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Single-cell RNAseq has allowed unprecedented insight into gene expression across different cell populations in normal tissue and disease states. However, almost all studies rely on annotated gene sets to capture gene expression levels and sequencing reads that do not align to known genes are discarded. Here, we discover thousands of long noncoding RNAs (lncRNAs) expressed in human mammary epithelial cells and analyze their expression in individual cells of the normal breast. We show that lncRNA expression alone can discriminate between luminal and basal cell types and define subpopulations of both compartments. Clustering cells based on lncRNA expression identified additional basal subpopulations, compared to clustering based on annotated gene expression, suggesting that lncRNAs can provide an additional layer of information to better distinguish breast cell subpopulations. In contrast, these breast-specific lncRNAs poorly distinguish brain cell populations, highlighting the need to annotate tissue-specific lncRNAs prior to expression analyses. We also identified a panel of 100 breast lncRNAs that could discern breast cancer subtypes better than protein-coding markers. Overall, our results suggest that lncRNAs are an unexplored resource for new biomarker and therapeutic target discovery in the normal breast and breast cancer subtypes.

Schistosomiasis is a disease that significantly impacts public health in the developing world. Effective diagnostics are urgently needed for improved control of this disease, but current diagnostic procedures lack the requisite sensitivity, portability and cost-effectiveness needed for use in resource-poor settings. We developed a novel assay for the detection ofSchistosoma japonicumusing the CRISPR mediated diagnostic platform SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing), combining recombinase polymerase amplification (RPA) with CRISPR and CRISPR-associated RNA-guided endoribonuclease Cas13 (CRISPR-Cas13). The assay was validated using 80 faecal samples obtained from a mouse model infected with the Philippine strain ofS. japonicum, as well as 38 clinical faecal and 37 serum samples obtained from subjects living in endemic areas forS. japonicumin Northern Samar, the Philippines. CRISPR-Cas13 mediated detection was determined via fluorescent readout or colorimetric readout on a lateral flow strip. Our results demonstrate that ourS. japonicumSHERLOCK assay is specific, sensitive and user-friendly. Although the assay does not require the specialized equipment or expertise necessary for real time PCR-based detection, which is currently the most sensitive approach for the diagnosis of helminthic infections, it achieved 93-100% sensitivity compared with the qPCR, as well as 100% specificity across all the human and animal samples tested. Although further optimisation is required before field-ready implementation, CRISPR-based nucleic acid detection shows great promise as the basis of a point-of-care (POC) diagnostic tool for clinical diagnosis and surveillance of schistosomiasis with potential extension to other helminthiases.Author SummaryParasitic helminths cause devastating diseases, including schistosomiasis, afflicting 1.5 billion people worldwide and representing a significant public health and economic burden. Currently available diagnostic tools for helminth infections are neither sufficiently sensitive nor field-friendly for use in resource-poor settings where infection is most prevalent, and advanced tools are are urgently needed for rapid mapping of helminthic diseases and monitoring control efforts. For the first time, we used theSchistosomabloodfluke model to successfully establish a diagnostic assay with the CRISPR-based nucleic acid detection platform SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing) by combining recombinase polymerase amplification (RPA) and CRISPR-Cas13 detection to diagnose schistosomiasis in humans and animals. We showed that the novel CRISPR-based assay, with its low cost of application, is capable of robust detection and is field-friendly. It exhibits similar diagnostic sensitivity as qPCR-based assays, which are currently the most sensitive approach for the diagnosis of helminthic infections, but with significantly reduced requirements for trained personnel and technical expensive equipment. OurS. japonicumSHERLOCK assay has the potential to fulfil key recommendations of the neglected tropical diseases (NTDs) 2021-2030 roadmap and the 2022 Guideline on the Control and Elimination of Human Schistosomiasis released recently by the World Health Organization.

Breast cancer is the most common malignancy among women. Approximately 25% of hereditary breast cancer cases can explained by rare pathogenic variants in a small number of moderate- to high-penetrance predisposition genes, and at least another 20% by common, low-risk variants. Next-generation sequencing and new risk prediction methods are providing more personalized options for breast cancer risk assessment. However, realizing the full clinical potential of the underlying genetics requires accurate inference of pathogenicity, comprehensive integration of all known risk factors and a better understanding of underlying biology. Here, we will discuss known and candidate breast cancer susceptibility variants, outline some of the available genetic risk assessment tools, and highlight recent publications identifying and functionally characterizing variants associated with breast cancer.

SummaryGenome-wide association studies (GWAS) have identified >200 loci associated with breast cancer (BC) risk. The majority of candidate causal variants (CCVs) are in non-coding regions and are likely to modulate cancer risk by regulating gene expression. We recently developed a scoring system, INQUISIT, to predict candidate risk genes at BC-risk loci. Here, we used pooled CRISPR activation and suppression screens to validate INQUISIT predictions, and to define the cancer phenotypes they mediate. We measured proliferation in 2D, 3D, and in immune-deficient mice, as well as the effect on the DNA damage response. We performed 60 CRISPR screens and identified 21 high-confidence INQUISIT predictions that mediate a cancer phenotype. We validated the direct regulation of a subset of genes by BC-risk variants using HiCHIP and CRISPRqtl. Furthermore, we show the utility of expression profiling for drug repurposing against these targets. We provide a platform for identifying gene targets of risk variants, and lay a blueprint of interventions for BC risk reduction and treatment.

BackgroundThe nucleocytoplasmic shuttling of ERK5 has gained recent attention as a regulator of its diverse roles in cancer progression but the exact mechanisms for this shuttling are still under investigation.MethodsUsing in vitro, in vivo and in silico studies, we investigated the roles of shorter ERK5 isoforms in regulating the nucleocytoplasmic shuttling of active phosphorylated-ERK5 (pERK5). Retrospective cohorts of primary and metastatic breast cancer cases were used to evaluate the association of the subcellular localization of pERK5 with clinicopathological features.ResultsExtranuclear localization of pERK5 was observed during cell migration in vitro and at the invasive fronts of metastatic tumors in vivo. The nuclear and extranuclear cell fractions contained different isoforms of pERK5, which are encoded by splice variants expressed in breast and other cancers in the TCGA data. One isoform, isoform-3, lacks the C-terminal transcriptional domain and the nuclear localization signal. The co-expression of isoform-3 and full-length ERK5 associated with high epithelial-to-mesenchymal transition (EMT) and poor patient survival. Experimentally, expressing isoform-3 with full-length ERK5 in breast cancer cells increased cell migration, drove EMT and led to tamoxifen resistance. In breast cancer patient samples, pERK5 showed variable subcellular localizations where its extranuclear localization associated with aggressive clinicopathological features, metastasis, and poor survival.ConclusionOur studies support a model of ERK5 nucleocytoplasmic shuttling driven by splice variants in an interplay between mesenchymal and epithelial states during metastasis. Using ERK5 as a biomarker and a therapeutic target should account for its splicing and context-dependent biological functions.Graphical AbstractERK5 isoform-3 expression deploys active ERK5 (pERK5) outside the nucleus to facilitate EMT and cell migration. In cells dominantly expressing isoform-1, pERK5 shuttles to the nucleus to drive cell expansion.

CRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. Here we report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. A CRISPR/Cas9-vector encoding gRNA X5 or X7 was introduced by electroporation into eggs recovered from livers of experimentally infected mice. Simultaneously, eggs were transfected with a ssODN donor encoding a stop codon in all six frames, flanked by 50 nt-long 5’- and 3’-homology arms matching the predicted Cas9-catalyzed double stranded break at X5 or X7. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programmed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, a significant decrease in circumoval granuloma size was observed in the lungs of the mice. Notably, there was an enhanced Th2 response involving IL-4, −5, −10, and-13 induced by lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, −13 and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes.Author SummarySchistosomiasis is the most devastating of the parasitic helminth diseases. Currently, no vaccines are available for human use and praziquantel is the only available treatment raising considerable concern that drug resistance will develop. A major challenge faced by the schistosomiasis research community is the lack of suitable tools to effectively characterise schistosome gene products as potential new drug and/or vaccine targets. We introduced CRISPR/Cas9 mediated editing into S. mansoni eggs targeting the gene encoding acetylcholinesterase (AChE), a recognized anthelminthic drug target. We found that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). This platform provides a unique opportunity to generate precise loss-of-function insertions into the schistosome genome. We pre-screened the activity of two guide RNAs of the AChE gene and compared/validated the mutation efficacy using next-generation sequencing analysis at the genomic level and phenotypic modifications at the protein level. That resulted in reduced AChE activity observed in AChE-edited eggs, and decreased lung circumoval granuloma size in mice injected with those edited eggs. The CRISPR/Cas9-genome editing system we established in this study provides a pivotal platform for gene functional studies to identify and test new anti-schistosome intervention targets, which can be extended to the other human schistosome species and other important parasitic helminths.

Breast cancer genome-wide association studies (GWASs) have identified 150 genomic risk regions containing more than 13,000 credible causal variants (CCVs). The CCVs are predominantly noncoding and enriched in regulatory elements. However, the genes underlying breast cancer risk associations are largely unknown. Here, we used genetic colocalization analysis to identify loci at which gene expression could potentially explain breast cancer risk phenotypes. Using data from the Breast Cancer Association Consortium (BCAC) and quantitative trait loci (QTL) from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Project (TCGA), we identify shared genetic relationships and reveal novel associations between cancer phenotypes and effector genes. Seventeen genes, including NTN4, were identified as potential mediators of breast cancer risk. For NTN4, we showed the rs61938093 CCV at this region was located within an enhancer element that physically interacts with the NTN4 promoter, and the risk allele reduced NTN4 promoter activity. Furthermore, knockdown of NTN4 in breast cells increased cell proliferation in vitro and tumor growth in vivo. These data provide evidence linking risk-associated variation to genes that may contribute to breast cancer predisposition.

Recent reports of CRISPR/Cas9 genome editing in parasitic helminths open up new avenues for research on these dangerous pathogens. However, the complex morphology and life cycles inherent to these parasites present obstacles for the efficient application of CRISPR/Cas9-targeted mutagenesis. This is especially true with the trematode flukes where only modest levels of gene mutation efficiency have been achieved. Current major challenges in the application of CRISPR/Cas9 for study of parasitic worms thus lie in enhancing gene mutation efficiency and overcoming issues involved in host passage so that mutated parasites survive. Strategies developed for CRISPR/Cas9 studies on Caenorhabditis elegans, protozoa and mammalian cells, including novel delivery methods, the choice of selectable markers, and refining mutation precision represent novel tactics whereby these impediments can be overcome. Furthermore, employing CRISPR/Cas9-mediated gene drive to interfere with vector transmission represents a novel approach for the control of parasitic worms that is worthy of further exploration.

Depletion of BRCA1 protein in mouse mammary glands results in defects in lactational development and increased susceptibility to mammary cancer. Extensive work has focussed on the role of BRCA1 in the normal breast and in the development of breast cancer, the cell of origin for BRCA1 tumours and the protein-coding genes altered in BRCA1 deficient cells. However, the role of non-coding RNAs in BRCA1-deficient cells is poorly understood. To evaluate miRNA expression in BRCA1 deficient mammary cells, RNA sequencing was performed on the mammary glands of Brca1 knockout mice. We identified 140 differentially expressed miRNAs, 9 of which were also differentially expressed in human BRCA1 breast tumours or familial non-BRCA1 patients and during normal gland development. We show that BRCA1 binds to putativecis-elements in promoter regions of the miRNAs and regulates their expression, and that four miRNAs (miR-29b-1-5p, miR-664, miR-16-2 and miR-744) significantly stratified the overall survival of basal-like tumours. Importantly the prognostic value of miR-29b-1-5p was higher in significance than several commonly used clinical biomarkers. These results emphasize the role of Brca1 in modulating expression of miRNAs and highlights the potential for BRCA1 regulated miRNAs to be informative biomarkers associated with BRCA1 loss and survival in breast cancer.

// Sonja Helbig 1, 2 , Leesa Wockner 3 , Annick Bouendeu 1 , Ursula Hille-Betz 1 , Karen McCue 2 , Juliet D. French 2 , Stacey L. Edwards 2 , Hilda A. Pickett 4 , Roger R. Reddel 5 , Georgia Chenevix-Trench 2 , Thilo Dork 1, * and Jonathan Beesley 2, * 1 Gynaecology Research Unit, Hannover Medical School, Hannover, Germany 2 Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, Australia 3 Statistics Unit, QIMR Berghofer Medical Research Institute, Brisbane, Australia 4 Telomere Length Regulation Unit, Children’s Medical Research Institute, University of Sydney, Westmead, Australia 5 Cancer Research Unit, Children’s Medical Research Institute, University of Sydney, Westmead, Australia * Shared senior authors Correspondence to: Sonja Helbig, email: Helbig.Sonja@mh-hannover.de Keywords: telomerase, SNP, breast carcinoma, GABPA, GWAS Received: November 04, 2016 Accepted: May 02, 2017 Published: May 26, 2017 ABSTRACT The multi-cancer susceptibility locus at 5p15.33 includes TERT , encoding the telomerase catalytic subunit. Genome-wide association studies (GWAS) have identified six single nucleotide polymorphisms (SNPs) in the TERT promoter associated with decreased breast cancer risk, although the precise causal variants and their mechanisms of action have remained elusive. Luciferase reporter assays indicated that the protective haplotype reduced TERT promoter activity in human mammary epithelial and cancer cells in an estrogen-independent manner. Using single variant constructs, we identified rs3215401 and rs2853669 as likely functional variants. Silencing of MYC decreased TERT promoter activity but neither MYC nor ETS2 silencing conferred allele-specificity. In chromatin immunoprecipitation experiments, the ETS protein GABPA, but not ETS2 or ELF1, bound rs2853669 in an allele-specific manner in mammary epithelial cells. Investigation of open chromatin in mammoplasty samples suggested involvement of three additional variants, though not rs3215401 or rs2853669. Chromosome conformation capture revealed no interaction of the TERT promoter with regulatory elements in the locus, indicating limited local impact of candidate variants on the TERT promoter. Collectively, our functional studies of the TERT - CLPTM1L breast cancer susceptibility locus describe rs2853669 as a functional variant of this association signal among three other potentially causal variants and demonstrate the versatile mechanisms by which TERT promoter variants may affect breast cancer risk.

Background Genetic variants identified through genome-wide association studies (GWAS) are predominantly non-coding and typically attributed to altered regulatory elements such as enhancers and promoters. However, the contribution of non-coding RNAs to complex traits is not clear. Results Using targeted RNA sequencing, we systematically annotated multi-exonic non-coding RNA (mencRNA) genes transcribed from 1.5-Mb intervals surrounding 139 breast cancer GWAS signals and assessed their contribution to breast cancer risk. We identify more than 4000 mencRNA genes and show their expression distinguishes normal breast tissue from tumors and different breast cancer subtypes. Importantly, breast cancer risk variants, identified through genetic fine-mapping, are significantly enriched in mencRNA exons, but not the promoters or introns. eQTL analyses identify mencRNAs whose expression is associated with risk variants. Furthermore, chromatin interaction data identify hundreds of mencRNA promoters that loop to regions that contain breast cancer risk variants. Conclusions We have compiled the largest catalog of breast cancer-associated mencRNAs to date and provide evidence that modulation of mencRNAs by GWAS variants may provide an alternative mechanism underlying complex traits.

International audience; Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present.

Fine-mapping of breast cancer GWAS regions has identified 195 high confidence signals containing more than 5,000 credible causal variants (CCVs). The CCVs are predominantly noncoding and enriched in regulatory elements and thus may confer the risk by altering gene expression in cis. We analyzed allelic expression imbalance (AEI) of genes surrounding known breast cancer signals, using normal breast and breast tumor transcriptome data and imputed genotypes. Fourteen genes, including NTN4, were identified whose expression was associated with CCV genotype. We showed that CCVs at this signal were located within an enhancer that physically interacts with the NTN4 promoter. Furthermore, knockdown of NTN4 in breast cells increased cell proliferation in vitro and tumor growth in vivo. Here, we present the most comprehensive AEI analysis of breast cancer CCVs resulting in identification of new candidate risk genes.

GWAS have identified a breast cancer susceptibility locus on 2q35. Here we report the fine mapping of this locus using data from 101,943 subjects from 50 case-control studies. We genotype 276 SNPs using the ‘iCOGS’ genotyping array and impute genotypes for a further 1,284 using 1000 Genomes Project data. All but two, strongly correlated SNPs (rs4442975 G/T and rs6721996 G/A) are excluded as candidate causal variants at odds against >100:1. The best functional candidate, rs4442975, is associated with oestrogen receptor positive (ER+) disease with an odds ratio (OR) in Europeans of 0.85 (95% confidence interval=0.84−0.87; P=1.7 × 10−43) per t-allele. This SNP flanks a transcriptional enhancer that physically interacts with the promoter of IGFBP5 (encoding insulin-like growth factor-binding protein 5) and displays allele-specific gene expression, FOXA1 binding and chromatin looping. Evidence suggests that the g-allele confers increased breast cancer susceptibility through relative downregulation of IGFBP5, a gene with known roles in breast cell biology., Previous studies identified an association between the 2q35 locus and breast cancer. Here, the authors show that a SNP at 2q35, rs4442975, is associated with oestrogen receptor positive disease and suggest that this effect is mediated through the downregulation of a known breast cancer gene, IGFBP5.

The gene(s) whose expression is regulated by allergy risk variants is unknown for many loci identified through genome-wide association studies. Addressing this knowledge gap might point to new therapeutic targets for allergic disease. The aim of this study was to identify the target gene(s) and the functional variant(s) underlying the association between rs7009110 on chromosome 8q21 and allergies. Eight genes are located within 1 Mb of rs7009110. Multivariate association analysis of publicly available exon expression levels from lymphoblastoid cell lines (LCLs) identified a significant association between rs7009110 and the expression of a single gene, PAG1 (p = 0.0017), 732 kb away. Analysis of histone modifications and DNase I hypersensitive sites in LCLs identified four putative regulatory elements (PREs) in the region. Chromosome conformation capture confirmed that two PREs interacted with the PAG1 promoter, one in allele-specific fashion. To determine whether these PREs were functional, LCLs were transfected with PAG1 promoter-driven luciferase reporter constructs. PRE3 acted as a transcriptional enhancer for PAG1 exclusively when it carried the rs2370615:C allergy predisposing allele, a variant in complete linkage disequilibrium with rs7009110. As such, rs2370615, which overlaps RelA transcription factor (TF) binding in LCLs and was found to disrupt Foxo3a binding to PRE3, represents the putative functional variant in this locus. Our studies suggest that the risk-associated allele of rs2370615 predisposes to allergic disease by increasing PAG1 expression, which might promote B cell activation and have a pro-inflammatory effect. Inhibition of PAG1 expression or function might have therapeutic potential for allergic diseases.

Genome-wide association studies have identified SNPs near ZNF365 at 10q21.2 that are associated with both breast cancer risk and mammographic density. To identify the most likely causal SNPs, we fine mapped the association signal by genotyping 428 SNPs across the region in 89,050 European and 12,893 Asian case and control subjects from the Breast Cancer Association Consortium. We identified four independent sets of correlated, highly trait-associated variants (iCHAVs), three of which were located within ZNF365. The most strongly risk-associated SNP, rs10995201 in iCHAV1, showed clear evidence of association with both estrogen receptor (ER)-positive (OR = 0.85 [0.82-0.88]) and ER-negative (OR = 0.87 [0.82-0.91]) disease, and was also the SNP most strongly associated with percent mammographic density. iCHAV2 (lead SNP, chr10: 64,258,684:D) and iCHAV3 (lead SNP, rs7922449) were also associated with ER-positive (OR = 0.93 [0.91-0.95] and OR = 1.06 [1.03-1.09]) and ER-negative (OR = 0.95 [0.91-0.98] and OR = 1.08 [1.04-1.13]) disease. There was weaker evidence for iCHAV4, located 5' of ADO, associated only with ER-positive breast cancer (OR = 0.93 [0.90-0.96]). We found 12, 17, 18, and 2 candidate causal SNPs for breast cancer in iCHAVs 1-4, respectively. Chromosome conformation capture analysis showed that iCHAV2 interacts with the ZNF365 and NRBF2 (more than 600 kb away) promoters in normal and cancerous breast epithelial cells. Luciferase assays did not identify SNPs that affect transactivation of ZNF365, but identified a protective haplotype in iCHAV2, associated with silencing of the NRBF2 promoter, implicating this gene in the etiology of breast cancer.

Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.

The 10q26 locus in the second intron of FGFR2 is the locus most strongly associated with estrogen-receptor-positive breast cancer in genome-wide association studies. We conducted fine-scale mapping in case-control studies genotyped with a custom chip (iCOGS), comprising 41 studies (n = 89,050) of European ancestry, 9 Asian ancestry studies (n = 13,983), and 2 African ancestry studies (n = 2,028) from the Breast Cancer Association Consortium. We identified three statistically independent risk signals within the locus. Within risk signals 1 and 3, genetic analysis identified five and two variants, respectively, highly correlated with the most strongly associated SNPs. By using a combination of genetic fine mapping, data on DNase hypersensitivity, and electrophoretic mobility shift assays to study protein-DNA binding, we identified rs35054928, rs2981578, and rs45631563 as putative functional SNPs. Chromatin immunoprecipitation showed that FOXA1 preferentially bound to the risk-associated allele (C) of rs2981578 and was able to recruit ERα to this site in an allele-specific manner, whereas E2F1 preferentially bound the risk variant of rs35054928. The risk alleles were preferentially found in open chromatin and bound by Ser5 phosphorylated RNA polymerase II, suggesting that the risk alleles are associated with changes in transcription. Chromatin conformation capture demonstrated that the risk region was able to interact with the promoter of FGFR2, the likely target gene of this risk region. A role for FOXA1 in mediating breast cancer susceptibility at this locus is consistent with the finding that the FGFR2 risk locus primarily predisposes to estrogen-receptor-positive disease. © 2013 The American Society of Human Genetics.

Genome-wide association studies (GWASs) have enabled the discovery of common genetic variation contributing to normal and pathological traits and clinical drug responses, but recognizing the precise targets of these associations is now the major challenge. Here, we review recent approaches to the functional follow-up of GWAS loci, including fine mapping of GWAS signal(s), prioritization of putative functional SNPs by the integration of genetic epidemiological and bioinformatic methods, and in vitro and in vivo experimental verification of predicted molecular mechanisms for identifying the targeted genes. The majority of GWAS-identified variants fall in noncoding regions of the genome. Therefore, this review focuses on strategies for assessing likely mechanisms affected by noncoding variants; such mechanisms include transcriptional regulation, noncoding RNA function, and epigenetic regulation. These approaches have already accelerated progress from genetic studies to biological knowledge and might ultimately guide the development of prognostic, preventive, and therapeutic measures.

Breast cancer risk is influenced by rare coding variants in susceptibility genes, such as BRCA1, and many common, mostly non-coding variants. However, much of the genetic contribution to breast cancer risk remains unknown. Here we report the results of a genome-wide association study of breast cancer in 122,977 cases and 105,974 controls of European ancestry and 14,068 cases and 13,104 controls of East Asian ancestry. We identified 65 new loci that are associated with overall breast cancer risk at P

Cancer sequencing predicts driver genes using recurrent protein-altering mutations, but detecting recurrence for non-coding mutations remains unsolved. Here, we present a convergence framework for recurrence analysis of non-coding mutations using three-dimensional co-localization of epigenomically-identified regions. We define the regulatory plexus of each gene as its cell-type-specific three-dimensional gene-regulatory neighborhood, inferred using Hi-C chromosomal interactions and chromatin state annotations. Using 16 matched tumor-normal prostate transcriptomes, we predict tumor-upregulated genes, and find enriched plexus mutations in distal regulatory regions normally repressed in prostate, suggesting out-of-context de-repression. Using 55 matched tumor-normal prostate genomes, we predict 15 driver genes by convergence of dispersed, low-frequency mutations into high-frequency dysregulation events along prostate-specific plexi, while controlling for mutational heterogeneity across regions, chromatin states, and patients. These putative drivers play roles in growth signaling, immune evasion, mitochondrial function, and vascularization, suggesting higher-order pathway-level convergence. We experimentally validate the PLCB4 plexus and its ability to affect the canonical PI3K cancer pathway.

The multi-cancer susceptibility locus at 5p15.33 includes TERT, encoding the telomerase catalytic subunit. Genome-wide association studies (GWAS) have identified six single nucleotide polymorphisms (SNPs) in the TERT promoter associated with decreased breast cancer risk, although the precise causal variants and their mechanisms of action have remained elusive. Luciferase reporter assays indicated that the protective haplotype reduced TERT promoter activity in human mammary epithelial and cancer cells in an estrogen-independent manner. Using single variant constructs, we identified rs3215401 and rs2853669 as likely functional variants. Silencing of MYC decreased TERT promoter activity but neither MYC nor ETS2 silencing conferred allele-specificity. In chromatin immunoprecipitation experiments, the ETS protein GABPA, but not ETS2 or ELF1, bound rs2853669 in an allele-specific manner in mammary epithelial cells. Investigation of open chromatin in mammoplasty samples suggested involvement of three additional variants, though not rs3215401 or rs2853669. Chromosome conformation capture revealed no interaction of the TERT promoter with regulatory elements in the locus, indicating limited local impact of candidate variants on the TERT promoter. Collectively, our functional studies of the TERT-CLPTM1L breast cancer susceptibility locus describe rs2853669 as a functional variant of this association signal among three other potentially causal variants and demonstrate the versatile mechanisms by which TERT promoter variants may affect breast cancer risk.

Predicting response to endocrine therapy and survival in oestrogen receptor positive breast cancer is a significant clinical challenge and novel prognostic biomarkers are needed. Long-range regulators of gene expression are emerging as promising biomarkers and therapeutic targets for human diseases, so we have explored the potential of distal enhancer elements of non-coding RNAs in the prognostication of breast cancer survival. HOTAIR is a long non-coding RNA that is overexpressed, promotes metastasis and is predictive of decreased survival. Here, we describe a long-range transcriptional enhancer of the HOTAIR gene that binds several hormone receptors and associated transcription factors, interacts with the HOTAIR promoter and augments transcription. This enhancer is dependent on Forkhead-Box transcription factors and functionally interacts with a novel alternate HOTAIR promoter. HOTAIR expression is negatively regulated by oestrogen, positively regulated by FOXA1 and FOXM1, and is inversely correlated with oestrogen receptor and directly correlated with FOXM1 in breast tumours. The combination of HOTAIR and FOXM1 enables greater discrimination of endocrine therapy responders and non-responders in patients with oestrogen receptor positive breast cancer. Consistent with this, HOTAIR expression is increased in cell-line models of endocrine resistance. Analysis of breast cancer gene expression data indicates that HOTAIR is co-expressed with FOXA1 and FOXM1 in HER2-enriched tumours, and these factors enhance the prognostic power of HOTAIR in aggressive HER2+ breast tumours. Our study elucidates the transcriptional regulation of HOTAIR, identifies HOTAIR and its regulators as novel biomarkers of patient response to endocrine therapy and corroborates the importance of transcriptional enhancers in cancer.

The 85-kb breast cancer-associated gene BRCA1 is an established tumor suppressor gene, but its regulation is poorly understood. We demonstrate by gene conformation analysis in both human cell lines and mouse mammary tissue that gene loops are imposed on BRCA1 between the promoter, introns, and terminator region. Significantly, association between the BRCA1 promoter and terminator regions change upon estrogen stimulation and during lactational development. Loop formation is transcription-dependent, suggesting that transcriptional elongation plays an active role in BRCA1 loop formation. We show that the BRCA1 terminator region can suppress estrogen-induced transcription and so may regulate BRCA1 expression. Significantly, BRCA1 promoter and terminator interactions vary in different breast cancer cell lines, indicating that defects in BRCA1 chromatin structure may contribute to dysregulated expression of BRCA1 seen in breast tumors.

Long noncoding RNAs (lncRNAs) are increasingly recognized to play major regulatory roles in development and disease. To identify novel regulators in breast biology, we identified differentially regulated lncRNAs during mouse mammary development. Among the highest and most differentially expressed was a transcript (Zfas1) antisense to the 5′ end of the protein-coding gene Znfx1. In vivo, Zfas1 RNA is localized within the ducts and alveoli of the mammary gland. Zfas1 intronically hosts three previously undescribed C/D box snoRNAs (SNORDs): Snord12, Snord12b, and Snord12c. In contrast to the general assumption that noncoding SNORD-host transcripts function only as vehicles to generate snoRNAs, knockdown of Zfas1 in a mammary epithelial cell line resulted in increased cellular proliferation and differentiation, while not substantially altering the levels of the SNORDs. In support of an independent function, we also found that Zfas1 is extremely stable, with a half-life >16 h. Expression analysis of the SNORDs revealed these were expressed at different levels, likely a result of distinct structures conferring differential stability. While there is relatively low primary sequence conservation between Zfas1 and its syntenic human ortholog ZFAS1, their predicted secondary structures have similar features. Like Zfas1, ZFAS1 is highly expressed in the mammary gland and is down-regulated in breast tumors compared to normal tissue. We propose a functional role for Zfas1/ ZFAS1 in the regulation of alveolar development and epithelial cell differentiation in the mammary gland, which, together with its dysregulation in human breast cancer, suggests ZFAS1 as a putative tumor suppressor gene.

To access publisher's full text version of this article click on the hyperlink at the bottom of the page We analyzed 3,872 common genetic variants across the ESR1 locus (encoding estrogen receptor α) in 118,816 subjects from three international consortia. We found evidence for at least five independent causal variants, each associated with different phenotype sets, including estrogen receptor (ER(+) or ER(-)) and human ERBB2 (HER2(+) or HER2(-)) tumor subtypes, mammographic density and tumor grade. The best candidate causal variants for ER(-) tumors lie in four separate enhancer elements, and their risk alleles reduce expression of ESR1, RMND1 and CCDC170, whereas the risk alleles of the strongest candidates for the remaining independent causal variant disrupt a silencer element and putatively increase ESR1 and RMND1 expression. National Institute for Health Research (NIHR) info:eu-repo/grantAgreement/EC/FP7/223175 Cancer Research UK C1287/A10118 C1287/A10710 C12292/A11174 C1281/A12014 C5047/A8384 C5047/A15007 C5047/A10692 C8197/A16565 US National Institutes of Health (NIH) CA128978 CA192393 CA116167 CA176785 US National Institutes of Health (NIH Specialized Program of Research Excellence (SPORE) in Breast Cancer) CA116201 Post-Cancer GWAS initiative 1U19 CA148537 1U19 CA148065 1U19 CA148112 Post-Cancer GWAS initiative (GAME-ON initiative) US Department of Defense W81XWH-10-1-0341 Canadian Institutes of Health Research (CIHR) Komen Foundation for the Cure Breast Cancer Research Foundation Ovarian Cancer Research Fund

Genome-wide association studies (GWAS) have identified markers within the WNT4 region on chromosome 1p36.12 showing consistent and strong association with increasing endometriosis risk. Fine mapping using sequence and imputed genotype data has revealed strong candidates for the causal SNPs within these critical regions; however, the molecular pathogenesis of these SNPs is currently unknown. We used gene expression data collected from whole blood from 862 individuals and endometrial tissue from 136 individuals from independent populations of European descent to examine the mechanism underlying endometriosis susceptibility. Association mapping results from 7,090 individuals (2,594 cases and 4,496 controls) supported rs3820282 as the SNP with the strongest association for endometriosis risk (P = 1.84 × 10−5, OR = 1.244 (1.126-1.375)). SNP rs3820282 is a significant eQTL in whole blood decreasing expression of LINC00339 (also known as HSPC157) and increasing expression of CDC42 (P = 2.0 ×10−54 and 4.5x10−4 respectively). The largest effects were for two LINC00339 probes (P = 2.0 ×10−54; 1.0 × 10−34). The eQTL for LINC00339 was also observed in endometrial tissue (P = 2.4 ×10−8) with the same direction of effect for both whole blood and endometrial tissue. There was no evidence for eQTL effects for WNT4. Chromatin conformation capture provides evidence for risk SNPs interacting with the promoters of both LINC00339 and CDC4 and luciferase reporter assays suggest the risk SNP rs12038474 is located in a transcriptional silencer for CDC42 and the risk allele increases expression of CDC42. However, no effect of rs3820282 was observed in the LINC00339 expression in Ishikawa cells. Taken together, our results suggest that SNPs increasing endometriosis risk in this region act through CDC42, but further functional studies are required to rule out inverse regulation of both LINC00339 and CDC42.

Breast cancer is a major health problem and understanding the genetic basis of this disease is crucial for predicting risk and developing effective targeted therapeutics. Several breast cancer predisposing genes have been identified, but mutations in the coding regions of these genes only accounts for a small proportion of risk. Research now suggests that combinations of multiple non-coding changes in breast cancer susceptibility genes, which cause moderate alterations in gene expression, will be responsible for the remaining inherited risk. These non-coding changes will include variants in proximal and distal transcriptional and post-transcriptional regulatory elements and may affect the levels and function of trans-acting factors, including proteins and RNAs, which act on these elements. Somatic changes in such elements and factors have also been associated with breast cancer progression. With the recent advent of techniques allowing the detection of long-range DNA interactions spanning the human genome, it has become increasingly clear that long-range regulatory elements constitute an important mechanism for gene regulation. Recent studies have identified several such elements that are important for regulating genes involved in breast cancer, raising the possibility that defects in these sequences may contribute to breast cancer predisposition and progression. In this review, we discuss the emerging functions of cis-regulatory elements and a subset of trans-acting factors in breast tumorigenesis. We also discuss some recent progress in our understanding of how dysregulation in these transcriptional components may contribute to breast cancer, and the potential implications for molecular diagnosis, prognosis prediction, and the treatment of this disease.

MicroRNAs (miRNAs) are small non-coding RNAs of ∼20 nt in length that are capable of modulating gene expression post-transcriptionally. Although miRNAs have been implicated in cancer, including breast cancer, the regulation of miRNA transcription and the role of defects in this process in cancer is not well understood. In this study we have mapped the promoters of 93 breast cancer-associated miRNAs, and then looked for associations between DNA methylation of 15 of these promoters and miRNA expression in breast cancer cells. The miRNA promoters with clearest association between DNA methylation and expression included a previously described and a novel promoter of the Hsa-mir-200b cluster. The novel promoter of the Hsa-mir-200b cluster, denoted P2, is located ∼2 kb upstream of the 5' stemloop and maps within a CpG island. P2 has comparable promoter activity to the previously reported promoter (P1), and is able to drive the expression of miR-200b in its endogenous genomic context. DNA methylation of both P1 and P2 was inversely associated with miR-200b expression in eight out of nine breast cancer cell lines, and in vitro methylation of both promoters repressed their activity in reporter assays. In clinical samples, P1 and P2 were differentially methylated with methylation inversely associated with miR-200b expression. P1 was hypermethylated in metastatic lymph nodes compared with matched primary breast tumours whereas P2 hypermethylation was associated with loss of either oestrogen receptor or progesterone receptor. Hypomethylation of P2 was associated with gain of HER2 and androgen receptor expression. These data suggest an association between miR-200b regulation and breast cancer subtype and a potential use of DNA methylation of miRNA promoters as a component of a suite of breast cancer biomarkers.

Disruption of the breast cancer susceptibility gene Brca1 results in defective lobular-alveolar development in the mammary gland and a predisposition to breast tumourigenesis in humans and in mice. Recent evidence suggests that BRCA1 loss in humans is associated with an expansion of the luminal progenitor cell compartment in the normal breast and tumours with a luminal progenitor-like expression profile. To further investigate the role of BRCA1 in the mammary gland, we examined the consequences of Brca1 loss in mouse mammary epithelial cells in vitro and in vivo. Here, we show that Brca1 loss is associated with defective morphogenesis of SCp2 and HC11 mouse mammary epithelial cell lines and that in the MMTV-Cre Brca1(Co/Co) mouse model of Brca1 loss, there is an accumulation of luminal progenitor (CD61(+)CD29(lo)CD24(+)) cells during pregnancy. By day 1 of lactation, there are marked differences in the expression of 1379 genes, with most significantly altered pathways and networks, including lactation, the immune response and cancer. One of the most differentially expressed genes was the luminal progenitor marker, c-kit. Immunohistochemical analysis revealed that the increase in c-kit levels is associated with an increase in c-kit positivity. Interestingly, an inverse association between Brca1 and c-kit expression was also observed during mammary epithelial differentiation, and small interfering RNA-mediated knockdown of Brca1 resulted in a significant increase in c-kit mRNA levels. We found no evidence that c-kit plays a direct role in regulating differentiation of HC11 cells, suggesting that Brca1-mediated induction of c-kit probably contributes to Brca1-associated tumourigenesis via another cellular process, and that c-kit is likely to be a marker rather than a mediator of defective lobular-alveolar development resulting from Brca1 loss.

Basal-like breast cancers arising in women carrying mutations in the BRCA1 gene, encoding the tumor suppressor protein BRCA1, are thought to develop from the mammary stem cell. To explore early cellular changes that occur in BRCA1 mutation carriers, we have prospectively isolated distinct epithelial subpopulations from normal mammary tissue and preneoplastic specimens from individuals heterozygous for a BRCA1 mutation. We describe three epithelial subsets including basal stem/progenitor, luminal progenitor and mature luminal cells. Unexpectedly, we found that breast tissue from BRCA1 mutation carriers harbors an expanded luminal progenitor population that shows factor-independent growth in vitro. Moreover, gene expression profiling revealed that breast tissue heterozygous for a BRCA1 mutation and basal breast tumors were more similar to normal luminal progenitor cells than any other subset, including the stem cell-enriched population. The c-KIT tyrosine kinase receptor (encoded by KIT) emerged as a key marker of luminal progenitor cells and was more highly expressed in BRCA1-associated preneoplastic tissue and tumors. Our findings suggest that an aberrant luminal progenitor population is a target for transformation in BRCA1-associated basal tumors .

BRCA1 is a breast cancer susceptibility gene that is down-regulated in a significant proportion of sporadic breast cancers. BRCA1 is posttranscriptionally regulated by RNA-binding proteins, the identities of which are unknown. HuR is an RNA binding protein implicated in posttranscriptional regulation of many genes and is overexpressed in sporadic breast cancer. To investigate the possibility that these two molecules are functionally linked in breast cancer, we performed bioinformatic analysis of the BRCA1 3′ untranslated region (UTR), RNA-protein assays with the HuR protein and the BRCA1 3′UTR, and immunohistochemical analysis of a cohort of breast tumors using antibodies against BRCA1 and HuR. Here, we describe the identification of two predicted HuR-binding sites in the BRCA1 3′UTR, one of which binds specifically to HuR. We also show that this interaction is disrupted by single nucleotide substitutions in the BRCA1 3′UTR and that endogenous HuR protein associates with BRCA1 transcripts in T47D and MCF7 breast cancer cells. Expression of ectopic HuR results in a significant decrease in BRCA1 protein expression and also BRCA1 3′UTR activity. Immunohistochemical analysis revealed that although BRCA1 and HuR expression were associated with some clinicopathologic features of the tumors, there was no statistically significant correlation between BRCA1 and HuR protein expression. These results identify the first posttranscriptional protein regulator of BRCA1 and have implications for understanding BRCA1 regulation in human breast cancer. [Cancer Res 2008;68(22):9469–78]

We conducted a meta-analysis of three endometrial cancer genome-wide association studies (GWAS) and two follow-up phases totaling 7,737 endometrial cancer cases and 37,144 controls of European ancestry. Genome-wide imputation and meta-analysis identified five new risk loci of genome-wide significance at likely regulatory regions on chromosomes 13q22.1 (rs11841589, near KLF5), 6q22.31 (rs13328298, in LOC643623 and near HEY2 and NCOA7), 8q24.21 (rs4733613, telomeric to MYC), 15q15.1 (rs937213, in EIF2AK4, near BMF) and 14q32.33 (rs2498796, in AKT1, near SIVA1). We also found a second independent 8q24.21 signal (rs17232730). Functional studies of the 13q22.1 locus showed that rs9600103 (pairwise r2 = 0.98 with rs11841589) is located in a region of active chromatin that interacts with the KLF5 promoter region. The rs9600103[T] allele that is protective in endometrial cancer suppressed gene expression in vitro, suggesting that regulation of the expression of KLF5, a gene linked to uterine development, is implicated in tumorigenesis. These findings provide enhanced insight into the genetic and biological basis of endometrial cancer.

Genome-wide association studies have found SNPs at 17q22 to be associated with breast cancer risk. To identify potential causal variants related to breast cancer risk, we performed a high resolution fine-mapping analysis that involved genotyping 517 SNPs using a custom Illumina iSelect array (iCOGS) followed by imputation of genotypes for 3,134 SNPs in more than 89,000 participants of European ancestry from the Breast Cancer Association Consortium (BCAC). We identified 28 highly correlated common variants, in a 53 Kb region spanning two introns of the STXBP4 gene, that are strong candidates for driving breast cancer risk (lead SNP rs2787486 (OR = 0.92; CI 0.90–0.94; P = 8.96 × 10−15)) and are correlated with two previously reported risk-associated variants at this locus, SNPs rs6504950 (OR = 0.94, P = 2.04 × 10−09, r2 = 0.73 with lead SNP) and rs1156287 (OR = 0.93, P = 3.41 × 10−11, r2 = 0.83 with lead SNP). Analyses indicate only one causal SNP in the region and several enhancer elements targeting STXBP4 are located within the 53 kb association signal. Expression studies in breast tumor tissues found SNP rs2787486 to be associated with increased STXBP4 expression, suggesting this may be a target gene of this locus., published version, peerReviewed

Assays to determine the pathogenicity of unclassified sequence variants in disease-associated genes include the analysis of lymphoblastoid cell lines (LCLs). We assessed the ability of several assays of LCLs to distinguish carriers of germline BRCA1 and BRCA2 gene mutations from mutation-negative controls to determine their utility for use in a diagnostic setting.Post-ionising radiation cell viability and micronucleus formation, and telomere length were assayed in LCLs carrying BRCA1 or BRCA2 mutations, and in unaffected mutation-negative controls.Post-irradiation cell viability and micronucleus induction assays of LCLs from individuals carrying pathogenic BRCA1 mutations, unclassified BRCA1 sequence variants or wildtype BRCA1 sequence showed significant phenotypic heterogeneity within each group. Responses were not consistent with predicted functional consequences of known pathogenic or normal sequences. Telomere length was also highly heterogeneous within groups of LCLs carrying pathogenic BRCA1 or BRCA2 mutations, and normal BRCA1 sequences, and was not predictive of mutation status.Given the significant degree of phenotypic heterogeneity of LCLs after gamma-irradiation, and the lack of association with BRCA1 or BRCA2 mutation status, we conclude that the assays evaluated in this study should not be used as a means of differentiating pathogenic and non-pathogenic sequence variants for clinical application. We suggest that a range of normal controls must be included in any functional assays of LCLs to ensure that any observed differences between samples reflect the genotype under investigation rather than generic inter-individual variation.

BRCA1 is a tumor suppressor that functions in controlling cell growth and maintaining genomic stability. BRCA1 has also been implicated in telomere maintenance through its ability to regulate the transcription of hTERT, the catalytic subunit of telomerase, resulting in telomere shortening, and to colocalize with the telomere-binding protein TRF1. The high incidence of nonreciprocal translocations in tumors arising from BRCA1 mutation carriers and Brca1-null mice also raises the possibility that BRCA1 plays a role in telomere protection. To date, however, the consequences for telomere status of disrupting BRCA1 have not been reported. To examine the role of BRCA1 in telomere regulation, we have expressed a dominant-negative mutant of BRCA1 (trBRCA1), known to disrupt multiple functions of BRCA1, in telomerase-positive mammary epithelial cells (SVCT) and telomerase-negative ALT cells (GM847). In SVCT cells, expression of trBRCA1 resulted in an increased incidence of anaphase bridges and in an increase in telomere length, but no change in telomerase activity. In GM847 cells, trBRCA1 also increased anaphase bridge formation but did not induce any change in telomere length. BRCA1 colocalized with TRF2 in telomerase-positive cells and with a small subset of ALT-associated PML bodies (APBs) in ALT cells. Together, these results raise the possibility that BRCA1 could play a role in telomere protection and suggest a potential mechanism for one of the phenotypes of BRCA1 deficient cells. (c) 2005 Wiley-Liss, Inc.

Large-scale transcriptomic studies have shown that the majority of the genome is transcribed, however, only a small proportion codes for protein (1). The majority of noncoding transcripts produced are long noncoding RNAs (lncRNAs), loosely defined as transcripts >200 nucleotides that do not code for protein.