Mutation detection by clonal sequencing of PCR amplicons and grouped read typing is applicable to clinical diagnostics.

We describe a sensitive technique for mutation detection using clonal sequencing. We analyzed DNA extracted from 13 cancer cell lines and 35 tumor samples and applied a novel approach to identify disease-associated somatic mutations. By matching reads against an index of known variants, noise can be dramatically reduced, enabling the detection and quantification of those variants, even when they are present at less than 1% of the total sequenced population; this is comparable to, or better than, current diagnostic methods. Following the identification or exclusion of known variants, unmatched reads are grouped for BLAST searching to identify novel variants or contaminants. Known variants, novel variants, and contaminants were readily identified in tumor tissue using this approach. Our approach also enables an estimation of the per-base sequencing error rate, providing a confidence threshold for interpretation of the results in the clinic. This novel approach has immediate applicability to clinical testing for disease-associated genetic variants.

In vitro functional effects of XPC gene rare variants from bladder cancer patients.

The XPC gene is involved in repair of bulky DNA adducts formed by carcinogenic metabolites and oxidative DNA damage, both known bladder cancer risk factors. Single nucleotide polymorphisms (SNPs) in XPC have been associated with increased bladder cancer risk. Recently, rarer genetic variants have been identified but it is difficult to ascertain which are of functional importance. During a mutation screen of XPC in DNA from 33 bladder tumour samples and matched blood samples, we identified five novel variants in the patients' germ line DNA. In a case-control study of 771 bladder cancer cases and 800 controls, c.905T>C (Phe302Ser), c.1177C>T (Arg393Trp), c.*156G>A [3' untranslated region (UTR)] and c.2251-37C>A (in an intronic C>G SNP site) were found to be rare variants, with a combined odds ratio of 3.1 (95% confidence interval 1.0-9.8, P=0.048) for carriage of one variant. The fifth variant was a 2% minor allele frequency SNP not associated with bladder cancer. The two non-synonymous coding variants were predicted to have functional effects using analytical algorithms; a reduced recruitment of GFP-tagged XPC plasmids containing either c.905T>C or c.1177C>T to sites of 408 nm wavelength laser-induced oxidative DNA damage was found in vitro. c.*156G>A appeared to be associated with reduced messenger RNA stability in an in vitro plasmid-based assay. Although the laser microbeam assay is relevant to a range of DNA repair genes, our 3' UTR assay based on Green fluorescent protein(GFP) has widespread applicability and could be used to assess any gene. These assays may be useful in determining which rare variants are functional, prior to large genotyping efforts.

A mutation hotspot at the p14ARF splice site.

Germline mutations of CDKN2A that affect the p16INK4a transcript have been identified in numerous melanoma pedigrees worldwide. In the UK, over 50% of pedigrees with three or more cases of melanoma have been found to carry mutations of CDKN2A. Mutations that affect p14ARF exon 1beta exclusively are very rare. This has led to the suggestion that it is p16INK4a and not p14ARF that plays the critical role in melanoma predisposition. We report the identification of a cluster of five different germline mutations at the p14ARF exon 1beta splice donor site in melanoma pedigrees. All the five splice site variants showed evidence of being causal mutations. Three of the variants were demonstrated to result in aberrant splicing of the p14ARF mRNA, confirming their role in melanoma predisposition. No other point mutations were identified in the coding region of p14ARF. The p14ARF transcript of CDKN2A is clearly important in disease predisposition in a subset of melanoma pedigrees. Curiously, the only mutations so far reported to affect p14ARF exon 1beta exclusively have been knockout mutations. Further investigation into the spectrum of mutations observed in this gene may help clarify the exact role of p14ARF in melanoma predisposition.

No Evidence for BRAF as a melanoma/nevus susceptibility gene.

Somatic mutations of BRAF have been identified in both melanoma tumors and benign nevi. Germ line mutations in BRAF have not been identified as causal in families predisposed to melanoma. However, a recent study suggested that a BRAF haplotype was associated with risk of sporadic melanoma in men. Polymorphisms or other variants in the BRAF gene may therefore act as candidate low-penetrance genes for nevus/melanoma susceptibility. We hypothesized that promoter variants would be the most likely candidates for determinants of risk. Using denaturing high-pressure liquid chromatography and sequencing, we screened peripheral blood DNA from 184 familial melanoma cases for BRAF promoter variants. We identified a promoter insertion/deletion in linkage disequilibrium with the previously described BRAF polymorphism in intron 11 (rs1639679) reported to be associated with melanoma susceptibility in males. We therefore investigated the contribution of this BRAF polymorphism to melanoma susceptibility in 581 consecutively recruited incident cases, 258 incident cases in a study of late relapse, 673 female general practitioner controls, and the 184 familial cases. We found no statistically significant difference in either genotype or allele frequencies between cases and controls overall or between male and female cases for the BRAF polymorphism in the two incident case series. Our results therefore suggest that the BRAF polymorphism is not significantly associated with melanoma and the promoter insertion/deletion linked with the polymorphism is not a causal variant. In addition, we found that there was no association between the BRAF genotype and mean total number of banal or atypical nevi in either the cases or controls.

Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis.

BACKGROUND: MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR-92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR-92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR-92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR-92 expression by qRT-PCR. Expression of ERß1, a direct miR-92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR-92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR-92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERß1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERß1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR-92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR-92 levels in NFs but not CAFs enhanced invasion of both MCF-7 and MDA-MB-231 breast cancer epithelial cells. CONCLUSIONS: miR-92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERß1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR-92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERß1 may not be the most important miR-92 target in breast cancer.

Ischemia- reperfusion injury and its influence on the epigenetic modification of the donor kidney genome.

BACKGROUND: In clinical transplantation, ischemia-reperfusion injury (I/RI) causes damage to DNA. We hypothesize that one form of damage is the demethylation of methylated cytosines in the donor genome caused by the oxidative environment created first by ischemia, and subsequently by reperfusion on transplantation. This study contributes to the understanding of how the short-lived and transient ischemic insult may influence chronic pathological changes that occur in clinical transplantation in the long term. METHODS: A model of I/RI and chronic rejection; Fisher to Fisher kidney transplant rendered cold-ischemic for 4 hr before transplantation, to induce antigen-independent chronic nephropathy over a 6-month period, was used. Tissue was assessed by histopathology and methylation by pyrosequencing analysis. RESULTS: An epigenetic map of the rat renal C3 promoter was produced, which identified methylated Cytosine phospho Guanine (CpG) sites coincident to cytokine response elements and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) binding sites. Pyrosequencing analysis showed that the tissue that had undergone 4 hr ischemia and reperfusion developed aberrant demethylation of cytosines in putative regulatory sites within the C3 promoter. CONCLUSION: These findings may describe a newly recognized phenomena in the field of transplantation. Aberrant demethylation has long been linked to the development of tumors, and our data suggest a similar mechanism of gene dysregulation that may be initiated by I/RI with acute and chronic effects. These data may contribute to a further understanding of how the short lived and transient ischemic insult influences chronic pathological changes that occur even in the absence of major histocompatibility complex disparity in transplantation.

SHV-34: an extended-spectrum beta-lactamase encoded by an epidemic plasmid.

OBJECTIVES: To elucidate the causes for treatment failure in children given extended-spectrum cephalosporins. METHODS: During April 1998-March 2000, 18 isolates of members of the family Enterobacteriaceae, fulfilling microbiological criteria for carriage of extended-spectrum beta-lactamases (ESBLs) and carrying blaSHV, were isolated from paediatric inpatients. The collection was subjected to a retrospective molecular analysis. RESULTS: Three species were represented in the collection: Citrobacter koseri (one isolate), Escherichia coli (one isolate) and Klebsiella pneumoniae (16 isolates). A common plasmid was found in these bacteria, as judged by restriction endonuclease digestion. This was able to transfer an ESBL phenotype from donors to a laboratory strain of E. coli. Nucleotide sequence analysis revealed that this phenotype was associated with a new variant in blaSHV encoding SHV-34. CONCLUSIONS: Analysis reveals the presence of an epidemic plasmid in this collection of bacteria. This carries a gene encoding the SHV-34 ESBL, described for the first time in this report. Nucleotide sequence analysis shows that there is a mutation from A-->G affecting the codon at amino acid position 64 (GAA-->GGA), changing the glutamic acid typically seen in this position to glycine.

Antibiotic resistance in general dental practice--a cause for concern?

This review examines the contribution dental prescribing makes to the selection of antibiotic resistance in bacteria of the oral flora. The antibiotics commonly used in dental prescribing in the UK are discussed, together with the problems of resistance in members of the oral flora. The antibiotic prescribing habits of general dental practitioners are then reviewed with respect to therapeutic prescriptions and those drugs that are prescribed prophylactically. Not all antibiotic prescriptions for dental problems are written by dentists; prescribing outside the dental profession is also considered. The review then considers the support available to dentists from clinical diagnostic microbiology laboratories. It concludes that better use of diagnostic services, surveillance and improvements in dental education are required now to lessen the impact of antibiotic resistance in the future.

The fate of antibiotic resistance marker genes in transgenic plant feed material fed to chickens.

We have examined the fate of an antibiotic resistance marker, incorporated into transgenic maize when fed to chicks. Plant-derived markers were found in the crops of five birds fed transgenic maize and in the stomach contents of two birds. The plant-derived marker gene was not found in the intestines. The survival of the antibiotic resistance marker gene mirrored that of plant DNA targets, demonstrating that it survives no better than other DNA and indicating that it is very unlikely that bacteria in the gut of chickens will be transformed to ampicillin resistance when the birds are fed transgenic maize.

Fate of genetically modified maize DNA in the oral cavity and rumen of sheep.

The polymerase chain reaction (PCR) technique was used to investigate the fate of a transgene in the rumen of sheep fed silage and maize grains from an insect-resistant maize line. A 1914-bp DNA fragment containing the entire coding region of the synthetic cryIA(b) gene was still amplifiable from rumen fluid sampled 5 h after feeding maize grains. The same target sequence, however, could not be amplified from rumen fluid sampled from sheep fed silage prepared from the genetically modified maize line. PCR amplification of a shorter (211-bp), yet still highly specific, target sequence was possible with rumen fluid sampled up to 3 and 24 h after feeding silage and maize grains, respectively. These findings indicate that intact transgenes from silage are unlikely to survive significantly in the rumen since a DNA sequence 211-bp long is very unlikely to transmit genetic information. By contrast, DNA in maize grains persists for a significant time and may, therefore, provide a source of transforming DNA in the rumen. In addition, we have examined the biological activity of plasmid DNA that had previously been exposed to the ovine oral cavity. Plasmid extracted from saliva sampled after incubation for 8 min was still capable of transforming competent Escherichia coli to kanamycin resistance, implying that DNA released from the diet within the mouth may retain sufficient biological activity for the transformation of competent oral bacteria.