Blood RNA analysis can increase clinical diagnostic rate and resolve variants of uncertain significance

Purpose Diagnosis of genetic disorders is hampered by large numbers of variants of uncertain significance (VUSs) identified through next-generation sequencing. Many such variants may disrupt normal RNA splicing. We examined effects on splicing of a large cohort of clinically identified variants and compared performance of bioinformatic splicing prediction tools commonly used in diagnostic laboratories. Methods Two hundred fifty-seven variants (coding and noncoding) were referred for analysis across three laboratories. Blood RNA samples underwent targeted reverse transcription polymerase chain reaction (RT-PCR) analysis with Sanger sequencing of PCR products and agarose gel electrophoresis. Seventeen samples also underwent transcriptome-wide RNA sequencing with targeted splicing analysis based on Sashimi plot visualization. Bioinformatic splicing predictions were obtained using Alamut, HSF 3.1, and SpliceAI software. Results Eighty-five variants (33%) were associated with abnormal splicing. The most frequent abnormality was upstream exon skipping (39/85 variants), which was most often associated with splice donor region variants. SpliceAI had greatest accuracy in predicting splicing abnormalities (0.91) and outperformed other tools in sensitivity and specificity. Conclusion Splicing analysis of blood RNA identifies diagnostically important splicing abnormalities and clarifies functional effects of a significant proportion of VUSs. Bioinformatic predictions are improving but still make significant errors. RNA analysis should therefore be routinely considered in genetic disease diagnostics

Facile synthesis and proposed mechanism of α,ω‐oxetanyl-telechelic poly(3-nitratomethyl-3-methyl oxetane) by an SN2(i) nitrato displacement method in basic media

The synthesis of a novel heterocyclic–telechelic polymer, α,ω-oxetanyl-telechelic poly(3-nitratomethyl-3-methyl oxetane), is described. Infrared spectroscopy (IR), gel permeation chromatography (GPC), and nuclear magnetic resonance (NMR) spectroscopy have been used to confirm the successful synthesis, demonstrating the presence of the telechelic-oxetanyl moieties. Synthesis of the terminal functionalities has been achieved via displacement of nitrato groups, in a manner similar to that employed with other leaving groups such as azido, bromo, and nitro, initiated by nucleophiles. In the present case, displacement occurs on the ends of a nitrato-functionalized polymer driven by the formation of sodium nitrate, which is supported by the polar aprotic solvent N,N-dimethyl formamide. The formation of an alkoxide at the polymer chain ends is favored and allows internal back-biting to the nearest carbon bearing the nitrato group, intrinsically in an SN2(i) reaction, leading to α,ω-oxetanyl functionalization. The telechelic-oxetanyl moieties have the potential to be cross-linked by chemical (e.g., acidic) or radiative (e.g., ultraviolet) curing methods without the use of high temperatures, usually below 100°C. This type of material was designed for future use as a contraband simulant, whereby it would form the predominant constituent of elastomeric composites comprising rubbery polymer with small quantities of solids, typically crystals of contraband substances, such as explosives or narcotics. This method also provides an alternative approach to ring closure and synthesis of heterocycles

Policing unacceptable protest in England and Wales: A case study of the policing of anti-fracking protests

In recent years public order policing policy in England and Wales has undergone significant changes. A ‘human rights compliant’ model of protest policing has been developed since 2009 and this article makes a contribution to the body of academic work considering the impact of these changes on operational policing. Drawing upon a longitudinal case study of the policing of protests against ‘fracking’ in Salford, Greater Manchester, in 2013-2014, the article contrasts post-2009 policy and academic discourses on protest policing with the experiences of anti-fracking protesters. To develop this assessment, the article also draws attention to previously unexplored definitions of acceptable and unacceptable protest set out by police in more recent policy, and considers the extent to which these definitions are reflected in the police response to anti-fracking protest. The article suggests that a police commitment to a human rights approach to protest facilitation is, at least in the case of anti-fracking protest, contingent on the focus and form of political activism

The impact of curriculum hierarchies on the development of professional self in teaching: student-teachers of drama negotiating issues of subject status at the interface between drama and English

At the level of policy the relative ‘value’ of subjects is determined by their official curriculum designation, creating a hierarchy of learning within which particular subjects are categorised as optional to the educational experience of young people. This situation is well-illustrated by the marginalised position of drama in the National Curriculum for England and Wales in which drama appears as an adjunct to the ‘core’ subject English. Yet at school level drama has survived as a discrete and reasonably embedded subject. Drawing on questionnaire and interview data, I investigate the effects of this mismatch on the emergence of pedagogical content knowledge, linked to notions of professional self, in drama student-teachers at one university in the UK. Findings indicate that the student-teachers, whilst not entirely eschewing a less-regulated relationship between the two subjects, view the curriculum for English and its accompanying assessment regime as an inadequate host for drama. In addition, they regard teacher autonomy over curriculum content and pedagogy as indicative of a high degree of professional expertise. This suggests that a case can be made for re-evaluating the nature of the relationship between drama and English and its representation in policy-constructed curricula

A novel de novo BRCA1 mutation in a Chinese woman with early onset breast cancer

Germline mutations in the two breast cancer susceptibility genes, BRCA1 and BRCA2 account for a significant portion of hereditary breast/ovarian cancer. De novo mutations such as multiple exon deletion are rarely occurred in BRCA1 and BRCA2. During our mutation screening for BRCA1/2 genes to Chinese women with risk factors for hereditary breast/ovarian cancer, we identified a novel germline mutation, consisting of a deletion from exons 1 to 12 in BRCA1 gene, in a patient diagnosed with early onset triple negative breast cancer with no family history of cancer. None of her parents carried the mutation and molecular analysis showed that this novel de novo germline mutation resulted in down-regulation of BRCA1 gene expression

Analysing the European Union's responses to organized crime through different securitization lenses

In the past 30 years, organized crime (OC) has shifted from being an issue of little, or no concern, to being considered one of the key security threats facing the European Union (EU), the economic and political fabric of its society and its citizens. The purpose of this article is to understand how OC has come to be understood as one of the major security threats in the EU, by applying different lenses of Securitization Theory (ST). More specifically, the research question guiding this article is whether applying different ST approaches can lead us to draw differing conclusions as to whether OC has been successfully securitized in the EU. Building on the recent literature that argues that this theoretical framework has branched out into different approaches, this article wishes to contrast two alternative views of how a security problem comes into being, in order to verify whether different approaches can lead to diverging conclusions regarding the same phenomenon. The purpose of this exercise is to contribute to the further development of ST by pointing out that the choice in approach bears direct consequences on reaching a conclusion regarding the successful character of a securitization process. Starting from a reflection on ST, the article proceeds with applying a “linguistic approach” to the case study, which it then contrasts with a “sociological approach”. The article proposes that although the application of a “linguistic approach” seems to indicate that OC has become securitized in the EU, it also overlooks a number of elements, which the “sociological approach” renders visible and which lead us to refute the initial conclusion

Molecular differences between ductal carcinoma in situ and adjacent invasive breast carcinoma: a multiplex ligation-dependent probe amplification study

Ductal carcinoma in situ (DCIS) accounts for approximately 20% of mammographically detected breast cancers. Although DCIS is generally highly curable, some women with DCIS will develop life-threatening invasive breast cancer, but the determinants of progression to infiltrating ductal cancer (IDC) are largely unknown. In the current study, we used multiplex ligation-dependent probe amplification (MLPA), a multiplex PCR-based test, to compare copy numbers of 21 breast cancer related genes between laser-microdissected DCIS and adjacent IDC lesions in 39 patients. Genes included in this study were ESR1, EGFR, FGFR1, ADAM9, IKBKB, PRDM14, MTDH, MYC, CCND1, EMSY, CDH1, TRAF4, CPD, MED1, HER2, CDC6, TOP2A, MAPT, BIRC5, CCNE1 and AURKA

A systematic analysis of splicing variants identifies new diagnoses in the 100,000 Genomes Project

Background Genomic variants which disrupt splicing are a major cause of rare genetic diseases. However, variants which lie outside of the canonical splice sites are difficult to interpret clinically. Improving the clinical interpretation of non-canonical splicing variants offers a major opportunity to uplift diagnostic yields from whole genome sequencing data. Methods Here, we examine the landscape of splicing variants in whole-genome sequencing data from 38,688 individuals in the 100,000 Genomes Project and assess the contribution of non-canonical splicing variants to rare genetic diseases. We use a variant-level constraint metric (the mutability-adjusted proportion of singletons) to identify constrained functional variant classes near exon–intron junctions and at putative splicing branchpoints. To identify new diagnoses for individuals with unsolved rare diseases in the 100,000 Genomes Project, we identified individuals with de novo single-nucleotide variants near exon–intron boundaries and at putative splicing branchpoints in known disease genes. We identified candidate diagnostic variants through manual phenotype matching and confirmed new molecular diagnoses through clinical variant interpretation and functional RNA studies. Results We show that near-splice positions and splicing branchpoints are highly constrained by purifying selection and harbour potentially damaging non-coding variants which are amenable to systematic analysis in sequencing data. From 258 de novo splicing variants in known rare disease genes, we identify 35 new likely diagnoses in probands with an unsolved rare disease. To date, we have confirmed a new diagnosis for six individuals, including four in whom RNA studies were performed. Conclusions Overall, we demonstrate the clinical value of examining non-canonical splicing variants in individuals with unsolved rare diseases