Mutations in tetratricopeptide repeat domain 7A result in a severe form of very early onset inflammatory bowel disease.

BACKGROUND & AIMS: Very early onset inflammatory bowel diseases (VEOIBD), including infant disorders, are a diverse group of diseases found in children younger than 6 years of age. They have been associated with several gene variants. Our aim was to identify the genes that cause VEOIBD. METHODS: We performed whole exome sequencing of DNA from 1 infant 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 TTC7A mutations in 2 unrelated families, each with 2 affected siblings. TTC7A interacts with EFR3 homolog B to regulate phosphatidylinositol 4-kinase 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. Phosphatidylinositol 4-kinase 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. CONCLUSIONS: 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 phosphatidylinositol 4-kinase-TTC7A-EFR3 homolog B pathway are involved in the pathogenesis of VEOIBD.

Chronic exposure to zinc chromate induces centrosome amplification and spindle assembly checkpoint bypass in human lung fibroblasts.

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 h 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. Furthermore, 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. Last, 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.

Zinc chromate induces chromosome instability and DNA double strand breaks in human lung cells.

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 cytotoxicity and genotoxicity of hexavalent chromium in medaka (Oryzias latipes) cells.

Chromium is an increasing health concern for aquatic environments, however, the mechanism of chromium toxicity in aquatic species is yet unknown. We used a medaka (Oryzias latipes) fin cell line to investigate the cytotoxicity and genotoxicity of sodium chromate, a soluble form of hexavalent chromium. We used a clonogenic cytotoxicity assay to measure sodium chromate cytotoxicity, gamma-H2A.X immunofluoresence to measure DNA double-strand breaks, and chromosome damage to measure clastogenicity. We found that sodium chromate is cytotoxic to medaka fin cells, with toxicity increasing in a concentration-dependent manner. Treatments of 0.5, 1, 5, 10, 25, 50 and 100 microM sodium chromate caused 100, 103.5, 87.8, 77.5, 40.9, 15 and 2.7% survival, respectively, relative to the control. We visualized DNA double-strand breaks in medaka cells through the formation of gamma-H2A.X foci. Breaks could be detected at concentrations as low as 1 microM. We also found that sodium chromate induces chromosomal aberrations, causing chromatid lesions and exchanges that increase with concentration. Treatments of 0, 1, 5, 10 and 25 microM sodium chromate damaged 10.3, 17, 32.3, 43 and 51.6% of metaphases and induced 13, 23, 44, 69 and 118 total aberrations in 100 metaphases, respectively. These data show that hexavalent chromium is both cytotoxic and genotoxic to fish cells. Our results set the context for future work in the medaka cell culture model and provide important tools for investigating mechanisms of toxicity in aquatic organisms.

Age-related loss of bone marrow pre-B- and immature B-lymphocytes in the autoimmune-prone flaky skin mutant mice.

Defective B-lymphopoiesis has been associated with development of auto-antibodies and auto-immunity in a number of autoimmune-prone strains of mice. The flaky skin (fsn) mutation results in development of chronic inflammation and auto-immunity. Associated with the development of auto-immunity is the hyperactivation of B-lymphocytes and production of auto-antibodies. We, therefore, undertook a detailed examination of B-lineage precursors in the bone marrow of fsn/fsn mice. We observed a rapid age-related loss of the pre-B and immature B cells. It was also noted that an accumulation of early precursor populations occurs coincident with the loss of Fr.D and Fr.E bone marrow B cell populations indicating a developmental block or accumulation of pro-B cells in 7 and 10 week old fsn/fsn mice. Our data suggests changes in the fsn/fsn bone-marrow microenvironment that results in senescence of B cell development.

The Tetratricopeptide repeat domain 7 gene is mutated in flaky skin mice: a model for psoriasis, autoimmunity, and anemia.

The flaky skin (fsn) mutation in mice causes pleiotropic abnormalities including psoriasiform dermatitis, anemia, hyper-IgE, and anti-dsDNA autoantibodies resembling those detected in systemic lupus erythematosus. The fsn mutation was mapped to an interval of 3.9 kb on chromosome 17 between D17Mit130 and D17Mit162. Resequencing of known and predicted exons and regulatory sequences from this region in fsn/fsn and wild-type mice indicated that the mutation is due to the insertion of an endogenous retrovirus (early transposon class) into intron 14 of the Tetratricopeptide repeat (TPR) domain 7 (Ttc7) gene. The insertion leads to reduced levels of wild-type Ttc7 transcripts in fsn mice and the insertion of an additional exon derived from the retrovirus into the majority of Ttc7 mRNAs. This disrupts one of the TPRs within TTC7 and may affect its interaction with an as-yet unidentified protein partner. The Ttc7 is expressed in multiple types of tissue including skin, kidney, spleen, and thymus, but is most abundant in germinal center B cells and hematopoietic stem cells, suggesting an important role in the development of immune system cells. Its role in immunologic and hematologic disorders should be further investigated.

Carcinogenic lead chromate induces DNA double-strand breaks in human lung cells.

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.

Hyperactivation and proliferation of lymphocytes from the spleens of flaky skin (fsn) mutant mice.

Mice homozygous for the flaky skin (fsn) single gene mutation have a severe hyperproliferative disease resulting in a complex phenotype, which includes widespread inflammation and autoimmunity. This study sought to characterize lymphocyte function of flaky skin mutant mice. Flaky skin lymphocytes show enhanced proliferation with in vitro mitogen stimulated spleen cells, as well as enriched splenic B- and T-cells. The production of IL-4 by fsn/fsn T-lymphocytes is increased dramatically compared with normal controls. Flaky skin lymphocytes exhibited increased responsiveness to IL-2, IL-4 and IL-7 in the absence of pre-activation, enhanced IgE production in response to ovalbumin immunization, and constitutive STAT6 activation. These data indicate that the cytokines IL-2, IL-4 and IL-7 likely contribute to the lymphocyte activation in fsn/fsn mutant mice. This lymphocyte hyperactivation may result in the development of systemic autoimmunity in fsn/fsn mutant mice.

Antinuclear autoantibodies in flaky skin (fsn) mutant mice.

Mice homozygous.for the flaky skin (fsn) single gene mutation have a severe hyperproliferative disease resulting complex phenotype, which includes widespread inflammation and autoimmunity. Flaky skin mice have several serological and pathological features that share similarities with the human systemic autoimmune disease systemic lupus erythematosus (SLE). Analyses of the antinuclear and anti-dsDNA autoantibodies in fsn/fsn mice indicate that they are low titer IgG antibodies. These low titer anti-dsDNA autoantibodies can ultimately form immune complex deposition in the glomeruli associated kidney damage. IgE antibodies were identified in the immune complex deposition, however their role in the pathology is not determined. It is hypothesized that the mechanism of autoantibody production and autoimmune disease pathogenesis in mice homozygous for the fsn mutation is initiated by non-specific polyclonal activation of B-lymphocytes resulting in the synthesis of low affinity autoantibodies.

Expansion of CD22lo B cells in the spleen of autoimmune-prone flaky skin mice.

Similar to murine models with compromised CD22/SHP-1 function, flaky skin (fsn) mutant mice exhibit lymphocyte hyperactivation and an autoimmune phenotype characterized by circulating autoantibodies to dsDNA and glomerulonephritis. Immunophenotyping of fsn/fsn splenic B cells was performed to determine if abnormalities in CD22 expression contributed to the phenotype. We identified an expansion of an IgM(bright) CD22lo population consistent with immature B-lymphocytes. While normal B-lymphocytes require IL-4 to achieve down-modulation of CD22 expression in response to BCR cross-linking, culture with anti-IgM alone led to reduced CD22 expression in fsn/fsn mice. Furthermore, when IL-4 was added to fsn/fsn cultures, no further reduction in CD22 expression was observed. This suggested that fsn/fsn B cells were pre-activated in vivo by chronic IL-4 exposure. A portion of these CD22lo cells expressed the B-1 surface marker CD11b. We contend that decreased activation thresholds among CD22lo B-lymphocytes contributes to the expansion of immature and B-1 B cell populations and to the development of autoimmune pathology in fsn/fsn mice.