Aula-MIMV-teorica-22-11-2024 PDF

27/11/2024 Mestrado Integrado em Medicina Veterinária MIMV – ICBAS Unidade curricular de Citogenética Animal Lucia M.W.G. Lacerda, Ph.D. Prof. Auxiliar Convidada ICBAS-Universidade do Porto Unidade de Bio...

27/11/2024 Mestrado Integrado em Medicina Veterinária MIMV – ICBAS Unidade curricular de Citogenética Animal Lucia M.W.G. Lacerda, Ph.D. Prof. Auxiliar Convidada ICBAS-Universidade do Porto Unidade de Bioquímica Genética, CGM - Centro de Genética Médica Jacinto Magalhães, CHUdSA - Centro Hospitalar e Universitário de Santo António [email protected] Clinical and Experimental Human Genomics group, UMIB-ICBAS-UP - Unidade Multidisciplinar de Investigação Biomédica (Unit for Multidisciplinary Research in Medicine) [email protected] 1  27/11/2024 Subject Approach/Study Objective To use the online knowledge database for research as a Survey prior literature and OMIA - Online Mendelian catalogue/ compendium of research findings, looking for Inheritance in Animals inherited disorders, other (single- trusted resources, peer-reviewed locus) traits, and associated genes research articles and variants in 588 animal species Presentations, 3 students/working group 1. Mouse 2. Rat 3. Rabbit Uses in Research Commonly Used Animal Models 4. Zebrafish Normative Biology (Principles of Animal Research for Graduate and 5. Amphibians and Reptiles Undergraduate Students. 2017 6. Birds Reproductive Physiology http://dx.doi.org/10.1016/B978-0-12-802151- 7. Other Small Mammals: Behavior 4.00007-4) Guinea Pigs Housing and Handling Hamsters Chinchillas Diseases Gerbils Armadillo Animal disease models  Animal disease models are non-human animals bearing pathologies that share features with a human disease  Disease models can either exist naturally or be induced artificially and are used for studying human disease with an experimental system 2  27/11/2024 Until now, many progresses in medical sciences have been achieved: Using animal  The discovery of numerous types of antibiotics for controlling models infectious disease and elimination some viral diseases like smallpox might be considered as one of researcher and indeed experimental animal's honor. associated with other  The development of blood transfusions, open heart surgery, and experimental models other life-saving techniques. gives an opportunity to There are many unsolved subjects including cancer, aging, accomplish many Alzheimer’s disease, and acquired immunodeficiency syndrome in findings in human front of the society. It is pivotal to: medicine  Use living animals in scientific research  Have ethical concerns over the animal's quality of life Animal models for human disease. 2020 I. Human diseases: in vivo and in vitro models, Animal Biotechnology. DOI: https://doi.org/10.1016/B978-0-12-811710-1.00008-2 The use of laboratory animals in research is of major ethical concern Guidelines endorse some essential principles for the care and use of animals for scientific projects: Replace animals with other methods such as mathematical models, computer simulations and in vitro biological systems Reducing the number of animals used to obtain valid results without unnecessary duplication Refining projects by selecting appropriate species and techniques to minimize pain or distress to animals using appropriate sedation or anesthesia 3Rs (Replacement, Reduction and Refinement) There is a wide spectrum of views on animal rights 3  27/11/2024  Mouse  Rat  Rabbit  Zebrafish  Amphibians and Reptiles Commonly Used  Birds Animal Models  Other Small Mammals: Guinea Pigs Uses in Research Normative Biology Hamsters Reproductive Physiology Chinchillas Behavior Gerbils Housing and Handling Armadillo Diseases Commonly Used Animal Models, Principles of Animal Research for Graduate and Undergraduate Students. 2017 http://dx.doi.org/10.1016/B978-0-12-802151-4.00007-4 Human neurodevelopmental disorders Direct cellular and molecular investigation of human brain cells cannot be undertaken on living patients affected by these disorders. Post-mortem and animal studies have provided substantial knowledge and important insights into human brain biology and pathology, Animal models can be both genetically and pharmacologically manipulated,  Not always able to recapitulate the human phenotype, in particular when behavioral, affective and cognitive changes are the principal phenotypes of the disorder 4  27/11/2024 Chromossomal and segmental aneuploidy Aneuploidy was defined as a deletion or duplication of a whole chromosome. This genomic abnormality is thought to occur in at least 5% of all clinically recognized pregnancies, usually resulting in spontaneous abortion Aneuploidy is thought to be usually highly deleterious because many genes are “dosage-sensitive” in that their expression is affected by their copy number in the genome, and changes in gene expression levels may result in altered phenotypes that can be lethal As well as whole chromosome aneuploidy, deletion of a few kilobases or megabases of DNA (microdeletion) or similarly a duplicated region (microduplication) within a chromosome can also result in changes in gene copy number Recent advances in genomic technologies have revealed the association of many of these segmental aneuploidies (microdeletions and duplications) with specific genetic syndromes and diseases Creating and studying MOUSE MODELS of aneuploidy and copy number variation Many aneuploidies are lethal Alterations in the copy Abnormalities of because the resulting number of sections of chromosome copy number abnormal gene dosage is chromosomes, which are are called aneuploidies and highly deleterious. also known as segmental make up a large health load Nevertheless, some whole aneuploidies, are also on the human population chromosome aneuploidies associated with deleterious can lead to live births. effects Models provide a whole Research in mouse models is animal system to investigate highlighting possible the complex phenotype- therapies that might help genotype interactions that alleviate the cognitive arise from alteration in the effects associated with copy number of genes changes in gene number 5  27/11/2024 Mouse models of Down syndrome Mouse models The ScientificWorld Journal, Volume 2012, of segmental doi:10.1100/2012/214078 Review Article Mouse Models of Aneuploidy, Sheppard,et al. aneuploidies 6  27/11/2024 Clinical features of mouse model segmental aneuploidies Mouse models of Prader-Willi syndrome (PWS) (deletion of paternal 15q11–13) and Angelman syndrome (AS) (deletion of maternal 15q11–13). PWS is also associated with chromosome 15 maternal disomy and AS with paternal chromosome 15 disomy:  Mouse models of these genetic changes have been reported, and both exhibit reduced viability and neonatal growth retardation.  Deficits in learning and memory have also been observed in a mouse model with a maternally inherited segmental deletions (Ube3a-Gabrb3) corresponding to part of the region lost in AS.  Mouse models deficient in the Ube3a and Gabrb3 PWS/AS candidate genes exhibit neurodevelopment and behavior changes, highlighting the key role these genes play in the syndromes.  Interestingly, maternal duplications of the PWS/AS associated region, 15q11–13, are associated with autism. Clinical features of mouse model segmental aneuploidies Mouse models of DiGeorge syndrome (deletion of 1.5–3Mb at 22q11) have been crucial to the molecular understanding of this condition.  A series of complementary mouse models with full or partial deletions of the region syntenic with 22q11 identified the key deleted gene, Tbx1, responsible for the syndrome’s deleterious phenotypes.  The 22q11 deletion is also the largest known genetic risk factors for schizophrenia, and the DiGeorge (DS) mouse models may also be useful to further understanding of this condition. Mouse models of the complete 3.7Mb deletion and duplication associated with Smith-Magenis (SMS) and Potocki-Lupski (PTLS) syndromes have helped identify one of the key dosage sensitive genes, Rai1. These models have also been used to investigate the relative effect of genomic rearrangement versus gene copy number change on gene expression. 7  27/11/2024 Structural variations copy number variants (CNVs) The human genome, with its enormous structural complexity, goes through a rapid evolution due to structural variations (SV), which contribute to extend the genetic diversity among individuals and generations. Structural variations can include genomic imbalances or inversions and balanced translocations: Insertions and deletions, which are generally found in a region of DNA measuring 1 kb or longer in size. Inversions, reciprocal translocations or copy-number-neutral insertions, which are balanced rearrangements and there is no loss or gain of genetic material. When these SV determine genomic imbalances, known as copy number variants (CNVs), they alter the euploid status of DNA by changing the copy number of chromosomes or chromosomal regions. The first studies on CNVs were carried out on engineered mice models, using the most frequently occurring CNVs, thus without showing the variability present in humans. Opposite roles of CNVs: EVOLUTION AND DISEASE Copy number variants (CNVs) can contribute to evolutionary trigger mechanisms as well as to the susceptibility and pathogenesis of several diseases Int. J. Mol. Sci. 2020, 21, 1860; doi:10.3390/ijms21051860 www.mdpi.com/journal/ijms 8  27/11/2024 Examples of disease phenotypes caused by the presence of copy number variants (CNVs) in coding and non-coding regions CNVs in coding regions could cause an alteration of: (a) copy number of dosage-sensitive genes (duplications and deletions) (b) genes involved in signal transduction pathways CNVs in non-coding regions could interfere with: (c) the positioning of cis-regulatory elements (deletion and duplication of enhancer or silencer elements) (d) the higher-order chromatin organization such as the structure of topologically associating domains (TADs) (deletion and duplication of boundaries). Int. J. Mol. Sci. 2020, 21, 1860; doi:10.3390/ijms21051860 The role of CNVs is controversial in neurodevelopmental/neuropsychiatric diseases The question is usually approached by analyzing whether: Consulting databases for correlations between CNVs and development of specific diseases: it is a de novo gene Schizophrenia mutation Size and content Autism Spectrum disorders or type (deletion and and Epileptic Encephalopathy duplication), Intellectual disabilities it is inherited, number Attention-deficit/hyperactivity disorder 9  27/11/2024 Their incomplete penetrance and variable expressivity make diagnosis difficult and hinder comprehension of the mechanistic Cutting-edge human bases of these disorders. genetics technology Additional elements such as co-presence of other CNVs, has allowed to identify genomic background and environmental factors are involved in determining the final phenotype associated with a CNV. copy number variations (CNVs) and has provided new insights for Genetically engineered animal models are helpful tools for understanding the behavioral consequences of CNVs. However, understanding causative the genetic background and the biology of these animal model systems have sometimes led to confusing results. mechanisms of human diseases New cellular models obtained through somatic cellular reprogramming technology that produce induced pluripotent stem cells (iPSCs) from human subjects are being used to explore the mechanisms involved in the pathogenic consequences of CNVs. Células estaminais pluripotentes induzidas iPSCs  A obtenção de células estaminais pluripotentes induzidas a partir da reprogramação directa de células somáticas foi um dos avanços mais importantes da biologia nos últimos anos.  As iPSCs são células somáticas que foram reprogramadas para um estado de pluripotência através da introdução de um conjunto de factores específicos.  Podem ser obtidas a partir de diferentes tipos de células, como os fibroblastos, queratinócitos, hepatócitos e células sanguíneas.  As iPSCs são similares às células estaminais embrionárias, quer na morfologia e na expressão de marcadores de pluripotência, como também na capacidade de desenvolver teratomas.  Como células estaminais pluripotentes podem diferenciar-se em todas as linhagens celulares, incluindo neurónios, células do sangue e células do coração. 10  27/11/2024 induced Pluripotent Stem Cells (iPSC) lines  Obtaining induced pluripotent stem cells from direct reprogramming of somatic cells is one of the most important biology advances in recent years.  The iPSCs are somatic cells that have been reprogrammed to a pluripotent state by introducing a number of specific factors.  These cells can be obtained from different types of cells, such as fibroblasts, keratinocytes, hepatocytes and blood cells.  iPSCs are similar to embryonic stem cells, both in morphology and expression of pluripotency markers, but also on the ability to develop teratomas.  As pluripotent stem cells, they can differentiate into all cell lines, including neurons, blood and heart cells. Mouse models and induced pluripotent stem cells (iPSC) lines carrying CNVs of chromosome 15q11-q13 In past years, a strong effort has done into developing mice models of neurodevelopmental/neuropsychiatric disorders, in the attempt to define behavioral assays reflecting their core symptoms—anomalies in social interaction, in communication, repetitive behavior and others. An increasing number of CNVs associated with disorders has been replicated in transgenic models; however, these systems have a questionable validity either by virtue of simply being non-human or because they are missing the genetic background typical of affected human subjects. On the left, mouse models syntenic to human chromosome 15q11-q13; on the right, iPSC lines obtained from patients carrying 15q11-q13 CNVs. Int. J. Mol. Sci. 2020, 21, 1860 11  27/11/2024 These patient-specific induced pluripotent stem cells (iPSCs) present several important advantages over other in vitro systems  Maintain both the primary genetic lesion and the genetic background from human individuals with confirmed diagnoses of CNV-dependent disorders;  Facilitate the study of larger deletions in complex genes containing internal promoters or polygenic cases which are currently difficult to engineer using genome editing technologies; Have changed the approach to studying neurodevelopmental/neurodegenerative CNV-dependent syndromes: In mouse models the effects of the CNVs were analyzed through behavioral studies associated with the anatomical aspects of the organs affected by the disease, whereas in the iPSCs, what is studied is the function of the associated genes, hence the therapy, rather than focusing on reversing behavioral aspects, will be based on targeting, reducing or increasing single proteins/enzymes/RNA which are functionally impaired because of the CNVs. Human stem cell models offer an opportunity to directly study the molecular and cellular mechanisms of diseases. iPSCs These are generated by reprogramming of somatic cells into pluripotent stem cells from which many cell types can be differentiated, including human- neurons and glial cells. induced Importantly, they can be easily obtained in the clinic from fibroblasts (skin biopsies), keratinocytes (hair roots), T lymphocytes (peripheral blood) and exfoliated renal epithelial cells from urine samples. Pluripotent Importantly, patient iPSCs enable the in vitro study of different cells types in isolation or co-culture in order to investigate cell function. Uniquely Stem Cells they can track the development profile of patient cell differentiation. derived from More recently the capacity of iPSCs to form 3D organoids has opened the possibility to investigate the interaction of multiple cell types in a more brain-like microenvironment. Methods for increasing reproducibility of brain patient cells organoid differentiation are improving substantially and being exploited to mechanistically dissect the effect of genetic lesions causing Autism Spectrum Disorders and Intellectual Deficit, as well as the role of specific genes and molecular modules key to human-specific neuronal differentiation trajectories and pathophysiology. 12  27/11/2024 Somatic cells can now be reprogrammed into pluripotent stem cells from a simple patient biopsy This technology allows researchers to:  use human neurons in vitro,  derived specifically from patients suffering from a specific disorder,  recapitulating the exact developmental events that are abnormal during the onset and the progression of the disorder. Copy number variants (CNVs): a powerful tool for iPSC-based modelling of ASD, Drakulic et al. Molecular Autism (2020) 11:42 https://doi.org/10.1186/s13229-020-00343-4 Natural and synthetic embryos side by side show comparable brain and heart formation. [Amadei and Handford] https://www.cam.ac.uk/storie s/model-embryo-from-stem- cells 13  27/11/2024 OMIA Nov-2024 Online Mendelian Inheritance in Animals (OMIA) is a catalogue/compendium of: inherited disorders, other (single-locus) traits, associated genes and variants in 588 animal species (other than human, mouse, rats, zebrafish and western clawed frog, which have their own resources) OMIA ID is binomial i.e. OMIA: xxxxxx-yyyy.., where: xxxxxx is the 6-digit number for a trait/disorder yyyy.. is the NCBI species taxonomy id (usually four digits, but sometimes longer) Inborn errors of metabolism are caused by genetic defects in Inherited intermediary metabolic pathways. metabolic The diagnosis of diseased animals can be achieved by searching for abnormal metabolites in body fluids, although such screening disorders (IMD) programmes have, until now, not been widely available to the small animal clinician. in companion A comprehensive battery of analytical tools exists for screening animals: for inborn metabolic diseases in humans which can be applied to animals and serve not only for the diagnosis of affected patients but also to detect asymptomatic carriers and further our understanding Searching for of metabolic pathways in dogs and cats. nature’s Moreover, naturally occurring animal models of inherited metabolic diseases provide a unique opportunity to study the mistakes biochemical and molecular pathogenesis of these disorders and to investigate possible therapeutic options. Vet J. 2007 September ; 174(2): 252–259. doi:10.1016/j.tvjl.2006.08.017 14  27/11/2024 The effect of an inherited enzyme defect in intermediary metabolism The accumulation of toxic metabolites (organic acids) and a decreased substrate for biosynthetic pathways give rise to clinical signs and energy deficiency Vet J. 2007 September ; 174(2): 252–259. doi:10.1016/j.tvjl.2006.08.017 The presence of an IMD in veterinary patients is still considered rare, but because of the common practice of inbreeding, some IMD have reached a high frequency in some breeds Inherited metabolic diseases have always been a domain of paediatric medicine; however, an ever-increasing number of descriptions from the animal kingdom have made it quite clear that IMD should also be considered in veterinary patients. This was recognized by Patterson, Jezyk, and colleagues in Philadelphia and led to the establishment of the Metabolic Genetics Disease Laboratory at the University of Pennsylvania. About 5000 inherited diseases are known in dogs and cats: Online Mendelian Inheritance in Animals http://omia.angis.org.au; Cambridge Inherited Disease in Dogs Database, http://www.vet.cam.ac.uk/idid) 15  27/11/2024 Clinical signs may be relatively non-specific in children and animals Generalised Growth Weight Vomiting Lethargy Seizures Dyspnea Skin skeletal corneal weakness retardation loss or abnormalities malformations clouding lack of (hair loss) and other weight ocular gain changes Are the clinical signs of an Inherited Inborn Error Metabolism in dogs and cats similar to those of affected children? Common presenting signs of acute inherited metabolic disease The classical life-threatening clinical presentation of acute May appear in affected animals, metabolic disease in neonates and but many may die before a young children (coma, metabolic definitive diagnosis acidosis, ketosis, encephalopathy, liver failure) The onset of clinical signs usually occurs early in life although some, such as some inherited eye Such signs may be part of the diseases, mucopolysaccharidosis puppy/kitten mortality complex type III, and Fanconi syndrome, do not appear until adulthood Vet J. 2007 September ; 174(2): 252–259. doi:10.1016/j.tvjl.2006.08.017 16  27/11/2024 A common presenting sign is failure-to-thrive as affected Common animals lag behind healthy litter-mates in their development, and they do not gain weight at a normal rate. presenting signs of Signs of metabolic (organic) acidosis (lethargy, coma, acute vomiting, anorexia) are likely to occur, but may not be recognized as such. Occasional reports of ‘classical’ organic acidurias (lactic aciduria and methylmalonic aciduria) in metabolic dogs with predominantly neurological signs. disease in affected Isolated methylmalonic aciduria is characteristic of vitamin B12 malabsorption in which patients present with a triad of failure-to-thrive, lethargy, and anorexia. If untreated, animals patients develop neurological signs of tremor, anxiety and convulsions. Examples of inherited neurodegenerative disease in companion animals Vet J. 2007 September ; 174(2): 252–259. doi:10.1016/j.tvjl.2006.08.017 17  27/11/2024 Inherited metabolic disease in companion animals Many inherited hematological disorders have been elegantly characterized and described, and functional or DNA carrier testing is available for some including bleeding disorders, immunodeficiencies, and erythrocyte defects Inherited diseases resulting from defects in the catabolism of proteins and complex carbohydrates represent classical examples of IMD; however, mention is also made of other disorders resulting from defects in the transport of intermediary metabolites. On the basis of relatively discrete clinical signs and simple metabolite analyses, a differential diagnosis list can be made, and further enzyme and molecular evaluation can lead to a definitive specific diagnosis. 18