A functional genomics pipeline to identify high-value asthma and allergy CpGs in the human methylome.

BACKGROUND: DNA methylation of cytosines at cytosine-phosphate-guanine (CpG) dinucleotides (CpGs) is a widespread epigenetic mark, but genome-wide variation has been relatively unexplored due to the limited representation of variable CpGs on commercial high-throughput arrays. OBJECTIVES: To explore this hidden portion of the epigenome, this study combined whole-genome bisulfite sequencing with in silico evidence of gene regulatory regions to design a custom array of high-value CpGs. This study focused on airway epithelial cells from children with and without allergic asthma because these cells mediate the effects of inhaled microbes, pollution, and allergens on asthma and allergic disease risk. METHODS: This study identified differentially methylated regions from whole-genome bisulfite sequencing in nasal epithelial cell DNA from a total of 39 children with and without allergic asthma of both European and African ancestries. This study selected CpGs from differentially methylated regions, previous allergy or asthma epigenome-wide association studies (EWAS), or genome-wide association study loci, and overlapped them with functional annotations for inclusion on a custom Asthma&Allergy array. This study used both the custom and EPIC arrays to perform EWAS of allergic sensitization (AS) in nasal epithelial cell DNA from children in the URECA (Urban Environment and Childhood Asthma) birth cohort and using the custom array in the INSPIRE [Infant Susceptibility to Pulmonary Infections and Asthma Following RSV Exposure] birth cohort. Each CpG on the arrays was assigned to its nearest gene and its promotor capture Hi-C interacting gene and performed expression quantitative trait methylation (eQTM) studies for both sets of genes. RESULTS: Custom array CpGs were enriched for intermediate methylation levels compared to EPIC CpGs. Intermediate methylation CpGs were further enriched among those associated with AS and for eQTMs on both arrays. CONCLUSIONS: This study revealed signature features of high-value CpGs and evidence for epigenetic regulation of genes at AS EWAS loci that are robust to race/ethnicity, ascertainment, age, and geography.

Transcriptome clarifies mechanisms of lesion genesis versus progression in models of Ccm3 cerebral cavernous malformations.

Cerebral cavernous malformations (CCMs) are dilated capillaries causing epilepsy and stroke. Inheritance of a heterozygous mutation in CCM3/PDCD10 is responsible for the most aggressive familial form of the disease. Here we studied the differences and commonalities between the transcriptomes of microdissected lesional neurovascular units (NVUs) from acute and chronic in vivo Ccm3/Pdcd10ECKO mice, and cultured brain microvascular endothelial cells (BMECs) Ccm3/Pdcd10ECKO.We identified 2409 differentially expressed genes (DEGs) in acute and 2962 in chronic in vivo NVUs compared to microdissected brain capillaries, as well as 121 in in vitro BMECs with and without Ccm3/Pdcd10 loss (fold change ≥ |2.0|; p < 0.05, false discovery rate corrected). A functional clustered dendrogram generated using the Euclidean distance showed that the DEGs identified only in acute in vivo NVUs were clustered in cellular proliferation gene ontology functions. The DEGs only identified in chronic in vivo NVUs were clustered in inflammation and immune response, permeability, and adhesion functions. In addition, 1225 DEGs were only identified in the in vivo NVUs but not in vitro BMECs, and these clustered within neuronal and glial functions. One miRNA mmu-miR-3472a was differentially expressed (FC = - 5.98; p = 0.07, FDR corrected) in the serum of Ccm3/Pdcd10+/- when compared to wild type mice, and this was functionally related as a putative target to Cand2 (cullin associated and neddylation dissociated 2), a DEG in acute and chronic lesional NVUs and in vitro BMECs. Our results suggest that the acute model is characterized by cell proliferation, while the chronic model showed inflammatory, adhesion and permeability processes. In addition, we highlight the importance of extra-endothelial structures in CCM disease, and potential role of circulating miRNAs as biomarkers of disease, interacting with DEGs. The extensive DEGs library of each model will serve as a validation tool for potential mechanistic, biomarker, and therapeutic targets.

Comprehensive transcriptome analysis of cerebral cavernous malformation across multiple species and genotypes.

The purpose of this study was to determine important genes, functions, and networks contributing to the pathobiology of cerebral cavernous malformation (CCM) from transcriptomic analyses across 3 species and 2 disease genotypes. Sequencing of RNA from laser microdissected neurovascular units of 5 human surgically resected CCM lesions, mouse brain microvascular endothelial cells, Caenorhabditis elegans with induced Ccm gene loss, and their respective controls provided differentially expressed genes (DEGs). DEGs from mouse and C. elegans were annotated into human homologous genes. Cross-comparisons of DEGs between species and genotypes, as well as network and gene ontology (GO) enrichment analyses, were performed. Among hundreds of DEGs identified in each model, common genes and 1 GO term (GO:0051656, establishment of organelle localization) were commonly identified across the different species and genotypes. In addition, 24 GO functions were present in 4 of 5 models and were related to cell-to-cell adhesion, neutrophil-mediated immunity, ion transmembrane transporter activity, and responses to oxidative stress. We have provided a comprehensive transcriptome library of CCM disease across species and for the first time to our knowledge in Ccm1/Krit1 versus Ccm3/Pdcd10 genotypes. We have provided examples of how results can be used in hypothesis generation or mechanistic confirmatory studies.

Genomics of Ovarian Cancer Progression Reveals Diverse Metastatic Trajectories Including Intraepithelial Metastasis to the Fallopian Tube.

Accumulating evidence has supported the fallopian tube rather than the ovary as the origin for high-grade serous ovarian cancer (HGSOC). To understand the relationship between putative precursor lesions and metastatic tumors, we performed whole-exome sequencing on specimens from eight HGSOC patient progression series consisting of serous tubal intraepithelial carcinomas (STIC), invasive fallopian tube lesions, invasive ovarian lesions, and omental metastases. Integration of copy number and somatic mutations revealed patient-specific patterns with similar mutational signatures and copy-number variation profiles across all anatomic sites, suggesting that genomic instability is an early event in HGSOC. Phylogenetic analyses supported STIC as precursor lesions in half of our patient cohort, but also identified STIC as metastases in 2 patients. Ex vivo assays revealed that HGSOC spheroids can implant in the fallopian tube epithelium and mimic STIC lesions. That STIC may represent metastases calls into question the assumption that STIC are always indicative of primary fallopian tube cancers. SIGNIFICANCE: We find that the putative precursor lesions for HGSOC, STIC, possess most of the genomic aberrations present in advanced cancers. In addition, a proportion of STIC represent intraepithelial metastases to the fallopian tube rather than the origin of HGSOC. Cancer Discov; 6(12); 1342-51. ©2016 AACR.See related commentary by Swisher et al., p. 1309This article is highlighted in the In This Issue feature, p. 1293.

The kinase activity of PKR represses inflammasome activity.

The protein kinase R (PKR) functions in the antiviral response by controlling protein translation and inflammatory cell signaling pathways. We generated a transgenic, knock-in mouse in which the endogenous PKR is expressed with a point mutation that ablates its kinase activity. This novel animal allows us to probe the kinase-dependent and -independent functions of PKR. We used this animal together with a previously generated transgenic mouse that is ablated for PKR expression to determine the role of PKR in regulating the activity of the cryopyrin inflammasome. Our data demonstrate that, in contradiction to earlier reports, PKR represses cryopyrin inflammasome activity. We demonstrate that this control is mediated through the established function of PKR to inhibit protein translation of constituents of the inflammasome to prevent initial priming during innate immune signaling. These findings identify an important role for PKR to dampen inflammation during the innate immune response and caution against the previously proposed therapeutic strategy to inhibit PKR to treat inflammation.

Notch1 Activation or Loss Promotes HPV-Induced Oral Tumorigenesis.

Viral oncogene expression is insufficient for neoplastic transformation of human cells, so human papillomavirus (HPV)-associated cancers will also rely upon mutations in cellular oncogenes and tumor suppressors. However, it has been difficult so far to distinguish incidental mutations without phenotypic impact from causal mutations that drive the development of HPV-associated cancers. In this study, we addressed this issue by conducting a functional screen for genes that facilitate the formation of HPV E6/E7-induced squamous cell cancers in mice using a transposon-mediated insertional mutagenesis protocol. Overall, we identified 39 candidate driver genes, including Notch1, which unexpectedly was scored by gain- or loss-of-function mutations that were capable of promoting squamous cell carcinogenesis. Autochthonous HPV-positive oral tumors possessing an activated Notch1 allele exhibited high rates of cell proliferation and tumor growth. Conversely, Notch1 loss could accelerate the growth of invasive tumors in a manner associated with increased expression of matrix metalloproteinases and other proinvasive genes. HPV oncogenes clearly cooperated with loss of Notch1, insofar as its haploinsufficiency accelerated tumor growth only in HPV-positive tumors. In clinical specimens of various human cancers, there was a consistent pattern of NOTCH1 expression that correlated with invasive character, in support of our observations in mice. Although Notch1 acts as a tumor suppressor in mouse skin, we found that oncogenes enabling any perturbation in Notch1 expression promoted tumor growth, albeit via distinct pathways. Our findings suggest caution in interpreting the meaning of putative driver gene mutations in cancer, and therefore therapeutic efforts to target them, given the significant contextual differences in which such mutations may arise, including in virus-associated tumors.

Homology-mediated end-capping as a primary step of sister chromatid fusion in the breakage-fusion-bridge cycles.

Breakage-fusion-bridge (BFB) cycle is a series of chromosome breaks and duplications that could lead to the increased copy number of a genomic segment (gene amplification). A critical step of BFB cycles leading to gene amplification is a palindromic fusion of sister chromatids following the rupture of a dicentric chromosome during mitosis. It is currently unknown how sister chromatid fusion is produced from a mitotic break. To delineate the process, we took an integrated genomic, cytogenetic and molecular approach for the recurrent MCL1 amplicon at chromosome 1 in human tumor cells. A newly developed next-generation sequencing-based approach identified a cluster of palindromic fusions within the amplicon at ∼50-kb intervals, indicating a series of breaks and fusions by BFB cycles. The physical location of the amplicon (at the end of a broken chromosome) further indicated BFB cycles as underlying processes. Three palindromic fusions were mediated by the homologies between two nearby inverted Alu repeats, whereas the other two fusions exhibited microhomology-mediated events. Such breakpoint sequences indicate that homology-mediated fold-back capping of broken ends followed by DNA replication is an underlying mechanism of sister chromatid fusion. Our results elucidate nucleotide-level events during BFB cycles and end processing for naturally occurring mitotic breaks.

Identification of disease-associated DNA methylation in B cells from Crohn's disease and ulcerative colitis patients.

BACKGROUND: Changes in the methylation status of inflammatory bowel disease (IBD)-associated genes could significantly alter levels of gene expression, thereby contributing to disease onset and progression. We previously identified seven disease-associated DNA methylation loci from intestinal tissues of IBD patients using the Illumina GoldenGate BeadArray assay. AIMS: In this study, we extended this approach to identify IBD-associated changes in DNA methylation in B cells from 18 IBD patients [9 Crohn's disease (CD) and 9 ulcerative colitis (UC)]. B cell DNA methylation markers are particularly favorable for diagnosis due to the convenient access to peripheral blood. METHODS: We examined DNA methylation profiles of B cell lines using the Illumina GoldenGate BeadArray assay. Disease-associated CpGs/genes with changes in DNA methylation were identified by comparison of methylation profiles between B cell lines from IBD patients and their siblings without IBD. BeadArray data were validated using a bisulfite polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) method. To verify that observed changes in DNA methylation were not due to virus transformation, we compared specific CpG DNA methylation levels of GADD45A and POMC between B cell lines and matching peripheral blood B lymphocytes from five individuals. RESULTS: Using this approach with strict statistical analysis, we identified 11 IBD-associated CpG sites, 14 CD-specific CpG sites, and 24 UC-specific CpG sites with methylation changes in B cells. CONCLUSIONS: IBD- and subtype-specific changes in DNA methylation were identified in B cells from IBD patients. Many of these genes have important immune and inflammatory response functions including several loci within the interleukin (IL)-12/IL-23 pathway.

CCL2 expression in primary ovarian carcinoma is correlated with chemotherapy response and survival outcomes.

CCL2, a chemokine, is expressed in normal human ovarian epithelium but down-regulated in ovarian adenocarcinomas. The association of CCL2 expression with chemotherapy response, invasion and survival outcomes was studied in patients with primary ovarian cancer (OC) and in ovarian cancer cell lines (OCCLs). Tumor specimens (>80% tumor) from patients with primary, advanced serous OC obtained at the time of cytoreductive surgery was used to isolate total RNA. The CCL2 gene expression evaluated by RT-PCR was investigated in relation to chemo-response/clinical outcomes in the OC patients and to sensitivity to cisplatin/paclitaxel in the OCCLs. In vitro invasion was measured by matrigel invasion and matrixmetallo-proteinase-9 (MMP-9) zymogram assays. Thirty-seven patients were included. In multivariable analyses that adjusted for the impact of debulking status, the CCL2 mRNA expression was correlated with objective complete response (p = 0.01), chemosensitivity (p = 0.04), and progression-free survival (PFS; p = 0.006). These findings were corroborated in vitro in the OCCLs. The cells expressing higher levels of CCL2 were more sensitive to paclitaxel and cisplatin as compared to those lines expressing lower levels of this chemokine. Up-regulation of CCL2 in the PAT-7 cell line further enhanced the response of these cells to paclitaxel (p = 0.0001) and led to decreased invasion (p = 0.0009). Increased ovarian tumoral expression of CCL2 is associated with improved chemoresponse and survival outcomes, and higher levels of CCL2 in ovarian cancer cell lines are associated with increased chemosensitivity and decreased invasion in vitro.

Genes regulated by Nkx2-3 in siRNA-mediated knockdown B cells: implication of endothelin-1 in inflammatory bowel disease.

Nkx2-3 gene variants are strongly associated with inflammatory bowel disease (IBD) and its expression is up-regulated in Crohn's disease (CD). However, the nature of its role underlying IBD pathogenesis is unknown. We investigated the genes regulated by Nkx2-3 using cDNA microarray. A small interfering RNA (siRNA)-mediated knockdown of Nkx2-3 in a B cell line from a CD patient was generated. Gene expression was profiled on high-density cDNA microarrays representing over 25,000 genes. Ingenuity pathway analysis (IPA) was used to identify gene networks according to biological functions and associated pathways. Expression profiling analysis by cDNA microarray showed that 125 genes were regulated by Nkx2-3 knockdown (fold change >or=3.0, p<0.01), among which 51 genes were immune and inflammatory response genes. Microarray results were validated by RT-PCR and further confirmed in a B cell line expressing siRNA of Nkx2-3 from an additional CD patient. The results showed that Nkx2-3 was up-regulated (p<0.05) and EDN1 was down-regulated (p<0.05) in B cell lines from CD patients. mRNA expression levels of Nkx2-3 were negatively correlated with those of EDN1 (r=-0.6044, p<0.05). EDN1 was also down-regulated in intestinal tissues from UC patients (p<0.05). Our present results demonstrate that a decrease in Nkx2-3 gene expression level can profoundly alter the expression of genes and cellular functions relevant to the pathogenesis and progression of IBD, such as EDN1.

Differential expression in clear cell renal cell carcinoma identified by gene expression profiling.

PURPOSE: Gene expression profiling has been shown to provide prognostic information on patients with solitary sporadic renal cell carcinoma. To our knowledge there is no reliable way to differentiate synchronous renal metastasis from bilateral primary tumors in patients with bilateral renal cell carcinoma. We present data using a custom kidney cancer cDNA array that can predict the outcome in patients with unilateral and bilateral renal cell carcinoma. MATERIALS AND METHODS: Fresh frozen tissue from 38 clear cell renal cell carcinomas was analyzed using a cancer cDNA array containing 3,966 genes relevant to cancer or kidney development. Median followup was 5.3 years. Cancer recurred in 12 patients (43%) and 11 (39%) had died by the last followup. RESULTS: Using a training data set of 8 tumors a 44 gene expression profile distinguishing aggressive and indolent clear cell renal cell carcinoma was identified. Of 29 single clear cell renal cell carcinomas 16 and 13 were predicted to be indolent and aggressive, respectively, by the gene expression profile. Recurrence-free survival at 5 years was 68% and 42% in these 2 groups, respectively (p = 0.032). Clear cell renal cell carcinoma classified as indolent or aggressive according to SSIGN (stage, size, grade and necrosis) score showed a 5-year recurrence-free survival rate of 78% and 42%, respectively (p = 0.021). On Cox proportional hazards analysis the gene expression profile was not an independent predictor of recurrence-free survival after accounting for SSIGN score. Gene expression profile classification correlated with cancer specific survival at 5 years in 4 of 4 patients with metachronous clear cell renal cell carcinoma but in only 2 of 4 with bilateral synchronous clear cell renal cell carcinoma. CONCLUSIONS: Gene expression profiling using a kidney cancer relevant cDNA array can differentiate between aggressive and indolent clear cell renal cell carcinomas. Gene expression profile results may be most useful for unilateral clear cell renal cell carcinoma when results are discordant with predictions of tumor behavior based on standard clinicopathological features. In addition, gene expression profiling can provide prognostic information that may help characterize tumors of unknown clinical stage, such as bilateral metachronous clear cell renal cell carcinoma.

GPRC6A null mice exhibit osteopenia, feminization and metabolic syndrome.

BACKGROUND: GPRC6A is a widely expressed orphan G-protein coupled receptor that senses extracellular amino acids, osteocalcin and divalent cations in vitro. The physiological functions of GPRC6A are unknown. METHODS/PRINCIPAL FINDINGS: In this study, we created and characterized the phenotype of GPRC6A(-/-) mice. We observed complex metabolic abnormalities in GPRC6A(-/-) mice involving multiple organ systems that express GPRC6A, including bone, kidney, testes, and liver. GPRC6A(-/-) mice exhibited hepatic steatosis, hyperglycemia, glucose intolerance, and insulin resistance. In addition, we observed high expression of GPRC6A in Leydig cells in the testis. Ablation of GPRC6A resulted in feminization of male GPRC6A(-/-) mice in association with decreased lean body mass, increased fat mass, increased circulating levels of estradiol, and reduced levels of testosterone. GPRC6A was also highly expressed in kidney proximal and distal tubules, and GPRC6A(-/-) mice exhibited increments in urine Ca/Cr and PO(4)/Cr ratios as well as low molecular weight proteinuria. Finally, GPRC6A(-/-) mice exhibited a decrease in bone mineral density (BMD) in association with impaired mineralization of bone. CONCLUSIONS/SIGNIFICANCE: GPRC6A(-/-) mice have a metabolic syndrome characterized by defective osteoblast-mediated bone mineralization, abnormal renal handling of calcium and phosphorus, fatty liver, glucose intolerance and disordered steroidogenesis. These findings suggest the overall function of GPRC6A may be to coordinate the anabolic responses of multiple tissues through the sensing of extracellular amino acids, osteocalcin and divalent cations.

Reduced expression of autotaxin predicts survival in uveal melanoma.

AIM: In an effort to identify patients with uveal melanoma at high risk of metastasis, the authors undertook correlation of gene expression profiles with histopathology data and tumour-related mortality. METHODS: The RNA was isolated from 27 samples of uveal melanoma from patients who had consented to undergo enucleation, and transcripts profiled using a cDNA array comprised of sequence-verified cDNA clones representing approximately 4000 genes implicated in cancer development. Two multivariate data mining techniques--hierarchical cluster analysis and multidimensional scaling--were used to investigate the grouping structure in the gene expression data. Cluster analysis was performed with a subset of 10,000 randomly selected genes and the cumulative contribution of all the genes in making the correct grouping was recorded. RESULTS: Hierarchical cluster analysis and multidimensional scaling revealed two distinct classes. When correlated with the data on metastasis, the two molecular classes corresponded very well to the survival data for the 27 patients. Thirty two discrete genes (corresponding to 44 probe sets) that correctly defined the molecular classes were selected. A single gene (ectonucleotide pyrophosphatase/phosphodiesterase 2; autotaxin) could classify the molecular types. The expression pattern was confirmed using real-time quantitative PCR. CONCLUSIONS: Gene expression profiling identifies two distinct prognostic classes of uveal melanoma. Underexpression of autotaxin in class 2 uveal melanoma with a poor prognosis needs to be explored further.

Identification of a novel extracellular cation-sensing G-protein-coupled receptor.

The C family G-protein-coupled receptors contain members that sense amino acid and extracellular cations, of which calcium-sensing receptor (CASR) is the prototypic extracellular calcium-sensing receptor. Some cells, such as osteoblasts in bone, retain responsiveness to extracellular calcium in CASR-deficient mice, consistent with the existence of another calcium-sensing receptor. We examined the calcium-sensing properties of GPRC6A, a newly identified member of this family. Alignment of GPRC6A with CASR revealed conservation of both calcium and calcimimetic binding sites. In addition, calcium, magnesium, strontium, aluminum, gadolinium, and the calcimimetic NPS 568 resulted in a dose-dependent stimulation of GPRC6A overexpressed in human embryonic kidney cells 293 cells. Also, osteocalcin, a calcium-binding protein highly expressed in bone, dose-dependently stimulated GPRC6A activity in the presence of calcium but inhibited the calcium-dependent activation of CASR. Coexpression of beta-arrestins 1 and 2, regulators of G-protein signaling RGS2 or RGS4, the RhoA inhibitor C3 toxin, the dominant negative Galpha(q)-(305-359) minigene, and pretreatment with pertussis toxin inhibited activation of GPRC6A by extracellular cations. Reverse transcription-PCR analyses showed that mouse GPRC6A is widely expressed in mouse tissues, including bone, calvaria, and the osteoblastic cell line MC3T3-E1. These data suggest that in addition to sensing amino acids, GPRC6A is a cation-, calcimimetic-, and osteocalcin-sensing receptor and a candidate for mediating extracellular calcium-sensing responses in osteoblasts and possibly other tissues.

A transcriptional signaling pathway in the IFN system mediated by 2'-5'-oligoadenylate activation of RNase L.

Virus replication in higher vertebrates is restrained by IFNs that cause cells to transcribe genes encoding antiviral proteins, such as 2'-5' oligoadenylate synthetases. 2'-5' oligoadenylate synthetase is stimulated by dsRNA to produce 5'-phosphorylated, 2'-5'-linked oligoadenylates (2-5A), whose function is to activate RNase L. Although RNase L is required for a complete IFN antiviral response and mutations in the RNase L gene (RNASEL or HPC1) increase prostate cancer rates, it is unknown how 2-5A affects these biological endpoints through its receptor, RNase L. Presently, we show that 2-5A activation of RNase L produces a remarkable stimulation of transcription (>/=20-fold) for genes that suppress virus replication and prostate cancer. Unexpectedly, exposure of DU145 prostate cancer cells to physiologic levels of 2-5A (0.1 muM) induced approximately twice as many RNA species as it down-regulated. Among the 2-5A-induced genes are several IFN-stimulated genes, including IFN-inducible transcript 1/P56, IFN-inducible transcript 2/P54, IL-8, and IFN-stimulated gene 15. 2-5A also potently elevated RNA for macrophage inhibitory cytokine-1/nonsteroidal antiinflammatory drug-activated gene-1, a TGF-beta superfamily member implicated as an apoptotic suppressor of prostate cancer. Transcriptional signaling to the macrophage inhibitory cytokine-1/nonsteroidal antiinflammatory drug-activated gene-1 promoter by 2-5A was deficient in HeLa cells expressing a nuclease-dead mutant of RNase L and was dependent on the mitogen-activated protein kinases c-Jun N-terminal kinase and extracellular signal-regulated kinase, both of which were activated in response to 2-5A treatments. Because 2-5A and RNase L participate in defenses against viral infections and prostate cancer, our findings have implications for basic cellular mechanisms that control major pathogenic processes.

Neuropathogenic forms of huntingtin and androgen receptor inhibit fast axonal transport.

Huntington's and Kennedy's disease are autosomal dominant neurodegenerative diseases caused by pathogenic expansion of polyglutamine tracts. Expansion of glutamine repeats must in some way confer a gain of pathological function that disrupts an essential cellular process and leads to loss of affected neurons. Association of huntingtin with vesicular structures raised the possibility that axonal transport might be altered. Here we show that polypeptides containing expanded polyglutamine tracts, but not normal N-terminal huntingtin or androgen receptor, directly inhibit both fast axonal transport in isolated axoplasm and elongation of neuritic processes in intact cells. Effects were greater with truncated polypeptides and occurred without detectable morphological aggregates.