Defining predictors of responsiveness to advanced therapies in Crohn's disease and ulcerative colitis: protocol for the IBD-RESPONSE and nested CD-metaRESPONSE prospective, multicentre, observational cohort study in precision medicine.

INTRODUCTION: Characterised by chronic inflammation of the gastrointestinal tract, inflammatory bowel disease (IBD) symptoms including diarrhoea, abdominal pain and fatigue can significantly impact patient's quality of life. Therapeutic developments in the last 20 years have revolutionised treatment. However, clinical trials and real-world data show primary non-response rates up to 40%. A significant challenge is an inability to predict which treatment will benefit individual patients.Current understanding of IBD pathogenesis implicates complex interactions between host genetics and the gut microbiome. Most cohorts studying the gut microbiota to date have been underpowered, examined single treatments and produced heterogeneous results. Lack of cross-treatment comparisons and well-powered independent replication cohorts hampers the ability to infer real-world utility of predictive signatures.IBD-RESPONSE will use multi-omic data to create a predictive tool for treatment response. Future patient benefit may include development of biomarker-based treatment stratification or manipulation of intestinal microbial targets. IBD-RESPONSE and downstream studies have the potential to improve quality of life, reduce patient risk and reduce expenditure on ineffective treatments. METHODS AND ANALYSIS: This prospective, multicentre, observational study will identify and validate a predictive model for response to advanced IBD therapies, incorporating gut microbiome, metabolome, single-cell transcriptome, human genome, dietary and clinical data. 1325 participants commencing advanced therapies will be recruited from ~40 UK sites. Data will be collected at baseline, week 14 and week 54. The primary outcome is week 14 clinical response. Secondary outcomes include clinical remission, loss of response in week 14 responders, corticosteroid-free response/remission, time to treatment escalation and change in patient-reported outcome measures. ETHICS AND DISSEMINATION: Ethical approval was obtained from the Wales Research Ethics Committee 5 (ref: 21/WA/0228). Recruitment is ongoing. Following study completion, results will be submitted for publication in peer-reviewed journals and presented at scientific meetings. Publications will be summarised at www.ibd-response.co.uk. TRIAL REGISTRATION NUMBER: ISRCTN96296121.

Gut mucosa dissociation protocols influence cell type proportions and single-cell gene expression levels.

Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of the cellular landscape of organs. Most single-cell protocols require fresh material, which limits sample size per experiment, and consequently, introduces batch effects. This is especially true for samples acquired through complex medical procedures, such as intestinal mucosal biopsies. Moreover, the tissue dissociation procedure required for obtaining single cells is a major source of noise; different dissociation procedures applied to different compartments of the tissue induce artificial gene expression differences between cell subsets. To overcome these challenges, we have developed a one-step dissociation protocol and demonstrated its use on cryopreserved gut mucosal biopsies. Using flow cytometry and scRNA-seq analysis, we compared this one-step dissociation protocol with the current gold standard, two-step collagenase digestion, and an adaptation of a recently published alternative, three-step cold-active Bacillus licheniformus protease digestion. Both cell viability and cell type composition were comparable between the one-step and two-step collagenase dissociation, with the former being more time-efficient. The cold protease digestion resulted in equal cell viability, but better preserves the epithelial cell types. Consequently, to analyze the rarer cell types, such as glial cells, larger total biopsy cell numbers are required as input material. The multi-step protocols affected cell types spanning multiple compartments differently. In summary, we show that cryopreserved gut mucosal biopsies can be used to overcome the logistical challenges and batch effects in large scRNA-seq studies. Furthermore, we demonstrate that using cryopreserved biopsies digested using a one-step collagenase protocol enables large-scale scRNA-seq, FACS, organoid generation and intraepithelial lymphocyte expansion.

Somatic Evolution in Non-neoplastic IBD-Affected Colon.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease associated with increased risk of gastrointestinal cancers. We whole-genome sequenced 446 colonic crypts from 46 IBD patients and compared these to 412 crypts from 41 non-IBD controls from our previous publication on the mutation landscape of the normal colon. The average mutation rate of affected colonic epithelial cells is 2.4-fold that of healthy colon, and this increase is mostly driven by acceleration of mutational processes ubiquitously observed in normal colon. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimeter-scale clonal expansions. We discovered non-synonymous mutations in ARID1A, FBXW7, PIGR, ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, under positive selection in IBD. These results suggest distinct selection mechanisms in the colitis-affected colon and that somatic mutations potentially play a causal role in IBD pathogenesis.

MEK1/2 inhibitor withdrawal reverses acquired resistance driven by BRAFV600E amplification whereas KRASG13D amplification promotes EMT-chemoresistance.

Acquired resistance to MEK1/2 inhibitors (MEKi) arises through amplification of BRAFV600E or KRASG13D to reinstate ERK1/2 signalling. Here we show that BRAFV600E amplification and MEKi resistance are reversible following drug withdrawal. Cells with BRAFV600E amplification are addicted to MEKi to maintain a precise level of ERK1/2 signalling that is optimal for cell proliferation and survival, and tumour growth in vivo. Robust ERK1/2 activation following MEKi withdrawal drives a p57KIP2-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death, selecting against those cells with amplified BRAFV600E. p57KIP2 expression is required for loss of BRAFV600E amplification and reversal of MEKi resistance. Thus, BRAFV600E amplification confers a selective disadvantage during drug withdrawal, validating intermittent dosing to forestall resistance. In contrast, resistance driven by KRASG13D amplification is not reversible; rather ERK1/2 hyperactivation drives ZEB1-dependent epithelial-to-mesenchymal transition and chemoresistance, arguing strongly against the use of drug holidays in cases of KRASG13D amplification.

Genome-wide association of multiple complex traits in outbred mice by ultra-low-coverage sequencing.

Two bottlenecks impeding the genetic analysis of complex traits in rodents are access to mapping populations able to deliver gene-level mapping resolution and the need for population-specific genotyping arrays and haplotype reference panels. Here we combine low-coverage (0.15×) sequencing with a new method to impute the ancestral haplotype space in 1,887 commercially available outbred mice. We mapped 156 unique quantitative trait loci for 92 phenotypes at a 5% false discovery rate. Gene-level mapping resolution was achieved at about one-fifth of the loci, implicating Unc13c and Pgc1a at loci for the quality of sleep, Adarb2 for home cage activity, Rtkn2 for intensity of reaction to startle, Bmp2 for wound healing, Il15 and Id2 for several T cell measures and Prkca for bone mineral content. These findings have implications for diverse areas of mammalian biology and demonstrate how genome-wide association studies can be extended via low-coverage sequencing to species with highly recombinant outbred populations.

A Genome-Wide Association Study for Regulators of Micronucleus Formation in Mice.

In mammals the regulation of genomic instability plays a key role in tumor suppression and also controls genome plasticity, which is important for recombination during the processes of immunity and meiosis. Most studies to identify regulators of genomic instability have been performed in cells in culture or in systems that report on gross rearrangements of the genome, yet subtle differences in the level of genomic instability can contribute to whole organism phenotypes such as tumor predisposition. Here we performed a genome-wide association study in a population of 1379 outbred Crl:CFW(SW)-US_P08 mice to dissect the genetic landscape of micronucleus formation, a biomarker of chromosomal breaks, whole chromosome loss, and extranuclear DNA. Variation in micronucleus levels is a complex trait with a genome-wide heritability of 53.1%. We identify seven loci influencing micronucleus formation (false discovery rate <5%), and define candidate genes at each locus. Intriguingly at several loci we find evidence for sexual dimorphism in micronucleus formation, with a locus on chromosome 11 being specific to males.

Mouse models of colorectal cancer as preclinical models.

In this review, we discuss the application of mouse models to the identification and pre-clinical validation of novel therapeutic targets in colorectal cancer, and to the search for early disease biomarkers. Large-scale genomic, transcriptomic and epigenomic profiling of colorectal carcinomas has led to the identification of many candidate genes whose direct contribution to tumourigenesis is yet to be defined; we discuss the utility of cross-species comparative 'omics-based approaches to this problem. We highlight recent progress in modelling late-stage disease using mice, and discuss ways in which mouse models could better recapitulate the complexity of human cancers to tackle the problem of therapeutic resistance and recurrence after surgical resection.

A high-throughput in vivo micronucleus assay for genome instability screening in mice.

We describe a sensitive, robust, high-throughput method for quantifying the formation of micronuclei, markers of genome instability, in mouse erythrocytes. Micronuclei are whole chromosomes or chromosome segments that have been separated from the nucleus. Other methods of detection rely on labor-intensive, microscopy-based techniques. Here we describe a 2-d, 96-well plate-based flow cytometric method of micronucleus scoring that is simple enough for a research technician experienced in flow cytometry to perform. The assay detects low levels of genome instability that cannot be readily identified by classic phenotyping, using 25 µl of blood. By using this assay, we have screened >10,000 blood samples and discovered novel genes that contribute to vertebrate genome maintenance, as well as novel disease models and mechanisms of genome instability disorders. We discuss experimental design considerations, including statistical power calculation, we provide troubleshooting tips and we discuss factors that contribute to a false-positive increase in the number of micronucleated red blood cells and to experimental variability.

Genetic screens in mice for genome integrity maintenance and cancer predisposition.

Genome instability is a feature of nearly all cancers and can be exploited for therapy. In addition, a growing number of genome maintenance genes have been associated with developmental disorders. Efforts to understand the role of genome instability in these processes will be greatly facilitated by a more comprehensive understanding of their genetic network. We highlight recent genetic screens in model organisms that have assisted in the discovery of novel regulators of genome stability and focus on the contribution of mice as a model organism to understanding the role of genome instability during embryonic development, tumour formation and cancer therapy.

Mcph1-deficient mice reveal a role for MCPH1 in otitis media.

Otitis media is a common reason for hearing loss, especially in children. Otitis media is a multifactorial disease and environmental factors, anatomic dysmorphology and genetic predisposition can all contribute to its pathogenesis. However, the reasons for the variable susceptibility to otitis media are elusive. MCPH1 mutations cause primary microcephaly in humans. So far, no hearing impairment has been reported either in the MCPH1 patients or mouse models with Mcph1 deficiency. In this study, Mcph1-deficient (Mcph1(tm1a) (/tm1a) ) mice were produced using embryonic stem cells with a targeted mutation by the Sanger Institute's Mouse Genetics Project. Auditory brainstem response measurements revealed that Mcph1(tm1a) (/tm1a) mice had mild to moderate hearing impairment with around 70% penetrance. We found otitis media with effusion in the hearing-impaired Mcph1(tm1a) (/tm1a) mice by anatomic and histological examinations. Expression of Mcph1 in the epithelial cells of middle ear cavities supported its involvement in the development of otitis media. Other defects of Mcph1(tm1a) (/tm1a) mice included small skull sizes, increased micronuclei in red blood cells, increased B cells and ocular abnormalities. These findings not only recapitulated the defects found in other Mcph1-deficient mice or MCPH1 patients, but also revealed an unexpected phenotype, otitis media with hearing impairment, which suggests Mcph1 is a new gene underlying genetic predisposition to otitis media.

Disruption of mouse Cenpj, a regulator of centriole biogenesis, phenocopies Seckel syndrome.

Disruption of the centromere protein J gene, CENPJ (CPAP, MCPH6, SCKL4), which is a highly conserved and ubiquitiously expressed centrosomal protein, has been associated with primary microcephaly and the microcephalic primordial dwarfism disorder Seckel syndrome. The mechanism by which disruption of CENPJ causes the proportionate, primordial growth failure that is characteristic of Seckel syndrome is unknown. By generating a hypomorphic allele of Cenpj, we have developed a mouse (Cenpj(tm/tm)) that recapitulates many of the clinical features of Seckel syndrome, including intrauterine dwarfism, microcephaly with memory impairment, ossification defects, and ocular and skeletal abnormalities, thus providing clear confirmation that specific mutations of CENPJ can cause Seckel syndrome. Immunohistochemistry revealed increased levels of DNA damage and apoptosis throughout Cenpj(tm/tm) embryos and adult mice showed an elevated frequency of micronucleus induction, suggesting that Cenpj-deficiency results in genomic instability. Notably, however, genomic instability was not the result of defective ATR-dependent DNA damage signaling, as is the case for the majority of genes associated with Seckel syndrome. Instead, Cenpj(tm/tm) embryonic fibroblasts exhibited irregular centriole and centrosome numbers and mono- and multipolar spindles, and many were near-tetraploid with numerical and structural chromosomal abnormalities when compared to passage-matched wild-type cells. Increased cell death due to mitotic failure during embryonic development is likely to contribute to the proportionate dwarfism that is associated with CENPJ-Seckel syndrome.

Increased tumorigenesis associated with loss of the tumor suppressor gene Cadm1.

BACKGROUND: CADM1 encodes an immunoglobulin superfamily (IGSF) cell adhesion molecule. Inactivation of CADM1, either by promoter hypermethylation or loss of heterozygosity, has been reported in a wide variety of tumor types, thus it has been postulated as a tumor suppressor gene. FINDINGS: We show for the first time that Cadm1 homozygous null mice die significantly faster than wildtype controls due to the spontaneous development of tumors at an earlier age and an increased tumor incidence of predominantly lymphomas, but also some solid tumors. Tumorigenesis was accelerated after irradiation of Cadm1 mice, with the reduced latency in tumor formation suggesting there are genes that collaborate with loss of Cadm1 in tumorigenesis. To identify these co-operating genetic events, we performed a Sleeping Beauty transposon-mediated insertional mutagenesis screen in Cadm1 mice, and identified several common insertion sites (CIS) found specifically on a Cadm1-null background (and not wildtype background). CONCLUSION: We confirm that Cadm1 is indeed a bona fide tumor suppressor gene and provide new insights into genetic partners that co-operate in tumorigenesis when Cadm1-expression is lost.

Nuclear receptor binding protein 1 regulates intestinal progenitor cell homeostasis and tumour formation.

Genetic screens in simple model organisms have identified many of the key components of the conserved signal transduction pathways that are oncogenic when misregulated. Here, we identify H37N21.1 as a gene that regulates vulval induction in let-60(n1046gf), a strain with a gain-of-function mutation in the Caenorhabditis elegans Ras orthologue, and show that somatic deletion of Nrbp1, the mouse orthologue of this gene, results in an intestinal progenitor cell phenotype that leads to profound changes in the proliferation and differentiation of all intestinal cell lineages. We show that Nrbp1 interacts with key components of the ubiquitination machinery and that loss of Nrbp1 in the intestine results in the accumulation of Sall4, a key mediator of stem cell fate, and of Tsc22d2. We also reveal that somatic loss of Nrbp1 results in tumourigenesis, with haematological and intestinal tumours predominating, and that nuclear receptor binding protein 1 (NRBP1) is downregulated in a range of human tumours, where low expression correlates with a poor prognosis. Thus NRBP1 is a conserved regulator of cell fate, that plays an important role in tumour suppression.

High levels of RNA-editing site conservation amongst 15 laboratory mouse strains.

BACKGROUND: Adenosine-to-inosine (A-to-I) editing is a site-selective post-transcriptional alteration of double-stranded RNA by ADAR deaminases that is crucial for homeostasis and development. Recently the Mouse Genomes Project generated genome sequences for 17 laboratory mouse strains and rich catalogues of variants. We also generated RNA-seq data from whole brain RNA from 15 of the sequenced strains. RESULTS: Here we present a computational approach that takes an initial set of transcriptome/genome mismatch sites and filters these calls taking into account systematic biases in alignment, single nucleotide variant calling, and sequencing depth to identify RNA editing sites with high accuracy. We applied this approach to our panel of mouse strain transcriptomes identifying 7,389 editing sites with an estimated false-discovery rate of between 2.9 and 10.5%. The overwhelming majority of these edits were of the A-to-I type, with less than 2.4% not of this class, and only three of these edits could not be explained as alignment artifacts. We validated 24 novel RNA editing sites in coding sequence, including two non-synonymous edits in the Cacna1d gene that fell into the IQ domain portion of the Cav1.2 voltage-gated calcium channel, indicating a potential role for editing in the generation of transcript diversity. CONCLUSIONS: We show that despite over two million years of evolutionary divergence, the sites edited and the level of editing at each site is remarkably consistent across the 15 strains. In the Cds2 gene we find evidence for RNA editing acting to preserve the ancestral transcript sequence despite genomic sequence divergence.

Cancer gene discovery in the mouse.

Developments in high-throughput genome analysis and in computational tools have made it possible to rapidly profile entire cancer genomes with basepair resolution. In parallel with these advances, mouse models of cancer have evolved into powerful tools for cancer gene discovery. Here we discuss some of the approaches that may be used for cancer gene identification in the mouse and discuss how a cross-species 'oncogenomics' approach to cancer gene discovery represents a powerful strategy for finding genes that drive tumorigenesis.

Effect of dexamethasone on postoperative morbidity after dental rehabilitation in children.

PURPOSE: Dexamethasone reduces postoperative morbidity after adenotonsillectomy, strabismus surgery, and third molar extraction. Our hypothesis was that dexamethasone would reduce pain and other morbidity in children undergoing dental surgery for up to 24 hr postoperatively. METHODS: A triple-blinded, randomized, controlled trial was carried out on 200 children undergoing prolonged dental rehabilitation under general anesthesia. Subjects were randomized into two groups: group D, given dexamethasone 0.3 mg·kg(-1); group S, given normal saline. The primary outcome measure was pain over 24 hr as evaluated by a parental 0-10 numerical rating scale (NRS). Key secondary outcomes included oral intake on a four-point scale at 24 hr and the overall incidence of postoperative vomiting (POV). Analysis for the primary outcome consisted of comparison of means in the NRS with the Wilcoxon rank sum test and for occurrence of POV with Fisher's test. RESULTS: After eliminating 22 subjects for protocol violations and withdrawals, 178 subjects were analyzed. There was no significant difference in pain scores (NRS) at 24 hr or the worst NRS experienced over the preceding 24 hr. There was no difference in the quality of oral intake between the groups. There was a significant difference in the percentage of patients who vomited during the first 24 hr: eight of 91 in group S and one of 87 in group D. Therefore, 7.74% more vomited in group S (P = 0.045), with a 95% confidence interval of 0.32 to15.16 for the difference in percentages. CONCLUSIONS: Dexamethasone, 0.3 mg·kg(-1), did not reduce pain over 24 hr in healthy children undergoing dental rehabilitation under general anesthesia. The quality of oral intake was also unaffected by dexamethasone at 24 hr. Dexamethasone did produce a significant reduction in postdischarge vomiting, beyond the incidence found with ondansetron alone.

The critical role of histone H2A-deubiquitinase Mysm1 in hematopoiesis and lymphocyte differentiation.

Stem cell differentiation and lineage specification depend on coordinated programs of gene expression, but our knowledge of the chromatin-modifying factors regulating these events remains incomplete. Ubiquitination of histone H2A (H2A-K119u) is a common chromatin modification associated with gene silencing, and controlled by the ubiquitin-ligase polycomb repressor complex 1 (PRC1) and H2A-deubiquitinating enzymes (H2A-DUBs). The roles of H2A-DUBs in mammalian development, stem cells, and hematopoiesis have not been addressed. Here we characterized an H2A-DUB targeted mouse line Mysm1(tm1a/tm1a) and demonstrated defects in BM hematopoiesis, resulting in lymphopenia, anemia, and thrombocytosis. Development of lymphocytes was impaired from the earliest stages of their differentiation, and there was also a depletion of erythroid cells and a defect in erythroid progenitor function. These phenotypes resulted from a cell-intrinsic requirement for Mysm1 in the BM. Importantly, Mysm1(tm1a/tm1a) HSCs were functionally impaired, and this was associated with elevated levels of reactive oxygen species, γH2AX DNA damage marker, and p53 protein in the hematopoietic progenitors. Overall, these data establish a role for Mysm1 in the maintenance of BM stem cell function, in the control of oxidative stress and genetic stability in hematopoietic progenitors, and in the development of lymphoid and erythroid lineages.

Mouse genomic variation and its effect on phenotypes and gene regulation.

We report genome sequences of 17 inbred strains of laboratory mice and identify almost ten times more variants than previously known. We use these genomes to explore the phylogenetic history of the laboratory mouse and to examine the functional consequences of allele-specific variation on transcript abundance, revealing that at least 12% of transcripts show a significant tissue-specific expression bias. By identifying candidate functional variants at 718 quantitative trait loci we show that the molecular nature of functional variants and their position relative to genes vary according to the effect size of the locus. These sequences provide a starting point for a new era in the functional analysis of a key model organism.

Disruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia.

The evolutionarily conserved SLX4 protein, a key regulator of nucleases, is critical for DNA damage response. SLX4 nuclease complexes mediate repair during replication and can also resolve Holliday junctions formed during homologous recombination. Here we describe the phenotype of the Btbd12 knockout mouse, the mouse ortholog of SLX4, which recapitulates many key features of the human genetic illness Fanconi anemia. Btbd12-deficient animals are born at sub-Mendelian ratios, have greatly reduced fertility, are developmentally compromised and are prone to blood cytopenias. Btbd12(-/-) cells prematurely senesce, spontaneously accumulate damaged chromosomes and are particularly sensitive to DNA crosslinking agents. Genetic complementation reveals a crucial requirement for Btbd12 (also known as Slx4) to interact with the structure-specific endonuclease Xpf-Ercc1 to promote crosslink repair. The Btbd12 knockout mouse therefore establishes a disease model for Fanconi anemia and genetically links a regulator of nuclease incision complexes to the Fanconi anemia DNA crosslink repair pathway.