Philip W. Garrett-engele

Plasma MicroRNAs as sensitive and specific biomarkers of tissue injury.

BACKGROUND MicroRNAs (miRNAs) are endogenous, small noncoding RNAs. Because of their size, abundance, tissue specificity, and relative stability in plasma, miRNAs hold promise as unique accessible biomarkers to monitor tissue injury. METHODS We investigated the use of liver-, muscle- and brain-specific miRNAs as circulating biomarkers of tissue injury. We used a highly sensitive quantitative PCR assay to measure specific miRNAs (miR-122, miR-133a, and miR-124) in plasma samples from rats treated with liver or muscle toxicants and from a rat surgical model of stroke. RESULTS We observed increases in plasma concentrations of miR-122, miR-133a, and miR-124 corresponding to injuries in liver, muscle, and brain, respectively. miR-122 and miR-133a illustrated specificity for liver and muscle toxicity, respectively, because they were not detectable in the plasma of animals with toxicity to the other organ. This result contrasted with the results for alanine aminotransferase (ALT) and aspartate aminotransferase, which were both increased with either organ toxicity. Furthermore, miR-122 exhibited a diagnostic sensitivity superior to that of ALT when the results were correlated to the liver histopathologic results. The miR-124 concentration increased in the plasma of rats 8 h after surgery to produce brain injury and peaked at 24 h, while the miR-122 and miR-133a concentrations remained at baseline values. CONCLUSIONS These results demonstrate that tissue-specific miRNAs may serve as diagnostically sensitive plasma biomarkers of tissue injury.

Experimental annotation of the human genome using microarray technology.

The most important product of the sequencing of a genome is a complete, accurate catalogue of genes and their products, primarily messenger RNA transcripts and their cognate proteins. Such a catalogue cannot be constructed by computational annotation alone; it requires experimental validation on a genome scale. Using ‘exon’ and ‘tiling’ arrays fabricated by ink-jet oligonucleotide synthesis, we devised an experimental approach to validate and refine computational gene predictions and define full-length transcripts on the basis of co-regulated expression of their exons. These methods can provide more accurate gene numbers and allow the detection of mRNA splice variants and identification of the tissue- and disease-specific conditions under which genes are expressed. We apply our technique to chromosome 22q under 69 experimental condition pairs, and to the entire human genome under two experimental conditions. We discuss implications for more comprehensive, consistent and reliable genome annotation, more efficient, full-length complementary DNA cloning strategies and application to complex diseases.

Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes.

Modern medicine faces the challenge of developing safer and more effective therapies to treat human diseases. Many drugs currently in use were discovered without knowledge of their underlying molecular mechanisms. Understanding their biological targets and modes of action will be essential to design improved second-generation compounds. Here, we describe the use of a genome-wide pool of tagged heterozygotes to assess the cellular effects of 78 compounds in Saccharomyces cerevisiae. Specifically, lanosterol synthase in the sterol biosynthetic pathway was identified as a target of the antianginal drug molsidomine, which may explain its cholesterol-lowering effects. Further, the rRNA processing exosome was identified as a potential target of the cell growth inhibitor 5-fluorouracil. This genome-wide screen validated previously characterized targets or helped identify potentially new modes of action for over half of the compounds tested, providing proof of this principle for analyzing the modes of action of clinically relevant compounds.

A comprehensive transcript index of the human genome generated using microarrays and computational approaches

BackgroundComputational and microarray-based experimental approaches were used to generate a comprehensive transcript index for the human genome. Oligonucleotide probes designed from approximately 50,000 known and predicted transcript sequences from the human genome were used to survey transcription from a diverse set of 60 tissues and cell lines using ink-jet microarrays. Further, expression activity over at least six conditions was more generally assessed using genomic tiling arrays consisting of probes tiled through a repeat-masked version of the genomic sequence making up chromosomes 20 and 22.ResultsThe combination of microarray data with extensive genome annotations resulted in a set of 28,456 experimentally supported transcripts. This set of high-confidence transcripts represents the first experimentally driven annotation of the human genome. In addition, the results from genomic tiling suggest that a large amount of transcription exists outside of annotated regions of the genome and serves as an example of how this activity could be measured on a genome-wide scale.ConclusionsThese data represent one of the most comprehensive assessments of transcriptional activity in the human genome and provide an atlas of human gene expression over a unique set of gene predictions. Before the annotation of the human genome is considered complete, however, the previously unannotated transcriptional activity throughout the genome must be fully characterized.

Evolutionarily conserved and diverged alternative splicing events show different expression and functional profiles.

To better decipher the functional impact of alternative splicing, we classified alternative splicing events in 10 818 pairs of human and mouse genes based on conservation at genome and transcript levels. Expression levels of conserved alternative splices in human and mouse expressed sequence tag databases show strong correlation, indicating that alternative splicing is similarly regulated in both species. A total of 43% (8921) of mouse alternative splices could be found in the human genome but not in human transcripts. Five of eleven tested mouse predictions were observed in human tissues, demonstrating that mouse transcripts provide a valuable resource for identifying alternative splicing events in human genes. Combining gene-specific measures of conserved and diverged alternative splicing with both gene classification based on Gene Ontology (GO) and microarray-determined gene expression in 52 diverse human tissues and cell lines, we found conserved alternative splicing most enriched in brain-expressed signaling pathways. Diverged alternative splicing is more prevalent in testis and cancerous cell line up-regulated processes, including protein biosynthesis, responses to stress and responses to endogenous stimuli. Using conservation as a surrogate for functional significance, these results suggest that alternative splicing plays an important role in enhancing the functional capacity of central nervous systems, while non-functional splicing more frequently occurs in testis and cell lines, possibly as a result of cellular stress and rapid proliferation.

Genetic validation of whole-transcriptome sequencing for mapping expression affected by cis-regulatory variation.

BackgroundIdentifying associations between genotypes and gene expression levels using microarrays has enabled systematic interrogation of regulatory variation underlying complex phenotypes. This approach has vast potential for functional characterization of disease states, but its prohibitive cost, given hundreds to thousands of individual samples from populations have to be genotyped and expression profiled, has limited its widespread application.ResultsHere we demonstrate that genomic regions with allele-specific expression (ASE) detected by sequencing cDNA are highly enriched for cis- acting expression quantitative trait loci (cis- eQTL) identified by profiling of 500 animals in parallel, with up to 90% agreement on the allele that is preferentially expressed. We also observed widespread noncoding and antisense ASE and identified several allele-specific alternative splicing variants.ConclusionMonitoring ASE by sequencing cDNA from as little as one sample is a practical alternative to expression genetics for mapping cis-acting variation that regulates RNA transcription and processing.

Circulating miR-122 as a potential biomarker of liver disease

AIM Expression profiles indicate that miR-122 is specifically and abundantly expressed in liver. This study sought to determine miR-122 plasma concentrations in 15 apparently healthy subjects and 30 patients with liver disease, and clarify whether plasma miR-122 correlates with ALT. MATERIALS & METHODS miR-122 was measured by quantitative PCR in healthy volunteers and patients with liver disease. RESULTS ALT was increased in two out of 15 (13%) apparently healthy subjects and 17 out of 30 (57%) liver disease patients. In healthy subjects, median miR-122 plasma concentration was 51.7 copies/20 pg RNA (range 16.0-312.0). In liver disease patients, median miR-122 was significantly elevated to 202.3 copies/20 pg RNA (range 20.9-1160.0; Mann-Whitney test between median concentrations; p = 0.0016). CONCLUSION This small proof-of-principle study suggests that miR-122 may be a potential plasma biomarker of liver damage.

ACC2 Is Expressed at High Levels Human White Adipose and Has an Isoform with a Novel N-Terminus

Acetyl-CoA carboxylases ACC1 and ACC2 catalyze the carboxylation of acetyl-CoA to malonyl-CoA, regulating fatty-acid synthesis and oxidation, and are potential targets for treatment of metabolic syndrome. Expression of ACC1 in rodent lipogenic tissues and ACC2 in rodent oxidative tissues, coupled with the predicted localization of ACC2 to the mitochondrial membrane, have suggested separate functional roles for ACC1 in lipogenesis and ACC2 in fatty acid oxidation. We find, however, that human adipose tissue, unlike rodent adipose, expresses more ACC2 mRNA relative to the oxidative tissues muscle and heart. Human adipose, along with human liver, expresses more ACC2 than ACC1. Using RT-PCR, real-time PCR, and immunoprecipitation we report a novel isoform of ACC2 (ACC2.v2) that is expressed at significant levels in human adipose. The protein generated by this isoform has enzymatic activity, is endogenously expressed in adipose, and lacks the N-terminal sequence. Both ACC2 isoforms are capable of de novo lipogenesis, suggesting that ACC2, in addition to ACC1, may play a role in lipogenesis. The results demonstrate a significant difference in ACC expression between human and rodents, which may introduce difficulties for the use of rodent models for development of ACC inhibitors.

Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis

Increased levels of HMG‐CoA reductase induce cell type‐ and isozyme‐specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear‐associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non‐overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutants sensitivity to elevated HMG‐CoA reductase and inability to assemble normal karmellae. The largest class of HMG‐CoA reductase‐sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation. Copyright

Abstract LB-012: Single cell RNA-sequencing of multiple myeloma clinical patients for identifying novel immuno-oncology drug targets

Recent advances in single cell genomic technologies have enabled unbiased quantification of cellular states within complex tissues, mechanisms of differentiation, biomarker presentation in cellular subsets; yet their applications in evaluating the response to therapeutic interventions are still underexplored. In our study, we apply single cell RNA sequencing (scRNA-seq) to uncover mechanisms and drug targets for Multiple Myeloma(MM), a malignancy of plasma cells with estimates of 30,770 new cases and 12,770 deaths in 2018 within US alone. Using the 10x Genomics scRNA-seq platform, we profiled ten clinical MM patients, pre- and post-treatment with four standard of care chemotherapy regimens (VAD, VCD, VMP, MP; A=Adriamycin; C=Cyclophosphamide; D=Dexamethasone; M=Melphalan; P=Prednisone; V=Velcade). We sequenced a total of 106,000 cells and determined differential gene expression upon treatment at single-cell resolution comparing 37,926 pre- vs 44,842 post-treatment cells. We identify activation of immune cells with increased expression of IL1B, CD14, CD52, CCL4, IFITM3, PSMA4; and repression of translational machinery with reduced expression of PABPC1, PABPC4, FUS, POLR2A in post-treatment (abs(log2 FC >0.25) and P-value 100) using oligo conjugated antibodies while retaining single cell resolution. Transcriptome and/or proteome analysis of bulk tumor often fails to detect signature of rare cell populations. Our study here highlights the use of single cell readouts for identifying novel oncology mechanisms, drug targets and drug combinations for more efficacious immunotherapies. Citation Format: Namit Kumar, Vanessa M. Peterson, Kelvin X. Zhang, Lixia Li, Philip W. Garrett-Engele, Raymond J. Moniz, Joel A. Klappenbach. Single cell RNA-sequencing of multiple myeloma clinical patients for identifying novel immuno-oncology drug targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-012.