Alan Aitchison

Targeted disruption of the 3p12 gene, Dutt1/Robo1, predisposes mice to lung adenocarcinomas and lymphomas with methylation of the gene promoter.

The DUTT1 gene is located on human chromosome 3, band p12, within a region of nested homozygous deletions in breast and lung tumors. It is therefore a candidate tumor suppressor gene in humans and is the homologue (ROBO1) of the Drosophila axonal guidance receptor gene, Roundabout. We have shown previously that mice with a targeted homozygous deletion within the Dutt1/Robo1 gene generally die at birth due to incomplete lung development: survivors die within the first year of life with epithelial bronchial hyperplasia as a common feature. Because Dutt1/Robo1 heterozygous mice develop normally, we have determined their tumor susceptibility. Mice with a targeted deletion within one Dutt1/Robo1 allele spontaneously develop lymphomas and carcinomas in their second year of life with a 3-fold increase in incidence compared with controls: invasive lung adenocarcinomas are by far the predominant carcinoma. In addition to the mutant allele, loss of heterozygosity analysis indicates that these tumors retain the structurally normal allele but with substantial methylation of the gene’s promoter. Substantial reduction of Dutt1/Robo1 protein expression in tumors is observed by Western blotting and immunohistochemistry. This suggests that Dutt1/Robo1 is a classic tumor suppressor gene requiring inactivation of both alleles to elicit tumorigenesis in these mice.

Impaired transforming growth factor β signalling in Barrett’s carcinogenesis due to frequent SMAD4 inactivation

Background and aims: Transforming growth factor β (TGF-β) is frequently involved in gastrointestinal carcinogenesis although its contribution to oesophageal adenocarcinoma (AC) and its precursor Barrett’s oesophageal epithelium (BE) metaplasia are unclear. Methods: Expression of TGF-β signalling components was assessed by reverse transcription-polymerase chain reaction (PCR), western blot, and immunohistochemistry in oesophageal endoscopic biopsies and cell lines. Genomic alterations in SMAD4 were characterised by fluorescence in situ hybridisation, methylation specific PCR, and sequencing. Functional integrity of TGF-β signalling was assessed by characterisation of p21 and proliferation status. Smad4 negative BIC-1 cells were transiently transfected with smad4 and TGF-β responsiveness evaluated. Results:smad4 mRNA expression was progressively reduced in the metaplasia-dysplasia-adenocarcinoma sequence (p<0.01). A quarter of AC samples displayed an abnormal Smad4 protein isoform, with no corresponding changes in gene sequence or organisation. Methylation of smad4 has not been described previously but we found promoter methylation in 70% of primary AC samples. In 6/8 oesophageal cell lines, chromosomal rearrangements affected the smad4 locus. Lack of smad4 expression in BIC-1 cells occurred secondary to loss of one copy and extensive deletion of the second allele’s promoter region. TGF-β dependent induction of p21 and downregulation of minichromosome maintenance protein 2 was lost in >80% of BE and AC. TGF-β failed to inhibit proliferation in 5/8 oesophageal cell lines. In BIC-1, the antiproliferative response was restored following transient transfection of smad4 cDNA. Conclusions: In BE carcinogenesis, downregulation of Smad4 occurs due to several different mechanisms, including methylation, deletion, and protein modification. Frequent alterations in TGF-β signalling lead to a functionally significant impairment of TGF-β mediated growth suppression.

Promoter methylation correlates with reduced Smad4 expression in advanced prostate cancer

Transforming growth factor‐β (TGF‐β) is a potent growth inhibitor in a wide range of cell types. A transducer of TGF‐β signaling known as Mothers against decapentaplegic homologue 4 (Smad4) is a known tumor suppressor found on chromosome 18q21.1 and is typically inactivated by deletion or mutation in pancreatic and colorectal cancers. The purpose of the article is to investigate Smad4 expression, gene copy number and methylation status in advanced cases of prostate cancer.

Colonization with enterotoxigenic Bacteroides fragilis is associated with early-stage colorectal neoplasia

Background Enterotoxigenic Bacteroides fragilis (ETBF) is a toxin-producing bacteria thought to possibly promote colorectal carcinogenesis by modulating the mucosal immune response and inducing epithelial cell changes. Here, we aim to examine the association of colonic mucosal colonization with ETBF and the presence of a range of lesions on the colonic neoplastic spectrum. Methods Mucosal tissue from up to four different colonic sites was obtained from a consecutive series of 150 patients referred for colonoscopy. The presence and relative abundance of the B. fragilis toxin gene (bft) in each tissue sample was determined using quantitative PCR, and associations with clinicopathological characteristics were analysed. Findings We found a high concordance of ETBF between different colonic sites (86%). Univariate analysis showed statistically significant associations between ETBF positivity and the presence of low-grade dysplasia (LGD), tubular adenomas (TA), and serrated polyps (P-values of 0.007, 0.027, and 0.007, respectively). A higher relative abundance of ETBF was significantly associated with LGD and TA (P-values of < 0.0001 and 0.025, respectively). Increased ETBF positivity and abundance was also associated with left-sided biopsies, compared to those from the right side of the colon. Conclusion Our results showing association of ETBF positivity and increased abundance with early-stage carcinogenic lesions underlines its importance in the development of colorectal cancer, and we suggest that detection of ETBF may be a potential marker of early colorectal carcinogenesis.

G-quadruplex structures and CpG methylation cause drop-out of the maternal allele in polymerase chain reaction amplification of the imprinted MEST gene promoter.

We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5′ end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs) in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a T m of greater than 99°C in polymerase chain reaction (PCR) buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings.

Comparison of Fecal Inflammatory Markers in Crohn's Disease.

Background:Fecal biomarkers are used increasingly to monitor Crohns disease (CD). However, the relative accuracy of different markers in identifying inflammation has been poorly evaluated. We evaluated fecal calprotectin (FC), lactoferrin (FL), and S100A12 (FS) using endoscopic validation in a prospective study of the progression of CD after intestinal resection. Methods:Data were collected from 135 participants in a prospective, randomized, controlled trial aimed at preventing postoperative CD recurrence. Three hundred nineteen stool samples were tested for FC, FL, and FS preoperatively and 6, 12, and 18 months after resection. Colonoscopy was performed at 6 and/or 18 months. Endoscopic recurrence was assessed blindly using the Rutgeerts score. C-reactive protein (CRP) and Crohns Disease Activity Index (CDAI) were assessed. Results:FC, FL, and FS concentrations were elevated preoperatively (median: 1347, 40.9, and 8.4 &mgr;g/g, respectively). At 6 months postoperatively, marker concentrations decreased (166, 3.0, 0.9 &mgr;g/g) and were higher in recurrent disease than remission (275 versus 72 &mgr;g/g, P < 0.001; 5.7 versus 1.6 &mgr;g/g, P = 0.007; 2.0 versus 0.8 &mgr;g/g, P = 0.188). FC > 135 &mgr;g/g, FL > 3.4 &mgr;g/g, and FS > 10.5 &mgr;g/g indicated endoscopic recurrence (score ≥ i2) with a sensitivity, specificity, and negative predictive value (NPV) of 0.87, 0.66, and 91%; 0.70, 0.68, and 81%; 0.91, 0.12, and 71%, respectively. FC and FL correlated significantly with the presence and severity of endoscopic recurrence, whereas FS, CRP and CDAI did not. Conclusions:FC was the optimal fecal marker for monitoring disease activity in postoperative CD and was superior to CRP and CDAI. FL offered modest sensitivity for detecting recurrent disease, whereas S100A12 was sensitive but had low specificity and NPV.

Exome sequencing and array-based comparative genomic hybridisation analysis of preferential 6-methylmercaptopurine producers

Preferential conversion of azathioprine or 6-mercaptopurine into methylated metabolites is a major cause of thiopurine resistance. To seek potentially Mendelian causes of thiopurine hypermethylation, we recruited 12 individuals who exhibited extreme therapeutic resistance while taking azathioprine or 6-mercaptopurine and performed whole-exome sequencing (WES) and copy-number variant analysis by array-based comparative genomic hybridisation (aCGH). Exome-wide variant filtering highlighted four genes potentially associated with thiopurine metabolism (ENOSF1 and NFS1), transport (SLC17A4) or therapeutic action (RCC2). However, variants of each gene were found only in two or three patients, and it is unclear whether these genes could influence thiopurine hypermethylation. Analysis by aCGH did not identify any unusual or pathogenic copy-number variants. This suggests that if causative mutations for the hypermethylation phenotype exist they may be heterogeneous, occurring in several different genes, or they may lie within regulatory regions not captured by WES. Alternatively, hypermethylation may arise from the involvement of multiple genes with small effects. To test this hypothesis would require recruitment of large patient samples and application of genome-wide association studies.

The effect of polymeric formula on enterocyte differentiation.

Exclusive enteral nutrition is established as an initial therapy to induce remission in active Crohn’s disease (CD), especially in children, but the mechanisms of action of this therapy are yet to be fully defined. Intestinal alkaline phosphatase (IAP), a recognised marker of enterocyte differentiation, is implicated in the innate gut immune response to enteric pathogens. Using the Caco-2 human adenocarcinoma cell line, this study showed that the incubation of human cells with a polymeric formula (PF) resulted in a dose-dependent increase in the expression of IAP on the cell surface. While further investigation is required to determine the pathway(s) involved, this finding suggests that cell surface-associated IAP may be an aspect of the gut’s innate immune response to pathogenic bacteria that is strengthened by PF in the setting of CD.

Telomere Length Measurement on the Roche LightCycler 480 Platform

BACKGROUND The average length of telomeres as measured in genomic DNA from human peripheral blood leukocytes is proving to be a potential biomarker of great interest, particularly with respect to studies of aging, specific diseases, and the effects of various stresses on overall health. AIMS The aim of this study was to establish an effective real-time quantitative polymerase chain reaction (qPCR) method for telomere length measurement on the Roche LightCycler® 480 (LC480) real-time PCR platform. METHODS Measurement of relative average telomere length was achieved by comparing products amplified from telomere-specific primers and single copy reference gene primers in a ratio (T/S). RESULTS Extensive testing led us to conclude that a modification of the original two-plate T/S assay was more compatible with this platform than the recently developed single-plate assay, and that choice of hot-start Taq polymerase and intercalating dye were critical factors. CONCLUSIONS This modified assay generates reliable measurements as judged by correlation with data derived by the telomeric restriction fragment Southern blot-based method.

PCR Detection of the Bacteroides fragilis Enterotoxin Gene Relies on Robust Primer Design.

Colonic carriage of enterotoxigenic Bacteroides fragilis (ETBF) is reportedly m ore common in people presenting with colorectal cancer (CRC) when compared to healthy controls (1-3), fuelling speculation that persistent carriage of these bacteria may “drive” colon carcinogenesis (4).…