PRUNE2 is a human prostate cancer suppressor regulated by the intronic long noncoding RNA PCA3.

Prostate cancer antigen 3 (PCA3) is the most specific prostate cancer biomarker but its function remains unknown. Here we identify PRUNE2, a target protein-coding gene variant, which harbors the PCA3 locus, thereby classifying PCA3 as an antisense intronic long noncoding (lnc)RNA. We show that PCA3 controls PRUNE2 levels via a unique regulatory mechanism involving formation of a PRUNE2/PCA3 double-stranded RNA that undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing. PRUNE2 expression or silencing in prostate cancer cells decreased and increased cell proliferation, respectively. Moreover, PRUNE2 and PCA3 elicited opposite effects on tumor growth in immunodeficient tumor-bearing mice. Coregulation and RNA editing of PRUNE2 and PCA3 were confirmed in human prostate cancer specimens, supporting the medical relevance of our findings. These results establish PCA3 as a dominant-negative oncogene and PRUNE2 as an unrecognized tumor suppressor gene in human prostate cancer, and their regulatory axis represents a unique molecular target for diagnostic and therapeutic intervention.

Strategies to design and analyze targeted sequencing data: cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium Targeted Sequencing Study.

BACKGROUND: Genome-wide association studies have identified thousands of genetic variants that influence a variety of diseases and health-related quantitative traits. However, the causal variants underlying the majority of genetic associations remain unknown. Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium Targeted Sequencing Study aims to follow up genome-wide association study signals and identify novel associations of the allelic spectrum of identified variants with cardiovascular-related traits. METHODS AND RESULTS: The study included 4231 participants from 3 CHARGE cohorts: the Atherosclerosis Risk in Communities Study, the Cardiovascular Health Study, and the Framingham Heart Study. We used a case-cohort design in which we selected both a random sample of participants and participants with extreme phenotypes for each of 14 traits. We sequenced and analyzed 77 genomic loci, which had previously been associated with ≥1 of 14 phenotypes. A total of 52 736 variants were characterized by sequencing and passed our stringent quality control criteria. For common variants (minor allele frequency ≥1%), we performed unweighted regression analyses to obtain P values for associations and weighted regression analyses to obtain effect estimates that accounted for the sampling design. For rare variants, we applied 2 approaches: collapsed aggregate statistics and joint analysis of variants using the sequence kernel association test. CONCLUSIONS: We sequenced 77 genomic loci in participants from 3 cohorts. We established a set of filters to identify high-quality variants and implemented statistical and bioinformatics strategies to analyze the sequence data and identify potentially functional variants within genome-wide association study loci.

Finding the missing honey bee genes: lessons learned from a genome upgrade.

BACKGROUND: The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. RESULTS: Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. CONCLUSIONS: Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.

Analysis of rare, exonic variation amongst subjects with autism spectrum disorders and population controls.

We report on results from whole-exome sequencing (WES) of 1,039 subjects diagnosed with autism spectrum disorders (ASD) and 870 controls selected from the NIMH repository to be of similar ancestry to cases. The WES data came from two centers using different methods to produce sequence and to call variants from it. Therefore, an initial goal was to ensure the distribution of rare variation was similar for data from different centers. This proved straightforward by filtering called variants by fraction of missing data, read depth, and balance of alternative to reference reads. Results were evaluated using seven samples sequenced at both centers and by results from the association study. Next we addressed how the data and/or results from the centers should be combined. Gene-based analyses of association was an obvious choice, but should statistics for association be combined across centers (meta-analysis) or should data be combined and then analyzed (mega-analysis)? Because of the nature of many gene-based tests, we showed by theory and simulations that mega-analysis has better power than meta-analysis. Finally, before analyzing the data for association, we explored the impact of population structure on rare variant analysis in these data. Like other recent studies, we found evidence that population structure can confound case-control studies by the clustering of rare variants in ancestry space; yet, unlike some recent studies, for these data we found that principal component-based analyses were sufficient to control for ancestry and produce test statistics with appropriate distributions. After using a variety of gene-based tests and both meta- and mega-analysis, we found no new risk genes for ASD in this sample. Our results suggest that standard gene-based tests will require much larger samples of cases and controls before being effective for gene discovery, even for a disorder like ASD.

Rare complete knockouts in humans: population distribution and significant role in autism spectrum disorders.

To characterize the role of rare complete human knockouts in autism spectrum disorders (ASDs), we identify genes with homozygous or compound heterozygous loss-of-function (LoF) variants (defined as nonsense and essential splice sites) from exome sequencing of 933 cases and 869 controls. We identify a 2-fold increase in complete knockouts of autosomal genes with low rates of LoF variation (≤ 5% frequency) in cases and estimate a 3% contribution to ASD risk by these events, confirming this observation in an independent set of 563 probands and 4,605 controls. Outside the pseudoautosomal regions on the X chromosome, we similarly observe a significant 1.5-fold increase in rare hemizygous knockouts in males, contributing to another 2% of ASDs in males. Taken together, these results provide compelling evidence that rare autosomal and X chromosome complete gene knockouts are important inherited risk factors for ASD.

Patterns and rates of exonic de novo mutations in autism spectrum disorders.

Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified. To identify further genetic risk factors, here we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n = 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant, and the overall rate of mutation is only modestly higher than the expected rate. In contrast, the proteins encoded by genes that harboured de novo missense or nonsense mutations showed a higher degree of connectivity among themselves and to previous ASD genes as indexed by protein-protein interaction screens. The small increase in the rate of de novo events, when taken together with the protein interaction results, are consistent with an important but limited role for de novo point mutations in ASD, similar to that documented for de novo copy number variants. Genetic models incorporating these data indicate that most of the observed de novo events are unconnected to ASD; those that do confer risk are distributed across many genes and are incompletely penetrant (that is, not necessarily sufficient for disease). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case-control study provide strong evidence in favour of CHD8 and KATNAL2 as genuine autism risk factors.

The Drosophila melanogaster Genetic Reference Panel.

A major challenge of biology is understanding the relationship between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determines our ability to predict phenotypes from genotypes and to understand how evolutionary forces shape variation within and between species. Previous efforts to dissect the genotype-phenotype map were based on incomplete genotypic information. Here, we describe the Drosophila melanogaster Genetic Reference Panel (DGRP), a community resource for analysis of population genomics and quantitative traits. The DGRP consists of fully sequenced inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomal regions and the X chromosome, evidence for positive and negative selection, and rapid evolution of the X chromosome. Many variants in novel genes, most at low frequency, are associated with quantitative traits and explain a large fraction of the phenotypic variance. The DGRP facilitates genotype-phenotype mapping using the power of Drosophila genetics.

Frequent somatic mutations in MAP3K5 and MAP3K9 in metastatic melanoma identified by exome sequencing.

We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.

Whole-exome sequencing identifies compound heterozygous mutations in WDR62 in siblings with recurrent polymicrogyria.

Polymicrogyria is a disorder of neuronal development resulting in structurally abnormal cerebral hemispheres characterized by over-folding and abnormal lamination of the cerebral cortex. Polymicrogyria is frequently associated with severe neurologic deficits including intellectual disability, motor problems, and epilepsy. There are acquired and genetic causes of polymicrogyria, but most patients with a presumed genetic etiology lack a specific diagnosis. Here we report using whole-exome sequencing to identify compound heterozygous mutations in the WD repeat domain 62 (WDR62) gene as the cause of recurrent polymicrogyria in a sibling pair. Sanger sequencing confirmed that the siblings both inherited 1-bp (maternal allele) and 2-bp (paternal allele) frameshift deletions, which predict premature truncation of WDR62, a protein that has a role in early cortical development. The probands are from a non-consanguineous family of Northern European descent, suggesting that autosomal recessive PMG due to compound heterozygous mutation of WDR62 might be a relatively common cause of PMG in the population. Further studies to identify mutation frequency in the population are needed.

Targeted enrichment beyond the consensus coding DNA sequence exome reveals exons with higher variant densities.

BACKGROUND: Enrichment of loci by DNA hybridization-capture, followed by high-throughput sequencing, is an important tool in modern genetics. Currently, the most common targets for enrichment are the protein coding exons represented by the consensus coding DNA sequence (CCDS). The CCDS, however, excludes many actual or computationally predicted coding exons present in other databases, such as RefSeq and Vega, and non-coding functional elements such as untranslated and regulatory regions. The number of variants per base pair (variant density) and our ability to interrogate regions outside of the CCDS regions is consequently less well understood. RESULTS: We examine capture sequence data from outside of the CCDS regions and find that extremes of GC content that are present in different subregions of the genome can reduce the local capture sequence coverage to less than 50% relative to the CCDS. This effect is due to biases inherent in both the Illumina and SOLiD sequencing platforms that are exacerbated by the capture process. Interestingly, for two subregion types, microRNA and predicted exons, the capture process yields higher than expected coverage when compared to whole genome sequencing. Lastly, we examine the variation present in non-CCDS regions and find that predicted exons, as well as exonic regions specific to RefSeq and Vega, show much higher variant densities than the CCDS. CONCLUSIONS: We show that regions outside of the CCDS perform less efficiently in capture sequence experiments. Further, we show that the variant density in computationally predicted exons is more than 2.5-times higher than that observed in the CCDS.

Whole-genome sequencing for optimized patient management.

Whole-genome sequencing of patient DNA can facilitate diagnosis of a disease, but its potential for guiding treatment has been under-realized. We interrogated the complete genome sequences of a 14-year-old fraternal twin pair diagnosed with dopa (3,4-dihydroxyphenylalanine)-responsive dystonia (DRD; Mendelian Inheritance in Man #128230). DRD is a genetically heterogeneous and clinically complex movement disorder that is usually treated with l-dopa, a precursor of the neurotransmitter dopamine. Whole-genome sequencing identified compound heterozygous mutations in the SPR gene encoding sepiapterin reductase. Disruption of SPR causes a decrease in tetrahydrobiopterin, a cofactor required for the hydroxylase enzymes that synthesize the neurotransmitters dopamine and serotonin. Supplementation of l-dopa therapy with 5-hydroxytryptophan, a serotonin precursor, resulted in clinical improvements in both twins.

Deep resequencing reveals excess rare recent variants consistent with explosive population growth.

Accurately determining the distribution of rare variants is an important goal of human genetics, but resequencing of a sample large enough for this purpose has been unfeasible until now. Here, we applied Sanger sequencing of genomic PCR amplicons to resequence the diabetes-associated genes KCNJ11 and HHEX in 13,715 people (10,422 European Americans and 3,293 African Americans) and validated amplicons potentially harbouring rare variants using 454 pyrosequencing. We observed far more variation (expected variant-site count ∼578) than would have been predicted on the basis of earlier surveys, which could only capture the distribution of common variants. By comparison with earlier estimates based on common variants, our model shows a clear genetic signal of accelerating population growth, suggesting that humanity harbours a myriad of rare, deleterious variants, and that disease risk and the burden of disease in contemporary populations may be heavily influenced by the distribution of rare variants.

Whole exome capture in solution with 3 Gbp of data.

We have developed a solution-based method for targeted DNA capture-sequencing that is directed to the complete human exome. Using this approach allows the discovery of greater than 95% of all expected heterozygous singe base variants, requires as little as 3 Gbp of raw sequence data and constitutes an effective tool for identifying rare coding alleles in large scale genomic studies.

Apoptosis, autophagy, accelerated senescence and reactive oxygen in the response of human breast tumor cells to adriamycin.

Although the primary response to Adriamycin (doxorubicin) in p53 mutant MDA-MB231 and p53 null MCF-7/E6 breast tumor cells is apoptotic cell death, the residual surviving population appears to be in a state of senescence, based on cell morphology, beta galactosidase staining, induction of p21(waf1/cip1) and down regulation of cdc2/cdk1. Suppression of apoptosis in MDA-MB231 and MCF-7/E6 cells treated with Adriamycin using the broad spectrum caspase inhibitor, zvad-Fmk, results in substantial induction of autophagy. Overall sensitivity to Adriamycin, measured by clonogenic survival, is not altered in the cells undergoing autophagy, consistent with autophagy contributing to cell death in response to Adriamycin. The free radical scavengers, glutathione and N-acetyl cysteine attenuate the accelerated senescence response to Adriamycin in MCF-7 cells as well as in MDA-MB231 and MCF-7/E6 cells, but protect primarily the MCF-7 cells, indicating that reactive oxygen is unlikely to be directly responsible for Adriamycin toxicity in breast tumor cells. Expression of caspase 3 or induced expression of c-myc in MCF-7 cells fails to abrogate accelerated senescence induced by Adriamycin. Taken together, these studies suggest that accelerated senescence induced by Adriamycin is similar in cells with wild type p53 and in cells lacking functional p53 with regard to the upregulation of p21(waf1/cip1), down regulation of cdc2 and the involvement of reactive oxygen species. Furthermore, accelerated senescence, autophagy and apoptosis all appear to be effective in suppressing self-renewal capacity in breast tumor cells exposed to Adriamycin.

Autophagic cell death, polyploidy and senescence induced in breast tumor cells by the substituted pyrrole JG-03-14, a novel microtubule poison.

JG-03-14, a substituted pyrrole that inhibits microtubule polymerization, was screened against MCF-7 (p53 wild type), MDA-MB231 (p53 mutant), MCF-7/caspase 3 and MCF-7/ADR (multidrug resistant) breast tumor cell lines. Cell viability and growth inhibition were assessed by the crystal violet dye assay. Apoptosis was evaluated by the TUNEL assay, cell cycle distribution by flow cytometry, autophagy by acridine orange staining of vesicle formation, and senescence based on beta-galactosidase staining and cell morphology. Our studies indicate that exposure to JG-03-14, at a concentration of 500 nM, induces time-dependent cell death in the MCF-7 and MDA-MB231 cell lines. In MCF-7 cells, a residual surviving cell population was found to be senescent; in contrast, there was no surviving senescent population in treated MDA-MB231 cells. No proliferative recovery was detected over a period of 15 days post-treatment in either cell line. Both the TUNEL assay and FLOW cytometry indicated a relatively limited degree of apoptosis (<10%) in response to drug treatment in MCF-7 cells with more extensive apoptosis (but <20%) in MDA-MB231 cells; acidic vacuole formation indicative of autophagic cell death was relatively extensive in both MCF-7 and MDA-MB231 cells. In addition, JG-03-14 induced the formation of a large hyperdiploid cell population in MDA-MB231 cells. JG-03-14 also demonstrated pronounced anti-proliferative activity in MCF-7/caspase 3 cells and in the MCF-7/ADR cell line. The observation that JG-03-14 promotes autophagic cell death and also retains activity in tumor cells expressing the multidrug resistance pump indicates that novel microtubule poisons of the substituted pyrroles class may hold promise in the treatment of breast cancer.

Downregulation of TSLC1 and DAL-1 expression occurs frequently in breast cancer.

TSLC1 and DAL-1 are tumor suppressor genes involved in cell adhesion. In this study, we examined the expression and methylation pattern of these genes in breast cancer cell lines and primary breast carcinomas. TSLC1 expression was lost in 5 of 8 (63%) and DAL-1 expression was lost in 6 of 8 (75%) breast cancer cell lines, respectively. Downregulation of TSLC1 expression was observed in 43 of 50 (86%) and of DAL-1 expression in 26 of 55 (47%) primary breast carcinomas. TSLC1 methylation was found in 4 of 8 (50%) and DAL-1 methylation was observed in 6 of 8 (75%) breast cancer cell lines, respectively. Of 95 primary breast carcinomas 46 (48%) were TSLC1 methylated and 26 (27%) were DAL-1 methylated. Twenty of 43 (47%) and 10 of 26 (38%) primary breast cancer samples which showed downregulation of TSLC1 and DAL-1 expression were unmethylated for these genes. Re-expression of TSLC1 and DAL-1 was observed after treatment of BT-20 cells with 5-aza-2'-deoxycytidine and TSA. Samples from patients with grade 3 tumors were more frequently TSLC1 and TSLC1 and/or DAL-1 methylated than samples from patients with grade 1 and 2 tumors (P = 0.032, P = 0.023). Moreover, TSLC1 methylation correlated with loss of both ER and PgR staining (P = 0.011, P = 0.02). Our findings suggest that TSLC1 and DAL-1 are involved in the pathogenesis of breast cancer and are frequently inactivated by methylation.

Potentiation of radiation sensitivity in breast tumor cells by the vitamin D3 analogue, EB 1089, through promotion of autophagy and interference with proliferative recovery.

1,25-Dihydroxyvitamin D(3) and vitamin D(3) analogues, such as EB 1089, potentiate the response to ionizing radiation in breast tumor cells. The current studies address the basis for this interaction by evaluating DNA damage and repair, the effect of interference with reactive oxygen generation, the involvement of p53 and caspase-3, signaling through c-myc, as well as the induction of senescence and multiple modes of cell death. EB 1089 failed to increase the extent of radiation-induced DNA damage or to attenuate the rate of DNA repair. The reactive oxygen scavengers N-acetyl-l-cysteine and reduced glutathione failed to protect the cells from the promotion of cell death by EB 1089 and radiation. Whereas MCF-7 cells expressing caspase-3 showed significant apoptosis with radiation alone as well as with EB 1089 followed by radiation, EB 1089 maintained its ability to confer susceptibility to radiation-induced cell killing, in large part by interference with proliferative recovery. In contrast, in breast tumor cells lacking p53, where radiation promoted extensive apoptosis and the cells failed to recover after radiation treatment, EB 1089 failed to influence the effect of radiation. EB 1089 treatment interfered with radiation-induced suppression of c-myc; however, induction of c-myc did not prevent senescence by radiation alone or radiation-induced cell death promoted by EB 1089. EB 1089 did not increase the extent of micronucleation, indicative of mitotic catastrophe, induced by radiation alone. However, EB 1089 did promote extensive autophagic cell death in the irradiated cells. Taken together, these studies suggest that the effect of EB 1089 treatment on the radiation response is related in part to enhanced promotion of autophagic cell death and in part to interference with the proliferative recovery that occurs with radiation alone in p53 wild-type breast tumor cells.

The tumor suppressor DAL-1/4.1B and protein methylation cooperate in inducing apoptosis in MCF-7 breast cancer cells.

BACKGROUND: DAL-1 (Differentially Expressed in Adenocarcinoma of the Lung)/4.1B is a member of the protein 4.1 superfamily that has been shown to suppress growth in lung, breast and brain tumor cells. In the case of the caspase-3 deficient MCF-7 breast cancer cells, this growth suppression has been shown to be partially mediated by the induction of apoptosis. However the exact mechanism of action of DAL-1/4.1B is unknown. Recently, protein arginine N-methyltransferase 3 (PRMT3) was identified as a DAL-1/4.1B interacting protein. Protein arginine methyltransferases (PRMTs) posttranslationally methylate the arginine residues of proteins, a modification which has been implicated in the regulation of multiple cellular processes including nuclear-cytoplasmic transport, signal transduction, and transcription. RESULTS: To investigate the role of protein methylation in cell death induced by DAL-1/4.1B, DAL-1/4.1B-inducible MCF-7 cells were examined for apoptosis and caspase activation in the absence and presence of the protein methylation inhibitor adenosine dialdehyde (AdOX). Flow cytometry analysis revealed that apoptosis was primarily associated with the activation of caspase 8, and inhibition of this activation blocked the ability of DAL-1/4.1B to induce cell death. CONCLUSION: These results suggest that protein methylation cooperates with DAL-1/4.1B-associated caspase 8-specific activation to induce apoptosis in breast cancer cells.

Protein 4.1B expression is induced in mammary epithelial cells during pregnancy and regulates their proliferation.

4.1B is a member of the protein 4.1 superfamily of proteins that link transmembrane proteins to the actin cytoskeleton. The 4.1B gene localizes to chromosome 18p11.3, which undergoes loss of heterozygosity in mammary tumors. Here, we examine the expression of 4.1B in murine mammary epithelium and find that 4.1B is dramatically upregulated in mammary epithelial cells during pregnancy when there is extensive cell proliferation. In contrast, 4.1B is not expressed in virgin, lactating, or involuting mammary epithelium. To examine the consequence of 4.1B loss on mammary epithelial cell proliferation, we analysed mammary glands in 4.1B-null mice. 4.1B loss results in a significant increase in mammary epithelial cell proliferation during pregnancy, but has no effect on mammary epithelial cell proliferation, in virgin or involuting mice. Furthermore, we show that 4.1B inhibits the proliferation of mammary epithelial cell lines by inducing a G1 cell cycle arrest, characterized by decreased cyclin A expression and reduced Rb phosphorylation, and accompanied by reduced erbB2 phosphorylation. This cell cycle arrest does not involve alterations in the activities of MAPK, JNK, or Akt. Collectively, our findings demonstrate that 4.1B regulates mammary epithelial cell proliferation during pregnancy and suggest that its loss may influence mammary carcinoma pathogenesis in multiparous women.

The tumor suppressor DAL-1/4.1B modulates protein arginine N-methyltransferase 5 activity in a substrate-specific manner.

We previously identified DAL-1/4.1B as a growth suppression protein involved in the pathogenesis of lung, breast, and meningioma tumors. Using yeast two-hybrid interaction cloning, protein arginine N-methyltransferase 3 (PRMT3) was originally identified as a DAL-1/4.1B-interacting protein. PRMTs catalyze the sequential transfer of methyl groups from S-adeonsyl-l-methionine to the guanidino nitrogens of arginine residues in proteins, the effect of which can include regulation of signal transduction, transcription regulation, and RNA transport, suggesting that modulating this event may have far-reaching impact. In this study, we assessed the impact of DAL-1/4.1B binding on the activity of another family member, PRMT5, both in vitro and in cells. In contrast to PRMT3, DAL-1/4.1B was found to mediate PRMT5 by either inhibiting (Sm proteins) or enhancing (myelin basic protein) protein methylation. We propose that this interaction between a tumor suppressor and a post-translational methylation enzyme is of biological importance in controlling tumorigenesis.