Sodium chromate at concentrations of 1, 2.5 and 5 microM induced 38, 78 and 107% relative increase in the tail integrated intensity ratio, respectively. We also show that genotoxic concentrations of lead chromate activate the ataxia telangiectasia mutated (ATM) protein, which is thought to play a central role in the early stages of DSB detection and controls cellular responses to this damage. The H2A.X protein becomes rapidly phosphorylated on residue serine 139 in cells when DSBs are introduced into the DNA by ionizing radiation. By using immunofluorescence, we found that lead chromate-induced concentration-dependent increases in phosphorylated H2A.X (r-H2A.X) foci formation with 0.1, 0.5, 1, 5 and 10 microg/cm2 lead chromate inducing a relative increase in the number of cells with r-H2A.X foci formation of 43, 51, 115 and 129%, respectively.
Hexavalent chromium (Cr(VI)) compounds are known human lung carcinogens. Solubility plays an important role in its carcinogenicity with the particulate or insoluble form being the most potent. Of the particulate Cr(VI) compounds, zinc chromate appears to be the most potent carcinogen, however, very few studies have investigated its carcinogenic mechanism. In this study, we investigated the ability of chronic exposure to zinc chromate to induce numerical chromosome instability. We found no increase in aneuploidy after a 24 hour exposure to zinc chromate, but with more chronic exposures, zinc chromate induced concentration- and time-dependent increases in aneuploidy in the form of hypodiploidy, hyperdiploidy and tetraploidy. Zinc chromate also induced centrosome amplification in a concentration- and time-dependent manner in both interphase and mitotic cells after chronic exposure, producing cells with centriolar defects. Further, chronic exposure to zinc chromate induced concentration- and time-dependent increases in spindle assembly checkpoint bypass with increases in centromere spreading, premature centromere division and premature anaphase. Lastly, we found that chronic exposure to zinc chromate induced a G2 arrest. All together, these data indicate that zinc chromate can induce chromosome instability after prolonged exposures.
Hexavalent chromium Cr(VI) is a respiratory toxicant and carcinogen, with solubility playing an important role in its carcinogenic potential. Zinc chromate, a water insoluble or ‘particulate’ Cr(VI) compound, has been shown to be carcinogenic in epidemiology studies and to induce tumors in experimental animals, but its genotoxicity is poorly understood. Our study shows that zinc chromate induced concentration-dependent increases in cytotoxicity, chromosome damage and DNA double strand breaks in human lung cells. In response to zinc chromate-induced breaks, MRE11 expression was increased and ATM and ATR were phosphorylated, indicating that the DNA double strand break repair system was initiated in the cells. In addition, our data show that zinc chromate-induced double strand breaks were only observed in the G2/M phase population, with no significant amount of double strand breaks observed in G1 and S phase cells. These data will aid in understanding the mechanisms of zinc chromate toxicity and carcinogenesis.
The autosomal recessive mutation "flaky skin" (fsn) causes pleiotropic abnormalities in the immune and hematopoietic systems accompanied by pathologic changes in the skin. Homozygotes (fsn/fsn) showed increased size and histological alterations in the spleen and lymph nodes. Abnormalities in lymphoid architecture of the spleen in fsn/fsn mice were accompanied by marked increases in total numbers of B cells, macrophages, and immature erythroid cells. Splenic B cells displayed elevated MHC class II expression. Serum IgE levels were greater than 100 microg/ml by 10 weeks of age, representing > 7000-fold increase compared with normal littermates. This increased IgE level was associated with elevated IL-4 production by spleen cells and with increased amounts of serum IL-4. Serum IgM, IgG1, and IgG2b levels were also increased in fsn/fsn mice while IgG3 was decreased. Autoimmunity in fsn/fsn mice was evidenced by glomerulonephritis accompanied by immune complex deposition in the kidneys, increased serum blood urea nitrogen levels, and the presence of circulating anti-double-stranded DNA autoantibodies. Pathological changes in the skin of fsn/fsn mice were characterized by epidermal hyperplasia and mixed dermal inflammation. Increased numbers of mast cells were also observed in the dermis of the truncal skin as well as in the epithelial stomach. These marked immunological abnormalities suggest that the fsn locus encodes a major immunoregulatory molecule important in multiple immune and hematopoietic functions.
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