Janet A. Warrington

Characterization of single-nucleotide polymorphisms in coding regions of human genes

Nature Genet. 14, 415– 420 (1996). Due to a cloning error, the sequence reported for ING1 was incorrect. The error appears to have been a result of a compression introducing a frameshift and of the ING1 gene encoding several differentially spliced isoforms that contain a common 3′ exon, one of whichis of a size very similar to that reported in the publication above.

Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes

The synthesis of fatty acids and cholesterol, the building blocks of membranes, is regulated by three membrane-bound transcription factors: sterol regulatory element-binding proteins (SREBP)-1a, -1c, and -2. Their function in liver has been characterized in transgenic mice that overexpress each SREBP isoform and in mice that lack all three nuclear SREBPs as a result of gene knockout of SREBP cleavage-activating protein (SCAP), a protein required for nuclear localization of SREBPs. Here, we use oligonucleotide arrays hybridized with RNA from livers of three lines of mice (transgenic for SREBP-1a, transgenic for SREBP-2, and knockout for SCAP) to identify genes that are likely to be direct targets of SREBPs in liver. A total of 1,003 genes showed statistically significant increased expression in livers of transgenic SREBP-1a mice, 505 increased in livers of transgenic SREBP-2 mice, and 343 showed decreased expression in Scap–/– livers. A subset of 33 genes met the stringent combinatorial criteria of induction in both SREBP transgenics and decreased expression in SCAP-deficient mice. Of these 33 genes, 13 were previously identified as direct targets of SREBP action. Of the remaining 20 genes, 13 encode enzymes or carrier proteins involved in cholesterol metabolism, 3 participate in fatty acid metabolism, and 4 have no known connection to lipid metabolism. Through application of stringent combinatorial criteria, the transgenic/knockout approach allows identification of genes whose activities are likely to be controlled directly by one family of transcription factors, in this case the SREBPs.

Mutations in the gene encoding cystatin B in progressive myoclonus epilepsy (EPM1)

Progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1) is an autosomal recessive inherited form of epilepsy, previously linked to human chromosome 21q22.3. The gene encoding cystatin B was shown to be localized to this region, and levels of messenger RNA encoded by this gene were found to be decreased in cells from affected individuals. Two mutations, a 3′ splice site mutation and a stop codon mutation, were identified in the gene encoding cystatin B in EPM1 patients but were not present in unaffected individuals. These results provide evidence that mutations in the gene encoding cystatin B are responsible for the primary defect in patients with EPM1.

Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse

Single-nucleotide polymorphisms (SNPs) have been the focus of much attention in human genetics because they are extremely abundant and well-suited for automated large-scale genotyping. Human SNPs, however, are less informative than other types of genetic markers (such as simple-sequence length polymorphisms or microsatellites) and thus more loci are required for mapping traits. SNPs offer similar advantages for experimental genetic organisms such as the mouse, but they entail no loss of informativeness because bi-allelic markers are fully informative in analysing crosses between inbred strains. Here we report a large-scale analysis of SNPs in the mouse genome. We characterized the rate of nucleotide polymorphism in eight mouse strains and identified a collection of 2,848 SNPs located in 1,755 sequence-tagged sites (STSs) using high-density oligonucleotide arrays. Three-quarters of these SNPs have been mapped on the mouse genome, providing a first-generation SNP map of the mouse. We have also developed a multiplex genotyping procedure by which a genome scan can be performed with only six genotyping reactions per animal.

BRCA1 transcriptionally regulates genes involved in breast tumorigenesis

Loss of function of BRCA1 caused by inherited mutation and tissue-specific somatic mutation leads to breast and ovarian cancer. Nearly all BRCA1 germ-line mutations involve truncation or loss of the C-terminal BRCT transcriptional activation domain, suggesting that transcriptional regulation is a critical function of the wild-type gene. The purpose of this project was to determine whether there is a link between the role of BRCA1 in transcriptional regulation and its role in tumor suppression. We developed a cell line (in which BRCA1 can be induced) and used microarray analysis to compare transcription profiles of epithelial cells with low endogenous levels of BRCA1 vs. transcription profiles of cells with 2–4-fold higher induced levels of expression of BRCA1. At these levels of expression, BRCA1 did not induce apoptosis. Undirected cluster analysis of six paired experiments revealed 373 genes, the expression of which was altered significantly and consistently by BRCA1 induction. Expression of 62 genes was altered more than 2-fold. BRCA1-regulated genes associated with breast tumorigenesis included the estrogen-responsive genes MYC and cyclin D1, which are overexpressed in many breast tumors; STAT1 and JAK1, key components of the cytokine signal transduction pathway; the extracellular matrix protein laminin 3A; ID4, an inhibitor of DNA-binding transcriptional activators, which in turn negatively regulates BRCA1 expression; and the prohormone stanniocalcin, expression of which is lost in breast tumor cells. Coordinated expression of BRCA1 with ID4 and with stanniocalcin was confirmed in primary breast and ovarian tumors.

Oral cancer in vivo gene expression profiling assisted by laser capture microdissection and microarray analysis.

Large scale gene expression profiling was carried out on laser capture microdissected (LCM) tumor and normal oral epithelial cells and analysed on high-density oligonucleotide microarrays. About 600 genes were found to be oral cancer associated. These oral cancer associated genes include oncogenes, tumor suppressors, transcription factors, xenobiotic enzymes, metastatic proteins, differentiation markers, and genes that have not been implicated in oral cancer. The database created provides a verifiable global profile of gene expression during oral carcinogenesis, revealing the potential role of known genes as well as genes that have not been previously implicated in oral cancer.

The External RNA Controls Consortium: a progress report

Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.Standard controls and best practice guidelines advance acceptance of data from research, preclinical and clinical laboratories by providing a means for evaluating data quality. The External RNA Controls Consortium (ERCC) is developing commonly agreed-upon and tested controls for use in expression assays, a true industry-wide standard control.

MOLECULAR GENETIC PROFILING OF GLEASON GRADE 4/5 PROSTATE CANCERS COMPARED TO BENIGN PROSTATIC HYPERPLASIA

PURPOSE Because Gleason grade 4/5 cancer is the primary cause of failure to cure prostate cancer, we examined the molecular profiles of this high grade cancer in search of potentially new therapeutic interventions as well as better serum markers than prostate specific antigen. MATERIALS AND METHODS We compared the gene expressions in fresh frozen tissues from 9 men with Gleason grade 4/5 cancer to 8 men with benign prostatic hyperplasia (BPH) treated with radical retropubic prostatectomy. Labeled complementary RNA from each of the 17 tissues was applied to HuGeneFL probe arrays representing approximately 6,800 genes (Affymetrix, Inc., Santa Clara, California). After removing all genes undetectable in BPH and grade 4/5 cancers, and transforming the data into a parametric distribution, we chose only those up and down-regulated genes with a p difference in fluorescence between grade 4/5 cancer and BPH of p <0.0005. This value reduced the data set to 40 up-regulated and 111 down-regulated genes. We then eliminated all genes that were not expressed in all 8 BPH and 9 grade 4/5 tissues, which produced a final set of 86 genes, of which 22 were up-regulated and 64 were down-regulated. RESULTS Cluster analysis cleanly separated men with grade 4/5 cancers from those with BPH. Only 17 of the 86 candidate genes (20%) were known to be prostate cancer related and 42 (49%) were related to other cancers. The most up-regulated gene is Hepsin, a trypsin-like serine protease with its enzyme catalytic domain oriented extracellularly. Prostate specific membrane antigen is the second most up-regulated gene (all other reports on prostate specific membrane antigen have been at the protein level). The genes for prostate specific antigen (hK3) and human glandular kallikrein2 (hK2) showed equivalent expression levels 10 times the average of other genes. Complete lists of all 22 up-regulated genes and 64 down-regulated genes, together with their locus on the chromosome, are presented in rank order. CONCLUSIONS We characterize for the first time 64 down-regulated and 22 up-regulated genes in Gleason grade 4/5 cancer, using the gene profile from BPH as control tissue. A number of interesting new genes, previously undescribed in prostate cancer, are presented as possibilities for further study.

Tissue Handling and Specimen Preparation in Surgical Pathology: Issues Concerning the Recovery of Nucleic Acids From Formalin-Fixed, Paraffin-Embedded Tissue

CONTEXT Expression profiling by microarrays and real-time polymerase chain reaction-based assays is a powerful tool for classification and prognostication of disease; however, it remains a research tool, largely reliant on frozen tissue. Limiting the utility of expression profiling is the isolation of quality nucleic acids from formalin-fixed, paraffin-embedded tissue. The collection, handling, and processing of tissue directly impacts the biomolecules that can be recovered from it. High-quality nucleic acids can be obtained from formalin-fixed, paraffin-embedded tissue, but greater attention to all steps in the process of tissue handling and preparation is required. OBJECTIVE To summarize the current state-of-the-art of preanalytic factors in tissue handling and processing as they impact the quality of RNA obtainable from formalin-fixed, paraffin-embedded tissue. The goals are to provide recommendations that will improve RNA quality for expression profiling from formalin-fixed, paraffin-embedded tissue and highlight areas for additional research. Tissue is an analyte and it must be handled in a standardized fashion to provide consistent results. DATA SOURCES The literature was reviewed. Consultation with industry and academic leaders in the use of RNA for expression profiling was obtained to identify areas for additional research. CONCLUSIONS Development of RNA-based assays from formalin-fixed, paraffin-embedded tissue is feasible. Greater attention to tissue handling and processing is essential to improve the quality of biospecimens for the development of robust RNA-based assays. Standardization of procedures and vigorous testing of alternative protocols are required to ensure that these assays function as designed.

Using RNA sample titrations to assess microarray platform performance and normalization techniques

We have assessed the utility of RNA titration samples for evaluating microarray platform performance and the impact of different normalization methods on the results obtained. As part of the MicroArray Quality Control project, we investigated the performance of five commercial microarray platforms using two independent RNA samples and two titration mixtures of these samples. Focusing on 12,091 genes common across all platforms, we determined the ability of each platform to detect the correct titration response across the samples. Global deviations from the response predicted by the titration ratios were observed. These differences could be explained by variations in relative amounts of messenger RNA as a fraction of total RNA between the two independent samples. Overall, both the qualitative and quantitative correspondence across platforms was high. In summary, titration samples may be regarded as a valuable tool, not only for assessing microarray platform performance and different analysis methods, but also for determining some underlying biological features of the samples.