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Specialist Secondary phenotyping (WP2)

Aims and Overview

A large number of interesting phenotypes will be uncovered by primary phenotyping at the mouse clinics (WP1). We propose that some of these new disease models will undergo more in-depth secondary phenotyping. We will establish a number of virtual secondary phenotyping centres across Europe consisting of networks of laboratories with the requisite expertise. The emphasis here will be on fostering discovery as well as further in-depth phenotyping of mouse mutant lines. Following primary phenotyping, the secondary phenotyping centres will be responsible for identifying and requesting relevant mutants taking into account the capacity available within each network. Data from secondary phenotyping will be entered into the EuroPhenome database.

It is important to emphasise that the tests offered by members of each virtual centre are not mutually exclusive. In many cases the bulk of secondary tests are available at each location and it would be expected that each member of a centre would carry out exhaustive analysis of mutant lines received. In other cases a particular member may provide sole access to a critical test and members would work together with an individual line being tested at more than one location. Each virtual centre will work together sharing results on individual mutant lines and completing the most appropriate set of tests depending upon ongoing findings. In addition, we will foster cross-talk between the centres that ensures that mice, which have been a focus of secondary phenotyping in one centre, can traffic to other centres for specific tests that may be of relevance. In this way the discovery process will drive in-depth secondary phenotyping of a number of mutant lines.

WP2.1 Clinical chemistry, haematology & metabolic

Western societies are witnessing a frightening increase in metabolic disorders, such as obesity, type 2 diabetes, hyperlipidemia and atherosclerosis, which are linked in good part to inappropriate nutritional and lifestyle habits, and favoured by genetic predisposition. One of these major chronic diseases is the metabolic syndrome, which reciprocally links alteration of glucose and lipid metabolisms to obesity, diabetes and cardiovascular diseases. The series of tests that will be provided in the secondary phenotyping program are particularly aimed at exploring the major parameters that reflect energy homeostasis. Two classes of tests will be provided. In a first series of tests, in-depth analyses of the metabolic status of the un-challenged animals will be carried out. The second series of tests are dynamic, i.e. homeostasis will be monitored during and after a challenge, as homeostasis in metabolism reflects a permanent and dynamic equilibrium.

First series tests:

·       Test 1: energy expenditure using indirect calorimetry, associated to activity recording, feeding and drinking behaviour and sampling of urine (an extension of the primary screen).

·       Test 2: detailed body composition, allowing measurement of the relative weight of bone, fat and lean mass, extending the primary screen and using non-invasive imaging such as EchoMRI

·       Test 3: endocrine profiling from blood samples, including measurement of lipoproteins and adipokines (such as leptin, adiponectin, resistin, and FIAF).

Second series tests:

·       Test 4: Analysis of the glycaemia control with a euglycaemic hyperinsulinaemic clamp. This test is today the best criteria for qualifying insulin resistance, which is at the core of the metabolic syndrome.

·       Test 5: Analysis of energy expenditure coupled to exercising. Alteration of muscle physiology is considered to be a very important event in the progressive alteration of the metabolic status of obese or ageing individuals, but little is known on the nature of the genetic factors that may predispose or protect against it.

·       Test 6: an evaluation in a global approach of the metabolite profile present in body fluids. This test will be performed on the urine collected during test 1.

Not only are metabolic diseases multifactorial, but they are also prone to a strong gender dimorphism. Thus, particular care will be given to compare observations in male and female subjects. Altogether, these approaches they will be a powerful route to identifying the mutant lines that are mouse models of obesity prone, insulin resistance, metabolic syndrome, and type 2 diabetes, encountered in human patients, so as to develop effective strategies for diagnosis, prevention, and therapy.

WP2.2 Cardiovascular

Cardiovascular diseases are the leading cause of death in Europe and in the Western world in general, accounting for over 40% of all deaths. Of these, coronary artery disease (CAD) and heart failure (CHF) are amongst the principal problems and, together with diabetes, they are increasing at an unprecedented rate.

We have introduced highly sensitive, specific and well established, primary screening methods for the detection of high blood pressure, left ventricular dysfunction and heart failure, i.e. tail cuff pressure, atrial natriuretic peptide and echocardiogram. The secondary screen will be a seamless continuation of the primary phenotyping; 15 - 20 selected lines identified by primary screening will be analysed further to identify functional and structural abnormalities. Careful 'clinical' phenotyping for paw oedema, ascites, pleural effusions and lung congestion will be carried out. Systolic and diastolic function will be evaluated using pressure-volume curves; invasive haemodynamic analysis under basal and stimulated conditions (isoproterenol or dobutamine) will be able to detect compromised contractile function of the heart.

Further in depth investigations will be carried out in selected lines depending on the profiles obtained; for example, it will be determined how the genetic modification affects the cardiovascular system under stress conditions, e.g. response to various hypertrophic stimuli or exercise; and changes in blood pressure, heart rate and locomotor activity will be continuously monitored by telemetry, detailed structural changes will be analysed by MRI. Metabolic analysis will be carried out in mice with left ventricular dysfunction/heart failure that also show a diabetic phenotype. Investigations will be carried out separately for male and female mice to detect sex-specific differences.

Vascular disease will be detected in the 'pathology' workpackage.

An effective primary screen for arrhythmias is presently not available and will therefore be part of a development project described elsewhere in this proposal (see WP3).

Taken together, EUMODIC will provide an effective and comprehensive primary and secondary screening programme for the commonest cardiovascular disorders, i.e. left ventricular dysfunction/heart failure, which is our focus, vascular disease (pathology screen), and arrhythmias (development of effective screening tests). The novel mouse models resulting from these screens will provide invaluable insights into the genetic basis of the major cardiovascular disorders and potential new treatment approaches by identifying novel pathways of disease which will be useful for developing drugs/gene therapies.

WP2.3 Respiratory

Respiratory diseases such as asthma, either allergic or not, is increasing in the western world, including Europe. Allergic asthma is a chronic disease characterised by reversible obstruction of the airways, bronchial hyperresponsiveness, oedema, infiltration of the lungs by inflammatory cells and mucus overproduction. Several genes involved in asthmatic responses have been identified, such as allergen-specific Th2 lymphocytes expressed IL-4, IL-5 and IL-13, or IgE. Although the early reaction due to IgE-mediated mast cell degranulation, with the release of preformed bioactive mediators may resolve, the repeated exposure to allergens promotes chronic inflammation leading to the long-term sequellae of asthma. Another major cause of severe airway disease is inflammation of the airway, often associated with life-threatening infection by Gram negative bacteria or presence of endotoxins. Moreover, inhaled endotoxin may play an important role in the development and progression of airway inflammation in asthma. Pathologic changes induced by endotoxin inhalation include bronchospasm, airflow obstruction, recruitment of inflammatory cells, injury of the alveolar epithelium and disruption of pulmonary capillary integrity leading to protein-rich fluid leak in the alveolar space. Most pathologic features of human airway inflammation have also been observed in experimental lung injury models. In mice, aerogenic exposure to endotoxins induces pulmonary inflammation with recruitment and activation of macrophages and neutrophils in the airways, local TNF production, alveolar-capillary leak and direct bronchoconstriction. In this workpackage, we will analyse mutant mice with a confirmed respiratory phenotype derived from the primary screen in terms of spontaneous airway function airway, response to endotoxins or allergic asthma. Neutrophil recruitment in the airways and lung damage will be monitored after endotoxin administration. Allergic asthma response in immunised mice to antigen will be determined by airway hyperresponsiveness, eosinophil infiltration in the airways and mucus hypersecretion in the lung. In addition chronic and fibrotic inflammation in response to bleomycin will be assessed as a model of chronic obstructive pulmonary disease. Therefore, this work package proposes to characterise airway function in naďve mutant mice, as well as their ability to develop changes in airway function and inflammation in disease conditions, and should lead to identification of novel disease-modifying genes.

WP2.4 Infection & immune response

Infectious diseases are still a major cause of morbidity and mortality worldwide. Until today only a few host-defence genes have been characterized that are involved in the control of immune response to different classes of pathogens. Despite the identification of some genes responsible for human primary immunodeficiencies, many genetically caused clinical immune disorders still remain unexplained. Primary immunodeficiency diseases can predispose individuals to different sets of infection, allergy, autoimmunity and cancer depending on which genes are affected. Although, in recent years, knowledge about immune system functions has been acquired using transgenic mouse models, there is still a shortage of mouse mutants to study basic mechanisms of immunity and infection in more detail. In this workpackage we will address key responses of the immune system in EUCOMM mouse mutants using a battery of phenotyping assays for different innate and adaptive immune functions. The host defence against bacterial pathogens will be tested at the HZI and CNRS using in vivo infection challenge with Yersinia enterocolitica (Y.e.), Listeria monocytogenes (L.m.) and Pseudomonas aeruginosa (P.a.). Using these diverse mouse infection models, the host reaction against extracellular, Gram-negative bacteria (Y.e. & P.a.) and intracellular Gram-positive L.m. will be tested. Phenotyping efforts will focus on host response mechanisms in different organ systems (Y.e., intestinal mucosa; P.a., lung epithelium; L.m., systemic infection). Infection with Plasmodium berghei, a pathogen causing cerebral malaria in mice, will be performed to investigate the host-defence against parasites. With these different classes of pathogens distinct host reactions (innate, adaptive, Th1 versus Th2 cell responses) will be evaluated that are important for many infectious diseases (pathogen induced enterocolitis, pneumonia & malaria). At Ani.Rhone-Alpes a contact hypersensitivity test will be undertaken that addresses deregulated inflammatory responses in mutants that might be caused by defects in innate (NK, mast cell) or adaptive (CD4 & CD8 T cells) immune responses. In addition, the production of autoantibodies, and the composition of lymphocytes in primary and secondary lymphoid organs will be evaluated. In summary, the 2° line phenotyping screens in WP2.4 will detect defects in immune cell development, immune cell effector functions and immune cell homeostasis in EUCOMM mutants that might be very relevant for different human immune / infectious diseases.

WP2.5 Behaviour, cognition & nervous system

Almost every aspect of human personality, temperament, cognitive function and psychiatric dysfunction is dependent on genetic and epigenetic factors. Effects of heritability account for 30 to 70% of the total variance and are highly reproducible between societies and cultures [6]. Psychiatric disorders of childhood onset like Autism, Mental Retardation and Attention Deficit Hyperactivity Disorder (ADHD) are believed to result from abnormalities in brain development [1,5,7].

Most adult neuropsychiatric illnesses have complex phenotypes and are apparently polygenic. There is a considerable overlap of symptoms between diseases, e.g. cognitive deficits can occur in Autism, Schizophrenia, ADHD, Mental Retardation, Dementia and Parkinsonism. Thus, to understand the genetic factors underlying psychiatric diseases, it is most useful to dissect complex phenotypes into components, called endophenotypes [8]. A neurobiological or physiological characteristic, e.g. a sensorimotor gating deficit, may occur as the result of single gene effects. Therefore our strategy is to apply a range of specialised, well established neurobehavioral, neurophysiological and neuroanatomical analytic methods,, which are known to be indicative of endophenotypes associated with neurological and psychiatric diseases like Anxiety and Mood disorders, Parkinsonism, Mental Retardation and Dementia, Epilepsy, Schizophrenia, Autism and ADHD [2-4].

These analyses will be performed on selected behaviourally conspicuous mutant lines detected in the primary screen. We will analyse sufficient numbers of mutants and littermate control mice of both sexes.

WP2.6 Sensory

Audition and balance

Hearing loss is the most commonly occurring handicap in humans. About one in 1,000 children are affected by severe deafness at birth and up to 60% of these cases are attributable to genetic defect. Because the auditory system of mice and humans is conserved, studies using mouse models have been able to predict several human deafness genes. Progressive sensorineural hearing loss is now found to be associated with some specific genes and a number mouse models have so far been found. 

Due to the broad similarity between the human and mouse auditory system, analysis of auditory mouse mutants has provided both valuable insights in to the function of the mammalian inner ear and has contributed to the identification of candidate deafness-causing genes in humans.  For the testing of auditory function in mice we are planning to use functional tests routinely performed in clinical environments. Depending upon the level and the type of impairment found in mutant mice, classical histology will be performed on the inner ear, supplemented when necessary by a sophisticated battery of morphological and immunohistochemical assays that are used in most laboratories for deciphering the relation between mutations and deafness phenotypes.

With the functional tests employed, it will be possible to find the functional consequence of a mutation, either a conductive or sensory impairment or both. Genes may affect inner ear development, and the inner ear will be investigated by appropriate histological tests as well as a 3D reconstruction of the embryonic ear in order to observe the target(s) by a gene of a specific component of the inner ear. The pathological effects of a gene can be followed up to cellular level by appropriate assays (electron microscopy) or at the protein level by immunohistochemistry.

Vision

Eye disease is common. Approximately 50 million people worldwide are estimated to be blind, and three times that number to have significant visual impairment. Genetic factors play a role in most if not all eye disease, with the genetic component ranging from 100% to a minor fraction. All mutants will be examined in WP1 by slit lamp biomicroscopy and indirect ophthalmoscopy at age 12 weeks. Those in which defects are observed will be subject to longitudinal study under WP2.6. Eyes will be examined from weaning (3 weeks) to 26 weeks to observe onset and progression of the defect. Histopathology will be performed on eyes from critical stages.

Mice do not normally have a strong reliance on visual cues, and so tests for vision need to be robust. The optokinetic response (OKR) is an involuntary response to a moving grating that is shown by all vertebrates and is a good assay in the mouse not only for vision per se, but also for visual acuity. Mutants with retinal defects in particular will be tested for OKR and visual acuity assessed.

Distortion of the globe of the eye is an indicator of raised intraocular pressure (IOP); unlike humans the mouse globe is plastic and distorts under increased IOP. Mutants in which globe distortion is observed, either through slit lamp examination or the dysmorphology screen, will be tested for altered IOP by tonometry. The key feature of glaucoma, caused by raised IOP, is cupping of the optic nerve head, indicating nerve damage. Those mutants that show globe distortion will undergo histological examination of the optic nerve to assess damage.

WP2.7 Skelotomuscular

In human, skeletomuscular diseases encompass a series of defects including developmental defects and more chronic and progressive disorders, such as osteoporosis or rheumatoid arthritis. With an incidence of 20% in the adult population and a higher prevalence in women and in older age groups, they represent a major cause of physical disabilities worldwide (World Health Report 2001). A direct consequence is a huge burden corresponding to direct and indirect long-term disability and morbidity costs.

WP2.7 focuses on skeletomuscular defects in order to highlight the genetic origin of these diseases using the mouse as a model organism. WP2.7 will provide a more detailed phenotypic analysis of mouse mutants with altered bone parameters found in the primary screen for the use as model systems for human bone and cartilage related diseases. We will concentrate our effort on four classes of disorders which include: 1) developmental patterning defects (e.g. polydactylism and syndactylism), 2) metabolic and growth defects (e.g. osteogenesis imperfecta, osteomalacia), 3) modelling and remodelling defects (e.g. osteoporosis, osteopetrosis), and 4) aging and immune system defects (e.g. arthritis, sponsirosis, ruptured disks). Mouse mutants will be intimately analyzed for medical relevant bone and cartilage parameters using a variety of techniques from bone radiography analysis to cellular assays.

The secondary tests include a more refined bone structure analysis by volumetric monitoring of bone density, mass and architecture of appendicular skeleton and tail vertebra. This will be achieved by µCT (micro computed tomography), [see SOP at EMPReSS website and refs. 6,7,10,12] and pQCT (peripheral quantitative computed tomography) analysis [2,4,5,8] and high resolution bone analysis by bone scintigraphy which might result in new models for human osteoporosis, osteopenia, osteogenesis imperfecta, scoliosis or osteoarthritis. Plastic deformation and fracture analysis by three point bend test [3,9] might lead to new models for human osteomalacia or rickets. Lines that display a phenotype in the first assays will then be further analysed to provide dynamic information on bone metabolism. This might result in new models for defects in development and homeostasis of bone and cartilage (bone formation and resorption). These tests include histomorphometry for bone formation rate and resorption areas, biochemical markers of bone metabolism [1,13], skeletal preparation [see SOP at EMPReSS website, see ref. 11]. To characterise defaults affecting bone cells (osteoblast and osteoclasts) in vitro studies will be performed.

WP2.8 Pathology & cancer

Inbred strains are the raw material for the generation of genetically engineered mice that have become indispensable tools for cancer research, and for the identification of genes involved in human diseases. The systematic morphological analysis of mouse models in the Pathology and Cancer phenotyping work package aims: 1) to support the discovery of new genes and genes´ functions, 2) to disclose the pathways and processes through which these genes influence the development of human diseases, and 3) to validate a mouse model as representative of a specific human disease.  Comparative mouse to human validation in cancer is an unavoidable step. These cancer models will further permit us to study the consecutive genetic steps involved in the initiation and progression of cancer, to identify tumor-cell of origin, to define markers for early diagnosis, and to learn about potential molecular targets for a therapeutic approach or test new tumor intervention strategies [1]. A high quality histopathological assessment, together with the combination of the most modern ancillary techniques used for human pathology, such as tissue arrays, immunohistochemistry (IHC), Comparative Genomic Hybridization (CGH), Spectral Karyotyping analysis (SKY) and expression array analysis, were selected to help in the interpretation of mouse models and to ease the comparison with their human counterparts. Immunohistochemistry (IHC) is essential for the morphological subclassification of neoplasias [2,3] which is the basis for the analysis of the genetic alterations that predispose to cancer. Additionally, IHC is also crucial to decipher the signalling pathways of normal development and differentiation, whose alterations can lead to human diseases [4]. Clonal analysis of lymphoid proliferations can help to understand the early events of lymphomagenesis [5]. The analysis of chromosomal changes with CGH and SKY has helped to understand the complex haplotypes in the most widely used inbred lines, as well as, the chromosomal aberrations in cancer models [6-8]. In the three-year period, we plan to analyze 10-15 cancer models and 10-15 interesting mouse models of human diseases derived from secondary screens.  

In vivo imaging

Medical in vivo imaging is being increasingly employed in small animals in order to provide anatomical, functional and molecular information in living animals and in humans. A major benefit of implementation of medical in vivo imaging tools is the reduction in the number of mice used in technical experiments. A wide range of imaging approaches are available including ultrasound, X-ray imaging, MRI, TEP, SPECT and optical (bioluminescence, fluorescence, confocal). Therefore, imaging is an increasingly important approach for the biologist to facilitate the validation of animal models of human diseases, and the evaluation of therapeutic strategies. Since genetically-modified mice are a major tool for drug development for humans, medical imaging will facilitate the translation of functional information in transgenic mice to human systems.

We are proposing several different validated tools which will be used in WPs such as musculo-skeletal, cardiovascular and oncology. Within this WP, we also aim at implementing a database of newly developed in vivo imaging methods within and outside the network which is available for biologists. This database will provide information on the available methods, the obtained parameters and their normal values, the standardized conditions for the image acquisition and analysis.