Elevated Epidermal Ornithine Decarboxylase Activity Suppresses Contact Hypersensitivity

Previous reports have shown that elevated polyamine biosynthesis is sufficient to promote skin tumorigenesis in susceptible mouse strains. We hypothesized that increased activity of epidermal ornithine decarboxylase (ODC), a key regulatory enzyme in polyamine biosynthesis, may suppress the cutaneous immune response in addition to stimulating proliferation. Using an ODCER transgenic mouse model in which ODC is targeted to the epidermis, we examined the effect of ODC overexpression in keratinocytes on a classic contact hypersensitivity (CHS) response. Compared with normal littermate mice, ODCER transgenic mice showed reduced ear swelling, reduced neutrophil infiltration, and decreased migration of fluorescein isothiocyanate-loaded dendritic cells (DCs) to draining lymph nodes following hapten elicitation of CHS. In addition, elevated epidermal ODC activity suppressed the levels of cytokines keratinocyte-derived chemokine, monocyte chemoattractant protein-1, interleukin-6 (IL-6), and IL-10. Adoptive transfer of lymphocytes from sensitized ODCER transgenic or normal littermate mice to naive ODCER transgenic or wild-type mice indicated that elevated epidermal ODC activity suppresses both the sensitization and elicitation phases of CHS. The specific ODC inhibitor, α-difluoromethylornithine, abrogated all suppressive effects of ODC in CHS reactions. Collectively, these data suggest that the immunosuppression promoted by increased epidermal ODC is mediated by a reduction in cytokine levels, which suppresses DC migration and reduces immune cell infiltration to the site of hapten application

Translational development of difluoromethylornithine (DFMO) for the treatment of neuroblastoma

Neuroblastoma is a childhood tumor in which MYC oncogenes are commonly activated to drive tumor progression. Survival for children with high-risk neuroblastoma remains poor despite treatment that incorporates high-dose chemotherapy, stem cell support, surgery, radiation therapy and immunotherapy. More effective and less toxic treatments are sought and one approach under clinical development involves re-purposing the anti-protozoan drug difluoromethylornithine (DFMO; Eflornithine) as a neuroblastoma therapeutic. DFMO is an irreversible inhibitor of ornithine decarboxylase (Odc), a MYC target gene, bona fide oncogene, and the rate-limiting enzyme in polyamine synthesis. DFMO is approved for the treatment of Trypanosoma brucei gambiense encephalitis (“African sleeping sickness”) since polyamines are essential for the proliferation of these protozoa. However, polyamines are also critical for mammalian cell proliferation and the finding that MYC coordinately regulates all aspects of polyamine metabolism suggests polyamines may be required to support cancer promotion by MYC. Pre-emptive blockade of polyamine synthesis is sufficient to block tumor initiation in an otherwise fully penetrant transgenic mouse model of neuroblastoma driven by MYCN, underscoring the necessity of polyamines in this process. Moreover, polyamine depletion regimens exert potent anti-tumor activity in pre-clinical models of established neuroblastoma as well, in combination with numerous chemotherapeutic agents and even in tumors with unfavorable genetic features such as MYCN, ALK or TP53 mutation. This has led to the testing of DFMO in clinical trials for children with neuroblastoma. Current trial designs include testing lower dose DFMO alone (2,000 mg/m2/day) starting at the completion of standard therapy, or higher doses combined with chemotherapy (up to 9,000 mg/m2/day) for patients with relapsed disease that has progressed. In this review we will discuss important considerations for the future design of DFMO-based clinical trials for neuroblastoma, focusing on the need to better define the principal mechanisms of anti-tumor activity for polyamine depletion regimens. Putative DFMO activities that are both cancer cell intrinsic (targeting the principal oncogenic driver, MYC) and cancer cell extrinsic (altering the tumor microenvironment to support anti-tumor immunity) will be discussed. Understanding the mechanisms of DFMO activity are critical in determining how it might be best leveraged in upcoming clinical trials. This mechanistic approach also provides a platform by which iterative pre-clinical testing using translational tumor models may complement our clinical approaches

High resolution mapping and positional cloning of ENU-induced mutations in the Rw region of mouse chromosome 5

<p>Abstract</p> <p>Background</p> <p>Forward genetic screens in mice provide an unbiased means to identify genes and other functional genetic elements in the genome. Previously, a large scale ENU mutagenesis screen was conducted to query the functional content of a ~50 Mb region of the mouse genome on proximal Chr 5. The majority of phenotypic mutants recovered were embryonic lethals.</p> <p>Results</p> <p>We report the high resolution genetic mapping, complementation analyses, and positional cloning of mutations in the target region. The collection of identified alleles include several with known or presumed functions for which no mutant models have been reported (<it>Tbc1d14</it>, <it>Nol14</it>, <it>Tyms</it>, <it>Cad</it>, <it>Fbxl5</it>, <it>Haus3</it>), and mutations in genes we or others previously reported (<it>Tapt1</it>, <it>Rest</it>, <it>Ugdh</it>, <it>Paxip1</it>, <it>Hmx1, Otoe, Nsun7</it>). We also confirmed the causative nature of a homeotic mutation with a targeted allele, mapped a lethal mutation to a large gene desert, and localized a spermiogenesis mutation to a region in which no annotated genes have coding mutations. The mutation in <it>Tbc1d14 </it>provides the first implication of a critical developmental role for RAB-GAP-mediated protein transport in early embryogenesis.</p> <p>Conclusion</p> <p>This collection of alleles contributes to the goal of assigning biological functions to all known genes, as well as identifying novel functional elements that would be missed by reverse genetic approaches.</p

The Human Phenotype Ontology in 2024: phenotypes around the world.

The Human Phenotype Ontology (HPO) is a widely used resource that comprehensively organizes and defines the phenotypic features of human disease, enabling computational inference and supporting genomic and phenotypic analyses through semantic similarity and machine learning algorithms. The HPO has widespread applications in clinical diagnostics and translational research, including genomic diagnostics, gene-disease discovery, and cohort analytics. In recent years, groups around the world have developed translations of the HPO from English to other languages, and the HPO browser has been internationalized, allowing users to view HPO term labels and in many cases synonyms and definitions in ten languages in addition to English. Since our last report, a total of 2239 new HPO terms and 49235 new HPO annotations were developed, many in collaboration with external groups in the fields of psychiatry, arthrogryposis, immunology and cardiology. The Medical Action Ontology (MAxO) is a new effort to model treatments and other measures taken for clinical management. Finally, the HPO consortium is contributing to efforts to integrate the HPO and the GA4GH Phenopacket Schema into electronic health records (EHRs) with the goal of more standardized and computable integration of rare disease data in EHRs

Phenotypic Characterization of EIF2AK4 Mutation Carriers in a Large Cohort of Patients Diagnosed Clinically With Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 (BMPR2) are the commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene (EIF2AK4) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH. METHODS: Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource-Rare Diseases study. Heterozygous variants in BMPR2 and biallelic EIF2AK4 variants with a minor allele frequency of <1:10 000 in control data sets and predicted to be deleterious (by combined annotation-dependent depletion, PolyPhen-2, and sorting intolerant from tolerant predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured. RESULTS: Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in BMPR2 were identified in 130 patients (14.8%). Biallelic mutations in EIF2AK4 were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic EIF2AK4 mutations. These patients had a reduced transfer coefficient for carbon monoxide (Kco; 33% [interquartile range, 30%-35%] predicted) and younger age at diagnosis (29 years; interquartile range, 23-38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without EIF2AK4 mutations. However, radiological assessment alone could not accurately identify biallelic EIF2AK4 mutation carriers. Patients with PAH with biallelic EIF2AK4 mutations had a shorter survival. CONCLUSIONS: Biallelic EIF2AK4 mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low Kco and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation

Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in $\textit{CHM}$ in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.This work was supported by The National Institute for Health Research England (NIHR) for the NIHR BioResource – Rare Diseases project (grant number RG65966). The Moorfields Eye Hospital cohort of patients and clinical and imaging data were ascertained and collected with the support of grants from the National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital, National Health Service Foundation Trust, and UCL Institute of Ophthalmology, Moorfields Eye Hospital Special Trustees, Moorfields Eye Charity, the Foundation Fighting Blindness (USA), and Retinitis Pigmentosa Fighting Blindness. M.M. is a recipient of an FFB Career Development Award. E.M. is supported by UCLH/UCL NIHR Biomedical Research Centre. F.L.R. and D.G. are supported by Cambridge NIHR Biomedical Research Centre

Telomerecat: A ploidy-agnostic method for estimating telomere length from whole genome sequencing data.

Telomere length is a risk factor in disease and the dynamics of telomere length are crucial to our understanding of cell replication and vitality. The proliferation of whole genome sequencing represents an unprecedented opportunity to glean new insights into telomere biology on a previously unimaginable scale. To this end, a number of approaches for estimating telomere length from whole-genome sequencing data have been proposed. Here we present Telomerecat, a novel approach to the estimation of telomere length. Previous methods have been dependent on the number of telomeres present in a cell being known, which may be problematic when analysing aneuploid cancer data and non-human samples. Telomerecat is designed to be agnostic to the number of telomeres present, making it suited for the purpose of estimating telomere length in cancer studies. Telomerecat also accounts for interstitial telomeric reads and presents a novel approach to dealing with sequencing errors. We show that Telomerecat performs well at telomere length estimation when compared to leading experimental and computational methods. Furthermore, we show that it detects expected patterns in longitudinal data, repeated measurements, and cross-species comparisons. We also apply the method to a cancer cell data, uncovering an interesting relationship with the underlying telomerase genotype

Comprehensive Cancer-Predisposition Gene Testing in an Adult Multiple Primary Tumor Series Shows a Broad Range of Deleterious Variants and Atypical Tumor Phenotypes.

Multiple primary tumors (MPTs) affect a substantial proportion of cancer survivors and can result from various causes, including inherited predisposition. Currently, germline genetic testing of MPT-affected individuals for variants in cancer-predisposition genes (CPGs) is mostly targeted by tumor type. We ascertained pre-assessed MPT individuals (with at least two primary tumors by age 60 years or at least three by 70 years) from genetics centers and performed whole-genome sequencing (WGS) on 460 individuals from 440 families. Despite previous negative genetic assessment and molecular investigations, pathogenic variants in moderate- and high-risk CPGs were detected in 67/440 (15.2%) probands. WGS detected variants that would not be (or were not) detected by targeted resequencing strategies, including low-frequency structural variants (6/440 [1.4%] probands). In most individuals with a germline variant assessed as pathogenic or likely pathogenic (P/LP), at least one of their tumor types was characteristic of variants in the relevant CPG. However, in 29 probands (42.2% of those with a P/LP variant), the tumor phenotype appeared discordant. The frequency of individuals with truncating or splice-site CPG variants and at least one discordant tumor type was significantly higher than in a control population (χ2 = 43.642; p ≤ 0.0001). 2/67 (3%) probands with P/LP variants had evidence of multiple inherited neoplasia allele syndrome (MINAS) with deleterious variants in two CPGs. Together with variant detection rates from a previous series of similarly ascertained MPT-affected individuals, the present results suggest that first-line comprehensive CPG analysis in an MPT cohort referred to clinical genetics services would detect a deleterious variant in about a third of individuals.JW is supported by a Cancer Research UK Cambridge Cancer Centre Clinical Research Training Fellowship. Funding for the NIHR BioResource – Rare diseases project was provided by the National Institute for Health Research (NIHR, grant number RG65966). ERM acknowledges support from the European Research Council (Advanced Researcher Award), NIHR (Senior Investigator Award and Cambridge NIHR Biomedical Research Centre), Cancer Research UK Cambridge Cancer Centre and Medical Research Council Infrastructure Award. The University of Cambridge has received salary support in respect of EM from the NHS in the East of England through the Clinical Academic Reserve. The views expressed are those of the authors and not necessarily those of the NHS or Department of Health. DGE is an NIHR Senior Investigator and is supported by the all Manchester NIHR Biomedical Research Centre