High-depth, high-accuracy microsatellite genotyping enables precision lung cancer risk classification.

There remains a large discrepancy between the known genetic contributions to cancer and that which can be explained by genomic variants, both inherited and somatic. Recently, understudied repetitive DNA regions called microsatellites have been identified as genetic risk markers for a number of diseases including various cancers (breast, ovarian and brain). In this study, we demonstrate an integrated process for identifying and further evaluating microsatellite-based risk markers for lung cancer using data from the cancer genome atlas and the 1000 genomes project. Comparing whole-exome germline sequencing data from 488 TCGA lung cancer samples to germline exome data from 390 control samples from the 1000 genomes project, we identified 119 potentially informative microsatellite loci. These loci were found to be able to distinguish between cancer and control samples with sensitivity and specificity ratios over 0.8. Then these loci, supplemented with additional loci from other cancers and controls, were evaluated using a target enrichment kit and sample-multiplexed nextgen sequencing. Thirteen of the 119 risk markers were found to be informative in a well powered study (>0.99 for a 0.95 confidence interval) using high-depth (579x±315) nextgen sequencing of 30 lung cancer and 89 control samples, resulting in sensitivity and specificity ratios of 0.90 and 0.94, respectively. When 8 loci harvested from the bioinformatic analysis of other cancers are added to the classifier, then the sensitivity and specificity rise to 0.93 and 0.97, respectively. Analysis of the genes harboring these loci revealed two genes (ARID1B and REL) and two significantly enriched pathways (chromatin organization and cellular stress response) suggesting that the process of lung carcinogenesis is linked to chromatin remodeling, inflammation, and tumor microenvironment restructuring. We illustrate that high-depth sequencing enables a high-precision microsatellite-based risk classifier analysis approach. This microsatellite-based platform confirms the potential to create clinically actionable diagnostics for lung cancer.

Microsatellite genotyping reveals a signature in breast cancer exomes.

Genomic instability at microsatellite loci is a hallmark of many cancers, including breast cancer. However, much of the genomic variation and many of the hereditary components responsible for breast cancer remain undetected. We hypothesized that variation at microsatellites could provide additional genomic markers for breast cancer risk assessment. A total of 1,345 germline and tumor DNA samples from individuals diagnosed with breast cancer, exome sequenced as part of The Cancer Genome Atlas, were analyzed for microsatellite variation. The comparison group for our analysis, representing healthy individuals, consisted of 249 females which were exome sequenced as part of the 1,000 Genomes Project. We applied our microsatellite-based genotyping pipeline to identify 55 microsatellite loci that can distinguish between the germline of individuals diagnosed with breast cancer and healthy individuals with a sensitivity of 88.4 % and a specificity of 77.1 %. Further, we identified additional microsatellite loci that are potentially useful for distinguishing between breast cancer subtypes, revealing a possible fifth subtype. These findings are of clinical interest as possible risk diagnostics and reveal genes that may be of potential therapeutic value, including genes previously not associated with breast cancer.

Population-scale analysis of human microsatellites reveals novel sources of exonic variation.

Using our microsatellite specific genotyping method, we analyzed tandem repeats, which are known to be highly variable with some recognized as biomarkers causative of disease, in over 500 individuals who were exon sequenced in a 1000 Genomes Project pilot study. We were able to genotype over 97% of the microsatellite loci in the targeted regions. A total of 25,115 variations were observed, including repeat length and single nucleotide polymorphisms, corresponding to an average of 45.6 variations per individual and a density of 1.1 variations per kilobase. Standard variant detection did not report 94.2% of the exonic repeat length variations in part because the alignment techniques are not ideal for repetitive regions. Additionally some standard variation detection tools rely on a database of known variations, making them less likely to call repeat length variations as only a small percent of these loci (~6000) have been accurately characterized. A subset of the hundreds of non-synonymous variations we identified was experimentally validated, indicating an accuracy of 96.5% for our microsatellite-based genotyping method, with some novel variants identified in genes associated with cancer. We propose that microsatellite-based genotyping be used as a part of large scale sequencing studies to identify novel variants.

Evaluation of microsatellite variation in the 1000 Genomes Project pilot studies is indicative of the quality and utility of the raw data and alignments.

We performed an analysis of global microsatellite variation on the two kindreds sequenced at high depth (~20×-60×) in the 1000 Genomes Project pilot studies because alterations in these highly mutable repetitive sequences have been linked with many phenotypes and disease risks. The standard alignment technique performs poorly in microsatellite regions as a consequence of low effective coverage (~1×-5×) resulting in 79% of the informative loci exhibiting non-Mendelian inheritance patterns. We used a more stringent approach in computing robust allelotypes resulting in 94.4% of the 1095 informative repeats conforming to traditional inheritance. The high-confidence allelotypes were analyzed to obtain an estimate of the minimum polymorphism rate as a function of motif length, motif sequence, and distribution within the genome.

Combating unethical publications with plagiarism detection services.

About 3,000 new citations that are highly similar to citations in previously published manuscripts that appear each year in the biomedical literature (Medline) alone. This underscores the importance for the opportunity for editors and reviewers to have detection system to identify highly similar text in submitted manuscripts so that they can then review them for novelty. New software-based services, both commercial and free, provide this capability. The availability of such tools provides both a way to intercept suspect manuscripts and serve as a deterrent. Unfortunately, the capabilities of these services vary considerably, mainly as a consequence of the availability and completeness of the literature bases to which new queries are compared. Most of the commercial software has been designed for detection of plagiarism in high school and college papers; however, there is at least 1 fee-based service (CrossRef) and 1 free service (etblast.org), which are designed to target the needs of the biomedical publication industry. Information on these various services, examples of the type of operability and output, and things that need to be considered by publishers, editors, and reviewers before selecting and using these services is provided.

A long AAAG repeat allele in the 5' UTR of the ERR-γ gene is correlated with breast cancer predisposition and drives promoter activity in MCF-7 breast cancer cells.

We sequenced the 5' UTR of the estrogen-related receptor gamma gene (ERR-γ) in ~500 patient and volunteer samples and found that longer alleles of the (AAAG)(n) microsatellite were statistically and significantly more likely to exist in the germlines of breast cancer patients when compared to healthy volunteers. This microsatellite region contains multiple binding sites for a number of transcription factors, and we hypothesized that the polymorphic AAAG-containing sequence in the 5' UTR region of ERR-γ might modulate expression of ERR-γ. We found that the 369 bp PCR product containing the AAAG repeat drove expression of a reporter gene in estrogen receptor positive breast cancer cells. Our results support a role for the 5' UTR region in ERR-γ expression, which is potentially mediated via binding to the variable tandem AAAG repeat, the length of which correlates with breast cancer pre-disposition. Our study indicates that the AAAG tetranucleotide repeat polymorphism in ERR-γ gene 5' UTR region may be a new biomarker for genetic susceptibility to breast cancer.

Comparative analysis of the early transcriptome of Brucella abortus--infected monocyte-derived macrophages from cattle naturally resistant or susceptible to brucellosis.

Brucellosis is a worldwide zoonotic infectious disease that has a significant economic impact on animal production and human public health. We characterized the gene expression profile of B. abortus-infected monocyte-derived macrophages (MDMs) from naïve cattle naturally resistant (R) or susceptible (S) to brucellosis using a cDNA microarray technology. Our data indicate that (1) B. abortus induced a slightly increased genome activation in R MDMs and a down-regulated transcriptome in S MDMs, during the onset of infection, (2) R MDMs had the ability to mount a type 1 immune response against B. abortus infection which was impaired in S cells, and (3) the host cell activity was not altered after 12 h post-B. abortus infection in R MDMs while the cell cycle was largely arrested in infected S MDMs at 12 h p.i. These results contribute to an improved understanding of how host responses may be manipulated to prevent infection by brucellae.

Global microsatellite content distinguishes humans, primates, animals, and plants.

Microsatellites are highly mutable, repetitive sequences commonly used as genetic markers, but they have never been studied en masse. Using a custom microarray to measure hybridization intensities of every possible repetitive nucleotide motif from 1-mers to 6-mers, we examined 25 genomes. Here, we show that global microsatellite content varies predictably by species, as measured by array hybridization signal intensities, correlating with established taxonomic relationships, and particular motifs are characteristic of one species versus another. For instance, hominid-specific microsatellite motifs were identified despite alignment of the human reference, Celera, and Venter genomic sequences indicating substantial variation (30-50%) among individuals. Differential microsatellite motifs were mainly associated with genes involved in developmental processes, whereas those found in intergenic regions exhibited no discernible pattern. This is the first description of a method for evaluating microsatellite content to classify individual genomes.

Construction and evaluation of an automated light directed protein-detecting microarray synthesizer.

We have designed, constructed, and evaluated an automated instrument that has produced high-density arrays with more than 30 000 peptide features within a 1.5 cm(2) area of a glass slide surface. These arrays can be used for high throughput library screening for protein binding ligands, for potential drug candidate molecules, or for discovering biomarkers. The device consists of a novel fluidics system, a relay control electrical system, an optics system that implements Texas Instruments' digital micromirror device (DMD), and a microwave source for accelerated synthesis of peptide arrays. The instrument implements two novel solid phase chemical synthesis strategies for producing peptide and peptoid arrays. Biotin-streptavidin and DNP anti-DNP (dinitrophenol) models of antibody small molecule interactions were used to demonstrate and evaluate the instrument's capability to produce high-density protein detecting arrays. Several screening assay and detection schemes were explored with various levels of efficiency and assays with sensitivity of 10 nM were also possible.

Heuristics for identification of acronym-definition patterns within text: towards an automated construction of comprehensive acronym-definition dictionaries.

OBJECTIVES: To develop an automated, accurate and scalable method by which acronym-definition pairs can be identified within text. Its primary advantage is in enabling information processing methods to resolve author-defined acronyms, but it also allows an automated creation of a reference work on acronym definitions. This has several advantages over manual or semi-automated methods, besides time and effort saved, such as enabling identification of relative frequencies for alternate acronyms and definitions as well as spelling, phrasing and hyphenation variants for a unique acronym-definition pair. It also aids users in identifying acronym/definition variants present in the literature that may not necessarily be in biomedical databases. METHODS: A set of heuristics to accurately locate and identify the boundaries of acronym-definition pairs was developed and refined in terms of precision and recall on subsets of MEDLINE records. These training sets were gradually increased in size and heuristics re-evaluated to ensure scalability. RESULTS: Our final set of Acronym Resolving General Heuristics (ARGH) had a sample-based estimated rate of 96.5 +/- 0.4% precision and 93.0 +/- 2.7% recall when tested on over 12 million MEDLINE records, identifying more than 174,000 unique acronyms and their 737,000 associated definitions. CONCLUSIONS: We estimate that as much as 36% of the acronyms in MEDLINE are associated with more than one definition and, conversely, up to 10% of definitions are associated with more than one acronym. The number of unique acronyms in MEDLINE is increasing at a rate of approximately 11,000 per year, while the number of definitions associated with them is growing at approximately four times that rate. Access to the ARGH database is available online at http://lethargy.swmed.edu/ARGH/argh.asp. The heuristic module and database are available upon request.

SNPCEQer: detecting SNPs in sequences generated by the Beckman CEQ2000 DNA Analysis System.

SNPCEQer identifies and reports SNPs in sequences obtained from the Beckman CEQ2000 DNA Analysis System. SNPCEQer aligns sequences obtained using CEQ2000 heterozygote detection analysis and reports discrepancies between individual sequences and the consensus sequence it generates from this set as SNPs when the individual base calls have high-quality values. SNPCEQer reported comparable numbers of SNPs to the UNIX-based PolyPhred (148 vs. 165, respectively) in regions amplified from eight genes. A total of 21 different SNPs was discovered. Each gene region was analyzed in 96-306 samples. SNPCEQer was designed to operate from Windows NT, making SNP detection more accessible to users without UNIX systems. SNPCEQer is available free of charge at http://innovation.swmed.edu.

MarC-V: a spreadsheet-based tool for analysis, normalization, and visualization of single cDNA microarray experiments.

The comprehensive analysis and visualization of data extracted from cDNA microarrays can be a time-consuming and error-prone process that becomes increasingly tedious with increased number of gene elements on a particular microarray. With the increasingly large number of gene elements on today's microarrays, analysis tools must be developed to meet this challenge. Here, we present MarC-V, a Microsoft Excel spreadsheet tool with Visual Basic macros to automate much of the visualization and calculation involved in the analysis process while providing the familiarity and flexibility of Excel. Automated features of this tool include (i) lower-bound thresholding, (ii) data normalization, (iii) generation of ratio frequency distribution plots, (iv) generation of scatter plots color-coded by expression level, (v) ratio scoring based on intensity measurements, (vi) filtering of data based on expression level or specific gene interests, and (vii) exporting data for subsequent multi-array analysis. MarC-V also has an importing function included for GenePix results (GPR) raw data files.

Searching for microsatellite mutations in coding regions in lung, breast, ovarian and colorectal cancers.

RepX represents a new informatics approach to probe the UniGene database for potentially polymorphic repeat sequences in the open reading frame (ORF) of genes, 56% of which were found to be actually polymorphic. We now have performed mutational analysis of 17 such sites in genes not found to be polymorphic (<0.03 frequency) in a large panel of human cancer genomic DNAs derived from 31 lung, 21 breast, seven ovarian, 21 (13 microsatellite instability (MSI)+ and eight MSI-) colorectal cancer cell lines. In the lung, breast and ovarian tumor DNAs we found no mutations (<0.03-0.04 rate of tumor associated open reading frame mutations) in these sequences. By contrast, 18 MSI+ colorectal cancers (13 cancer cell lines and five primary tumors) with mismatch repair defects exhibited six mutations in three of the 17 genes (SREBP-2, TAN-1, GR6) (P<0.000003 compared to all other cancers tested). We conclude that coding region microsatellite alterations are rare in lung, breast, ovarian carcinomas and MSI (-) colorectal cancers, but are relatively frequent in MSI (+) colorectal cancers with mismatch repair deficits.

Hyperspectral imaging: a novel approach for microscopic analysis.

BACKGROUND: The usefulness of the light microscope has been dramatically enhanced by recent developments in hardware and software. However, current technologies lack the ability to capture and analyze a high-resolution image representing a broad diversity of spectral signatures in a single-pass view. We show that hyperspectral imaging offers such a technology. METHODS AND RESULTS We developed a prototype hyperspectral imaging microscope capable of collecting the complete emission spectrum from a microscope slide. A standard epifluorescence microscope was optically coupled to an imaging spectrograph, with output recorded by a CCD camera. Software was developed for image acquisition and computer display of resultant X--Y images with spectral information. Individual images were captured representing Y-wavelength planes, with the stage successively moved in the X direction, allowing an image cube to be constructed from the compilation of generated scan files. This prototype instrument was tested with samples relevant to cytogenetic, histologic, cell fusion, microarray scanning, and materials science applications. CONCLUSIONS: Hyperspectral imaging microscopy permits the capture and identification of different spectral signatures present in an optical field during a single-pass evaluation, including molecules with overlapping but distinct emission spectra. This instrument can reduce dependence on custom optical filters and, in future imaging applications, should facilitate the use of new fluorophores or the simultaneous use of similar fluorophores.

Genome-wide responses to mitochondrial dysfunction.

Mitochondrial dysfunction can lead to diverse cellular and organismal responses. We used DNA microarrays to characterize the transcriptional responses to different mitochondrial perturbations in Saccharomyces cerevisiae. We examined respiratory-deficient petite cells and respiratory-competent wild-type cells treated with the inhibitors of oxidative phosphorylation antimycin, carbonyl cyanide m-chlorophenylhydrazone, or oligomycin. We show that respiratory deficiency, but not inhibition of mitochondrial ATP synthesis per se, induces a suite of genes associated with both peroxisomal activities and metabolite-restoration (anaplerotic) pathways that would mitigate the loss of a complete tricarboxylic acid cycle. The array data suggested, and direct microscopic observation of cells expressing a derivative of green fluorescent protein with a peroxisomal matrix-targeting signal confirmed, that respiratory deficiency dramatically induces peroxisome biogenesis. Transcript profiling of cells harboring null alleles of RTG1, RTG2, or RTG3, genes known to control signaling from mitochondria to the nucleus, suggests that there are multiple pathways of cross-talk between these organelles in yeast.

MAD: a suite of tools for microarray data management and processing.

SUMMARY: Microarray data management and processing (MAD) is a set of Windows integrated software for microarray analysis. It consists of a relational database for data storage with many user-interfaces for data manipulation, several text file parsers and Microsoft Excel macros for automation of data processing, and a generator to produce text files that are ready for cluster analysis. AVAILABILITY: Executable is available free of charge on http://pompous.swmed.edu. The source code is also available upon request.

Repeat polymorphisms within gene regions: phenotypic and evolutionary implications.

We have developed an algorithm that predicted 11,265 potentially polymorphic tandem repeats within transcribed sequences. We estimate that 22% (2,207/9,717) of the annotated clusters within UniGene contain at least one potentially polymorphic locus. Our predictions were tested by allelotyping a panel of approximately 30 individuals for 5% of these regions, confirming polymorphism for more than half the loci tested. Our study indicates that tandem-repeat polymorphisms in genes are more common than is generally believed. Approximately 8% of these loci are within coding sequences and, if polymorphic, would result in frameshifts. Our catalogue of putative polymorphic repeats within transcribed sequences comprises a large set of potentially phenotypic or disease-causing loci. In addition, from the anomalous character of the repetitive sequences within unannotated clusters, we also conclude that the UniGene cluster count substantially overestimates the number of genes in the human genome. We hypothesize that polymorphisms in repeated sequences occur with some baseline distribution, on the basis of repeat homogeneity, size, and sequence composition, and that deviations from that distribution are indicative of the nature of selection pressure at that locus. We find evidence of selective maintenance of the ability of some genes to respond very rapidly, perhaps even on intragenerational timescales, to fluctuating selective pressures.

High resolution chromosome 3p allelotyping of human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple, discontinuous sites of 3p allele loss and three regions of frequent breakpoints.

Allele loss involving chromosome arm 3p is one of the most frequent and earliest known genetic events in lung cancer pathogenesis and may affect several potential tumor suppressor gene regions. To further study the role of chromosome 3p allele loss in the pathogenesis of lung cancer, we performed high resolution loss of heterozygosity (LOH) studies on 97 lung cancer and 54 preneoplastic/preinvasive microdissected respiratory epithelial samples using a panel of 28 3p markers. Allelic losses of 3p were detected in 96% of the lung cancers and in 78% of the preneoplastic/preinvasive lesions. The allele losses were often multiple and discontinuous, with areas of LOH interspersed with areas of retention of heterozygosity. Most small cell lung carcinomas (91%) and squamous cell carcinomas (95%) demonstrated larger 3p segments of allele loss, whereas most (71%) of the adenocarcinomas and preneoplastic/preinvasive lesions had smaller chromosome areas of 3p allele loss. There was a progressive increase in the frequency and size of 3p allele loss regions with increasing severity of histopathological preneoplastic/preinvasive changes. In analyses of the specific parental allele lost comparing 42 preneoplastic/preinvasive foci with those lost in the lung cancer in the same patient (n = 10), the same parental allele was lost in 88% of 244 comparisons for 28 3p markers (P = 1.2 x 10(-36) for this occurring by chance). This indicates the occurrence of allele-specific loss in these foci similar to that seen in the tumor by a currently unknown mechanism. Analysis of all of the data indicated multiple regions of localized 3p allele loss including telomere-D3S1597, D3S1111-D3S2432, D3S2432-D3S1537, D3S1537, D3S1537-D3S1612, D3S4604/Luca19.1-D3S4622/Luca4.1, D3S4624/Luca2.1, D3S4624/Luca2.1-D3S1582, D3S1766, D3S1234-D3S1300 (FHIT/FRA3B region centered on D3S1300), D3S1284-D3S1577 (U2020/DUTT1 region centered on D3S1274), and D3S1511-centromere. A panel of six markers in the 600-kb 3p21.3 deletion region showed loss in 77% of the lung cancers, 70% of normal or preneoplastic/preinvasive lesions associated with lung cancer, and 49% of 47 normal, mildly abnormal, or preneoplastic/preinvasive lesions found in smokers without lung cancer; however, loss was seen in 0% of 18 epithelial samples from seven never smokers. The 600-kb 3p21.3 region and the 3p14.2 (FHIT/FRA3B) and 3p12 (U2020/DUTT1) regions were common, independent sites of breakpoints (retention of heterozygosity by some markers and LOH by other markers in the immediate region). We conclude that 3p allele loss is nearly universal in lung cancer pathogenesis; involves multiple, discrete, 3p LOH sites that often show a "discontinuous LOH" pattern in individual tumors; occurs in preneoplastic/preinvasive lesions in smokers with and without lung cancer (multiple lesions often lose the same parental allele); frequently involves breakpoints in at least three very small defined genomic regions; and appears to have allele loss and breakpoints first occurring in the 600-kb 3p21.3 region. These findings are consistent with previously reported LOH studies in a variety of tumors showing allele loss occurring by mitotic recombination and induced by oxidative damage.