YES1 amplification is a mechanism of acquired resistance to EGFR inhibitors identified by transposon mutagenesis and clinical genomics.

In ∼30% of patients with EGFR-mutant lung adenocarcinomas whose disease progresses on EGFR inhibitors, the basis for acquired resistance remains unclear. We have integrated transposon mutagenesis screening in an EGFR-mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify mechanisms of resistance to EGFR inhibitors. The most prominent candidate genes identified by insertions in or near the genes during the screen were MET, a gene whose amplification is known to mediate resistance to EGFR inhibitors, and the gene encoding the Src family kinase YES1. Cell clones with transposon insertions that activated expression of YES1 exhibited resistance to all three generations of EGFR inhibitors and sensitivity to pharmacologic and siRNA-mediated inhibition of YES1 Analysis of clinical genomic sequencing data from cases of acquired resistance to EGFR inhibitors revealed amplification of YES1 in five cases, four of which lacked any other known mechanisms of resistance. Preinhibitor samples, available for two of the five patients, lacked YES1 amplification. None of 136 postinhibitor samples had detectable amplification of other Src family kinases (SRC and FYN). YES1 amplification was also found in 2 of 17 samples from ALK fusion-positive lung cancer patients who had progressed on ALK TKIs. Taken together, our findings identify acquired amplification of YES1 as a recurrent and targetable mechanism of resistance to EGFR inhibition in EGFR-mutant lung cancers and demonstrate the utility of transposon mutagenesis in discovering clinically relevant mechanisms of drug resistance.

Retained mismatch repair protein expression occurs in approximately 6% of microsatellite instability-high cancers and is associated with missense mutations in mismatch repair genes.

Immunohistochemistry for mismatch repair protein expression is widely used as a surrogate for microsatellite instability status-an important signature for immunotherapy and germline testing. There are no systematic analyses examining the sensitivity of immunohistochemistry for microsatellite instability-high status. Mismatch repair immunohistochemistry and microsatellite instability testing were performed routinely as clinically validated assays. We classified germline/somatic mutation types as truncating (nonsense, frameshift, and in/del) versus missense and predicted pathogenicity of the latter. Discordant cases were compared with concordant groups: microsatellite instability-high/mismatch repair-deficient for mutation comparison and microsatellite stable/mismatch repair-proficient for immunohistochemical comparison. 32 of 443 (7%) microsatellite instability-high cases had immunohistochemistry. Four additional microsatellite instability-high research cases had discordant immunohistochemistry. Of 36 microsatellite instability-high cases with discordant immunohistochemistry, 30 were mismatch repair-proficient, while six (five MLH1 and one MSH2) retained expression of the defective mismatch repair protein and lost its partner. In microsatellite instability-high tumors with discordant immunohistochemistry, we observed an enrichment in deleterious missense mutations over truncating mutations, with 69% (25/36) of cases having pathogenic germline or somatic missense mutations, as opposed to only 19% (7/36) in a matched microsatellite instability-high group with concordant immunohistochemistry (p = 0.0007).  In microsatellite instability-high cases with discordant immunohistochemistry and MLH1 or PMS2 abnormalities, less cells showed expression (p = 0.015 and p = 0.00095, respectively) compared with microsatellite stable/mismatch repair-proficient cases. Tumor mutation burden, MSIsensor score, and truncating mismatch repair gene mutations were similar between microsatellite instability-high cases with concordant versus discordant immunohistochemical expression. Approximately 6% of microsatellite instability-high cases have retained mismatch repair protein expression and would be missed by immunohistochemistry-based testing, hindering patient access to immunotherapy. Another 1% of microsatellite instability-high cases show isolated loss of the defective gene's dimerization partner, which may lead to germline testing of the wrong gene. These cases are enriched for pathogenic mismatch repair missense mutations.

Gene Expression Signatures Characterized by Longitudinal Stability and Interindividual Variability Delineate Baseline Phenotypic Groups with Distinct Responses to Immune Stimulation.

Human immunity exhibits remarkable heterogeneity among individuals, which engenders variable responses to immune perturbations in human populations. Population studies reveal that, in addition to interindividual heterogeneity, systemic immune signatures display longitudinal stability within individuals, and these signatures may reliably dictate how given individuals respond to immune perturbations. We hypothesize that analyzing relationships among these signatures at the population level may uncover baseline immune phenotypes that correspond with response outcomes to immune stimuli. To test this, we quantified global gene expression in peripheral blood CD4+ cells from healthy individuals at baseline and following CD3/CD28 stimulation at two time points 1 mo apart. Systemic CD4+ cell baseline and poststimulation molecular immune response signatures (MIRS) were defined by identifying genes expressed at levels that were stable between time points within individuals and differential among individuals in each state. Iterative differential gene expression analyses between all possible phenotypic groupings of at least three individuals using the baseline and stimulated MIRS gene sets revealed shared baseline and response phenotypic groupings, indicating the baseline MIRS contained determinants of immune responsiveness. Furthermore, significant numbers of shared phenotype-defining sets of determinants were identified in baseline data across independent healthy cohorts. Combining the cohorts and repeating the analyses resulted in identification of over 6000 baseline immune phenotypic groups, implying that the MIRS concept may be useful in many immune perturbation contexts. These findings demonstrate that patterns in complex gene expression variability can be used to define immune phenotypes and discover determinants of immune responsiveness.

Identification of prognostic molecular biomarkers in 157 HPV-positive and HPV-negative squamous cell carcinomas of the oropharynx.

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been increasing due to high-risk HPV infection. We explored the significance of genetic alterations in HPV-positive (HPV-P) and HPV-negative (HPV-N) OPSCC patients on long-term outcome. A total of 157 cases of primary resected OPSCC diagnosed from 1978 to 2005 were subjected to a targeted exome sequencing by MSK-IMPACT™ interrogating somatic mutations in 410 cancer-related genes. Mutational profiles were correlated to recurrence and survival outcomes. OPSCC included 47% HPV-positive (HPV-P) and 53% HPV-negative (HPV-N) tumors arising in the base of tongue (BOT, 43%), palatine tonsil (30%) and soft palate (SP, 27%). HPV negative status, SP location and smoking were associated with poorer outcome. Poorer overall survival was found in NOTCH1-mutated HPV-P (p = 0.039), and in SOX2-amplified HPV-N cases (p = 0.036). Chromosomal arm gains in 8p and 8q, and 16q loss were more common in HPV-P (p = 0.005, 0.04 and 0.01, respectively), while 9p, 18q and 21q losses were more frequent in HPV-N OPSCC (p = 0.006, 0.002 and 0.01, respectively). Novel, potentially functional JAK3, MYC and EP300 intragenic deletions were found in HPV-P, and FOXP1, CDKN2A, CCND1 and RUNX1 intragenic deletions and one FGFR3 inversion were detected in HPV-N tumors. HPV-N/TP53-wild-type OPSCC harbored recurrent mutations in NOTCH1/3/4 (39%), PIK3CA, FAT1 and TERT. In comparison to their oral and laryngeal counterparts, HPV-N OPSCC were genetically distinct. In OPSCC, HPV status, tumor subsite and smoking determine outcome. Risk-stratification can be further refined based on the mutational signature, namely, NOTCH1 and SOX2 mutation status.

REVEL: An Ensemble Method for Predicting the Pathogenicity of Rare Missense Variants.

The vast majority of coding variants are rare, and assessment of the contribution of rare variants to complex traits is hampered by low statistical power and limited functional data. Improved methods for predicting the pathogenicity of rare coding variants are needed to facilitate the discovery of disease variants from exome sequencing studies. We developed REVEL (rare exome variant ensemble learner), an ensemble method for predicting the pathogenicity of missense variants on the basis of individual tools: MutPred, FATHMM, VEST, PolyPhen, SIFT, PROVEAN, MutationAssessor, MutationTaster, LRT, GERP, SiPhy, phyloP, and phastCons. REVEL was trained with recently discovered pathogenic and rare neutral missense variants, excluding those previously used to train its constituent tools. When applied to two independent test sets, REVEL had the best overall performance (p < 10-12) as compared to any individual tool and seven ensemble methods: MetaSVM, MetaLR, KGGSeq, Condel, CADD, DANN, and Eigen. Importantly, REVEL also had the best performance for distinguishing pathogenic from rare neutral variants with allele frequencies <0.5%. The area under the receiver operating characteristic curve (AUC) for REVEL was 0.046-0.182 higher in an independent test set of 935 recent SwissVar disease variants and 123,935 putatively neutral exome sequencing variants and 0.027-0.143 higher in an independent test set of 1,953 pathogenic and 2,406 benign variants recently reported in ClinVar than the AUCs for other ensemble methods. We provide pre-computed REVEL scores for all possible human missense variants to facilitate the identification of pathogenic variants in the sea of rare variants discovered as sequencing studies expand in scale.

Comprehensively evaluating cis-regulatory variation in the human prostate transcriptome by using gene-level allele-specific expression.

The identification of cis-acting regulatory variation in primary tissues has the potential to elucidate the genetic basis of complex traits and further our understanding of transcriptomic diversity across cell types. Expression quantitative trait locus (eQTL) association analysis using RNA sequencing (RNA-seq) data can improve upon the detection of cis-acting regulatory variation by leveraging allele-specific expression (ASE) patterns in association analysis. Here, we present a comprehensive evaluation of cis-acting eQTLs by analyzing RNA-seq gene-expression data and genome-wide high-density genotypes from 471 samples of normal primary prostate tissue. Using statistical models that integrate ASE information, we identified extensive cis-eQTLs across the prostate transcriptome and found that approximately 70% of expressed genes corresponded to a significant eQTL at a gene-level false-discovery rate of 0.05. Overall, cis-eQTLs were heavily concentrated near the transcription start and stop sites of affected genes, and effects were negatively correlated with distance. We identified multiple instances of cis-acting co-regulation by using phased genotype data and discovered 233 SNPs as the most strongly associated eQTLs for more than one gene. We also noted significant enrichment (25/50, p = 2E-5) of previously reported prostate cancer risk SNPs in prostate eQTLs. Our results illustrate the benefit of assessing ASE data in cis-eQTL analyses by showing better reproducibility of prior eQTL findings than of eQTL mapping based on total expression alone. Altogether, our analysis provides extensive functional context of thousands of SNPs in prostate tissue, and these results will be of critical value in guiding studies examining disease of the human prostate.

A FISH assay efficiently screens for BRAF gene rearrangements in pancreatic acinar-type neoplasms.

Approximately 1-2% of pancreatic neoplasms are acinar cell carcinomas. Recently, BRAF gene rearrangements were identified in over 20% of acinar-type neoplasms, which included both pure acinar cell carcinomas and mixed carcinomas with acinar differentiation, using next-generation sequencing-based platforms, providing a potential therapeutic target for patients with these neoplasms. Thus, it is clinically important to develop a rapid, cost- and material-efficient assay to screen for BRAF gene fusions in pancreatic acinar-type neoplasms. We developed a dual color, break-apart FISH assay to detect BRAF gene rearrangements in these neoplasms and evaluated its performance in comparison to next-generation sequencing-based studies. A blinded BRAF rearrangement FISH investigation was performed on 31 acinar-type neoplasms that had been studied previously by next-generation sequencing-based analysis as well as on 18 additional acinar-type neoplasms that were accrued over the past 2 years. In total, BRAF fusions were identified in 12/49 (24%) acinar-type neoplasms by FISH. BRAF fusion partners were uncovered by using targeted next-generation sequencing studies in 11 FISH-positive cases that had sufficient material for next-generation sequencing studies. SND1 was the most frequent fusion partner involved in BRAF-fusion acinar-type neoplasms (50%), followed by HERPUD1 (18%). No BRAF fusions were identified by next-generation sequencing in any of the FISH-negative cases investigated. FISH analysis showed that BRAF rearrangements were diffusely present across tumor-rich areas in BRAF-fusion acinar-type neoplasms, which is consistent with an oncogenic driver alteration pattern. Thus, we demonstrated that, in comparison to targeted next-generation sequencing-based technologies, the FISH assay is highly sensitive and specific as well as time- and cost-efficient in the detection of BRAF fusions in acinar-type neoplasms. The FISH assay can be easily implemented in diagnostic settings to identify acinar-type neoplasms patients potentially suitable for targeted therapy to inhibit MAPK pathway activity.

The genetic landscape of endometrial clear cell carcinomas.

Clear cell carcinoma of the endometrium is a rare type of endometrial cancer that is generally associated with an aggressive clinical behaviour. Here, we sought to define the repertoire of somatic genetic alterations in endometrial clear cell carcinomas (ECCs), and whether ECCs could be classified into the molecular subtypes described for endometrial endometrioid and serous carcinomas. We performed a rigorous histopathological review, immunohistochemical analysis and massively parallel sequencing targeting 300 cancer-related genes of 32 pure ECCs. Eleven (34%), seven (22%) and six (19%) ECCs showed abnormal expression patterns for p53, ARID1A, and at least one DNA mismatch repair (MMR) protein, respectively. Targeted sequencing data were obtained from 30 of the 32 ECCs included in this study, and these revealed that two ECCs (7%) were ultramutated and harboured mutations affecting the exonuclease domain of POLE. In POLE wild-type ECCs, TP53 (46%), PIK3CA (36%), PPP2R1A (36%), FBXW7 (25%), ARID1A (21%), PIK3R1 (18%) and SPOP (18%) were the genes most commonly affected by mutations; 18% and 11% harboured CCNE1 and ERBB2 amplifications, respectively, and 11% showed DAXX homozygous deletions. ECCs less frequently harboured mutations affecting CTNNB1 and PTEN but more frequently harboured PPP2R1A and TP53 mutations than non-POLE endometrioid carcinomas from The Cancer Genome Atlas (TCGA). Compared to endometrial serous carcinomas (TCGA), ECCs less frequently harboured TP53 mutations. When a surrogate model for the molecular-based TCGA classification was used, all molecular subtypes previously identified in endometrial endometrioid and serous carcinomas were present in the ECCs studied, including POLE, MMR-deficient, copy-number high (serous-like)/p53 abnormal, and copy-number low (endometrioid)/p53 wild-type, which were significantly associated with disease-free survival in univariate analysis. These findings demonstrate that ECCs constitute a histologically and genetically heterogeneous group of tumours with varying outcomes. Furthermore, our data suggest that the classification of ECCs as being generally 'high-grade' or 'type II' tumours may not be warranted. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Morphological characterization of colorectal cancers in The Cancer Genome Atlas reveals distinct morphology-molecular associations: clinical and biological implications.

The Cancer Genome Atlas data on colorectal carcinoma have provided a comprehensive view of the tumor's genomic alterations and their tumorigenic roles. Tumor morphology, however, has not been fully integrated into the analysis. The aim of this study was to explore relevant associations between tumor morphology and the newly characterized genomic alterations in colorectal carcinoma. Two hundred and seven colorectal carcinomas that had undergone whole-exome sequencing as part of The Cancer Genome Atlas project and had adequate virtual images in the cBioPortal for Cancer Genomics constituted our study population. Upon analysis, a tight association between 'microsatellite instability-high histology' and microsatellite instability-high (P<0.001) was readily detected and helped validate our image-based histology evaluation. Further, we showed, (1) among all histologies, the not otherwise specified type had the lowest overall mutation count (P<0.001 for entire cohort, P<0.03 for the microsatellite-instable group), and among the microsatellite-instable tumors, this type also correlated with fewer frameshift mutations in coding mononucleotide repeats of a defined set of relevant genes (P<0.01); (2) cytosine phosphate guanine island methylator phenotype-high colorectal cancers with or without microsatellite instability tended to have different histological patterns: the former more often mucinous and the latter more often not otherwise specified; (3) mucinous histology was associated with more frequent alterations in BRAF, PIK3CA, and the transforming growth factor-ß pathway when compared with non-mucinous histologies (P<0.001, P=0.01, and P<0.001, respectively); and (4) few colorectal cancers (<9%) exhibited upregulation of immune-inhibitory genes including major immune checkpoints; these tumors were primarily microsatellite-instable (up to 43%, vs <3% in microsatellite-stable group) and had distinctly non-mucinous histologies with a solid growth. These morphology-molecular associations are interesting and propose important clinical implications. The morphological patterns associated with alterations of immune checkpoint genes bear the potential to guide patient selection for clinical trials that target immune checkpoints in colorectal cancer, and provide directions for future studies.

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

Homozygous/Compound Heterozygous Triadin Mutations Associated With Autosomal-Recessive Long-QT Syndrome and Pediatric Sudden Cardiac Arrest: Elucidation of the Triadin Knockout Syndrome.

BACKGROUND: Long-QT syndrome (LQTS) may result in syncope, seizures, or sudden cardiac arrest. Although 16 LQTS-susceptibility genes have been discovered, 20% to 25% of LQTS remains genetically elusive. METHODS AND RESULTS: We performed whole-exome sequencing child-parent trio analysis followed by recessive and sporadic inheritance modeling and disease-network candidate analysis gene ranking to identify a novel underlying genetic mechanism for LQTS. Subsequent mutational analysis of the candidate gene was performed with polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing on a cohort of 33 additional unrelated patients with genetically elusive LQTS. After whole-exome sequencing and variant filtration, a homozygous p.D18fs*13 TRDN-encoded triadin frameshift mutation was discovered in a 10-year-old female patient with LQTS with a QTc of 500 milliseconds who experienced recurrent exertion-induced syncope/cardiac arrest beginning at 1 year of age. Subsequent mutational analysis of TRDN revealed either homozygous or compound heterozygous frameshift mutations in 4 of 33 unrelated cases of LQTS (12%). All 5 TRDN-null patients displayed extensive T-wave inversions in precordial leads V1 through V4, with either persistent or transient QT prolongation and severe disease expression of exercise-induced cardiac arrest in early childhood (≤3 years of age) and required aggressive therapy. The overall yield of TRDN mutations was significantly greater in patients ≤10 years of age (5 of 10, 50%) compared with older patients (0 of 24, 0%; P=0.0009). CONCLUSIONS: We identified TRDN as a novel underlying genetic basis for recessively inherited LQTS. All TRDN-null patients had strikingly similar phenotypes. Given the recurrent nature of potential lethal arrhythmias, patients fitting this phenotypic profile should undergo cardiac TRDN genetic testing.

TNNI3K mutation in familial syndrome of conduction system disease, atrial tachyarrhythmia and dilated cardiomyopathy.

Locus mapping has uncovered diverse etiologies for familial atrial fibrillation (AF), dilated cardiomyopathy (DCM), and mixed cardiac phenotype syndromes, yet the molecular basis for these disorders remains idiopathic in most cases. Whole-exome sequencing (WES) provides a powerful new tool for familial disease gene discovery. Here, synergistic application of these genomic strategies identified the pathogenic mutation in a familial syndrome of atrial tachyarrhythmia, conduction system disease (CSD), and DCM vulnerability. Seven members of a three-generation family exhibited the variably expressed phenotype, three of whom manifested CSD and clinically significant arrhythmia in childhood. Genome-wide linkage analysis mapped two equally plausible loci to chromosomes 1p3 and 13q12. Variants from WES of two affected cousins were filtered for rare, predicted-deleterious, positional variants, revealing an unreported heterozygous missense mutation disrupting the highly conserved kinase domain in TNNI3K. The G526D substitution in troponin I interacting kinase, with the most deleterious SIFT and Polyphen2 scores possible, resulted in abnormal peptide aggregation in vitro and in silico docking models predicted altered yet energetically favorable wild-type mutant dimerization. Ventricular tissue from a mutation carrier displayed histopathological hallmarks of DCM and reduced TNNI3K protein staining with unique amorphous nuclear and sarcoplasmic inclusions. In conclusion, mutation of TNNI3K, encoding a heart-specific kinase previously shown to modulate cardiac conduction and myocardial function in mice, underlies a familial syndrome of electrical and myopathic heart disease. The identified substitution causes a TNNI3K aggregation defect and protein deficiency, implicating a dominant-negative loss of function disease mechanism.

Comparison of the Life Cycles of Genetically Distant Species C and Species D Human Adenoviruses Ad6 and Ad26 in Human Cells.

UNLABELLED: Our understanding of adenovirus (Ad) biology is largely extrapolated from human species C Ad5. Most humans are immune to Ad5, so lower-seroprevalence viruses like human Ad6 and Ad26 are being tested as therapeutic vectors. Ad6 and Ad26 differ at the DNA level by 34%. To better understand how this might impact their biology, we examined the life cycle of the two viruses in human lung cells in vitro. Both viruses infected A549 cells with similar efficiencies, executed DNA replication with identical kinetics within 12 h, and began killing cells within 72 h. While Ad6-infected cells remained adherent until death, Ad26-infected cells detached within 12 h of infection but remained viable. Next-generation sequencing (NGS) of mRNA from infected cells demonstrated that viral transcripts constituted 1% of cellular mRNAs within 6 h and 8 to 16% within 12 h. Quantitative PCR and NGS revealed the activation of key early genes at 6 h and transition to late gene activation by 12 h by both viruses. There were marked differences in the balance of E1A and E1B activation by the two viruses and in the expression of E3 immune evasion mRNAs. Ad6 was markedly more effective at suppressing major histocompatibility complex class I (MHC I) display on the cell surface and in evading TRAIL-mediated apoptosis than was Ad26. These data demonstrate shared as well as divergent life cycles in these genetically distant human adenoviruses. An understanding of these differences expands the knowledge of alternative Ad species and may inform the selection of related Ads for therapeutic development. IMPORTANCE: A burgeoning number of adenoviruses (Ads) are being harnessed as therapeutics, yet the biology of these viruses is generally extrapolated from Ad2 and Ad5. Here, we are the first to compare the transcriptional programs of two genetically distant Ads by mRNA next-generation sequencing (NGS). Species C Ad6 and Ad26 are being pursued as lower-seroprevalence Ad vectors but differ at the DNA level by 34%. Head-to-head comparison in human lung cells by NGS revealed that the two viruses generally conform to our general understanding of the Ad transcriptional program. However, fine mapping revealed subtle and strong differences in how these two viruses execute these programs, including differences in the balance of E1A and E1B mRNAs and in E3 immune evasion genes. This suggests that not all adenoviruses behave like Ad2 and Ad5 and that they may have unique strategies to infect cells and evade the immune system.

Identification and characterization of a novel promoter for the human ANO1 gene regulated by the transcription factor signal transducer and activator of transcription 6 (STAT6).

Anoctamin-1 (Ano1) is a widely expressed protein responsible for endogenous Ca(2+)-activated Cl(-) currents. Ano1 is overexpressed in cancer. Differential expression of transcriptional variants is also found in other diseases. However, the mechanisms underlying regulation of Ano1 are unknown. This study identifies the Ano1 promoter and defines a mechanism for regulating its expression. Next-generation RNA sequencing (RNA-seq) analysis in human gastric muscle found a new exon upstream of the reported exon 1 and identified a promoter proximal to this new exon. Reporter assays in human embryonic kidney 293 cells showed a 6.7 ± 2.1-fold increase in activity over empty vector. Treatment with a known regulator of Ano1 expression, IL-4, increased promoter activity by 1.6 ± 0.02-fold over untreated cells. The promoter region contained putative binding sites for multiple transcription factors including signal transducer and activator of transcription 6 (STAT6), a downstream effector of IL-4. Chromatin immunoprecipitation (ChIP) experiments on T84 cells, which endogenously express Ano1, showed a 2.1 ± 0.12-fold increase in binding of STAT6 to P0 after IL-4 treatment. These results were confirmed by mutagenesis, expression, and RNA interference techniques. This work allows deeper understanding of the regulation of Ano1 in physiology and as a potential therapeutic target in a variety of diseases.

Gene expression patterns in CD4+ peripheral blood cells in healthy subjects and stage IV melanoma patients.

Melanoma patients exhibit changes in immune responsiveness in the local tumor environment, draining lymph nodes, and peripheral blood. Immune-targeting therapies are revolutionizing melanoma patient care increasingly, and studies show that patients derive clinical benefit from these newer agents. Nonetheless, predicting which patients will benefit from these costly therapies remains a challenge. In an effort to capture individual differences in immune responsiveness, we are analyzing patterns of gene expression in human peripheral blood cells using RNAseq. Focusing on CD4+ peripheral blood cells, we describe multiple categories of immune regulating genes, which are expressed in highly ordered patterns shared by cohorts of healthy subjects and stage IV melanoma patients. Despite displaying conservation in overall transcriptome structure, CD4+ peripheral blood cells from melanoma patients differ quantitatively from healthy subjects in the expression of more than 2000 genes. Moreover, 1300 differentially expressed genes are found in transcript response patterns following activation of CD4+ cells ex vivo, suggesting that widespread functional discrepancies differentiate the immune systems of healthy subjects and melanoma patients. While our analysis reveals that the transcriptome architecture characteristic of healthy subjects is maintained in cancer patients, the genes expressed differentially among individuals and across cohorts provide opportunities for understanding variable immune states as well as response potentials, thus establishing a foundation for predicting individual responses to stimuli such as immunotherapeutic agents.

Brain expression genome-wide association study (eGWAS) identifies human disease-associated variants.

Genetic variants that modify brain gene expression may also influence risk for human diseases. We measured expression levels of 24,526 transcripts in brain samples from the cerebellum and temporal cortex of autopsied subjects with Alzheimer's disease (AD, cerebellar n=197, temporal cortex n=202) and with other brain pathologies (non-AD, cerebellar n=177, temporal cortex n=197). We conducted an expression genome-wide association study (eGWAS) using 213,528 cisSNPs within ± 100 kb of the tested transcripts. We identified 2,980 cerebellar cisSNP/transcript level associations (2,596 unique cisSNPs) significant in both ADs and non-ADs (q<0.05, p=7.70 × 10(-5)-1.67 × 10(-82)). Of these, 2,089 were also significant in the temporal cortex (p=1.85 × 10(-5)-1.70 × 10(-141)). The top cerebellar cisSNPs had 2.4-fold enrichment for human disease-associated variants (p<10(-6)). We identified novel cisSNP/transcript associations for human disease-associated variants, including progressive supranuclear palsy SLCO1A2/rs11568563, Parkinson's disease (PD) MMRN1/rs6532197, Paget's disease OPTN/rs1561570; and we confirmed others, including PD MAPT/rs242557, systemic lupus erythematosus and ulcerative colitis IRF5/rs4728142, and type 1 diabetes mellitus RPS26/rs1701704. In our eGWAS, there was 2.9-3.3 fold enrichment (p<10(-6)) of significant cisSNPs with suggestive AD-risk association (p<10(-3)) in the Alzheimer's Disease Genetics Consortium GWAS. These results demonstrate the significant contributions of genetic factors to human brain gene expression, which are reliably detected across different brain regions and pathologies. The significant enrichment of brain cisSNPs among disease-associated variants advocates gene expression changes as a mechanism for many central nervous system (CNS) and non-CNS diseases. Combined assessment of expression and disease GWAS may provide complementary information in discovery of human disease variants with functional implications. Our findings have implications for the design and interpretation of eGWAS in general and the use of brain expression quantitative trait loci in the study of human disease genetics.

HiChIP: a high-throughput pipeline for integrative analysis of ChIP-Seq data.

BACKGROUND: Chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-Seq) has been widely used to identify genomic loci of transcription factor (TF) binding and histone modifications. ChIP-Seq data analysis involves multiple steps from read mapping and peak calling to data integration and interpretation. It remains challenging and time-consuming to process large amounts of ChIP-Seq data derived from different antibodies or experimental designs using the same approach. To address this challenge, there is a need for a comprehensive analysis pipeline with flexible settings to accelerate the utilization of this powerful technology in epigenetics research. RESULTS: We have developed a highly integrative pipeline, termed HiChIP for systematic analysis of ChIP-Seq data. HiChIP incorporates several open source software packages selected based on internal assessments and published comparisons. It also includes a set of tools developed in-house. This workflow enables the analysis of both paired-end and single-end ChIP-Seq reads, with or without replicates for the characterization and annotation of both punctate and diffuse binding sites. The main functionality of HiChIP includes: (a) read quality checking; (b) read mapping and filtering; (c) peak calling and peak consistency analysis; and (d) result visualization. In addition, this pipeline contains modules for generating binding profiles over selected genomic features, de novo motif finding from transcription factor (TF) binding sites and functional annotation of peak associated genes. CONCLUSIONS: HiChIP is a comprehensive analysis pipeline that can be configured to analyze ChIP-Seq data derived from varying antibodies and experiment designs. Using public ChIP-Seq data we demonstrate that HiChIP is a fast and reliable pipeline for processing large amounts of ChIP-Seq data.

MAP-RSeq: Mayo Analysis Pipeline for RNA sequencing.

BACKGROUND: Although the costs of next generation sequencing technology have decreased over the past years, there is still a lack of simple-to-use applications, for a comprehensive analysis of RNA sequencing data. There is no one-stop shop for transcriptomic genomics. We have developed MAP-RSeq, a comprehensive computational workflow that can be used for obtaining genomic features from transcriptomic sequencing data, for any genome. RESULTS: For optimization of tools and parameters, MAP-RSeq was validated using both simulated and real datasets. MAP-RSeq workflow consists of six major modules such as alignment of reads, quality assessment of reads, gene expression assessment and exon read counting, identification of expressed single nucleotide variants (SNVs), detection of fusion transcripts, summarization of transcriptomics data and final report. This workflow is available for Human transcriptome analysis and can be easily adapted and used for other genomes. Several clinical and research projects at the Mayo Clinic have applied the MAP-RSeq workflow for RNA-Seq studies. The results from MAP-RSeq have thus far enabled clinicians and researchers to understand the transcriptomic landscape of diseases for better diagnosis and treatment of patients. CONCLUSIONS: Our software provides gene counts, exon counts, fusion candidates, expressed single nucleotide variants, mapping statistics, visualizations, and a detailed research data report for RNA-Seq. The workflow can be executed on a standalone virtual machine or on a parallel Sun Grid Engine cluster. The software can be downloaded from http://bioinformaticstools.mayo.edu/research/maprseq/.

Histone deacetylase inhibition promotes osteoblast maturation by altering the histone H4 epigenome and reduces Akt phosphorylation.

Bone has remarkable regenerative capacity, but this ability diminishes during aging. Histone deacetylase inhibitors (HDIs) promote terminal osteoblast differentiation and extracellular matrix production in culture. The epigenetic events altered by HDIs in osteoblasts may hold clues for the development of new anabolic treatments for osteoporosis and other conditions of low bone mass. To assess how HDIs affect the epigenome of committed osteoblasts, MC3T3 cells were treated with suberoylanilide hydroxamic acid (SAHA) and subjected to microarray gene expression profiling and high-throughput ChIP-Seq analysis. As expected, SAHA induced differentiation and matrix calcification of osteoblasts in vitro. ChIP-Seq analysis revealed that SAHA increased histone H4 acetylation genome-wide and in differentially regulated genes, except for the 500 bp upstream of transcriptional start sites. Pathway analysis indicated that SAHA increased the expression of insulin signaling modulators, including Slc9a3r1. SAHA decreased phosphorylation of insulin receptor ß, Akt, and the Akt substrate FoxO1, resulting in FoxO1 stabilization. Thus, SAHA induces genome-wide H4 acetylation and modulates the insulin/Akt/FoxO1 signaling axis, whereas it promotes terminal osteoblast differentiation in vitro.

CAP-miRSeq: a comprehensive analysis pipeline for microRNA sequencing data.

BACKGROUND: miRNAs play a key role in normal physiology and various diseases. miRNA profiling through next generation sequencing (miRNA-seq) has become the main platform for biological research and biomarker discovery. However, analyzing miRNA sequencing data is challenging as it needs significant amount of computational resources and bioinformatics expertise. Several web based analytical tools have been developed but they are limited to processing one or a pair of samples at time and are not suitable for a large scale study. Lack of flexibility and reliability of these web applications are also common issues. RESULTS: We developed a Comprehensive Analysis Pipeline for microRNA Sequencing data (CAP-miRSeq) that integrates read pre-processing, alignment, mature/precursor/novel miRNA detection and quantification, data visualization, variant detection in miRNA coding region, and more flexible differential expression analysis between experimental conditions. According to computational infrastructure, users can install the package locally or deploy it in Amazon Cloud to run samples sequentially or in parallel for a large number of samples for speedy analyses. In either case, summary and expression reports for all samples are generated for easier quality assessment and downstream analyses. Using well characterized data, we demonstrated the pipeline's superior performances, flexibility, and practical use in research and biomarker discovery. CONCLUSIONS: CAP-miRSeq is a powerful and flexible tool for users to process and analyze miRNA-seq data scalable from a few to hundreds of samples. The results are presented in the convenient way for investigators or analysts to conduct further investigation and discovery.