Precision medicine screening using whole-genome sequencing and advanced imaging to identify disease risk in adults.

Reducing premature mortality associated with age-related chronic diseases, such as cancer and cardiovascular disease, is an urgent priority. We report early results using genomics in combination with advanced imaging and other clinical testing to proactively screen for age-related chronic disease risk among adults. We enrolled active, symptom-free adults in a study of screening for age-related chronic diseases associated with premature mortality. In addition to personal and family medical history and other clinical testing, we obtained whole-genome sequencing (WGS), noncontrast whole-body MRI, dual-energy X-ray absorptiometry (DXA), global metabolomics, a new blood test for prediabetes (Quantose IR), echocardiography (ECHO), ECG, and cardiac rhythm monitoring to identify age-related chronic disease risks. Precision medicine screening using WGS and advanced imaging along with other testing among active, symptom-free adults identified a broad set of complementary age-related chronic disease risks associated with premature mortality and strengthened WGS variant interpretation. This and other similarly designed screening approaches anchored by WGS and advanced imaging may have the potential to extend healthy life among active adults through improved prevention and early detection of age-related chronic diseases (and their risk factors) associated with premature mortality.

Fast and accurate HLA typing from short-read next-generation sequence data with xHLA.

The HLA gene complex on human chromosome 6 is one of the most polymorphic regions in the human genome and contributes in large part to the diversity of the immune system. Accurate typing of HLA genes with short-read sequencing data has historically been difficult due to the sequence similarity between the polymorphic alleles. Here, we introduce an algorithm, xHLA, that iteratively refines the mapping results at the amino acid level to achieve 99-100% four-digit typing accuracy for both class I and II HLA genes, taking only [Formula: see text]3 min to process a 30× whole-genome BAM file on a desktop computer.

Identification of individuals by trait prediction using whole-genome sequencing data.

Prediction of human physical traits and demographic information from genomic data challenges privacy and data deidentification in personalized medicine. To explore the current capabilities of phenotype-based genomic identification, we applied whole-genome sequencing, detailed phenotyping, and statistical modeling to predict biometric traits in a cohort of 1,061 participants of diverse ancestry. Individually, for a large fraction of the traits, their predictive accuracy beyond ancestry and demographic information is limited. However, we have developed a maximum entropy algorithm that integrates multiple predictions to determine which genomic samples and phenotype measurements originate from the same person. Using this algorithm, we have reidentified an average of >8 of 10 held-out individuals in an ethnically mixed cohort and an average of 5 of either 10 African Americans or 10 Europeans. This work challenges current conceptions of personal privacy and may have far-reaching ethical and legal implications.

The blood DNA virome in 8,000 humans.

The characterization of the blood virome is important for the safety of blood-derived transfusion products, and for the identification of emerging pathogens. We explored non-human sequence data from whole-genome sequencing of blood from 8,240 individuals, none of whom were ascertained for any infectious disease. Viral sequences were extracted from the pool of sequence reads that did not map to the human reference genome. Analyses sifted through close to 1 Petabyte of sequence data and performed 0.5 trillion similarity searches. With a lower bound for identification of 2 viral genomes/100,000 cells, we mapped sequences to 94 different viruses, including sequences from 19 human DNA viruses, proviruses and RNA viruses (herpesviruses, anelloviruses, papillomaviruses, three polyomaviruses, adenovirus, HIV, HTLV, hepatitis B, hepatitis C, parvovirus B19, and influenza virus) in 42% of the study participants. Of possible relevance to transfusion medicine, we identified Merkel cell polyomavirus in 49 individuals, papillomavirus in blood of 13 individuals, parvovirus B19 in 6 individuals, and the presence of herpesvirus 8 in 3 individuals. The presence of DNA sequences from two RNA viruses was unexpected: Hepatitis C virus is revealing of an integration event, while the influenza virus sequence resulted from immunization with a DNA vaccine. Age, sex and ancestry contributed significantly to the prevalence of infection. The remaining 75 viruses mostly reflect extensive contamination of commercial reagents and from the environment. These technical problems represent a major challenge for the identification of novel human pathogens. Increasing availability of human whole-genome sequences will contribute substantial amounts of data on the composition of the normal and pathogenic human blood virome. Distinguishing contaminants from real human viruses is challenging.

Deep sequencing of 10,000 human genomes.

We report on the sequencing of 10,545 human genomes at 30×-40× coverage with an emphasis on quality metrics and novel variant and sequence discovery. We find that 84% of an individual human genome can be sequenced confidently. This high-confidence region includes 91.5% of exon sequence and 95.2% of known pathogenic variant positions. We present the distribution of over 150 million single-nucleotide variants in the coding and noncoding genome. Each newly sequenced genome contributes an average of 8,579 novel variants. In addition, each genome carries on average 0.7 Mb of sequence that is not found in the main build of the hg38 reference genome. The density of this catalog of variation allowed us to construct high-resolution profiles that define genomic sites that are highly intolerant of genetic variation. These results indicate that the data generated by deep genome sequencing is of the quality necessary for clinical use.

A gene expression signature of CD34+ cells to predict major cytogenetic response in chronic-phase chronic myeloid leukemia patients treated with imatinib.

In chronic-phase chronic myeloid leukemia (CML) patients, the lack of a major cytogenetic response (< 36% Ph(+) metaphases) to imatinib within 12 months indicates failure and mandates a change of therapy. To identify biomarkers predictive of imatinib failure, we performed gene expression array profiling of CD34(+) cells from 2 independent cohorts of imatinib-naive chronic-phase CML patients. The learning set consisted of retrospectively selected patients with a complete cytogenetic response or more than 65% Ph(+) metaphases within 12 months of imatinib therapy. Based on analysis of variance P less than .1 and fold difference 1.5 or more, we identified 885 probe sets with differential expression between responders and nonresponders, from which we extracted a 75-probe set minimal signature (classifier) that separated the 2 groups. On application to a prospectively accrued validation set, the classifier correctly predicted 88% of responders and 83% of nonresponders. Bioinformatics analysis and comparison with published studies revealed overlap of classifier genes with CML progression signatures and implicated beta-catenin in their regulation, suggesting that chronic-phase CML patients destined to fail imatinib have more advanced disease than evident by morphologic criteria. Our classifier may allow directing more aggressive therapy upfront to the patients most likely to benefit while sparing good-risk patients from unnecessary toxicity.

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.

Comprehensive analysis of loss of heterozygosity events in glioblastoma using the 100K SNP mapping arrays and comparison with copy number abnormalities defined by BAC array comparative genomic hybridization.

We have undertaken an extensive high-resolution analysis of loss of heterozygosity (LOH) in 30 high grade gliomas using the Affymetrix 100K SNP mapping array. Only 70% of LOH events were accompanied by a copy number loss (CNA(loss)), and of the other 30%, the distal region of 17p preferentially showed copy number neutral (CNN)-associated LOH. Combined analysis of CNA(loss) and LOH using MergeLevels analysis software predicts whether the observed losses occurred on a diploid or tetraploid background. In a side-by-side comparison between SNP and bacterial artificial chromosome (BAC) arrays, the overall identification of CNAs was similar on both platforms. The resolution provided by the SNP arrays, however, allowed a considerably more accurate definition of breakpoints as well as defining small events within the cancer genomes, which could not be detected on BAC arrays. CNN LOH was only detected by the SNP arrays, as was ploidy prediction. From our analysis, therefore, it is clear that simultaneously defining CNAs and CNN-LOH using the SNP platform provides a higher resolution and more complete analysis of the genetic events that have occurred within tumor cells. Our extensive analysis of SNP array data has also allowed an objective assessment of threshold LOH scores that can accurately predict LOH. This capability has important implications for interpretation of LOH events since they have consistently been used to localize potential tumor suppressor genes within the cancer genome.

The balance of reproducibility, sensitivity, and specificity of lists of differentially expressed genes in microarray studies.

BACKGROUND: Reproducibility is a fundamental requirement in scientific experiments. Some recent publications have claimed that microarrays are unreliable because lists of differentially expressed genes (DEGs) are not reproducible in similar experiments. Meanwhile, new statistical methods for identifying DEGs continue to appear in the scientific literature. The resultant variety of existing and emerging methods exacerbates confusion and continuing debate in the microarray community on the appropriate choice of methods for identifying reliable DEG lists. RESULTS: Using the data sets generated by the MicroArray Quality Control (MAQC) project, we investigated the impact on the reproducibility of DEG lists of a few widely used gene selection procedures. We present comprehensive results from inter-site comparisons using the same microarray platform, cross-platform comparisons using multiple microarray platforms, and comparisons between microarray results and those from TaqMan - the widely regarded "standard" gene expression platform. Our results demonstrate that (1) previously reported discordance between DEG lists could simply result from ranking and selecting DEGs solely by statistical significance (P) derived from widely used simple t-tests; (2) when fold change (FC) is used as the ranking criterion with a non-stringent P-value cutoff filtering, the DEG lists become much more reproducible, especially when fewer genes are selected as differentially expressed, as is the case in most microarray studies; and (3) the instability of short DEG lists solely based on P-value ranking is an expected mathematical consequence of the high variability of the t-values; the more stringent the P-value threshold, the less reproducible the DEG list is. These observations are also consistent with results from extensive simulation calculations. CONCLUSION: We recommend the use of FC-ranking plus a non-stringent P cutoff as a straightforward and baseline practice in order to generate more reproducible DEG lists. Specifically, the P-value cutoff should not be stringent (too small) and FC should be as large as possible. Our results provide practical guidance to choose the appropriate FC and P-value cutoffs when selecting a given number of DEGs. The FC criterion enhances reproducibility, whereas the P criterion balances sensitivity and specificity.

Ploidy status and copy number aberrations in primary glioblastomas defined by integrated analysis of allelic ratios, signal ratios and loss of heterozygosity using 500K SNP Mapping Arrays.

BACKGROUND: Genomic hybridization platforms, including BAC-CGH and genotyping arrays, have been used to estimate chromosome copy number (CN) in tumor samples by detecting the relative strength of genomic signal. The methods rely on the assumption that the predominant chromosomal background of the samples is diploid, an assumption that is frequently incorrect for tumor samples. In addition to generally greater resolution, an advantage of genotyping arrays over CGH arrays is the ability to detect signals from individual alleles, allowing estimation of loss-of-heterozygosity (LOH) and allelic ratios to enhance the interpretation of copy number alterations. Copy number events associated with LOH potentially have the same genetic consequences as deletions. RESULTS: We have utilized allelic ratios to detect patterns that are indicative of higher ploidy levels. An integrated analysis using allelic ratios, total signal and LOH indicates that many or most of the chromosomes from 24 glioblastoma tumors are in fact aneuploid. Some putative whole-chromosome losses actually represent trisomy, and many apparent sub-chromosomal losses are in fact relative losses against a triploid or tetraploid background. CONCLUSION: These results suggest a re-interpretation of previous findings based only on total signal ratios. One interesting observation is that many single or multiple-copy deletions occur at common putative tumor suppressor sites subsequent to chromosomal duplication; these losses do not necessarily result in LOH, but nonetheless occur in conspicuous patterns. The 500 K Mapping array was also capable of detecting many sub-mega base losses and gains that were overlooked by CGH-BAC arrays, and was superior to CGH-BAC arrays in resolving regions of complex CN variation.

CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues.

Cavities on a proteins surface as well as specific amino acid positioning within it create the physicochemical properties needed for a protein to perform its function. CASTp (http://cast.engr.uic.edu) is an online tool that locates and measures pockets and voids on 3D protein structures. This new version of CASTp includes annotated functional information of specific residues on the protein structure. The annotations are derived from the Protein Data Bank (PDB), Swiss-Prot, as well as Online Mendelian Inheritance in Man (OMIM), the latter contains information on the variant single nucleotide polymorphisms (SNPs) that are known to cause disease. These annotated residues are mapped to surface pockets, interior voids or other regions of the PDB structures. We use a semi-global pair-wise sequence alignment method to obtain sequence mapping between entries in Swiss-Prot, OMIM and entries in PDB. The updated CASTp web server can be used to study surface features, functional regions and specific roles of key residues of proteins.

Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA.

BACKGROUND: The interpretability of microarray data can be affected by sample quality. To systematically explore how RNA quality affects microarray assay performance, a set of rat liver RNA samples with a progressive change in RNA integrity was generated by thawing frozen tissue or by ex vivo incubation of fresh tissue over a time course. RESULTS: Incubation of tissue at 37 degrees C for several hours had little effect on RNA integrity, but did induce changes in the transcript levels of stress response genes and immune cell markers. In contrast, thawing of tissue led to a rapid loss of RNA integrity. Probe sets identified as most sensitive to RNA degradation tended to be located more than 1000 nucleotides upstream of their transcription termini, similar to the positioning of control probe sets used to assess sample quality on Affymetrix GeneChip(R) arrays. Samples with RNA integrity numbers less than or equal to 7 showed a significant increase in false positives relative to undegraded liver RNA and a reduction in the detection of true positives among probe sets most sensitive to sample integrity for in silico modeled changes of 1.5-, 2-, and 4-fold. CONCLUSION: Although moderate levels of RNA degradation are tolerated by microarrays with 3'-biased probe selection designs, in this study we identify a threshold beyond which decreased specificity and sensitivity can be observed that closely correlates with average target length. These results highlight the value of annotating microarray data with metrics that capture important aspects of sample quality.

Identification of interspecies concordance of mechanisms of arsenic-induced bladder cancer.

Exposure to arsenic causes cancer by inducing a variety of responses that affect the expression of genes associated with numerous biological pathways leading to altered cell growth and proliferation, signaling, apoptosis and oxidative stress response. Affymetrix GeneChip arrays were used to detect gene expression changes following dimethylarsinic acid (DMA) exposure to human bladder cells (UROtsa) or rat bladder cells (MYP3) and rat bladder epithelium in vivo at comparable doses. Using different experimental models coupled with transcriptional profiling allowed investigation of the correlation of mechanisms of DMA-induced toxicity between in vitro and in vivo treatment and across species. Our observations suggest that DMA-induced gene expression in UROtsa cells is distinct from that observed in the MYP3 cells. Principal component analysis shows a more distinct separation by treatment and dose in MYP3 cells as compared to UROtsa cells. However, at the level of pathways and biological networks, DMA affects both common and unique processes in the bladder transitional cells of human and rats. Twelve pathways were found common between human in vitro, rat in vitro and rat in vivo systems. These included signaling pathways involved in adhesion, cellular growth and differentiation. Fifty-five genes found to be commonly expressed between rat in vivo and rat in vitro systems were involved in diverse functions such as cell cycle regulation, lipid metabolism and protein degradation. Many of the genes, processes and pathways have previously been associated with arsenic-induced toxicity. Our finding reiterates and also identifies new biological processes that might provide more information regarding the mechanisms of DMA-induced toxicity. The results of our analysis further suggest that gene expression profiles can address pertinent issues of relevance to risk assessment, namely interspecies extrapolation of mechanistic information as well as comparison of in vitro to in vivo response.

Use of a mixed tissue RNA design for performance assessments on multiple microarray formats.

The comparability and reliability of data generated using microarray technology would be enhanced by use of a common set of standards that allow accuracy, reproducibility and dynamic range assessments on multiple formats. We designed and tested a complex biological reagent for performance measurements on three commercial oligonucleotide array formats that differ in probe design and signal measurement methodology. The reagent is a set of two mixtures with different proportions of RNA for each of four rat tissues (brain, liver, kidney and testes). The design provides four known ratio measurements of >200 reference probes, which were chosen for their tissue-selectivity, dynamic range coverage and alignment to the same exemplar transcript sequence across all three platforms. The data generated from testing three biological replicates of the reagent at eight laboratories on three array formats provides a benchmark set for both laboratory and data processing performance assessments. Close agreement with target ratios adjusted for sample complexity was achieved on all platforms and low variance was observed among platforms, replicates and sites. The mixed tissue design produces a reagent with known gene expression changes within a complex sample and can serve as a paradigm for performance standards for microarrays that target other species.

Whole-genome sequencing identifies common-to-rare variants associated with human blood metabolites.

Genetic factors modifying the blood metabolome have been investigated through genome-wide association studies (GWAS) of common genetic variants and through exome sequencing. We conducted a whole-genome sequencing study of common, low-frequency and rare variants to associate genetic variations with blood metabolite levels using comprehensive metabolite profiling in 1,960 adults. We focused the analysis on 644 metabolites with consistent levels across three longitudinal data collections. Genetic sequence variations at 101 loci were associated with the levels of 246 (38%) metabolites (P ≤ 1.9 × 10-11). We identified 113 (10.7%) among 1,054 unrelated individuals in the cohort who carried heterozygous rare variants likely influencing the function of 17 genes. Thirteen of the 17 genes are associated with inborn errors of metabolism or other pediatric genetic conditions. This study extends the map of loci influencing the metabolome and highlights the importance of heterozygous rare variants in determining abnormal blood metabolic phenotypes in adults.

Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array.

BACKGROUND: Alternative splicing is a mechanism for increasing protein diversity by excluding or including exons during post-transcriptional processing. Alternatively spliced proteins are particularly relevant in oncology since they may contribute to the etiology of cancer, provide selective drug targets, or serve as a marker set for cancer diagnosis. While conventional identification of splice variants generally targets individual genes, we present here a new exon-centric array (GeneChip Human Exon 1.0 ST) that allows genome-wide identification of differential splice variation, and concurrently provides a flexible and inclusive analysis of gene expression. RESULTS: We analyzed 20 paired tumor-normal colon cancer samples using a microarray designed to detect over one million putative exons that can be virtually assembled into potential gene-level transcripts according to various levels of prior supporting evidence. Analysis of high confidence (empirically supported) transcripts identified 160 differentially expressed genes, with 42 genes occupying a network impacting cell proliferation and another twenty nine genes with unknown functions. A more speculative analysis, including transcripts based solely on computational prediction, produced another 160 differentially expressed genes, three-fourths of which have no previous annotation. We also present a comparison of gene signal estimations from the Exon 1.0 ST and the U133 Plus 2.0 arrays. Novel splicing events were predicted by experimental algorithms that compare the relative contribution of each exon to the cognate transcript intensity in each tissue. The resulting candidate splice variants were validated with RT-PCR. We found nine genes that were differentially spliced between colon tumors and normal colon tissues, several of which have not been previously implicated in cancer. Top scoring candidates from our analysis were also found to substantially overlap with EST-based bioinformatic predictions of alternative splicing in cancer. CONCLUSION: Differential expression of high confidence transcripts correlated extremely well with known cancer genes and pathways, suggesting that the more speculative transcripts, largely based solely on computational prediction and mostly with no previous annotation, might be novel targets in colon cancer. Five of the identified splicing events affect mediators of cytoskeletal organization (ACTN1, VCL, CALD1, CTTN, TPM1), two affect extracellular matrix proteins (FN1, COL6A3) and another participates in integrin signaling (SLC3A2). Altogether they form a pattern of colon-cancer specific alterations that may particularly impact cell motility.

Cistrome: an integrative platform for transcriptional regulation studies.

The increasing volume of ChIP-chip and ChIP-seq data being generated creates a challenge for standard, integrative and reproducible bioinformatics data analysis platforms. We developed a web-based application called Cistrome, based on the Galaxy open source framework. In addition to the standard Galaxy functions, Cistrome has 29 ChIP-chip- and ChIP-seq-specific tools in three major categories, from preliminary peak calling and correlation analyses to downstream genome feature association, gene expression analyses, and motif discovery. Cistrome is available at http://cistrome.org/ap/.

DNA copy number analysis in gastrointestinal stromal tumors using gene expression microarrays.

We report a method, Expression-Microarray Copy Number Analysis (ECNA) for the detection of copy number changes using Affymetrix Human Genome U133 Plus 2.0 arrays, starting with as little as 5 ng input genomic DNA. An analytical approach was developed using DNA isolated from cell lines containing various X-chromosome numbers, and validated with DNA from cell lines with defined deletions and amplifications in other chromosomal locations. We applied this method to examine the copy number changes in DNA from 5 frozen gastrointestinal stromal tumors (GIST). We detected known copy number aberrations consistent with previously published results using conventional or BAC-array CGH, as well as novel changes in GIST tumors. These changes were concordant with results from Affymetrix 100K human SNP mapping arrays. Gene expression data for these GIST samples had previously been generated on U133A arrays, allowing us to explore correlations between chromosomal copy number and RNA expression levels. One of the novel aberrations identified in the GIST samples, a previously unreported gain on 1q21.1 containing the PEX11B gene, was confirmed in this study by FISH and was also shown to have significant differences in expression pattern when compared to a control sample. In summary, we have demonstrated the use of gene expression microarrays for the detection of genomic copy number aberrations in tumor samples. This method may be used to study copy number changes in other species for which RNA expression arrays are available, e.g. other mammals, plants, etc., and for which SNPs have not yet been mapped.

CYP4F2 genetic variant alters required warfarin dose.

Warfarin is an effective, commonly prescribed anticoagulant used to treat and prevent thrombotic events. Because of historically high rates of drug-associated adverse events, warfarin remains underprescribed. Further, interindividual variability in therapeutic dose mandates frequent monitoring until target anticoagulation is achieved. Genetic polymorphisms involved in warfarin metabolism and sensitivity have been implicated in variability of dose. Here, we describe a novel variant that influences warfarin requirements. To identify additional genetic variants that contribute to warfarin requirements, screening of DNA variants in additional genes that code for drug-metabolizing enzymes and drug transport proteins was undertaken using the Affymetrix drug-metabolizing enzymes and transporters panel. A DNA variant (rs2108622; V433M) in cytochrome P450 4F2 (CYP4F2) was associated with warfarin dose in 3 independent white cohorts of patients stabilized on warfarin representing diverse geographic regions in the United States and accounted for a difference in warfarin dose of approximately 1 mg/day between CC and TT subjects. Genetic variation of CYP4F2 was associated with a clinically relevant effect on warfarin requirement.

Massively parallel pathogen identification using high-density microarrays.

Identification of microbial pathogens in clinical specimens is still performed by phenotypic methods that are often slow and cumbersome, despite the availability of more comprehensive genotyping technologies. We present an approach based on whole-genome amplification and resequencing microarrays for unbiased pathogen detection. This 10 h process identifies a broad spectrum of bacterial and viral species and predicts antibiotic resistance and pathogenicity and virulence profiles. We successfully identify a variety of bacteria and viruses, both in isolation and in complex mixtures, and the high specificity of the microarray distinguishes between different pathogens that cause diseases with overlapping symptoms. The resequencing approach also allows identification of organisms whose sequences are not tiled on the array, greatly expanding the repertoire of identifiable organisms and their variants. We identify organisms by hybridization of their DNA in as little as 1-4 h. Using this method, we identified Monkeypox virus and drug-resistant Staphylococcus aureus in a skin lesion taken from a child suspected of an orthopoxvirus infection, despite poor transport conditions of the sample, and a vast excess of human DNA. Our results suggest this technology could be applied in a clinical setting to test for numerous pathogens in a rapid, sensitive and unbiased manner.