High-throughput method for analyzing methylation of CpGs in targeted genomic regions.

A unique microarray-based method for determining the extent of DNA methylation has been developed. It relies on a selective enrichment of the regions to be assayed by target amplification by capture and ligation (mTACL). The assay is quantitatively accurate, relatively precise, and lends itself to high-throughput determination using nanogram amounts of DNA. The measurements using mTACLs are highly reproducible and in excellent agreement with those obtained by sequencing (r = 0.94). In the present work, the methylation status of >145,000 CpGs from 5,472 promoters in 221 samples was measured. The methylation levels of nearby CpGs are correlated, but the correlation falls off dramatically over several hundred base pairs. In some instances, nearby CpGs have very different levels of methylation. Comparison of normal and tumor samples indicates that in tumors, the promoter regions of genes involved in differentiation and signaling are preferentially hypermethylated, whereas those of housekeeping genes remain hypomethylated. mTACL is a platform for profiling the state of methylation of a large number of CpG in many samples in a cost-effective fashion, and is capable of scaling to much larger numbers of CpGs than those collected here.

Prediction of epigenetically regulated genes in breast cancer cell lines.

BACKGROUND: Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profiles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP). The pipeline (i) reduces the dimensionality of the methylation data, (ii) associates the reduced methylation data with gene expression data, and (iii) ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i) methylation sites are grouped across the genome to identify regions of interest, and (ii) methylation profiles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fixed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. RESULTS: Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically significant negative correlation between methylation profiles and gene expression in the panel of breast cancer cell lines. Subnetwork enrichment of these genes has identified 35 common regulators with 6 or more predicted markers. In addition to identifying epigenetically regulated genes, we show evidence of differentially expressed methylation patterns between the basal and luminal subtypes. CONCLUSIONS: Our results indicate that the proposed computational protocol is a viable platform for identifying epigenetically regulated genes. Our protocol has generated a list of predictors including COL1A2, TOP2A, TFF1, and VAV3, genes whose key roles in epigenetic regulation is documented in the literature. Subnetwork enrichment of these predicted markers further suggests that epigenetic regulation of individual genes occurs in a coordinated fashion and through common regulators.

Incidence and Preoperative Predictive Indicators of Incudal Necrosis in CSOM: A Prospective Study in a Tertiary Care Centre.

Both type of CSOM, tubotympanic which is considered safe as well as atticoantral which is considered unsafe may lead to erosion of the ossicular chain. Discontinuity of the ossicular chain is typically confirmed only during an operation. Knowing before surgery whether the patient has an ossicular discontinuity is important because it allows the surgeon to know the possibility of performing an ossiculoplasty and obtaining patient consent. The aims is to (1) study the incidence of incus necrosis in safe and unsafe CSOM. (2) Determine the preoperative predictive factors for incus necrosis. (3) Use angled otoscopes to determine the incidence of residual disease peroperative after conventional microscopic surgery. This is a prospective study carried out in the department of otorhinolaryngology, Govt Doon medical college, Dehradun from July 2014 to July 2016. A total of 100 patients who presented with CSOM and have not undergone any surgical procedure for the same were included in this study. Patients group was divided into cholesteatoma and non cholesteatoma group. Both groups were subdivided into intact and eroded incus group and were analysed in 11 parameters which were compared statistically. Incudal necrosis is more common in cholesteatoma group. In non cholesteatoma ears subtotal perforation with exposure of IS joint is reliable indicators of incudal necrosis. In non cholesteatoma group extension of cholesteatoma to tympanic sinus and mastoid and presence of persistent discharge are reliable indicators of necrosed incus. Moderate to moderately severe hearing loss indicate incudal necrosis in both groups therefore we conclude that these parameters can be reliably considered as predictors for incudal necrosis preoperatively.

Basal Cell Adenoma of Parotid Gland: Case Report and Review of Literature.

Basal cell adenoma (BCA) of the salivary gland is a rare neoplasm consists of a monomorphic population of basaloid epithelial cells, and it accounts for approximately 1-2 % of all salivary gland tumors. Its most frequent location is the parotid gland. It usually appears as a firm and mobile slow-growing mass. Histologically, isomorphic cells in nests and interlaced trabecules with a prominent basal membrane are observed. In contrast to pleomorphic adenoma, it tends to be multiple and its recurrence rate after surgical excision is high. Due to prognostic implications, differential diagnosis with basal cell adenocarcinoma, adenoid cystic carcinoma and basaloid squamous cell carcinoma is mandatory. We report a case of BCA of the parotid gland. We also review the literature and discuss the diagnosis and management of this rare entity.

The genome-wide expression response to telomerase deletion in Saccharomyces cerevisiae.

Loss of the protective function of telomeres has previously been hypothesized to cause a DNA damage response. Here, we report a genome-wide expression response, the telomerase deletion response (TDR), that occurs when telomeres can no longer be maintained by telomerase. The TDR shares features with other DNA damage responses and the environmental stress response. Unexpectedly, another feature of the TDR is the up-regulation of energy production genes, accompanied by a proliferation of mitochondria. Finally, a discrete set of genes, the "telomerase deletion signature", is uniquely up-regulated in the TDR but not under other conditions of stress and DNA damage that have been reported. The telomerase deletion signature genes define new candidates for involvement in cellular responses to altered telomere structure or function.