Search

Autonomous thyroid adenomas (ATAs) are a frequent cause of hyperthyroidism. Mutations in the genes encoding the TSH receptor (TSHR) or the Gs protein a subunit (GNAS) are found in approximately 70% of ATAs. The involvement of other genes and the pathogenesis of the remaining cases are presently unknown. Here, we performed whole-exome sequencing in 19 ATAs that were paired with normal DNA samples and identified a recurrent hot-spot mutation (c.1712A>G; p.Gln571Arg) in the enhancer of zeste homolog 1 (EZH1) gene, which codes for a catalytic subunit of the polycomb complex. Targeted screening in an independent cohort confirmed that this mutation occurs with high frequency (27%) in ATAs. EZH1 mutations were strongly associated with known (TSHR, GNAS) or presumed (adenylate cyclase 9 [ADCY9]) alterations in cAMP pathway genes. Furthermore, functional studies revealed that the p.Gln571Arg EZH1 mutation caused increased histone H3 trimethylation and increased proliferation of thyroid cells. In summary, this study revealed that a hot-spot mutation in EZH1 is the second most frequent genetic alteration in ATAs. The association between EZH1 and TSHR mutations suggests a 2-hit model for the pathogenesis of these tumors, whereby constitutive activation of the cAMP pathway and EZH1 mutations cooperate to induce the hyperproliferation of thyroid cells., Introduction Autonomous thyroid adenomas (ATAs) are a major cause of hyperthyroidism, especially in iodine-deficient areas (1, 2). The thyroid-stimulating hormone (TSH) plays a major role in controlling the function and [...]

The Cl/HC[O.sub.3] exchanger downregulated in adenoma (DRA) mediates electroneutral NaC1 absorption in the intestine together with the apical Na/H exchanger NHE3. Lipid rafts (LR) modulate transport activity and are involved in phosphatidylinositol 3-kinase (PI3-kinase)-dependent trafficking of NHE3. Although DRA and NHE3 interact via PDZ adaptor proteins of the NHERF family, the role of LR and PDZ proteins in the regulation of DRA is unknown. We examined the association of DRA with LR using the nonionic detergent Triton X-100. DRA cofractionated with LR independently of its PDZ binding motif. Furthermore, DRA interacts with PDZK1, E3KARP, and IKEPP in LR, although their localization within lipid rafts is independent of DRA. Disruption of LR integrity resulted in the disappearance of DRA from LR, in a decrease of its surface expression and in a reduction of its activity. In HEK cells the inhibition of DRA by LR disruption was entirely dependent on the presence of the PDZ interaction motif. In addition, in Caco-2/BBE cells the inhibition by LR disruption was more pronounced in wild-type DRA than in mutated DRA (DRA-ETKFminus; lacking the PDZ binding motif)-expressing cells. Inhibition of PI3-kinase decreased the activity and the cell surface expression of wild-type DRA but not of DRA-ETKFminus; the partitioning into LR was unaffected. Furthermore, simultaneous inhibition of PI3-kinase and disruption of LR did not further decrease DRA activity and cell surface expression compared with LR disruption only. These results suggest that the activity of DRA depends on its LR association, on its PDZ interaction, and on PI3-kinase activity. Cl/HC[O.sub.3] exchange; PDZK1; E3KARP; NHERF; IKEPP doi: 10.1152/ajpgi.00191.2010.

A loss-of-function mutation in the APC gene initiates colorectal carcinogenesis. Although the molecular mechanism of tumor initiation is complex, several modifier genes have been identified using mouse models, including the ApcMin mouse. Among the familial adenomatous polyposis mouse lines carrying a truncation mutation at codon 580 in Apc (Apc580D), one line (line 19-[Apc.sup.580D/+]) showed a remarkably reduced incidence of intestinal adenomas ( APC | modifier

Down-regulated in adenoma (DRA), also referred to as SLC26A3, is an intestinal anion transporter essential for intestinal chloride absorption. Mutations in DRA result in congenital chloride diarrhea. DRA expression has been shown to be induced by differentiation and to be modulated by cytokines. However, mechanisms of DRA gene transcription and its tissue-specific targeting have not yet been investigated. In this study, we cloned a 3,765-bp promoter fragment of human DRA gene and characterized its activity in human colonic LS 174T and Caco-2 human colon cell lines. Primer extension identified a single transcriptional initiation site that was identical in both colon cancer cell lines and normal colon. Although hepatic nuclear factor HNF-4 is involved in the basal activity of DRA promoter, sodium butyrate induces its activity in LS 174T cells via the binding of Yin Yang 1 (YY1) and GATA transcription factors to their respective cis-elements in promoter region. We also demonstrated a reduction in DRA promoter activity in Caco-2 cells by IFN-[gamma], suggesting that regulation of DRA promoter by IFN-[gamma] may contribute to the pathophysiology of intestinal inflammation. Furthermore, we showed that the DRA promoter fragment is sufficient to drive human growth hormone transgene expression specifically in villus epithelial cells of the small intestine and in differentiated upper crypt and surface epithelial ceils of the colon. Our studies provide evidence for the involvement of HNF-4, YY1, and GATA transcription factors in DRA expression in intestinal differentiated epithelial cells. SLC26A3; congenital chloride diarrhea; intestinal gene expression

Adequate folate availability is necessary to sustain normal DNA synthesis and normal patterns of DNA methylation and these features of DNA can be modified by methylenetetrahydrofolate reductase (MTHFR) C677T genotype. This study investigated the effect of MTHFR C677T genotype and daily supplementation with 5 mg folic acid and 1.25 mg vitamin B-12 on uracil misincorporation into DNA and promoter methylation. Subjects (n = 86) with a history of colorectal adenoma and MTHFR CC or TT genotype were randomly assigned to receive folic acid plus vitamin B-12 or placebo for 6 mo. Uracil misincorporation and promoter methylation of 6 tumor suppressor and DNA repair genes were assessed in DNA from rectal biopsies at baseline and after the intervention. The biomarkers did not differ between the treated group and the placebo group after 6 mo compared with baseline. The uracil concentration of DNA increased in the treated group (5.37 fmol/[micro]g DNA, P = 0.02), whereas it did not change in the placebo group (P = 0.42). The change from baseline of 4.01 fmol uracil/[micro]g DNA tended to differ between the groups (P = 0.16). An increase in promoter methylation tended to occur more often in the intervention group than in the placebo group (OR = 1.67; P = 0.08). This study suggests that supplementation with high doses of folic acid and vitamin B-12 may not favorably influence uracil incorporation and promoter methylation in subjects with previous colorectal adenomas. Because such alterations may potentially increase the risk of neoplastic transformation, more research is needed to fully define the consequences of these molecular alterations.

A multilocus test of genetic association is proposed, which can account for genetic effects that might be modified by variants in other genes or by environmental factors. The powerful advantages of this method is demonstrated both in analysis of data from a case-control study of the association between colorectal adenoma and DNA variants in the NAT2 genomic region that are well known to be related to a common biological phenotype and under different models of gene-gene interactions with use of simulated data.

Research has been conducted on the down regulated in adenoma gene product. The hypothesis that this gene product can bind to the PDZ domains of NHE3 building a structural link between Na (super)+ /H (super)+ and Cl (super)- /HCO (sub)3 (super)- exchangers has been tested and the results are reported.

We have examined the effects of inactivation of the p53 tumor suppressor gene on the incidence of apoptotic cell death in two stages of the adenoma-to-carcinoma progression in the intestine: in early adenomas where p53 mutations are rare and in highly dysplastic adenomas where loss of p53 occurs frequently. Homozygosity for an inactivating germ-line mutation of p53 had no effect on the incidence or the rate of progression of [Apc.sup.Min/+]-induced adenomas in mice and also did not affect the frequency of apoptosis in the cells of these adenomas. To examine the effect of p53 loss on apoptosis in late-stage adenomas, we compared the incidence of apoptotic cell death before and after the appearance of highly dysplastic cells in human colonic adenomas. The appearance of highly dysplastic cells, which usually coincides during colon tumor progression with loss of heterozygosity at the p53 locus, did not correlate with a reduction in the incidence of apoptosis. These studies suggest that p53 is only one of the genes that determine the incidence of apoptotic in colon carcinomas and that wild-type p53 retards the progression of many benign colonic adenoma to malignant carcinomas by mechanism(s) other than the promotion of apoptosis.

Mice heterozygous for the [Apc.sup.Min] (Min) mutation develop adenomas throughout the intestinal tract. Apc is believed to be involved in cell migration, adhesion, and polarity. Adenoma multiplicity and growth rate are modulated by an unlinked modifier locus, Mom1. The secretory phospholipase Pla2g2a is a candidate for Mom1. Here, we investigate the range of action of Apc and Mom1. Analysis of chimeric Min mice indicates that the actions of both Apc and Mom1 are localized within the cell lineage that gives rise to intestinal tumors.

The intestinal tumor multiplicity in mice heterozygous for [Apc.sup.Min] is strongly modulated by genetic background. On the sensitive C57BL/6J (B6) background, mice develop large numbers of intestinal adenomas. The AKR/J (AKR) strain carries alleles that correlate with a strong reduction in tumor multiplicity. To study the effect of one of these modifiers, Mom1, we have generated a mouse line in which the AKR allele of Mom1 is carried on the sensitive B6 genetic background. This strain was produced by using a marker-assisted selection method to eliminate unlinked AKR alleles more rapidly. The application and efficiency of this method are discussed. We used this strain to determine that Mom1 affects both tumor multiplicity and tumor size in a semi-dominant fashion.

The multiple intestinal neoplasia (Min) strain of mice has a mutation in the homolog of the human adenoamatous polyposis coli gene and develops multiple adenomas in the intestines. Loci studies show that the locus Mom1 (modifier of Min 1) is responsible for the genetic variation in the Min strain and the locus contains the gene which encodes for type II non-pancreatic phospholipase A2 (Pla2s). The Min mice probably do not produce Pla2s which is necessary for either protecting the intestine from dietary fatty acids or for maintaining the intestine's flora.

The intestinal tumors of B6-Min/+ mice, a mutant strain that develops multiple intestinal adenomas along the duodenal-to-colonic axis (DCA), were studied as to their cellular differentiation. Molecular markers specific for enterocytes, enteroendocrine, goblet and Paneth cells showed that a given tumor contains all cell types as well as undifferentiated cells, and expression of gene products by the cells was appropriate for the region on the DCA. These results indicate that adenomasmay each arise from single, mutated, multipotent stem cells in the intestinal crypts.