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Hailey-Hailey disease (OMIM#169600) is an autosomal dominantly inherited genodermatosis characterized by erosions in the flexural areas of the body. Hailey-Hailey disease is caused by variants in ATP2C1, but for ~10% of the patients, no causative variant is found in the coding region of ATP2C1. We aimed to determine the genetic cause of Hailey-Hailey disease in a family without a variant in the coding areas of ATP2C1. By genome sequencing and analysis of all exon and intron sequences of ATP2C1, we identified the variant c.532-560 T>G (NM_014382.5) in intron 7 of ATP2C1. The variant is predicted by in silico tools to create a new deep intronic donor splice site. Segregation analysis detected the variant in the three affected family members. RNA sequencing confirmed that the variant creates a new deep intronic donor splice site that gives rise to an alternative exon. The identified deep-intronic variant in ATP2C1 is the likely cause of Hailey-Hailey disease. This is to our knowledge the first report of a deep-intronic variant as the cause of Hailey-Hailey disease, which shows that the analysis of the intronic sequences of ATP2C1 could increase the genetic diagnostic yield for Hailey-Hailey disease patients., (© 2024 The Author(s). American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

The growth and development of the skeleton is regulated by bone morphogenetic proteins of which several are linked to genetic skeletal disorders. So far, no human skeletal malformations have been associated with variants in BMP5. Here, we report a patient with biallelic loss of function variants in BMP5 and a syndromic phenotype including skeletal dysostosis, dysmorphic features, hypermobility, laryngo-tracheo-bronchomalacia and atrioventricular septal defect. We discuss the phenotype in relation to the known tissue-specific expression of Bmp5 and similar morphological abnormalities previously reported in experimental animal models. Our findings suggest a new association between BMP5 variants and a range of developmental anomalies, involving ears, heart and skeleton, thereby increasing understanding of BMP5's role in human development., (© 2024 The Author(s). Clinical Genetics published by John Wiley & Sons Ltd.)

Genetic variants that affect mRNA splicing are a major cause of hereditary disorders, but the spliceogenicity of variants is challenging to predict. RNA diagnostics of clinically accessible tissues enable rapid functional characterization of splice-altering variants within their natural genetic context. However, this analysis cannot be offered to all individuals as one in five human disease genes are not expressed in easily accessible cell types. To overcome this problem, we have used CRISPR activation (CRISPRa) based on a dCas9-VPR mRNA-based delivery platform to induce expression of the gene of interest in skin fibroblasts from individuals with suspected monogenic disorders. Using this ex vivo splicing assay, we characterized the splicing patterns associated with germline variants in the myelin protein zero gene (MPZ), which is exclusively expressed in Schwann cells of the peripheral nerves, and the spastin gene (SPAST), which is predominantly expressed in the central nervous system. After overnight incubation, CRISPRa strongly upregulated MPZ and SPAST transcription in skin fibroblasts, which enabled splice variant profiling using reverse transcription polymerase chain reaction, next-generation sequencing, and long-read sequencing. Our investigations show proof of principle of a promising genetic diagnostic tool that involves CRISPRa to activate gene expression in easily accessible cells to study the functional impact of genetic variants. The procedure is easy to perform in a diagnostic laboratory with equipment and reagents all readily available., Competing Interests: Declaration of interests R.O.B. holds equity in Graphite Bio and UNIKUM Tx and is a paid consultant of UNIKUM Therapeutics., (Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Extensive research has explored the role of gut microbiota in colorectal cancer (CRC). Nonetheless, metatranscriptomic studies investigating the in situ functional implications of host-microbe interactions in CRC are scarce. Therefore, we characterized the influence of CRC core pathogens and biofilms on the tumor microenvironment (TME) in 40 CRC, paired normal, and healthy tissue biopsies using fluorescence in situ hybridization (FISH) and dual-RNA sequencing. FISH revealed that Fusobacterium spp . was associated with increased bacterial biomass and inflammatory response in CRC samples. Dual-RNA sequencing demonstrated increased expression of pro-inflammatory cytokines, defensins, matrix-metalloproteases, and immunomodulatory factors in CRC samples with high bacterial activity. In addition, bacterial activity correlated with the infiltration of several immune cell subtypes, including M2 macrophages and regulatory T-cells in CRC samples. Specifically, Bacteroides fragilis and Fusobacterium nucleatum correlated with the infiltration of neutrophils and CD4 + T-cells, respectively. The collective bacterial activity/biomass appeared to exert a more significant influence on the TME than core pathogens, underscoring the intricate interplay between gut microbiota and CRC. These results emphasize how biofilms and core pathogens shape the immune phenotype and TME in CRC while highlighting the need to extend the bacterial scope beyond CRC pathogens to advance our understanding and identify treatment targets.

Differential analysis of bulk RNA-seq data often suffers from lack of good controls. Here, we present a generative model that replaces controls, trained solely on healthy tissues. The unsupervised model learns a low-dimensional representation and can identify the closest normal representation for a given disease sample. This enables control-free, single-sample differential expression analysis. In breast cancer, we demonstrate how our approach selects marker genes and outperforms a state-of-the-art method. Furthermore, significant genes identified by the model are enriched in driver genes across cancers. Our results show that the in silico closest normal provides a more favorable comparison than control samples., (© 2023. The Author(s).)

KPTN-related disorder is an autosomal recessive disorder associated with germline variants in KPTN (previously known as kaptin), a component of the mTOR regulatory complex KICSTOR. To gain further insights into the pathogenesis of KPTN-related disorder, we analysed mouse knockout and human stem cell KPTN loss-of-function models. Kptn -/- mice display many of the key KPTN-related disorder phenotypes, including brain overgrowth, behavioural abnormalities, and cognitive deficits. By assessment of affected individuals, we have identified widespread cognitive deficits (n = 6) and postnatal onset of brain overgrowth (n = 19). By analysing head size data from their parents (n = 24), we have identified a previously unrecognized KPTN dosage-sensitivity, resulting in increased head circumference in heterozygous carriers of pathogenic KPTN variants. Molecular and structural analysis of Kptn-/- mice revealed pathological changes, including differences in brain size, shape and cell numbers primarily due to abnormal postnatal brain development. Both the mouse and differentiated induced pluripotent stem cell models of the disorder display transcriptional and biochemical evidence for altered mTOR pathway signalling, supporting the role of KPTN in regulating mTORC1. By treatment in our KPTN mouse model, we found that the increased mTOR signalling downstream of KPTN is rapamycin sensitive, highlighting possible therapeutic avenues with currently available mTOR inhibitors. These findings place KPTN-related disorder in the broader group of mTORC1-related disorders affecting brain structure, cognitive function and network integrity., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Polygenic risk scores (PRS) identify at-risk individuals for many common diseases. A discussion of strengths and limitations is carried out in this review. PRS complement traditional genetic testing and have shown utility in establishing a proper diagnosis and guiding primary and secondary prevention. Some individuals with high PRS have risks similar to those with monogenic predisposition. Limitations include potential misinterpretations, problems with application across ancestries, and limited usefulness in low-heritability traits. Despite its shortcomings PRS are predicted to play major roles in the future of personal medicine and genetic testing., (Published under Open Access CC-BY-NC-BD 4.0. https://creativecommons.org/licenses/by-nc-nd/4.0/.)

Background & Aims: Colonic adenomatous polyps, or adenomas, are frequent precancerous lesions and the origin of most cases of colorectal adenocarcinoma. However, we know from epidemiologic studies that although most colorectal cancers (CRCs) originate from adenomas, only a small fraction of adenomas (3%-5%) ever progress to cancer. At present, there are no molecular markers to guide follow-up surveillance programs., Methods: We profiled, by mass spectrometry-based proteomics combined with machine learning analysis, a selected cohort of formalin-fixed, paraffin-embedded high-grade (HG) adenomas with long clinical follow-up, collected as part of the Danish national screening program. We grouped subjects in the cohort according to their subsequent history of findings: a nonmetachronous advanced neoplasia group (G0), with no new HG adenomas or CRCs up to 10 years after polypectomy, and a metachronous advanced neoplasia group (G1) where individuals developed a new HG adenoma or CRC within 5 years of diagnosis., Results: We generated a proteome dataset from 98 selected HG adenoma samples, including 20 technical replicates, of which 45 samples belonged to the nonmetachronous advanced neoplasia group and 53 to the metachronous advanced neoplasia group. The clear distinction of these 2 groups seen in a uniform manifold approximation and projection plot indicated that the information contained within the abundance of the ∼5000 proteins was sufficient to predict the future occurrence of HG adenomas or development of CRC., Conclusions: We performed an in-depth analysis of quantitative proteomic data from 98 resected adenoma samples using various novel algorithms and statistical packages and found that their proteome can predict development of metachronous advanced lesions and progression several years in advance., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

BOADICEA is a comprehensive risk prediction model for breast and/or ovarian cancer (BC/OC) and for carrying pathogenic variants (PVs) in cancer susceptibility genes. In addition to BRCA1 and BRCA2, BOADICEA version 6 includes PALB2, CHEK2, ATM, BARD1, RAD51C and RAD51D. To validate its predictions for these genes, we conducted a retrospective study including 2033 individuals counselled at clinical genetics departments in Denmark. All counselees underwent comprehensive genetic testing by next generation sequencing on suspicion of hereditary susceptibility to BC/OC. Likelihoods of PVs were predicted from information about diagnosis, family history and tumour pathology. Calibration was examined using the observed-to-expected ratio (O/E) and discrimination using the area under the receiver operating characteristics curve (AUC). The O/E was 1.11 (95% CI 0.97-1.26) for all genes combined. At sub-categories of predicted likelihood, the model performed well with limited misestimation at the extremes of predicted likelihood. Discrimination was acceptable with an AUC of 0.70 (95% CI 0.66-0.74), although discrimination was better for BRCA1 and BRCA2 than for the other genes in the model. This suggests that BOADICEA remains a valid decision-making aid for determining which individuals to offer comprehensive genetic testing for hereditary susceptibility to BC/OC despite suboptimal calibration for individual genes in this population., (© 2023. The Author(s).)

Mutations, which result in amino acid substitutions, influence the stability of proteins and their binding to biomolecules. A molecular understanding of the effects of protein mutations is both of biotechnological and medical relevance. Empirical free energy functions that quickly estimate the free energy change upon mutation (ΔΔG) can be exploited for systematic screenings of proteins and protein complexes. In silico saturation mutagenesis can guide the design of new experiments or rationalize the consequences of known mutations. Often software such as FoldX, while fast and reliable, lack the necessary automation features to apply them in a high-throughput manner. We introduce MutateX, a software to automate the prediction of ΔΔGs associated with the systematic mutation of each residue within a protein, or protein complex to all other possible residue types, using the FoldX energy function. MutateX also supports ΔΔG calculations over protein ensembles, upon post-translational modifications and in multimeric assemblies. At the heart of MutateX lies an automated pipeline engine that handles input preparation, parallelization and outputs publication-ready figures. We illustrate the MutateX protocol applied to different case studies. The results of the high-throughput scan provided by our tools can help in different applications, such as the analysis of disease-associated mutations, to complement experimental deep mutational scans, or assist the design of variants for industrial applications. MutateX is a collection of Python tools that relies on open-source libraries. It is available free of charge under the GNU General Public License from https://github.com/ELELAB/mutatex., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The YTH domain family member 3 gene (YTHDF3) encodes a reader of the abundant N6-methyladenosine (m 6 A) modification of eukaryotic mRNA, which plays an essential role in regulating mRNA stability and is necessary to achieve normal development of the central nervous system in animal models. YTHDF3 has not previously been implicated in Mendelian disease despite a high probability of loss of function intolerance and statistical evidence of enrichment for gene-disruptive de novo variants in large-scale studies of individuals with intellectual disability and/or developmental delay. We report four individuals with deletion of 8q12.3, deletion size 1.38-2.60 Mb, encompassing YTHDF3, three of them were de novo, and in one case, the inheritance was unknown. Common features of the individuals (age range, 4-22 years) were developmental delay and/or intellectual disability. Two individuals underwent squint surgery. We suggest that haploinsufficiency of YTHDF3 causes a neurodevelopmental disorder with developmental delay and intellectual disability of variable degree., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Canine distemper virus (CDV) and phocine distemper virus (PDV) are major pathogens to terrestrial and marine mammals. Yet little is known about the timing and geographical origin of distemper viruses and to what extent it was influenced by environmental change and human activities. To address this, we (i) performed the first comprehensive time-calibrated phylogenetic analysis of the two distemper viruses, (ii) mapped distemper antibody and virus detection data from marine mammals collected between 1972 and 2018, and (iii) compiled historical reports on distemper dating back to the eighteenth century. We find that CDV and PDV diverged in the early seventeenth century. Modern CDV strains last shared a common ancestor in the nineteenth century with a marked radiation during the 1930s-1950s. Modern PDV strains are of more recent origin, diverging in the 1970s-1980s. Based on the compiled information on distemper distribution, the diverse host range of CDV and basal phylogenetic placement of terrestrial morbilliviruses, we hypothesize a terrestrial CDV-like ancestor giving rise to PDV in the North Atlantic. Moreover, given the estimated timing of distemper origin and radiation, we hypothesize a prominent role of environmental change such as the Little Ice Age, and human activities like globalization and war in distemper virus evolution.

Despite significant advancements in breast cancer (BC) research, clinicians lack robust serological protein markers for accurate diagnostics and tumor stratification. Tumor interstitial fluid (TIF) accumulates aberrantly externalized proteins within the local tumor space, which can potentially gain access to the circulatory system. As such, TIF may represent a valuable starting point for identifying relevant tumor-specific serological biomarkers. The aim of the study was to perform comprehensive proteomic profiling of TIF to identify proteins associated with BC tumor status and subtype. A liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of 35 TIFs of three main subtypes: luminal (19), Her2 (4), and triple-negative (TNBC) (12) resulted in the identification of > 8800 proteins. Unsupervised hierarchical clustering segregated the TIF proteome into two major clusters, luminal and TNBC/Her2 subgroups. High-grade tumors enriched with tumor infiltrating lymphocytes (TILs) were also stratified from low-grade tumors. A consensus analysis approach, including differential abundance analysis, selection operator regression, and random forest returned a minimal set of 24 proteins associated with BC subtypes, receptor status, and TIL scoring. Among them, a panel of 10 proteins, AGR3, BCAM, CELSR1, MIEN1, NAT1, PIP4K2B, SEC23B, THTPA, TMEM51, and ULBP2, was found to stratify the tumor subtype-specific TIFs. In particular, upregulation of BCAM and CELSR1 differentiates luminal subtypes, while upregulation of MIEN1 differentiates Her2 subtypes. Immunohistochemistry analysis showed a direct correlation between protein abundance in TIFs and intratumor expression levels for all 10 proteins. Sensitivity and specificity were estimated for this protein panel by using an independent, comprehensive breast tumor proteome dataset. The results of this analysis strongly support our data, with eight of the proteins potentially representing biomarkers for stratification of BC subtypes. Five of the most representative proteomics databases currently available were also used to estimate the potential for these selected proteins to serve as putative serological markers., (© 2020 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Pathogenic variants in STK11, also designated as LKB1, cause Peutz-Jeghers syndrome, which is a rare autosomal dominant disorder characterized by mucocutaneous pigmentation changes, polyposis, and a high risk of cancer., Methods: A male meeting the clinical diagnostic criteria for Peutz-Jeghers syndrome underwent next-generation sequencing. To validate the predicted splicing impact of a detected STK11 variant, we performed RNA-Seq on mRNA extracted from patient-derived Epstein-Barr virus-transformed lymphocytes treated with cycloheximide to inhibit nonsense-mediated decay ex vivo., Results: Blood testing identified a novel single-nucleotide substitution, NM_000455.4:c.735-10C>A, at the end of the 3' polypyrimidine tract of intron five in STK11. RNA-Seq confirmed a predicted eight base pair insertion in the mRNA transcript. Following inhibition of nonsense-mediated decay, the out-of-frame insertion was detected in 50% of all RNA-Seq reads. This confirmed a strong, deleterious splicing impact of the variant., Conclusion: We characterized a novel likely pathogenic germline variant in intron five of STK11 associated with Peutz-Jeghers syndrome. The study highlights RNA-Seq as a useful supplement in hereditary cancer predisposition testing., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Background: Studies on tumor-secreted microRNAs point to a functional role of these in cellular communication and reprogramming of the tumor microenvironment. Uptake of tumor-secreted microRNAs by neighboring cells may result in the silencing of mRNA targets and, in turn, modulation of the transcriptome. Studying miRNAs externalized from tumors could improve cancer patient diagnosis and disease monitoring and help to pinpoint which miRNA-gene interactions are central for tumor properties such as invasiveness and metastasis., Methods: Using a bioinformatics approach, we analyzed the profiles of secreted tumor and normal interstitial fluid (IF) microRNAs, from women with breast cancer (BC). We carried out differential abundance analysis (DAA), to obtain miRNAs, which were enriched or depleted in IFs, from patients with different clinical traits. Subsequently, miRNA family enrichment analysis was performed to assess whether any families were over-represented in the specific sets. We identified dysregulated genes in tumor tissues from the same cohort of patients and constructed weighted gene co-expression networks, to extract sets of co-expressed genes and co-abundant miRNAs. Lastly, we integrated miRNAs and mRNAs to obtain interaction networks and supported our findings using prediction tools and cancer gene databases., Results: Network analysis showed co-expressed genes and miRNA regulators, associated with tumor lymphocyte infiltration. All of the genes were involved in immune system processes, and many had previously been associated with cancer immunity. A subset of these, BTLA, CXCL13, IL7R, LAMP3, and LTB, was linked to the presence of tertiary lymphoid structures and high endothelial venules within tumors. Co-abundant tumor interstitial fluid miRNAs within this network, including miR-146a and miR-494, were annotated as negative regulators of immune-stimulatory responses. One co-expression network encompassed differences between BC subtypes. Genes differentially co-expressed between luminal B and triple-negative breast cancer (TNBC) were connected with sphingolipid metabolism and predicted to be co-regulated by miR-23a. Co-expressed genes and TIF miRNAs associated with tumor grade were BTRC, CHST1, miR-10a/b, miR-107, miR-301a, and miR-454., Conclusion: Integration of IF miRNAs and mRNAs unveiled networks associated with patient clinicopathological traits, and underlined molecular mechanisms, specific to BC sub-groups. Our results highlight the benefits of an integrative approach to biomarker discovery, placing secreted miRNAs within a biological context.

With the improvement of -omics and next-generation sequencing (NGS) methodologies, along with the lowered cost of generating these types of data, the analysis of high-throughput biological data has become standard both for forming and testing biomedical hypotheses. Our knowledge of how to normalize datasets to remove latent undesirable variances has grown extensively, making for standardized data that are easily compared between studies. Here we present the CAncer bioMarker Prediction Pipeline (CAMPP), an open-source R-based wrapper (https://github.com/ELELAB/CAncer-bioMarker-Prediction-Pipeline -CAMPP) intended to aid bioinformatic software-users with data analyses. CAMPP is called from a terminal command line and is supported by a user-friendly manual. The pipeline may be run on a local computer and requires little or no knowledge of programming. To avoid issues relating to R-package updates, a renv .lock file is provided to ensure R-package stability. Data-management includes missing value imputation, data normalization, and distributional checks. CAMPP performs (I) k-means clustering, (II) differential expression/abundance analysis, (III) elastic-net regression, (IV) correlation and co-expression network analyses, (V) survival analysis, and (VI) protein-protein/miRNA-gene interaction networks. The pipeline returns tabular files and graphical representations of the results. We hope that CAMPP will assist in streamlining bioinformatic analysis of quantitative biological data, whilst ensuring an appropriate bio-statistical framework., Competing Interests: The authors have declared that no competing interests exist.

Cancer driver gene alterations influence cancer development, occurring in oncogenes, tumor suppressors, and dual role genes. Discovering dual role cancer genes is difficult because of their elusive context-dependent behavior. We define oncogenic mediators as genes controlling biological processes. With them, we classify cancer driver genes, unveiling their roles in cancer mechanisms. To this end, we present Moonlight, a tool that incorporates multiple -omics data to identify critical cancer driver genes. With Moonlight, we analyze 8000+ tumor samples from 18 cancer types, discovering 3310 oncogenic mediators, 151 having dual roles. By incorporating additional data (amplification, mutation, DNA methylation, chromatin accessibility), we reveal 1000+ cancer driver genes, corroborating known molecular mechanisms. Additionally, we confirm critical cancer driver genes by analysing cell-line datasets. We discover inactivation of tumor suppressors in intron regions and that tissue type and subtype indicate dual role status. These findings help explain tumor heterogeneity and could guide therapeutic decisions.

Background: Preoperative genetic testing affects the surgical decision-making among women with breast cancer. To avoid breast-conserving surgery and to offer the possibility of mastectomy with immediate reconstruction in high-risk patients, genetic testing for pathogenic variants in BRCA1 or BRCA2 and a pedigree-based familial breast cancer risk assessment was offered to younger women with breast cancer in Denmark. We evaluated the impact of the risk stratification through genetic counseling on the uptake of contralateral prophylactic mastectomy (CPM). Material and methods: The prospective cohort study included all women with unilateral breast cancer before the age of 45 who participated in a genetic counseling program during their primary diagnostics in the Central Denmark Region (2013-2018). Each patient was followed from the time of the genetic test result to the end of follow-up to estimate the long-term uptake of CPM as a competing risk-adjusted cumulative incidence. We compared the uptake of CPM between the various genetic risk categories, ages of onset, and family histories in a multivariable Cox proportional hazards regression model, reporting hazard ratios (HR) with two-sided 95% confidence intervals (CIs). Results: 156 females, aged 21-44, learned their genetic test result within a median of 92 days [interquartile range (IQR): 75-114]. The maximal follow-up was 3.8 years (median 1.8; IQR: 0.49-2.5), after which 33% (95% CI: 24-42%) of the patients had undergone CPM. The uptake of CPM was inversely associated with the age of onset (HR 0.92; 95% CI: 0.86-0.98) and significantly higher among BRCA carriers (HR 2.9; 95% CI: 1.3-6.8) and patients from the high risk of breast cancer families (HR 5.6; 95% CI: 1.9-16) compared to the lower genetic risk categories. Conclusion: The risk stratification obtained through genetic counseling had a considerable impact on the surgical decision-making among younger women with breast cancer at long-term follow-up.

Apoptosis is an essential defensive mechanism against tumorigenesis. Proteins of the B-cell lymphoma-2 (Bcl-2) family regulate programmed cell death by the mitochondrial apoptosis pathway. In response to intracellular stress, the apoptotic balance is governed by interactions of three distinct subgroups of proteins; the activator/sensitizer BH3 (Bcl-2 homology 3)-only proteins, the pro-survival, and the pro-apoptotic executioner proteins. Changes in expression levels, stability, and functional impairment of pro-survival proteins can lead to an imbalance in tissue homeostasis. Their overexpression or hyperactivation can result in oncogenic effects. Pro-survival Bcl-2 family members carry out their function by binding the BH3 short linear motif of pro-apoptotic proteins in a modular way, creating a complex network of protein-protein interactions. Their dysfunction enables cancer cells to evade cell death. The critical role of Bcl-2 proteins in homeostasis and tumorigenesis, coupled with mounting insight in their structural properties, make them therapeutic targets of interest. A better understanding of gene expression, mutational profile, and molecular mechanisms of pro-survival Bcl-2 proteins in different cancer types, could help to clarify their role in cancer development and may guide advancement in drug discovery. Here, we shed light on the pro-survival Bcl-2 proteins in breast cancer using different bioinformatic approaches, linking -omics with structural data. We analyzed the changes in the expression of the Bcl-2 proteins and their BH3-containing interactors in breast cancer samples. We then studied, at the structural level, a selection of interactions, accounting for effects induced by mutations found in the breast cancer samples. We find two complexes between the up-regulated Bcl2A1 and two down-regulated BH3-only candidates (i.e., Hrk and Nr4a1) as targets associated with reduced apoptosis in breast cancer samples for future experimental validation. Furthermore, we predict L99R, M75R as damaging mutations altering protein stability, and Y120C as a possible allosteric mutation from an exposed surface to the BH3-binding site., Competing Interests: The authors have declared that no competing interests exist.

The Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) calculates the probability that a woman carries a pathogenic variant in BRCA1 or BRCA2 based on her pedigree and the population frequencies of pathogenic alleles of BRCA1 (0.0006394) and BRCA2 (0.00102) in the United Kingdom (UK). BOADICEA allows the clinician to define the population frequencies of pathogenic alleles of BRCA1 and BRCA2 for other populations but only includes preset values for the Ashkenazy Jewish and Icelandic populations. Among 173 early-onset breast cancer pedigrees in Denmark, BOADICEA discriminated well between carriers and non-carriers of pathogenic variants (area under the receiver operating characteristics curve: 0.81; 95% CI 0.74-0.86) but underestimated the frequency of carriers of pathogenic variants in BRCA1 or BRCA2 as measured by the observed-to-expected ratio (O/E 1.83; 95% CI 1.18-2.84). This reflects findings from older studies of BOADICEA in UK, German, Italian, and Chinese populations, all accounting for the different calibration for different carrier probabilities. To improve the performance of BOADICEA for non-UK populations, we developed a method to derive population frequencies of pathogenic alleles of BRCA1 and BRCA2. Compared to the UK population frequencies, we estimated the Danish population frequencies of pathogenic alleles to be higher for BRCA1 (0.0015; 95% CI 0.00064-0.0034) and lower for BRCA2 (0.00052; 95% CI 0.00018-0.0017) after adjusting for the different calibration of BOADICEA for different carrier probabilities. Incorporating additional population frequencies into BOADICEA could improve its performance for non-UK populations.

Background: Genomic initiatives such as The Cancer Genome Atlas (TCGA) contain data from -omics profiling of thousands of tumor samples, which may be used to decipher cancer signaling, and related alterations. Managing and analyzing data from large-scale projects, such as TCGA, is a demanding task. It is difficult to dissect the high complexity hidden in genomic data and to account for inter-tumor heterogeneity adequately., Methods: In this study, we used a robust statistical framework along with the integration of diverse bioinformatic tools to analyze next-generation sequencing data from more than 1000 patients from two different lung cancer subtypes, i.e., the lung adenocarcinoma (LUAD) and the squamous cell carcinoma (LUSC)., Results: We used the gene expression data to identify co-expression modules and differentially expressed genes to discriminate between LUAD and LUSC. We identified a group of genes which could act as specific oncogenes or tumor suppressor genes in one of the two lung cancer types, along with two dual role genes. Our results have been validated against other transcriptomics data of lung cancer patients., Conclusions: Our integrative approach allowed us to identify two key features: a substantial up-regulation of genes involved in O-glycosylation of mucins in LUAD, and a compromised immune response in LUSC. The immune-profile associated with LUSC might be linked to the activation of three oncogenic pathways, which promote the evasion of the antitumor immune response. Collectively, our results provide new future directions for the design of target therapies in lung cancer.

Objective: To investigate the effects of centralizing the acute stroke services in the Central Denmark Region (CDR)., Methods: The CDR (1.3 million inhabitants) centralized acute stroke care from 6 to 2 designated acute stroke units with 7-day outpatient clinics. We performed a prospective "before-and-after" cohort study comparing all strokes from the CDR with strokes in the rest of Denmark to discover underlying general trends, adopting a difference-in-differences approach. The population comprised 22,141 stroke cases hospitalized from May 2011 to April 2012 and May 2013 to April 2014., Results: Centralization was associated with a significant reduction in length of acute hospital stay from a median of 5 to 2 days with a length-of-stay ratio of 0.53 (95% confidence interval 0.38-0.75, data adjusted) with no corresponding change seen in the rest of Denmark. Similarly, centralization led to a significant increase in strokes with same-day admission (mainly outpatients), whereas this remained unchanged in the rest of Denmark. We observed a significant improvement in quality of care captured in 11 process performance measures in both the CDR and the rest of Denmark. Centralization was associated with a nonsignificant increase in thrombolysis rate. We observed a slight increase in readmissions at day 30, but this was not significantly different from the general trend. Mortality at days 30 and 365 remained unchanged, as in the rest of Denmark., Conclusions: Centralizing acute stroke care in the CDR significantly reduced the length of acute hospital stay without compromising quality. Readmissions and mortality stayed comparable to the rest of Denmark., (© 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Particular N-glycan structures are known to be associated with breast malignancies by coordinating various regulatory events within the tumor and corresponding microenvironment, thus implying that N-glycan patterns may be used for cancer stratification and as predictive or prognostic biomarkers. However, the association between N-glycans secreted by breast tumor and corresponding clinical relevance remain to be elucidated. We profiled N-glycans by HILIC UPLC across a discovery dataset composed of tumor interstitial fluids (TIF, n = 85), paired normal interstitial fluids (NIF, n = 54) and serum samples (n = 28) followed by independent evaluation, with the ultimate goal of identifying tumor-related N-glycan patterns in blood of patients with breast cancer. The segregation of N-linked oligosaccharides revealed 33 compositions, which exhibited differential abundances between TIF and NIF. TIFs were depleted of bisecting N-glycans, which are known to play essential roles in tumor suppression. An increased level of simple high mannose N-glycans in TIF strongly correlated with the presence of tumor infiltrating lymphocytes within tumor. At the same time, a low level of highly complex N-glycans in TIF inversely correlated with the presence of infiltrating lymphocytes within tumor. Survival analysis showed that patients exhibiting increased TIF abundance of GP24 had better outcomes, whereas low levels of GP10, GP23, GP38, and coreF were associated with poor prognosis. Levels of GP1, GP8, GP9, GP14, GP23, GP28, GP37, GP38, and coreF were significantly correlated between TIF and paired serum samples. Cross-validation analysis using an independent serum dataset supported the observed correlation between TIF and serum, for five of nine N-glycan groups: GP8, GP9, GP14, GP23, and coreF. Collectively, our results imply that profiling of N-glycans from proximal breast tumor fluids is a promising strategy for determining tumor-derived glyco-signature(s) in the blood. N-glycans structures validated in our study may serve as novel biomarkers to improve the diagnostic and prognostic stratification of patients with breast cancer., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Intrinsically disordered proteins (IDPs) are involved in many pivotal cellular processes including phosphorylation and signalling. The structural and functional effects of phosphorylation of IDPs remain poorly understood and difficult to predict. Thus, a need exists to identify motifs that confer phosphorylation-dependent perturbation of the local preferences for forming e.g. helical structures as well as motifs that do not. The disordered distal tail of the Na + /H + exchanger 1 (NHE1) is six-times phosphorylated (S693, S723, S726, S771, T779, S785) by the mitogen activated protein kinase 2 (MAPK1, ERK2). Using NMR spectroscopy, we found that two out of those six phosphorylation sites had a stabilizing effect on transient helices. One of these was further investigated by circular dichroism and NMR spectroscopy as well as by molecular dynamic simulations, which confirmed the stabilizing effect and resulted in the identification of a short linear motif for helix stabilisation: [S/T]-P-{3}-[R/K] where [S/T] is the phosphorylation-site. By analysing IDP and phosphorylation site databases we found that the motif is significantly enriched around known phosphorylation sites, supporting a potential wider-spread role in phosphorylation-mediated regulation of intrinsically disordered proteins. The identification of such motifs is important for understanding the molecular mechanism of cellular signalling, and is crucial for the development of predictors for the structural effect of phosphorylation; a tool of relevance for understanding disease-promoting mutations that for example interfere with signalling for instance through constitutive active and often cancer-promoting signalling., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

SCAN domains in zinc-finger transcription factors are crucial mediators of protein-protein interactions. Up to 240 SCAN-domain encoding genes have been identified throughout the human genome. These include cancer-related genes, such as the myeloid zinc finger 1 ( MZF1 ), an oncogenic transcription factor involved in the progression of many solid cancers. The mechanisms by which SCAN homo- and heterodimers assemble and how they alter the transcriptional activity of zinc-finger transcription factors in cancer and other diseases remain to be investigated. Here, we provide the first description of the conformational ensemble of the MZF1 SCAN domain cross-validated against NMR experimental data, which are probes of structure and dynamics on different timescales. We investigated the protein-protein interaction network of MZF1 and how it is perturbed in different cancer types by the analyses of high-throughput proteomics and RNASeq data. Collectively, we integrated many computational approaches, ranging from simple empirical energy functions to all-atom microsecond molecular dynamics simulations and network analyses to unravel the effects of cancer-related substitutions in relation to MZF1 structure and interactions.

Background Thrombolysis with intravenous recombinant tissue-type plasminogen activator improves functional outcome in acute ischemic stroke. Few studies have investigated the effects of thrombolysis in a real-world setting. We evaluated the impact of thrombolysis on long-term hospital bed day use and the risk of readmission due to stroke-related complications. Methods We conducted a register-based nationwide propensity score-matched follow-up study among ischemic stroke patients in Denmark (2004-2011). Thrombolysed patients were propensity-score matched with non-thrombolysed acute ischemic stroke patients admitted to stroke centers not yet offering thrombolysis in 2004-2006. The outcomes were length of the stroke admission, total all-cause hospital bed day use during the first year after the stroke, and the long-term risk of readmissions. Thrombolysed and non-thrombolysed patients were compared using multivariable log-linear regression and Cox regression. Results We identified 1095 thrombolysed and 1095 propensity score matched eligible but non-thrombolysed acute ischemic stroke patients. The median length of the stroke admission was 9 days in the thrombolysed group and 13 days in the non-thrombolysed group (adjusted geometric mean ratio, 0.88; 95% CI: 0.78-1.00). The median all-cause hospital bed day use within the first year was 12 days in the thrombolysed group and 19 days in the non-thrombolysed group (adjusted geometric mean ratio, 0.82; 95% CI: 0.73-0.92). There was no significant difference in the overall risk of readmission (adjusted hazard ratio, 0.91; 95% CI: 0.79-1.04); however, thrombolysis was associated with reduced risk of pneumonia (adjusted hazard ratio, 0.59; 95% CI: 0.35-0.97). Conclusions Thrombolysis in ischemic stroke was associated with lower long-term hospital bed day use and decreased risk of readmission due to pneumonia.

Plant oil is an essential dietary and bio-energy resource. Despite this, the effects of climate change on plant oil quality remain to be elucidated. The present study is the first to show changes in oil quality and quantity of four rapeseed cultivars in climate scenarios with elevated [CO2], [O3] and temperature (T) combined and as single factors. The combination of environmental factors resembled IPCC's 'business as usual' emission scenario predicted for late this century. Generally, the climate scenarios reduced the average amounts of the six fatty acids (FAs) analysed, though in some treatments single FAs remained unchanged or even increased. Most reduced was the FA essential for human nutrition, C18:3-ω3, which decreased by 39% and 45% in the combined scenarios with elevated [CO2]+T+[O3] and [CO2]+T, respectively. Average oil content decreased 3-17%. When [CO2] and T were elevated concurrently, the seed biomass was reduced by half, doubling the losses in FAs and oil content. This corresponded to a 58% reduction in the oil yield per hectare, and C18:3-ω3 decreased by 77%. Furthermore, the polyunsaturated FAs were significantly decreased. The results indicate undesirable consequences for production and health benefits of rapeseed oil with future climate change. The results also showed strong interactive effects of CO2, T and O3 on oil quality, demonstrating why prediction of climate effects requires experiments with combined factors and should not be based on extrapolation from single factor experiments., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

We recently described that the autoimmune, central nervous system disease, multiple sclerosis (MS), is genetically associated with the human endogenous retroviral locus, HERV-Fc1, in Scandinavians. A number of dominant human genes encoding factors that restrict retrovirus replication have been known for a long time. Today human restriction genes for retroviruses include amongst others TRIMs, APOBEC3s, BST2 and TREXs. We have therefore looked for a role of these retroviral restriction genes in MS using genetic epidemiology. We here report that markers in two TRIMs, TRIM5 and TRIM22 and a marker in BST2, associated statistically with the risk of getting MS, while markers in or near APOBEC3s and TREXs showed little or no effect. This indicates that the two TRIMs and BST2 influence the risk of disease and thus supports the hypothesis of a viral involvement.

The possibility that retroviruses play a role in multiple sclerosis (MS) has long been considered; accumulating findings suggest this to be most likely in the form of human endogenous retroviruses (HERVs). A genetic test series of fifty endogenous retroviral loci for association with MS in Danes showed SNP markers near a specific endogenous retroviral locus, HERV-Fc1 located on the X-chromosome, to be positive. Bout Onset MS was associated with the HERV-Fc1 locus, while a rarer form, Primary Progressive MS, was not. Moreover, HERV-Fc1 Gag RNA in plasma was increased 4-fold in patients with recent history of attacks, relative to patients in a stable state and to healthy controls.Finally, genetic variations in restriction genes for retroviruses influence the risk of MS, providing further support for a role of retroviral elements in disease.We speculate that endogenous retroviruses may activate the innate immune system in a variety of ways, involving the host proteins, TRIMs, TLRs, TREXs and STING. Observations in HIV-positive patients suggest that antiretroviral drugs can curb MS. Thus, these new findings regarding the etiology and pathogenesis of MS, suggest alternative ways to challenge autoimmune diseases.

Unlabelled: A dipstick test for microalbuminuria was compared with urinary albumin excretion and urinary albumin concentration. Elevated urinary albumin excretion was defined as > 30 mg/24 h, elevated urinary albumin concentration in early morning urine as = > 20 mg/l and sticks = > 20 mg/l were considered positive. 1,071 samples with urinary albumin concentration 20-200 mg/l from 258 diabetic subjects were evaluated in three settings: I. 3 trained nurses testing samples from day-clinic diabetic patients. Sticks v.s. urinary albumin excretion sensitivity 86%, specificity 97%, predictive value of negative test 97%, correlation coefficient 0.79. Values for same setting but v.s. urinary albumin concentration were almost identical. II. 1 laboratory technician testing not-hospitalised diabetic patients: Sticks v.s. urinary albumin concentration: sensitivity 91%, specificity 85%, predictive value of negative test 95%, correlation coefficient 0.82. III. 58 general practitioners testing not-hospitalized diabetic patients: Sticks v.s. urinary albumin concentration: sensitivity 66%, specificity 92%, predictive value of negative test 83%, correlation coefficient 0.70., Conclusions: In the hands of trained nurses and laboratory technician the Micral-Test showed good correlation with urinary albumin excretion and urinary albumin concentration for day-clinic and not-hospitalized patients and can be recommended as a screening tool. General practitioners obtained a lower sensitivity probably due to lack of experience and incorrect handling of the sticks leading to systematical errors. Training in the use of the stick must be emphasized, since under such circumstances the results are satisfactory.