A Multicomponent Primary-Care Intervention for Preventing Falls in Older Adults Living in the Community: The PREMIO Study.

BACKGROUND: Falls are a common cause of morbidity and functional impairment in the elderly and represent a significant health problem. General practitioners (GPs) are the first point of contact for health issues and may provide preventive services. The randomized clinical trial PREMIO was conducted by GPs to evaluate the effects of a multicomponent intervention for the prevention of falls in older adults aged ≥ 65 years at high risk of falling. METHODS: 117 GPs enrolled 1757 patients (1116 F, 641 M) and randomized them into 2 groups (intervention and control). The intervention group received medical and behavioral counseling, home risk-factor assessment, a physical-activity program and nutritional counseling. The control group received only the nutritional counseling. Both groups were followed for one year. The primary outcome was the rate of falls at home over 12 months. RESULTS: 1225 patients completed the study. Subjects receiving the intervention had, on average, fewer falls at home (percentage change -31.2%, p < 0.02) and fewer total falls (-26.0%, p < 0.02), although the reduction in the number of fallers was small (-3.9%, p = 0.05). Among the secondary endpoints, rates of general hospital or emergency-department admission and GP visits showed slight improvements (not statistically significant), while the risk of fractures was unexpectedly increased in the intervention group compared to the controls (odds ratio 2.39, p = 0.023). CONCLUSIONS: Future studies and public-health interventions to prevent domestic falls among community-dwelling older people at high risk of falling could benefit from a multicomponent approach including medication review, physical exercise and home risk assessment and should include assessment of risk factors for fractures.

Detailed characterizations of cranial nerve anatomy in E14.5 mouse embryos/fetuses and their use as reference for diagnosing subtle, but potentially lethal malformations in mutants.

Careful phenotype analysis of genetically altered mouse embryos/fetuses is vital for deciphering the function of pre- and perinatally lethal genes. Usually this involves comparing the anatomy of mutants with that of wild types of identical developmental stages. Detailed three dimensional information on regular cranial nerve (CN) anatomy of prenatal mice is very scarce. We therefore set out to provide such information to be used as reference data and selected mutants to demonstrate its potential for diagnosing CN abnormalities. Digital volume data of 152 wild type mice, harvested on embryonic day (E)14.5 and of 18 mutants of the Col4a2, Arid1b, Rpgrip1l and Cc2d2a null lines were examined. The volume data had been created with High Resolution Episcopic Microscopy (HREM) as part of the deciphering the mechanisms of developmental disorders (DMDD) program. Employing volume and surface models, oblique slicing and digital measuring tools, we provide highly detailed anatomic descriptions of the CNs and measurements of the diameter of selected segments. Specifics of the developmental stages of E14.5 mice and anatomic norm variations were acknowledged. Using the provided data as reference enabled us to objectively diagnose CN abnormalities, such as abnormal formation of CN3 (Col4a2), neuroma of the motor portion of CN5 (Arid1b), thinning of CN7 (Rpgrip1l) and abnormal topology of CN12 (Cc2d2a). Although, in a first glimpse perceived as unspectacular, defects of the motor CN5 or CN7, like enlargement or thinning can cause death of newborns, by hindering feeding. Furthermore, abnormal topology of CN12 was recently identified as a highly reliable marker for low penetrating, but potentially lethal defects of the central nervous system.

The venous system of E14.5 mouse embryos-reference data and examples for diagnosing malformations in embryos with gene deletions.

Approximately one-third of randomly produced knockout mouse lines produce homozygous offspring, which fail to survive the perinatal period. The majority of these die around or after embryonic day (E)14.5, presumably from cardiovascular insufficiency. For diagnosing structural abnormalities underlying death and diseases and for researching gene function, the phenotype of these individuals has to be analysed. This makes the creation of reference data, which define normal anatomy and normal variations the highest priority. While such data do exist for the heart and arteries, they are still missing for the venous system. Here we provide high-quality descriptive and metric information on the normal anatomy of the venous system of E14.5 embryos. Using high-resolution digital volume data and 3D models from 206 genetically normal embryos, bred on the C57BL/6N background, we present precise descriptive and metric information of the venous system as it presents itself in each of the six developmental stages of E14.5. The resulting data shed new light on the maturation and remodelling of the venous system at transition of embryo to foetal life and provide a reference that can be used for detecting venous abnormalities in mutants. To explore this capacity, we analysed the venous phenotype of embryos from 7 knockout lines (Atp11a, Morc2a, 1700067K01Rik, B9d2, Oaz1, Celf4 and Coro1c). Careful comparisons enabled the diagnosis of not only simple malformations, such as dual inferior vena cava, but also complex and subtle abnormalities, which would have escaped diagnosis in the absence of detailed, stage-specific referenced data.

Artefacts in Volume Data Generated with High Resolution Episcopic Microscopy (HREM).

High resolution episcopic microscopy (HREM) produces digital volume data by physically sectioning histologically processed specimens, while capturing images of the subsequently exposed block faces. Our study aims to systematically define the spectrum of typical artefacts inherent to HREM data and to research their effect on the interpretation of the phenotype of wildtype and mutant mouse embryos. A total of 607 (198 wildtypes, 409 mutants) HREM data sets of mouse embryos harvested at embryonic day (E) 14.5 were systematically and comprehensively examined. The specimens had been processed according to essentially identical protocols. Each data set comprised 2000 to 4000 single digital images. Voxel dimensions were 3 × 3 × 3 µm3. Using 3D volume models and virtual resections, we identified a number of characteristic artefacts and grouped them according to their most likely causality. Furthermore, we highlight those that affect the interpretation of embryo data and provide examples for artefacts mimicking tissue defects and structural pathologies. Our results aid in optimizing specimen preparation and data generation, are vital for the correct interpretation of HREM data and allow distinguishing tissue defects and pathologies from harmless artificial alterations. In particular, they enable correct diagnosis of pathologies in mouse embryos serving as models for deciphering the mechanisms of developmental disorders.

Hypoglossal Nerve Abnormalities as Biomarkers for Central Nervous System Defects in Mouse Lines Producing Embryonically Lethal Offspring.

An essential step in researching human central nervous system (CNS) disorders is the search for appropriate mouse models that can be used to investigate both genetic and environmental factors underlying the etiology of such conditions. Identification of murine models relies upon detailed pre- and post-natal phenotyping since profound defects are not only the result of gross malformations but can be the result of small or subtle morphological abnormalities. The difficulties in identifying such defects are compounded by the finding that many mouse lines show quite a variable penetrance of phenotypes. As a result, without analysis of large numbers, such phenotypes are easily missed. Indeed for null mutations, around one-third have proved to be pre- or perinatally lethal, their analysis resting entirely upon phenotyping of accessible embryonic stages.To simplify the identification of potentially useful mouse mutants, we have conducted three-dimensional phenotype analysis of approximately 500 homozygous null mutant embryos, produced from targeting a variety of mouse genes and harvested at embryonic day 14.5 as part of the "Deciphering the Mechanisms of Developmental Disorders" www.dmdd.org.uk program. We have searched for anatomical features that have the potential to serve as biomarkers for CNS defects in such genetically modified lines. Our analysis identified two promising biomarker candidates. Hypoglossal nerve (HGN) abnormalities (absent, thin, and abnormal topology) and abnormal morphology or topology of head arteries are both frequently associated with the full spectrum of morphological CNS defects, ranging from exencephaly to more subtle defects such as abnormal nerve cell migration. Statistical analysis confirmed that HGN abnormalities (especially those scored absent or thin) indeed showed a significant correlation with CNS defect phenotypes. These results demonstrate that null mutant lines showing HGN abnormalities are also highly likely to produce CNS defects whose identification may be difficult as a result of morphological subtlety or low genetic penetrance.

The Col4a2em1(IMPC)Wtsi mouse line: lessons from the Deciphering the Mechanisms of Developmental Disorders program.

The Deciphering the Mechanisms of Developmental Disorders (DMDD) program uses a systematic and standardised approach to characterise the phenotype of embryos stemming from mouse lines, which produce embryonically lethal offspring. Our study aims to provide detailed phenotype descriptions of homozygous Col4a2em1(IMPC)Wtsi mutants produced in DMDD and harvested at embryonic day 14.5. This shall provide new information on the role Col4a2 plays in organogenesis and demonstrate the capacity of the DMDD database for identifying models for researching inherited disorders. The DMDD Col4a2em1(IMPC)Wtsi mutants survived organogenesis and thus revealed the full spectrum of organs and tissues, the development of which depends on Col4a2 encoded proteins. They showed defects in the brain, cranial nerves, visual system, lungs, endocrine glands, skeleton, subepithelial tissues and mild to severe cardiovascular malformations. Together, this makes the DMDD Col4a2em1(IMPC)Wtsi line a useful model for identifying the spectrum of defects and for researching the mechanisms underlying autosomal dominant porencephaly 2 (OMIM # 614483), a rare human disease. Thus we demonstrate the general capacity of the DMDD approach and webpage as a valuable source for identifying mouse models for rare diseases.

Common and distinct transcriptional signatures of mammalian embryonic lethality.

The Deciphering the Mechanisms of Developmental Disorders programme has analysed the morphological and molecular phenotypes of embryonic and perinatal lethal mouse mutant lines in order to investigate the causes of embryonic lethality. Here we show that individual whole-embryo RNA-seq of 73 mouse mutant lines (>1000 transcriptomes) identifies transcriptional events underlying embryonic lethality and associates previously uncharacterised genes with specific pathways and tissues. For example, our data suggest that Hmgxb3 is involved in DNA-damage repair and cell-cycle regulation. Further, we separate embryonic delay signatures from mutant line-specific transcriptional changes by developing a baseline mRNA expression catalogue of wild-type mice during early embryogenesis (4-36 somites). Analysis of transcription outside coding sequence identifies deregulation of repetitive elements in Morc2a mutants and a gene involved in gene-specific splicing. Collectively, this work provides a large scale resource to further our understanding of early embryonic developmental disorders.

Mammalian Hbs1L deficiency causes congenital anomalies and developmental delay associated with Pelota depletion and 80S monosome accumulation.

Hbs1 has been established as a central component of the cell's translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition.

Visualising the Cardiovascular System of Embryos of Biomedical Model Organisms with High Resolution Episcopic Microscopy (HREM).

The article will briefly introduce the high-resolution episcopic microscopy (HREM) technique and will focus on its potential for researching cardiovascular development and remodelling in embryos of biomedical model organisms. It will demonstrate the capacity of HREM for analysing the cardiovascular system of normally developed and genetically or experimentally malformed zebrafish, frog, chick and mouse embryos in the context of the whole specimen and will exemplarily show the possibilities HREM offers for comprehensive visualisation of the vasculature of adult human skin. Finally, it will provide examples of the successful application of HREM for identifying cardiovascular malformations in genetically altered mouse embryos produced in the deciphering the mechanisms of developmental disorders (DMDD) program.

The International Mouse Phenotyping Consortium (IMPC): a functional catalogue of the mammalian genome that informs conservation.

The International Mouse Phenotyping Consortium (IMPC) is building a catalogue of mammalian gene function by producing and phenotyping a knockout mouse line for every protein-coding gene. To date, the IMPC has generated and characterised 5186 mutant lines. One-third of the lines have been found to be non-viable and over 300 new mouse models of human disease have been identified thus far. While current bioinformatics efforts are focused on translating results to better understand human disease processes, IMPC data also aids understanding genetic function and processes in other species. Here we show, using gorilla genomic data, how genes essential to development in mice can be used to help assess the potentially deleterious impact of gene variants in other species. This type of analyses could be used to select optimal breeders in endangered species to maintain or increase fitness and avoid variants associated to impaired-health phenotypes or loss-of-function mutations in genes of critical importance. We also show, using selected examples from various mammal species, how IMPC data can aid in the identification of candidate genes for studying a condition of interest, deliver information about the mechanisms involved, or support predictions for the function of genes that may play a role in adaptation. With genotyping costs decreasing and the continued improvements of bioinformatics tools, the analyses we demonstrate can be routinely applied.

Placentation defects are highly prevalent in embryonic lethal mouse mutants.

Large-scale phenotyping efforts have demonstrated that approximately 25-30% of mouse gene knockouts cause intrauterine lethality. Analysis of these mutants has largely focused on the embryo and not the placenta, despite the crucial role of this extraembryonic organ for developmental progression. Here we screened 103 embryonic lethal and sub-viable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders program for placental phenotypes. We found that 68% of knockout lines that are lethal at or after mid-gestation exhibited placental dysmorphologies. Early lethality (embryonic days 9.5-14.5) is almost always associated with severe placental malformations. Placental defects correlate strongly with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests that a considerable number of factors that cause embryonic lethality when ablated have primary gene function in trophoblast cells. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes that govern placenta formation.

Corrigendum: High-throughput discovery of novel developmental phenotypes.

This corrects the article DOI: 10.1038/nature19356.

Morphology, topology and dimensions of the heart and arteries of genetically normal and mutant mouse embryos at stages S21-S23.

Accurate identification of abnormalities in the mouse embryo depends not only on comparisons with appropriate, developmental stage-matched controls, but also on an appreciation of the range of anatomical variation that can be expected during normal development. Here we present a morphological, topological and metric analysis of the heart and arteries of mouse embryos harvested on embryonic day (E)14.5, based on digital volume data of whole embryos analysed by high-resolution episcopic microscopy (HREM). By comparing data from 206 genetically normal embryos, we have analysed the range and frequency of normal anatomical variations in the heart and major arteries across Theiler stages S21-S23. Using this, we have identified abnormalities in these structures among 298 embryos from mutant mouse lines carrying embryonic lethal gene mutations produced for the Deciphering the Mechanisms of Developmental Disorders (DMDD) programme. We present examples of both commonly occurring abnormal phenotypes and novel pathologies that most likely alter haemodynamics in these genetically altered mouse embryos. Our findings offer a reference baseline for identifying accurately abnormalities of the heart and arteries in embryos that have largely completed organogenesis.

Dataset of Sgo1 expression in cardiac, gastrointestinal, hepatic and neuronal tissue in mouse.

The data shown in this article are related to the research article entitled "Characterization of Sgo1 expression pattern in developing and adult mouse" (Song et al., 2017) [3]. The article provides novel data on Sgo1 gene expression pattern utilizing Sgo1_LacZ_Knock in mouse line and immunohistochemistry in wild type mice. The data presents Sgo1 expression pattern during development, and in post-developmental proliferative and quiescent tissue. The article describes following tissues: developing heart, neural tube, adult colon, cerebellum, cerebral cortex, liver, and testis.

Integrated genome and transcriptome sequencing identifies a noncoding mutation in the genome replication factor DONSON as the cause of microcephaly-micromelia syndrome.

While next-generation sequencing has accelerated the discovery of human disease genes, progress has been largely limited to the "low hanging fruit" of mutations with obvious exonic coding or canonical splice site impact. In contrast, the lack of high-throughput, unbiased approaches for functional assessment of most noncoding variants has bottlenecked gene discovery. We report the integration of transcriptome sequencing (RNA-seq), which surveys all mRNAs to reveal functional impacts of variants at the transcription level, into the gene discovery framework for a unique human disease, microcephaly-micromelia syndrome (MMS). MMS is an autosomal recessive condition described thus far in only a single First Nations population and causes intrauterine growth restriction, severe microcephaly, craniofacial anomalies, skeletal dysplasia, and neonatal lethality. Linkage analysis of affected families, including a very large pedigree, identified a single locus on Chromosome 21 linked to the disease (LOD > 9). Comprehensive genome sequencing did not reveal any pathogenic coding or canonical splicing mutations within the linkage region but identified several nonconserved noncoding variants. RNA-seq analysis detected aberrant splicing in DONSON due to one of these noncoding variants, showing a causative role for DONSON disruption in MMS. We show that DONSON is expressed in progenitor cells of embryonic human brain and other proliferating tissues, is co-expressed with components of the DNA replication machinery, and that Donson is essential for early embryonic development in mice as well, suggesting an essential conserved role for DONSON in the cell cycle. Our results demonstrate the utility of integrating transcriptomics into the study of human genetic disease when DNA sequencing alone is not sufficient to reveal the underlying pathogenic mutation.

Characterization of Sgo1 expression in developing and adult mouse.

SGO1 has been characterized in its function in correct cell division and its role in centrosome cohesion in the nucleus. However, its organ-specific maturation-related expression pattern in vivo remains largely uncharacterized. Here, we show clear SGO1 expression in post-developmental neuronal cells and cytoplasmic localisation in nucleated cells with a transgenic mice model and immunohistochemistry of wild type mice. We demonstrate extranuclear expression of Sgo1 in the developing heart and gut, which have been shown to be dysregulated in humans with homozygous SGO1 mutation. Additionally, we show Sgo1 expression in select population of retinal cells in developing and post-developmental retina. Our expression analysis strongly suggests that the function of SGO1 goes beyond its well characterized role in cell division.

Genome-wide in vivo screen identifies novel host regulators of metastatic colonization.

Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.

Highly variable penetrance of abnormal phenotypes in embryonic lethal knockout mice.

Background: Identifying genes that are essential for mouse embryonic development and survival through term is a powerful and unbiased way to discover possible genetic determinants of human developmental disorders. Characterising the changes in mouse embryos that result from ablation of lethal genes is a necessary first step towards uncovering their role in normal embryonic development and establishing any correlates amongst human congenital abnormalities. Methods: Here we present results gathered to date in the Deciphering the Mechanisms of Developmental Disorders (DMDD) programme, cataloguing the morphological defects identified from comprehensive imaging of 220 homozygous mutant and 114 wild type embryos from 42 lethal and subviable lines, analysed at E14.5. Results: Virtually all mutant embryos show multiple abnormal phenotypes and amongst the 42 lines these affect most organ systems. Within each mutant line, the phenotypes of individual embryos form distinct but overlapping sets. Subcutaneous edema, malformations of the heart or great vessels, abnormalities in forebrain morphology and the musculature of the eyes are all prevalent phenotypes, as is loss or abnormal size of the hypoglossal nerve.Conclusions: Overall, the most striking finding is that no matter how profound the malformation, each phenotype shows highly variable penetrance within a mutant line. These findings have challenging implications for efforts to identify human disease correlates.

PRMT5 is essential for the maintenance of chondrogenic progenitor cells in the limb bud.

During embryonic development, undifferentiated progenitor cells balance the generation of additional progenitor cells with differentiation. Within the developing limb, cartilage cells differentiate from mesodermal progenitors in an ordered process that results in the specification of the correct number of appropriately sized skeletal elements. The internal pathways by which these cells maintain an undifferentiated state while preserving their capacity to differentiate is unknown. Here, we report that the arginine methyltransferase PRMT5 has a crucial role in maintaining progenitor cells. Mouse embryonic buds lacking PRMT5 have severely truncated bones with wispy digits lacking joints. This novel phenotype is caused by widespread cell death that includes mesodermal progenitor cells that have begun to precociously differentiate into cartilage cells. We propose that PRMT5 maintains progenitor cells through its regulation of Bmp4 Intriguingly, adult and embryonic stem cells also require PRMT5 for maintaining pluripotency, suggesting that similar mechanisms might regulate lineage-restricted progenitor cells during organogenesis.

Synthetic lethality between PAXX and XLF in mammalian development.

PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf-/- mice, Paxx-/- mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4-/- and Lig4-/- mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals.