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To determine the effects of single nucleotide polymorphisms (SNPs) identified in a genome-wide association study of progressive supranuclear palsy (PSP), we tested their association with brain gene expression, CpG methylation and neuropathology. In 175 autopsied PSP subjects, we performed associations between seven PSP risk variants and temporal cortex levels of 20 genes in-cis, within ±100 kb. Methylation measures were collected using reduced representation bisulfite sequencing in 43 PSP brains. To determine whether SNP/expression associations are due to epigenetic modifications, CpG methylation levels of associated genes were tested against relevant variants. Quantitative neuropathology endophenotypes were tested for SNP associations in 422 PSP subjects. Brain levels of LRRC37A4 and ARL17B were associated with rs8070723; MOBP with rs1768208 and both ARL17A and ARL17B with rs242557. Expression associations for LRRC37A4 and MOBP were available in an additional 100 PSP subjects. Meta-analysis revealed highly significant associations for PSP risk alleles of rs8070723 and rs1768208 with higher LRRC37A4 and MOBP brain levels, respectively. Methylation levels of one CpG in the 3' region of ARL17B associated with rs242557 and rs8070723. Additionally, methylation levels of an intronic ARL17A CpG associated with rs242557 and that of an intronic MOBP CpG with rs1768208. MAPT and MOBP region risk alleles also associated with higher levels of neuropathology. Strongest associations were observed for rs242557/coiled bodies and tufted astrocytes; and for rs1768208/coiled bodies and tau threads. These findings suggest that PSP variants at MAPT and MOBP loci may confer PSP risk via influencing gene expression and tau neuropathology. MOBP, LRRC37A4, ARL17A and ARL17B warrant further assessment as candidate PSP risk genes. Our findings have implications for the mechanism of action of variants at some of the top PSP risk loci.

Advantages of RNA-Seq over array based platforms are quantitative gene expression and discovery of expressed single nucleotide variants (eSNVs) and fusion transcripts from a single platform, but the sensitivity for each of these characteristics is unknown. We measured gene expression in a set of manually degraded RNAs, nine pairs of matched fresh-frozen, and FFPE RNA isolated from breast tumor with the hybridization based, NanoString nCounter (226 gene panel) and with whole transcriptome RNA-Seq using RiboZeroGold ScriptSeq V2 library preparation kits. We performed correlation analyses of gene expression between samples and across platforms. We then specifically assessed whole transcriptome expression of lincRNA and discovery of eSNVs and fusion transcripts in the FFPE RNA-Seq data. For gene expression in the manually degraded samples, we observed Pearson correlations of >0.94 and >0.80 with NanoString and ScriptSeq protocols, respectively. Gene expression data for matched fresh-frozen and FFPE samples yielded mean Pearson correlations of 0.874 and 0.783 for NanoString (226 genes) and ScriptSeq whole transcriptome protocols respectively, p<2x10(-16). Specifically for lincRNAs, we observed superb Pearson correlation (0.988) between matched fresh-frozen and FFPE pairs. FFPE samples across NanoString and RNA-Seq platforms gave a mean Pearson correlation of 0.838. In FFPE libraries, we detected 53.4% of high confidence SNVs and 24% of high confidence fusion transcripts. Sensitivity of fusion transcript detection was not overcome by an increase in depth of sequencing up to 3-fold (increase from ~56 to ~159 million reads). Both NanoString and ScriptSeq RNA-Seq technologies yield reliable gene expression data for degraded and FFPE material. The high degree of correlation between NanoString and RNA-Seq platforms suggests discovery based whole transcriptome studies from FFPE material will produce reliable expression data. The RiboZeroGold ScriptSeq protocol performed particularly well for lincRNA expression from FFPE libraries, but detection of eSNV and fusion transcripts was less sensitive.

Background: Gene profiling methods have been widely useful for delineating changes in gene expression as an approach for gaining insight into the mechanism of rejection or disease pathology. Herein, we use gene profiling to compare changes in gene expression associated with different orthotopic cardiac xenotransplantation (OCXTx) outcomes and to identify potential effects of OCXTx on cardiac physiology., Methods: We used the Affymetrix GeneChip Porcine Genomic Array to characterize three types of orthotopic cardiac xenograft outcomes: 1) rejected hearts that underwent delayed xenograft rejection (DXR); 2) survivor hearts in which the xenograft was not rejected and recipient death was due to model complications; and 3) hearts which failed to provide sufficient circulatory support within the first 48 h of transplant, termed "perioperative cardiac xenograft dysfunction" (PCXD). Gene expression in each group was compared to control, not transplanted pig hearts, and changes in gene expression > 3 standard deviations (±3SD) from the control samples were analyzed. A bioinformatics analysis was used to identify enrichments in genes involved in Kyoto Encyclopedia of Genes and Genomes pathways and gene ontogeny molecular functions. Changes in gene expression were confirmed by quantitative RT-PCR., Results: The ±3SD data set contained 260 probes, which minimally exhibited a 3.5-fold change in gene expression compared to control pig hearts. Hierarchical cluster analysis segregated rejected, survivor and PCXD samples, indicating a unique change in gene expression for each group. All transplant outcomes shared a set of 21 probes with similarly altered expression, which were indicative of ongoing myocardial inflammation and injury. Some outcome-specific changes in gene expression were identified. Bioinformatics analysis detected an enrichment of genes involved in protein, carbohydrate and branched amino acid metabolism, extracellular matrix-receptor interactions, focal adhesion, and cell communication., Conclusions: This is the first genome wide assessment of changes in cardiac gene expression after OCXTx. Hierarchical cluster analysis indicates a unique gene profile for each transplant outcome but additional samples will be required to define the unique classifier probe sets. Quantitative RT-PCR confirmed that all transplants exhibited strong evidence of ongoing inflammation and myocardial injury consistent with the effects of cytokines and vascular antibody-mediated inflammation. This was also consistent with bioinformatic analysis suggesting ongoing tissue repair in survivor and PCXD samples. Bioinformatics analysis suggests for the first time that xenotransplantation may affect cardiac metabolism in survivor and rejected samples. This study highlights the potential utility of molecular analysis to monitor xenograft function, to identify new molecular markers and to understand processes, which may contribute to DXR., (© 2011 John Wiley & Sons A/S.)

Ferroptosis is a newly identified cell death mechanism and potential biomarker for hepatocellular carcinoma (HCC) therapy; however, its clinical relevance and underlying mechanism remain unclear. In this study, transcriptome and methylome data from 374 HCC cases were investigated for 41 ferroptosis‐related genes to identify ferroptosis activity‐associated subtypes. These subtypes were further investigated for associations with clinical and pathological variables, gene mutation landscapes, deregulated pathways and tumour microenvironmental immunity. A gene expression signature and predictive model were developed and validated using an additional 232 HCC cases from another independent cohort. Two distinct ferroptosis phenotypes (Ferroptosis‐H and Ferroptosis‐L) were identified according to ferroptosis gene expression and methylation in the patients with HCC. Patients with the Ferroptosis‐H had worse overall and disease‐specific survival, and the molecular subtypes were significantly associated with different clinical characteristics, mRNA expression patterns, tumour mutation profiles and microenvironmental immune status. Furthermore, a 15‐gene ferroptosis‐related prognostic model (FPM) for HCC was developed and validated which demonstrated accurate risk stratification ability. A nomogram included the FPM risk score, ECOG PS and hepatitis B status was developed for eventual clinical translation. Our results suggest that HCC subtypes defined by ferroptosis gene expression and methylation may be used to stratify patients for clinical decision‐making. [ABSTRACT FROM AUTHOR]

Purpose Spliced variant forms of androgen receptor were recently identified in castration resistant prostate cancer cell lines and clinical samples. We identified the cistrome and gene signature of androgen receptor splice variants in castration resistant prostate cancer cell lines and determined the clinical significance of androgen receptor splice variant regulated genes. Materials and Methods The castration resistant prostate cancer cell line 22Rv1, which expresses full-length androgen receptor and androgen receptor splice variants endogenously, was used as the research model. We established 22Rv1-ARFL – /ARV + and 22Rv1-ARFL – /ARV – through RNA interference. Chromatin immunoprecipitation coupled with next generation sequencing and microarray techniques were used to identify the cistrome and gene expression profiles of androgen receptor splice variants in the absence of androgen. Results Androgen receptor splice variant binding sites were identified in 22Rv1-ARFL – /ARV + . A gene set was regulated uniquely by androgen receptor splice variants but not by full-length androgen receptor in the absence of androgen. Integrated analysis revealed that some genes were directly modulated by androgen receptor splice variants. Unsupervised clustering analysis showed that the androgen receptor splice variant gene signature differentiated benign from malignant prostate tissue as well as localized prostate cancer from metastatic castration resistant prostate cancer specimens. Some genes that were modulated uniquely by androgen receptor splice variants also correlated with histological grade and biochemical failure. Conclusions Androgen receptor splice variants can bind to DNA independent of full-length androgen receptor in the absence of androgen and modulate a unique set of genes that is not regulated by full-length androgen receptor. The androgen receptor splice variant gene signature correlates with disease progression. It distinguishes primary cancer from castration resistant prostate cancer specimens and benign from malignant prostate specimens. [ABSTRACT FROM AUTHOR]

Objectives: The sequencing by the PolyA selection is the most common approach for library preparation. With limited amount or degraded RNA, alternative protocols such as the NuGEN have been developed. However, it is not yet clear how the different library preparations affect the downstream analyses of the broad applications of RNA sequencing. Methods and Materials: Eight human mammary epithelial cell (HMEC) lines with high quality RNA were sequenced by Illumina’s mRNA-Seq PolyA selection and NuGEN ENCORE library preparation. The following analyses and comparisons were conducted: 1) the numbers of genes captured by each protocol; 2) the impact of protocols on differentially expressed gene detection between biological replicates; 3) expressed single nucleotide variant (SNV) detection; 4) non-coding RNAs, particularly lincRNA detection; and 5) intragenic gene expression. Results: Sequences from the NuGEN protocol had lower (75%) alignment rate than the PolyA (over 90%). The NuGEN protocol detected fewer genes (12–20% less) with a significant portion of reads mapped to non-coding regions. A large number of genes were differentially detected between the two protocols. About 17–20% of the differentially expressed genes between biological replicates were commonly detected between the two protocols. Significantly higher numbers of SNVs (5–6 times) were detected in the NuGEN samples, which were largely from intragenic and intergenic regions. The NuGEN captured fewer exons (25% less) and had higher base level coverage variance. While 6.3% of reads were mapped to intragenic regions in the PolyA samples, the percentages were much higher (20–25%) for the NuGEN samples. The NuGEN protocol did not detect more known non-coding RNAs such as lincRNAs, but targeted small and “novel” lincRNAs. Conclusion: Different library preparations can have significant impacts on downstream analysis and interpretation of RNA-seq data. The NuGEN provides an alternative for limited or degraded RNA but it has limitations for some RNA-seq applications. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) are a large class of gene transcripts that do not code proteins; however, their functions are largely unknown and many new lncRNAs are yet to be discovered. Taking advantage of our previously developed, super-fast, novel lncRNA discovery pipeline, UClncR, and rich resources of GTEx RNA-seq data, we performed systematic novel lincRNA discovery for over 8000 samples across 30 tissue types. We conducted novel detection for each major tissue type first and then consolidated the novel discoveries from all tissue types. These novel lincRNs were profiled and analyzed along with known genes to identify tissue-specific genes in 30 major human tissue types. Thirteen sub-brain regions were also analyzed in a similar manner. Our analysis revealed thousands to tens of thousands of novel lincRNAs for each tissue type. These lincRNAs could define each tissue type's identity and demonstrated their reliability and tissue-specific expression. Tissue-specific genes were identified for each major tissue type and sub-brain region. The tissue-specific genes clearly defined each respective tissue's unique function and could be used to expand the interpretation of non-coding SNPs from genome-wide association (GWAS) studies. [ABSTRACT FROM AUTHOR]

Lung adenocarcinomas from never smokers account for approximately 15 to 20% of all lung cancers and these tumors often carry genetic alterations that are responsive to targeted therapy. Here we examined mutation status in 10 oncogenes among 89 lung adenocarcinomas from never smokers. We also screened for oncogene fusion transcripts in 20 of the 89 tumors by RNA-Seq. In total, 62 tumors had mutations in at least one of the 10 oncogenes, including EGFR (49 cases, 55%), K-ras (5 cases, 6%), BRAF (4 cases, 5%), PIK3CA (3 cases, 3%), and ERBB2 (4 cases, 5%). In addition to ALK fusions identified by IHC/FISH in four cases, two previously known fusions involving EZR- ROS1 and KIF5B-RET were identified by RNA-Seq as well as a third novel fusion transcript that was formed between exons 1-9 of SND1 and exons 2 to 3′ end of BRAF. This in-frame fusion was observed in 3/89 tested tumors and 2/64 additional never smoker lung adenocarcinoma samples. Ectopic expression of SND1-BRAF in H1299 cells increased phosphorylation levels of MEK/ERK, cell proliferation, and spheroid formation compared to parental mock-transfected control. Jointly, our results suggest a potential role of the novel BRAF fusion in lung cancer development and therapy. [ABSTRACT FROM AUTHOR]

Abstract: Lung adenocarcinoma is the most common type of primary lung cancer. The purpose of this study was to delineate gene expression patterns for survival prediction in lung adenocarcinoma. Gene expression profiles of 82 (discovery set) and 442 (validation set 1) lung adenocarcinoma tumor tissues were analyzed using a systems biology-based network approach. We also examined the expression profiles of 78 adjacent normal lung tissues from 82 patients. We found a significant correlation of an expression module with overall survival (adjusted hazard ratio or HR=1.71; 95% CI=1.06–2.74 in discovery set; adjusted HR=1.26; 95% CI=1.08–1.49 in validation set 1). This expression module contained genes enriched in the biological process of the cell cycle. Interestingly, the cell cycle gene module and overall survival association were also significant in normal lung tissues (adjusted HR=1.91; 95% CI, 1.32–2.75). From these survival-related modules, we further defined three hub genes (UBE2C, TPX2, and MELK) whose expression-based risk indices were more strongly associated with poor 5-year survival (HR=3.85, 95% CI=1.34–11.05 in discovery set; HR=1.72, 95% CI=1.21–2.46 in validation set 1; and HR=3.35, 95% CI=1.08–10.04 in normal lung set). The 3-gene prognostic result was further validated using 92 adenocarcinoma tumor samples (validation set 2); patients with a high-risk gene signature have a 1.52-fold increased risk (95% CI, 1.02–2.24) of death than patients with a low-risk gene signature. These results suggest that a network-based approach may facilitate discovery of key genes that are closely linked to survival in patients with lung adenocarcinoma. [Copyright &y& Elsevier]

Lung cancer occurs in never-smokers. Epigenetic changes in lung cancer potentially represent important diagnostic, prognostic, and therapeutic targets. We compared DNA methylation profiles of 28 adenocarcinomas of the lungs of never-smokers with paired adjacent nonmalignant lung tissue. We correlated differential methylation changes with gene expression changes from the same 28 sample pairs. Using principal component analysis, we observed a distinct separation in methylation profiles between tumor and adjacent nonmalignant lung tissue. Tumors were generally hypomethylated compared with adjacent nonmalignant tissue. Of 1,906 CpG sites differentially methylated between tumor and nonmalignant tissue, 1,198 were within classically defined CpG islands where tumors were hypermethylated compared with nonmalignant tissue. A total of 708 sites were outside CpG islands where tumors were hypomethylated compared with nonmalignant tissue. There were significant differences in expression of 351 genes (23%) of the 1,522 genes matched to the differentially methylated CpG sites. Genes that were not significantly differentially expressed and were hypermethylated within CpG sites were enriched for homeobox genes. These results suggest that the methylation profiles of lung adenocarcinomas of never-smokers and adjacent nonmalignant lung tissue are significantly different. Despite the differential methylation of homeobox genes, no significant changes in expression of these genes were detected. [ABSTRACT FROM AUTHOR]