Genome-wide screening reveals the genetic basis of mammalian embryonic eye development.

BACKGROUND: Microphthalmia, anophthalmia, and coloboma (MAC) spectrum disease encompasses a group of eye malformations which play a role in childhood visual impairment. Although the predominant cause of eye malformations is known to be heritable in nature, with 80% of cases displaying loss-of-function mutations in the ocular developmental genes OTX2 or SOX2, the genetic abnormalities underlying the remaining cases of MAC are incompletely understood. This study intended to identify the novel genes and pathways required for early eye development. Additionally, pathways involved in eye formation during embryogenesis are also incompletely understood. This study aims to identify the novel genes and pathways required for early eye development through systematic forward screening of the mammalian genome. RESULTS: Query of the International Mouse Phenotyping Consortium (IMPC) database (data release 17.0, August 01, 2022) identified 74 unique knockout lines (genes) with genetically associated eye defects in mouse embryos. The vast majority of eye abnormalities were small or absent eyes, findings most relevant to MAC spectrum disease in humans. A literature search showed that 27 of the 74 lines had previously published knockout mouse models, of which only 15 had ocular defects identified in the original publications. These 12 previously published gene knockouts with no reported ocular abnormalities and the 47 unpublished knockouts with ocular abnormalities identified by the IMPC represent 59 genes not previously associated with early eye development in mice. Of these 59, we identified 19 genes with a reported human eye phenotype. Overall, mining of the IMPC data yielded 40 previously unimplicated genes linked to mammalian eye development. Bioinformatic analysis showed that several of the IMPC genes colocalized to several protein anabolic and pluripotency pathways in early eye development. Of note, our analysis suggests that the serine-glycine pathway producing glycine, a mitochondrial one-carbon donator to folate one-carbon metabolism (FOCM), is essential for eye formation. CONCLUSIONS: Using genome-wide phenotype screening of single-gene knockout mouse lines, STRING analysis, and bioinformatic methods, this study identified genes heretofore unassociated with MAC phenotypes providing models to research novel molecular and cellular mechanisms involved in eye development. These findings have the potential to hasten the diagnosis and treatment of this congenital blinding disease.

Equitable Expanded Carrier Screening Needs Indigenous Clinical and Population Genomic Data.

Expanded carrier screening (ECS) for recessive monogenic diseases requires prior knowledge of genomic variation, including DNA variants that cause disease. The composition of pathogenic variants differs greatly among human populations, but historically, research about monogenic diseases has focused mainly on people with European ancestry. By comparison, less is known about pathogenic DNA variants in people from other parts of the world. Consequently, inclusion of currently underrepresented Indigenous and other minority population groups in genomic research is essential to enable equitable outcomes in ECS and other areas of genomic medicine. Here, we discuss this issue in relation to the implementation of ECS in Australia, which is currently being evaluated as part of the national Government's Genomics Health Futures Mission. We argue that significant effort is required to build an evidence base and genomic reference data so that ECS can bring significant clinical benefit for many Aboriginal and/or Torres Strait Islander Australians. These efforts are essential steps to achieving the Australian Government's objectives and its commitment "to leveraging the benefits of genomics in the health system for all Australians." They require culturally safe, community-led research and community involvement embedded within national health and medical genomics programs to ensure that new knowledge is integrated into medicine and health services in ways that address the specific and articulated cultural and health needs of Indigenous people. Until this occurs, people who do not have European ancestry are at risk of being, in relative terms, further disadvantaged.

Molecular characterization and genetic subclassification comparison of diffuse large B-cell lymphoma (DLBCL) - real-life experience with 74 cases.

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity. Lately, several algorithms achieving therapeutically and prognostically relevant DLBCL subclassification have been published. METHODS: A cohort of 74 routine DLBCL cases was broadly characterized by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) of the BCL2, BCL6 and MYC loci, and comprehensive high throughput sequencing (HTS). Based on the genetic alterations found, cases were reclassified using two probabilistic tools - LymphGen and Two-step classifier, allowing for comparison of the two models. RESULTS: Hans and Tally's overall IHC-based subclassification success rate was 96% and 82%, respectively. HTS and FISH data allowed the LymphGen algorithm to successfully classify 11/55 cases, (1 - BN2, 7 - EZB, 1 - MCD, and 2 - genetically composite EZB/N1). The total subclassification rate was 20%. On the other hand, the Two-step classifier categorized 36/55 cases, with 65.5% success (9 - BN2, 12 - EZB, 9 - MCD, 2 - N1, and 4 - ST2). Clinical correlations highlighted MCD as an aggressive subtype associated with higher relapse and mortality. CONCLUSIONS: The Two-step algorithm has a better success rate at subclassifying DLBCL cases based on genetic differences. Further improvement of the classifiers is required to increase the number of classifiable cases and thus prove their applicability in routine diagnostics.

Asian Mouse Mutagenesis Resource Association (AMMRA): mouse genetics and laboratory animal resources in the Asia Pacific.

The Asian Mouse Mutagenesis Resource Association (AMMRA) is a non-profit organization consisting of major resource and research institutions with rodent expertise from within the Asia Pacific region. For more than a decade, aiming to support biomedical research and stimulate international collaboration, AMMRA has always been a friendly and passionate ally of Asian and Australian member institutions devoted to sharing knowledge, exchanging resources, and promoting biomedical research. AMMRA is also missioned to global connection by working closely with the consortiums such as the International Mouse Phenotyping Consortium and the International Mouse Strain Resource. This review discusses the emergence of AMMRA and outlines its many roles and responsibilities in promoting, assisting, enriching research, and ultimately enhancing global life science research quality.

[Who must be referred for genetic counseling?] / Wer braucht eine genetische Beratung?

Who must be referred for genetic counseling ? Abstract. A family history of breast cancer and / or ovarian cancer is the main risk factor for a woman to develop breast (or / and ovarian cancer) herself. A detailed analysis of the family history by a oncogenetics specialist allows establishing the indication for genetic testing. Required criteria for testing in Switzerland are well defined. High-risk women can be identified by a mutation detection in predisposing gene like BRCA1 or BRCA2. Management of mutation carriers should comply with international guidelines.

Cauli: a mouse strain with an Ift140 mutation that results in a skeletal ciliopathy modelling Jeune syndrome.

Cilia are architecturally complex organelles that protrude from the cell membrane and have signalling, sensory and motility functions that are central to normal tissue development and homeostasis. There are two broad categories of cilia; motile and non-motile, or primary, cilia. The central role of primary cilia in health and disease has become prominent in the past decade with the recognition of a number of human syndromes that result from defects in the formation or function of primary cilia. This rapidly growing class of conditions, now known as ciliopathies, impact the development of a diverse range of tissues including the neural axis, craniofacial structures, skeleton, kidneys, eyes and lungs. The broad impact of cilia dysfunction on development reflects the pivotal position of the primary cilia within a signalling nexus involving a growing number of growth factor systems including Hedgehog, Pdgf, Fgf, Hippo, Notch and both canonical Wnt and planar cell polarity. We have identified a novel ENU mutant allele of Ift140, which causes a mid-gestation embryonic lethal phenotype in homozygous mutant mice. Mutant embryos exhibit a range of phenotypes including exencephaly and spina bifida, craniofacial dysmorphism, digit anomalies, cardiac anomalies and somite patterning defects. A number of these phenotypes can be attributed to alterations in Hedgehog signalling, although additional signalling systems are also likely to be involved. We also report the identification of a homozygous recessive mutation in IFT140 in a Jeune syndrome patient. This ENU-induced Jeune syndrome model will be useful in delineating the origins of dysmorphology in human ciliopathies.

Improving laboratory animal genetic reporting: LAG-R guidelines.

The biomedical research community addresses reproducibility challenges in animal studies through standardized nomenclature, improved experimental design, transparent reporting, data sharing, and centralized repositories. The ARRIVE guidelines outline documentation standards for laboratory animals in experiments, but genetic information is often incomplete. To remedy this, we propose the Laboratory Animal Genetic Reporting (LAG-R) framework. LAG-R aims to document animals' genetic makeup in scientific publications, providing essential details for replication and appropriate model use. While verifying complete genetic compositions may be impractical, better reporting and validation efforts enhance reliability of research. LAG-R standardization will bolster reproducibility, peer review, and overall scientific rigor.

Ribociclib-induced liver injury: a case report.

Background: Idiosyncratic drug-induced liver injury (DILI) is a rare, unpredictable hepatic adverse event and the most common cause of acute liver failure in Europe and the US. Ribociclib is a potent Cyclin-dependent kinase 4 and 6 (CDK4/6)-inhibitor administered for advanced hormone-receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer. Previous reports have shown hepatotoxicity without liver necrosis related to ribociclib. Case presentation: A 41-year-old female patient with primary metastatic HR-positive, HER2-negative breast cancer developed liver enzyme elevation under treatment with ribociclib. Ribociclib was withdrawn 8 weeks after initiation due to liver enzyme elevation. A liver biopsy, performed due to further enzyme increase (peak ALT 2836 U/l), onset of jaundice (peak bilirubin 353 µmol/l) and coagulopathy (INR 1.8) two weeks later, revealed acute hepatitis with 30% parenchymal necrosis. Roussel Uclaf Causality Assessment Method (RUCAM) score was 7 points (probable). Under treatment with prednisone (60mg), initiated 2 weeks after drug withdrawal, and subsequently N-acetylcysteine (Prescott regimen) liver enzymes normalized within 8 weeks along with prednisone tapering. Conclusion: This case illustrates the development of a severe idiosyncratic hepatocellular pattern DILI grade 3 (International DILI Expert Working Group) induced by ribociclib. Routine liver enzyme testing during therapy, immediate hepatologic work-up and treatment interruption in case of liver enzyme elevation are highly recommended. Corticosteroid treatment should be considered in cases of severe necroinflammation.

Analysis of genome-wide knockout mouse database identifies candidate ciliopathy genes.

We searched a database of single-gene knockout (KO) mice produced by the International Mouse Phenotyping Consortium (IMPC) to identify candidate ciliopathy genes. We first screened for phenotypes in mouse lines with both ocular and renal or reproductive trait abnormalities. The STRING protein interaction tool was used to identify interactions between known cilia gene products and those encoded by the genes in individual knockout mouse strains in order to generate a list of "candidate ciliopathy genes." From this list, 32 genes encoded proteins predicted to interact with known ciliopathy proteins. Of these, 25 had no previously described roles in ciliary pathobiology. Histological and morphological evidence of phenotypes found in ciliopathies in knockout mouse lines are presented as examples (genes Abi2, Wdr62, Ap4e1, Dync1li1, and Prkab1). Phenotyping data and descriptions generated on IMPC mouse line are useful for mechanistic studies, target discovery, rare disease diagnosis, and preclinical therapeutic development trials. Here we demonstrate the effective use of the IMPC phenotype data to uncover genes with no previous role in ciliary biology, which may be clinically relevant for identification of novel disease genes implicated in ciliopathies.

ENU mutagenesis screen to establish motor phenotypes in wild-type mice and modifiers of a pre-existing motor phenotype in tau mutant mice.

Modifier screening is a powerful genetic tool. While not widely used in the vertebrate system, we applied these tools to transgenic mouse strains that recapitulate key aspects of Alzheimer's disease (AD), such as tau-expressing mice. These are characterized by a robust pathology including both motor and memory impairment. The phenotype can be modulated by ENU mutagenesis, which results in novel mutant mouse strains and allows identifying the underlying gene/mutation. Here we discuss this strategy in detail. We firstly obtained pedigrees that modify the tau-related motor phenotype, with mapping ongoing. We further obtained transgene-independent motor pedigrees: (i) hyperactive, circling ENU 37 mice with a causal mutation in the Tbx1 gene-the complete knock-out of Tbx1 models DiGeorge Syndrome; (ii) ENU12/301 mice that show sudden jerky movements and tremor constantly; they have a causal mutation in the Kcnq1 gene, modelling aspects of the Romano-Ward and Jervell and Lange-Nielsen syndromes; and (iii) ENU16/069 mice with tremor and hypermetric gait that have a causal mutation in the Mpz (Myelin Protein Zero) gene, modelling Charcot-Marie-Tooth disease type 1 (CMT1B). Together, we provide evidence for a real potential of an ENU mutagenesis to dissect motor functions in wild-type and tau mutant mice.

Nivolumab in chemotherapy-resistant cervical cancer: report of a vulvitis as a novel immune-related adverse event and molecular analysis of a persistent complete response.

BACKGROUND: Treatment options for advanced cervical cancer are limited and patients experiencing recurrence after first-line cisplatin-based chemotherapy and bevacizumab have a poor prognosis. A recent phase II study in advanced cervical cancer has demonstrated a disease control rate of 68.4% with the immune checkpoint inhibitor nivolumab. By blocking immune checkpoints, immunotherapy puts the immune system into a state of hyper-activation that can cause immune-related adverse events. We present the clinical, pathological and molecular data of a patient with metastatic cervical cancer and progressive disease after second-line therapy. We report on the therapeutic response under third-line immunotherapy with nivolumab, the immune-related adverse events (IRAE), and their successful management. CASE PRESENTATION: We report the case of a 62-year-old woman who was diagnosed with advanced squamous cell carcinoma of the cervix with paraaortic lymph node metastases. After an initial combined radio-chemotherapy with cisplatin, she developed local and nodal (supraclavicular) recurrence. Second-line chemotherapy with 6 cycles of carboplatin, paclitaxel, and bevacizumab resulted in a partial response for 6 months. Checkpoint inhibition with nivolumab was started due to progression, leading to persistent complete remission. Immunotherapy was well tolerated for 8 months until the patient presented with an immune-related isolated vulvitis, which was successfully managed with topical corticosteroids. CONCLUSIONS: The persistent complete response after third-line treatment for relapsed chemotherapy-resistant cervical cancer presented in this case highlights the potential of immunotherapy for patients with advanced cervical cancer impressively. To our knowledge, this is the first report of an isolated immune-related vulvitis under nivolumab. This adverse event might be underdiagnosed and mistreated, however, it is of importance due to its impact on quality of life, sexual wellbeing and compliance of patients. Successful IRAE management may enable prolonged immune checkpoint inhibitor therapy. In the future, routine molecular tumour profiling is likely to aid in the stratification of cervical cancer patients for immunotherapy. Here, we provide the methylome data of a case with complete response.

Identification of genetic elements in metabolism by high-throughput mouse phenotyping.

Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic data of 2016 knockout mouse strains under the aegis of the International Mouse Phenotyping Consortium (IMPC) and find 974 gene knockouts with strong metabolic phenotypes. 429 of those had no previous link to metabolism and 51 genes remain functionally completely unannotated. We compared human orthologues of these uncharacterized genes in five GWAS consortia and indeed 23 candidate genes are associated with metabolic disease. We further identify common regulatory elements in promoters of candidate genes. As each regulatory element is composed of several transcription factor binding sites, our data reveal an extensive metabolic phenotype-associated network of co-regulated genes. Our systematic mouse phenotype analysis thus paves the way for full functional annotation of the genome.

Up-regulation of endothelial delta-like 4 expression correlates with vessel maturation in bladder cancer.

PURPOSE: Angiogenesis and vascular endothelial growth factor (VEGF) expression are associated with a poor outcome in bladder cancer. To understand more about the mechanisms, we studied the role of delta-like 4 (DLL4), an endothelial-specific ligand of the Notch signaling pathway, in bladder cancer angiogenesis. EXPERIMENTAL DESIGN: The expression of DLL4, CD34, and VEGF were studied in a cohort of 60 bladder tumors and 10 normal samples using quantitative PCR. In situ hybridization was used to study the pattern of DLL4 expression in 22 tumor and 9 normal samples. Serial sections were also stained for CD34 and alpha-smooth muscle actin (alpha-SMA) using conventional immunohistochemistry. RESULTS: The expression of DLL4 was significantly up-regulated in superficial (P < 0.01) and invasive (P < 0.05) bladder cancers. DLL4 expression significantly correlated with CD34 (P < 0.001) and VEGF (P < 0.001) expression. The in situ hybridization studies showed that DLL4 was highly expressed within bladder tumor vasculature. Additionally, DLL4 expression significantly correlated with vessel maturation as judged by periendothelial cell expression of alpha-SMA, 98.7% of DLL4-positive tumor vessels coexpressed alpha-SMA, compared with 64.5% of DLL4-negative tumor vessels (P < 0.001). High DLL4 expression may have prognostic value in superficial and invasive bladder. CONCLUSION: DLL4 expression is associated with vascular differentiation in bladder cancer; thus, targeting DLL4 may be a novel antiangiogenic therapy.

Nitric oxide and peroxynitrite cause irreversible increases in the K(m) for oxygen of mitochondrial cytochrome oxidase: in vitro and in vivo studies.

Mitochondrial cytochrome oxidase is competitively and reversibly inhibited by inhibitors that bind to ferrous heme, such as carbon monoxide and nitric oxide. In the case of nitric oxide, nanomolar levels inhibit cytochrome oxidase by competing with oxygen at the enzyme's heme-copper active site. This raises the K(m) for cellular respiration into the physiological range. This effect is readily reversible and may be a physiological control mechanism. Here we show that a number of in vitro and in vivo conditions result in an irreversible increase in the oxygen K(m). These include: treatment of the purified enzyme with peroxynitrite or high (microM) levels of nitric oxide; treatment of the endothelial-derived cell line, b.End5, with NO; activation of astrocytes by cytokines; reperfusion injury in the gerbil brain. Studies of cell respiration that fail to vary the oxygen concentration systematically are therefore likely to significantly underestimate the degree of irreversible damage to cytochrome oxidase.

Drug resistance in epilepsy: the role of the blood-brain barrier.

The blood-brain barrier (BBB) is formed by the endothelial cells lining the brain microvessels. Complex tight junctions linking adjacent endothelial cells make brain capillaries around 100 times tighter than peripheral capillaries to small hydrophilic molecules. As a result, drugs required to act in the brain, including anti-epileptic drugs (AEDs), have generally been made lipophilic, and are thus able to cross the brain endothelium via the lipid membranes. However, such lipophilic drugs are potential substrates for efflux carriers of the BBB, particularly P glycoprotein (Pgp), predominantly located on the endothelial luminal membrane. It is estimated that up to 50% of drug candidates may be substrates for Pgp. The barrier phenotype of the brain endothelium is induced and maintained by chemical factors released by brain cells, particularly perivascular astrocytic end feet. In several neuropathological conditions, the BBB is disturbed, either as a result of pathology of the endothelium, or of the cells responsible for barrier induction and maintenance. During epileptic attacks, there may be transient BBB opening in the epileptogenic focus. There is evidence that under such pathological conditions, 'second line defence' mechanisms in perivascular glia may be up-regulated, including expression of Pgp and other drug efflux transporters. This complicates interpretation of drug resistance in epilepsy, and therapeutic strategies.

Genome-wide ENU mutagenesis in combination with high density SNP analysis and exome sequencing provides rapid identification of novel mouse models of developmental disease.

BACKGROUND: Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU). METHODOLOGY/PRINCIPAL FINDINGS: ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1. CONCLUSIONS/SIGNIFICANCE: In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.

[Bell's palsy: idiopathic or infectious?]. / La paralysie de Bell: idiopathique ou infectieuse?

Bell's palsy (BP), is a disease of unknown etiology; it's thought to be idiopathic, although it seems to be linked with infections, in particular viral ones. Within three weeks, three cases of BP were admitted to our hospital. The investigations suggested different causes: Herpes simplex virus infection, neuroborreliosis and the side effect of an influenza vaccine. Based on the literature, guidelines were established for the diagnostic evaluation and management of cases of BP in our hospital. Criteria for cerebral radiologic imaging procedures and laboratory investigations are discussed. We propose therapeutic strategies adapted to the probable cause of BP. In conclusion BP is associated with several distinct disease entities and the description of Sir Bell in the XIXth century appears outdated. In any case of BP the aim should be to find a specific etiology in order to give appropriate treatment.

Interaction between flavonoids and the blood-brain barrier: in vitro studies.

There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood-brain barrier (BBB) using well-established in vitro models (brain endothelial cell lines and ECV304 monolayers co-cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevant in vivo metabolites, as well as the dietary anthocyanins and in vivo forms, cyanidin-3-rutinoside and pelargonidin-3-glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (Papp) of the citrus flavonoids, hesperetin and naringenin, across the in vitro BBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and its in vivo metabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity.