A CRISPR/Cas9 based engineering tool to activate expression of multiple genes individually or in any specific combination

Engineering of cells by overexpression or knock-down/out of individual genes has demonstrated that in most cases the manipulation of single genes is not sufficient to alter a cellular phenotype. Rather, multiple genes involved in a pathway need to be manipulated. Especially in mammalian cells such as CHO, where clonal variation is large, it has been difficult to unequivocally assess whether the observed change in phenotype is due to such clonal variation or the engineered gene. This can in part be overcome by testing multiple subclones, however, once it comes to engineering multiple genes and combinations thereof, the required workload quickly becomes prohibitive. We here present a simple technology for successive and/or specific activation of multiple genes integrated into a single genomic locus, which presents a potential solution to this problem. The technology consists of a vector containing multiple genes to be engineered or copies of the same gene. The promoters of these genes/gene copies are separated from the translation start site by repressor elements, flanked by individual guide RNA (gRNA) target sites. After integration of the construct into the genome and clone selection, these repressor elements can be removed by transfection with Cas9 and the corresponding pair of gRNAs that target the repressor of the gene(s) to be activated. Efficiency of target gene activation was in the range of 20-30% of the population for individual genes. Using 4 different fluorescent genes, the success of the technology was shown by activation of different combinations of these genes, followed by sorting of cells with the correct combination of required target genes activated. For pathway engineering studies, the selected genes can be expressed linked to these fluorescent genes e.g. via an IRES or a 2A self-cleaving peptide and cells with the desired co-expression pattern sorted, thus obviating the necessity to subclone for subsequent phenotypic characterization of the engineered cells. The technology provides a rapid procedure to assess the effect of gene combination on cellular behavior. Please click Additional Files below to see the full abstract

Sequencing-based breast cancer diagnostics as an alternative to routine biomarkers

Sequencing-based breast cancer diagnostics have the potential to replace routine biomarkers and provide molecular characterization that enable personalized precision medicine. Here we investigate the concordance between sequencing-based and routine diagnostic biomarkers and to what extent tumor sequencing contributes clinically actionable information. We applied DNA- and RNA-sequencing to characterize tumors from 307 breast cancer patients with replication in up to 739 patients. We developed models to predict status of routine biomarkers (ER, HER2,Ki-67, histological grade) from sequencing data. Non-routine biomarkers, including mutations in BRCA1, BRCA2 and ERBB2(HER2), and additional clinically actionable somatic alterations were also investigated. Concordance with routine diagnostic biomarkers was high for ER status (AUC = 0.95;AUC(replication) = 0.97) and HER2 status (AUC = 0.97;AUC(replication) = 0.92). The transcriptomic grade model enabled classification of histological grade 1 and histological grade 3 tumors with high accuracy (AUC = 0.98;AUC(replication) = 0.94). Clinically actionable mutations in BRCA1, BRCA2 and ERBB2(HER2) were detected in 5.5% of patients, while 53% had genomic alterations matching ongoing or concluded breast cancer studies. Sequencing-based molecular profiling can be applied as an alternative to histopathology to determine ER and HER2 status, in addition to providing improved tumor grading and clinically actionable mutations and molecular subtypes. Our results suggest that sequencing-based breast cancer diagnostics in a near future can replace routine biomarkersNonePublishe

What\u27s in a Phenotype?

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Genome-Wide Analyses for Osteosarcoma in Leonberger Dogs Reveal the CDKN2A/B Gene Locus as a Major Risk Locus

Dogs represent a unique spontaneous cancer model. Osteosarcoma (OSA) is the most common primary bone tumor in dogs (OMIA 001441-9615), and strongly resembles human forms of OSA. Several large- to giant-sized dog breeds, including the Leonberger, have a greatly increased risk of developing OSA. We performed genome-wide association analysis with high-density imputed SNP genotype data from 273 Leonberger cases with a median age of 8.1 [3.1–13.5] years and 365 controls older than eight years. This analysis revealed significant associations at the CDKN2A/B gene locus on canine chromosome 11, mirroring previous findings in other dog breeds, such as the greyhound, that also show an elevated risk for OSA. Heritability (h2SNP) was determined to be 20.6% (SE = 0.08; p-value = 5.7 × 10−4) based on a breed prevalence of 20%. The 2563 SNPs across the genome accounted for nearly all the h2SNP of OSA, with 2183 SNPs of small effect, 316 SNPs of moderate effect, and 64 SNPs of large effect. As with many other cancers it is likely that regulatory, non-coding variants underlie the increased risk for cancer development. Our findings confirm a complex genetic basis of OSA, moderate heritability, and the crucial role of the CDKN2A/B locus leading to strong cancer predisposition in dogs. It will ultimately be interesting to study and compare the known genetic loci associated with canine OSA in human OSA

Genome-wide analyses implicate 33 loci in heritable dog osteosarcoma, including regulatory variants near CDKN2A/B

Background: Canine osteosarcoma is clinically nearly identical to the human disease, but is common and highly heritable, making genetic dissection feasible. Results: Through genome-wide association analyses in three breeds (greyhounds, Rottweilers, and Irish wolfhounds), we identify 33 inherited risk loci explaining 55% to 85% of phenotype variance in each breed. The greyhound locus exhibiting the strongest association, located 150 kilobases upstream of the genes CDKN2A/B, is also the most rearranged locus in canine osteosarcoma tumors. The top germline candidate variant is found at a >90% frequency in Rottweilers and Irish wolfhounds, and alters an evolutionarily constrained element that we show has strong enhancer activity in human osteosarcoma cells. In all three breeds, osteosarcoma-associated loci and regions of reduced heterozygosity are enriched for genes in pathways connected to bone differentiation and growth. Several pathways, including one of genes regulated by miR124, are also enriched for somatic copy-number changes in tumors. Conclusions: Mapping a complex cancer in multiple dog breeds reveals a polygenic spectrum of germline risk factors pointing to specific pathways as drivers of disease

CCR2-V64I polymorphism is associated with increased risk of cervical cancer but not with HPV infection or pre-cancerous lesions in African women

<p>Abstract</p> <p>Background</p> <p>Cervical cancer, caused by specific oncogenic types of human papillomavirus (HPV), is the second most common cancer in women worldwide. A large number of young sexually active women get infected by HPV but only a small fraction of them have persistent infection and develop cervical cancer pointing to co- factors including host genetics that might play a role in outcome of the HPV infection. This study investigated the role of <it>CCR2-V64I </it>polymorphism in cervical cancer, pre-cancers and HPV infection in South African women resident in Western Cape. <it>CCR2-V64I </it>polymorphism has been previously reported to influence the progression to cervical cancer in some populations and has also been associated with decreased progression from HIV infection to AIDS.</p> <p>Methods</p> <p>Genotyping for <it>CCR2-V64I </it>was done by PCR-SSP in a case-control study of 446 women (106 black African and 340 mixed-ancestry) with histologically confirmed invasive cervical cancer and 1432 controls (322 black African and 1110 mixed-ancestry) group-matched (1:3) by age, ethnicity and domicile status. In the control women HPV was detected using the Digene Hybrid Capture II test and cervical disease was detected by cervical cytology.</p> <p>Results</p> <p>The <it>CCR2-64I </it>variant was significantly associated with cervical cancer when cases were compared to the control group (P = 0.001). Further analysis comparing selected groups within the controls showed that individuals with abnormal cytology and high grade squamous intraepitleial neoplasia (HSIL) did not have this association when compared to women with normal cytology. HPV infection also showed no association with <it>CCR2-64I </it>variant. Comparing SIL positive controls with the cases showed a significant association of <it>CCR2-64I </it>variant (P = 0.001) with cervical cancer.</p> <p>Conclusions</p> <p>This is the first study of the role of <it>CCR2-V64I </it>polymorphism in cervical cancer in an African population. Our results show that <it>CCR2-64I </it>variant is associated with the risk of cervical cancer but does not affect the susceptibility to HPV infection or HSIL in South African women of black and mixed-ancestry origin. This result implies that the role of CCR2 is important in invasive cancer of the cervix but not in HPV infection or in the development of pre-cancers.</p

Identification of two novel mammographic density loci at 6Q25.1

INTRODUCTION: Mammographic density (MD) is a strong heritable and intermediate phenotype for breast cancer, but much of its genetic variation remains unexplained. We performed a large-scale genetic association study including 8,419 women of European ancestry to identify MD loci. METHODS: Participants of three Swedish studies were genotyped on a custom Illumina iSelect genotyping array and percent and absolute mammographic density were ascertained using semiautomated and fully automated methods from film and digital mammograms. Linear regression analysis was used to test for SNP-MD associations, adjusting for age, body mass index, menopausal status and six principal components. Meta-analyses were performed by combining P values taking sample size, study-specific inflation factor and direction of effect into account. RESULTS: Genome-wide significant associations were observed for two previously identified loci: ZNF365 (rs10995194, P = 2.3 × 10(−8) for percent MD and P = 8.7 × 10(−9) for absolute MD) and AREG (rs10034692, P = 6.7 × 10(−9) for absolute MD). In addition, we found evidence of association for two variants at 6q25.1, both of which are known breast cancer susceptibility loci: rs9485370 in the TAB2 gene (P = 4.8 × 10(−9) for percent MD and P = 2.5 × 10(−8) for absolute MD) and rs60705924 in the CCDC170/ESR1 region (P = 2.2 × 10(−8) for absolute MD). Both regions have been implicated in estrogen receptor signaling with TAB2 being a potential regulator of tamoxifen response. CONCLUSIONS: We identified two novel MD loci at 6q25.1. These findings underscore the importance of 6q25.1 as a susceptibility region and provide more insight into the mechanisms through which MD influences breast cancer risk. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13058-015-0591-2) contains supplementary material, which is available to authorized users

Stressen inom läraryrket – en kvalitativ studie

Denna studie presenterar resultat från sex intervjuer av lärare inom gymnasiet och syftar till att undersöka deras uppfattning om lärarstress och stressorer inom yrket samt undersöka vilka metoder de använder för att hantera detta. Resultaten indikerar att lärarens stress sker beroende på deras engagemang, egen uppfattad arbetsbörda och personlig utveckling samt under tider med nationella prov och vid betygsättningar. De vanligaste stressorerna var den sociala kontakten med elever och kommunikationen med kollegor samt tiden till planering och feedback. Resultaten indikerade vidare att de intervjuade lärarna fick symptom som problem med sömnen, minskad kreativitet och svårigheter att skilja arbete från privatliv. Metoderna dessa lärare använde sig av för att hantera stressen var att prata med andra utanför skolans kontext samt att sysselsätta sig med annat på fritiden. Betydelsen studien har för läraryrket är att skapa en högre medvetenhet kring stressen och stressorernas ursprung samt att visa att träning i stresshantering behövs för att behålla lärare inom skolan. This study presents the results derived from six interviews conducted with teachers in the upper secondary school in Sweden about their perception on teacher stress and stressors. The purpose of the study is to review their perception on their own and which methods’ they use to handle their stress. The findings indicate that these teachers’ stress occurs depending on commitment, individual perceived workload and personal growth, and during times with national tests and grading. The most common stressors were social connections to students, communication between collegues and the time to planning and feedback. The Findings also indicated that the interviewed teachers’ symptoms from stress were sleepdistortions, loss of creativity and difficulty to distinguish professional from private life. The methods the interviewed teachers’ used to handle stress were to talk about it to others outside of their workplace and time to occupy the mind with non-school related activities. The effect this could create for the pedagogical community is higher conciousness about the origins of stress and that training over time is needed to help teachers’ stay in the school environment