Inativação do cromossoma X: implicações na patologia humana

2020/2021 Guilherme José Ruivo Pereira Inativação do cromossoma X: implicações na patologia humana X-Chromosome Inactivation: implications in human disease ABRIL 2021 Mestrado Integrado em Medicina Área: Genética Tipologia: Monografia Trabalho efetuado sob a Orientação de: Doutora Sofia Dória Príncipe dos Santos Cerveira Trabalho organizado de acordo com as normas da revista: Journal of Genetics published by the Indian Academy of Sciences Guilherme José Ruivo Pereira Inativação do Cromossoma X: implicações na patologia humana X-Chromosome Inactivation: implications in human disease ABRIL 2021 Dedicatória Aos meus pais Marcelo e Isabel, que deram todo o apoio que puderam (e até demasiado) durante o curso, a nível de tempo e disponibilidade, nos momentos bons e nos menos bons, física e emocionalmente. Ao meu irmão Henrique, pela paciência que teve, tanto ao compartilhar casa durante os últimos três anos, aguentando a minha falta de organização, como pelos momentos embaraçosos que passou por minha culpa. Aos meus avós José, Maria e Manuel, pelo suporte que sempre deram, quando foi preciso e quando não foi preciso, e por estarem presentes durante todo este tempo. À minha avó Fernanda, que foi a grande razão pela qual escolhi este curso, e que me acompanhou e ajudou a partir de um lugar melhor. X-Chromosome Inactivation: implications in human disease Running title: X-Chromosome inactivation in human disease Submission date: February 23, 2021 Authors: Guilherme Pereira and Sofia Dória Guilherme JR Pereira, Faculty of Medicine of the University of Porto, Portugal; [email protected]; +351 965868417 Sofia Dória (corresponding author), Department of Pathology, Genetics Service, Faculty of Medicine of the University of Porto, Portugal and I3S - Instituto de Investigação e Inovação em Saúde, University of Porto, Portugal; [email protected];+351220426715 Keywords: X-Chromosome inactivation, X-linked diseases, XIST, skewed inactivation, X chromosome rearrangements Abbreviations: XCI (X-Chromosome Inactivation), Xic (X inactivation center), XIST (X-inactive specific transcript), Xi (inactive X chromosome), Xce (X controlling Word Count: 2380 Number of tables: 0 Number of figures: 0 Number of references: 27 Conflicts of Interest: No conflicts of interest to be declared. Author contribution: Guilherme Pereira was responsible for the literature review, drafting and revision of the paper. Sofia Dória was responsible for the critical revision and for the final manuscript editing. 2 Abstract X-Chromosome Inactivation (XCI) is a process involved in the pathogenesis of several diseases. In this mini review we discuss the known mechanisms associated with XCI, when and how it initiate, spreads and maintain, as well as the mechanisms that allow some genes to escape from it. We address skewed XCI, the condition that happen when this process is not fully randomized, and its consequences to the phenotype of some pathologies. We debate about the known pathologies implicated, including X unbalanced rearrangements, X-autosomal balanced translocations, Turner and Klinefelter syndromes and also for X-linked diseases and his consequences for males and females. Some pathologies were discussed more in detail such as intellectual disability with a recognize relation with XCI. Finally, possible future implications for genomic therapy and treatment of patients and list areas that need further research on this topic were addressed. 3 Introduction In humans, female cells carry two X-chromosomes, whereas male cells have only one X chromosome plus a Y chromosome. Y chromosome contains less genetic material (approximately 70 genes) than the X chromosome (approximately 900-1500) (Disteche 2016). This contributes to a big imbalance between men and women regarding genes, imbalance that has two mechanisms for compensation: X chromosome upregulation or X chromosome inactivation (XCI) (Fang et al. 2019). Focusing on XCI, the first hypothesis was formulated by Mary Lyon in 1961, and it consisted on “that mosaic phenotype is due to the inactivation of one or other X chromosome early in embryonic development” (Disteche and Berletch 2015). This reasoning was proven to be accurate, first based on mice, and later confirmed as a law in all mammals. The so-called Lyon Law is still the most important contribute to this topic, more than 50 years after its formulation. It happens during early female embryonic development (Monk et al. 1987; Disteche and Berletch 2015) and it starts shortly after embryo implantation (Fang et al. 2019). The cells lineage maintain the initial chosen chromosome (either the paternal or the maternal X chromosome) throughout all the development till the adult age (Fang et al. 2019) while the other is silenced resulting in the so-called Barr body (Galupa and Heard 2018), a condensed heteropycnotic structure in the interphase nuclei of female cells (Disteche and Berletch 2015). This provides dosage compensation for eutherians between the sexes (Dixon-McDougall and Brown 2016). Logically, XCI randomness creates a coexistence between both chromosomes activated of an approximate ratio of 1:1. This is extremely relevant to phenotypic changes, as mutations that could lead to serious complications in males are compensated by another chromosome in 50% of the female cells (Cantone and Fisher 2017). This process of randomness follows a normal distribution, what almost mandatorily creates examples of very unbalanced ratios. This process is called “skewing of XCI”, and the extreme cases (over 90% of the cells with inactivation of the same chromosome) skewed X-chromosome inactivation (Cantone and Fisher 2017). Non-random inactivation also exists in special cases where a group of cells has a proliferative advantage (or disadvantage) based on Xchromosome mutations, what causes one to outgrow the other. 4 It is also important to refer that XCI is not a permanent state, as it can be reversed (Payer 2016). It is physiologic for example when it happens on haploid cells (like the spermatozoid – the paternal X is silenced till fertilization happens), but it can also be pathological (in some congenital or acquired diseases) (Disteche and Berletch 2015). Age-related X reactivation may also happen, and that this process could be related with neurological and oncological problems (Leppig et al. 2001). Methods To write this mini-review, two searches (first on 20/07/2020 and second on 23/02/2021) were conducted in PubMed, Scopus and Cochrane Library, including all articles written in English, Spanish and Portuguese. The searched was made using the following query: (X-chromosome inactivation [MeSH Major Topic]) OR (Lyon Hypotesis [MeSH Major Topic]). Inclusion criteria included studies made in humans and related with pathology while exclusion criteria included studies made in animals. Year of publication from the articles was not an exclusion criteria, but most recent studies were treated as more relevant. XCI regulation and function XCI comprises a series of highly organized events that result in the transcriptional silence of one of the two female X chromosomes. The process starts with the counting and choosing of the X-chromosome that will be silenced followed by three steps referred usually as: XCI initiation, XCI spreading and XCI maintenance. The initiation of XCI is controlled by a regulatory locus named X-inactivation centre (Xic) that contains clustered genes and regulatory sequences involved in the inactivation process. It is defined as the region that is necessary and sufficient to trigger XCI when present in two copies (Galupa and Heard 2018). The proximal limit of the human Xinactivation center (XIC) has been localized between Xq13.1 and Xq13.2 chromosome region. This region was found to have a gene that encodes noncoding RNA (IncRNA), named Xinactive specific transcript (XIST) that is transcribed only from the future inactive X chromosome (Xi). The process of silencing is still largely unknown but according to 11 Monk M, Boubelik M, Lehnert S. Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development. 1987;99(3):371-82. Monkhorst K, Jonkers I, Rentmeester E, Grosveld F, Gribnau J. X inactivation counting and choice is a stochastic process: evidence for involvement of an X-linked activator. Cell. 2008;132(3):410-21. Payer B. Developmental regulation of X-chromosome inactivation. Semin Cell Dev Biol. 2016;56:88-99. Pinter SF, Sadreyev RI, Yildirim E, Jeon Y, Ohsumi TK, Borowsky M, et al. Spreading of X chromosome inactivation via a hierarchy of defined Polycomb stations. Genome Res. 2012;22(10):1864-76. Plenge RM, Stevenson RA, Lubs HA, Schwartz CE, Willard HF. Skewed X-chromosome inactivation is a common feature of X-linked mental retardation disorders. Am J Hum Genet. 2002;71(1):168-73. Posynick BJ, Brown CJ. Escape From X-Chromosome Inactivation: An Evolutionary Perspective. Front Cell Dev Biol. 2019;7:241. Sierra I, Anguera MC. Enjoy the silence: X-chromosome inactivation diversity in somatic cells. Curr Opin Genet Dev. 2019;55:26-31. Shoukat HMH, Ghous G, Tarar ZI, Shoukat MM, Ajmal N. Skewed Inactivation of X Chromosome: A Cause of Hemophilia Manifestation in Carrier Females. Cureus. 2020;12(10):e11216. Sun Y, Luo Y, Qian Y, Chen M, Wang L, Li H, et al. Heterozygous Deletion of the SHOX Gene Enhancer in two Females With Clinical Heterogeneity Associating With Skewed XCI and Escaping XCI. Front Genet. 2019 ;10:1086. Yamoto K, Saitsu H, Fujisawa Y, Kato F, Matsubara K, Fukami M, et al. 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Indian Academy of Sciences, publisher of the journal, will acquire copyright over all published material. Authors may reproduce their published material elsewhere subsequently with the usual acknowledgement to ‘Journal of Genetics, published by Indian Academy of Sciences, Bengaluru’ and the volume and page details, but a request to do so will be appreciated and also serve to keep the editorial office informed. Third parties who wish to reproduce published material should write to the editorial office for permission. Scale for the Assessment of Narrative Review Articles Justification of the article’s important for the readership – 2 Page 2 – “X-Chromosome Inactivation (XCI) is a process involved in the pathogenesis of several diseases (…). We debate about the known pathologies implicated” Pages 8/9 – “It’s also relevant to refer a problem to be solved: usually all the studies were done with small cohorts, and there is a little amount of bibliography related to this topic. Moreover, every data should be taken carefully, and more studies with bigger cohorts should be performed, to distinguish which diseases are and which aren’t related with XCI, and what future treatments could be applied in that cases.” Page 9 – “XCI understanding, namely how it starts, spreads, maintains, and how escape from it happens can help us finding genomic treatments that will certainly be fundamental on the many diseases that get affected by this mechanism, from ultra-rare to common pathologies such as Down Syndrome, Turner Syndrome or Klinefelter Syndrome” Statement of concrete aims or formulate of questions – 2 Page 2 – “We address skewed XCI, the condition that happen when this process is not fully randomized, and its consequences to the phenotype of some pathologies. We debate about the known pathologies implicated (…) and his consequences for males and females.” Page 2 – “Finally, possible future implications for genomic therapy and treatment of patients and list areas that need further research on this topic were addressed.” Description of the literature search – 2 Page 4 – “To write this mini-review, two searches (first on 20/07/2020 and second on 23/02/2021) were conducted in PubMed, Scopus and Cochrane Library, including all articles written in English, Spanish and Portuguese. The searched was made using the following query: (X-chromosome inactivation [MeSH Major Topic]) OR (Lyon Hypotesis [MeSH Major Topic]). Inclusion criteria included studies made in humans and related with pathology while exclusion criteria included studies made in animals. Year of publication from the articles was not an exclusion criteria, but most recent studies were treated as more relevant.” Referencing – 2 Pages 4/5 – “The process of silencing is still largely unknown but according to literature XIST RNA may binding to concrete sites after starting in the XCI, beginning a cascade of epigenetic changes that results in the formation of the Barr body (Pinter et al. 2012; Payer 2016; Posynick and Brown 2019).” Page 6 – “One of the most intriguing questions regarding X inactivation is how (and why) some loci are able to escape gene silencing. In human, up to 25% of X-linked genes can escape from XCI - 15% of them constitutively and about 10% of them variably between individuals (Galupa and Heard 2018). This biallelic expression results in a higher dosage of these genes in females suggesting that these genes might have female-specific roles (Berletch et al. 2011)” Page 6 – “These genes that escape X-inactivation are responsible for many of the different phenotypes that some diseases can present. As such, and due to the fact that we have drawn a clear correlation between DNA methylation and escape from XCI (Payer 2016; Halmai et al. 2020) , we can now project that understanding this can have a lot of potential to create innovative ways of treatment for people that suffer from this X-linked disorders (Halmai et al. 2020).” Scientific reasoning – 2 Page 7 – “A correlation between Intellectual Disability (ID) and skewed XCI has been established (Plenge et al. 2002; Gieldon et al. 2017).” Appropriate presentation of data – 2 Non-applicable. Agradecimentos Agradeço à Professora Doutora Sofia Dória, pela disponibilidade total e ajuda na realização deste trabalho, desde a organização de reuniões, à procura de artigos e documentos, às incontáveis vezes em que esteve disponível para responder a dúvidas, até às numerosas revisões que realizou a todo o artigo. Agradeço também ao Serviço de Genética pela sugestão do tema. Agradeço aos meus pais, Marcelo e Isabel, pela enorme ajuda que me deram durante todo este curso, tanto económica como emocionalmente, nos momentos onde as coisas correram bem e nos momentos onde correram menos bem. Agradeço-lhes também a preocupação constante que tiveram comigo. Sem eles, ter-me-ia perdido no caminho demasiadas vezes, e certamente não conseguiria ter chegado tão longe. Agradeço ao meu irmão Henrique, pela paciência que demonstrou durante todo este tempo, por ter convivido comigo e partilhado casa durante estes últimos três anos, atravessando comigo todas as dificuldades que foram surgindo, e nunca abdicando de ser o meu confidente dos dias menos bons, por muito que isso lhe tirasse tempo para outros afazeres. Agradeço aos meus avós, Manuel, José e Maria, que me acompanharam a todo o momento, sempre interessados por mim, pelos meus problemas e pelos acontecimentos do curso, pelas orações incansáveis todos os dias, e por me terem apoiado durante estes seis anos. Agradeço à minha avó Fernanda, que como referi na dedicatória, foi a grande razão da escolha do meu curso, com todos os problemas de saúde que infelizmente a afetaram nos últimos vinte anos da sua vida. Tenho a certeza que está orgulhosa deste percurso, tal como sempre esteve dos seus dois netos. Agradeço à minha restante família, incontáveis pessoas que me ajudaram neste caminho, a partir dos vários países onde vivem, sempre com carinho e disponibilidade. Agradeço aos meus amigos Tiago Ribeiro, Tiago Oliveira e Tiago Aguiar, os famosos “Triagos”, que foram os ombros amigos em cada momento do meu curso, nos dias bons e maus, nas aventuras mais divertidas e nos passeios mais incríveis. Sem eles, tudo isto teria sido muito mais aborrecido. Agradeço aos meus restantes colegas de turma, a maioria dos quais estiveram comigo durante os seis anos que percorri nesta faculdade, que me ajudaram sempre. Agradeço aos amigos do basquetebol, aos colegas de curso que passaram próximos de mim em momentos concretos destes seis anos e aos meus companheiros de ERASMUS em Bochum. A todos eles, o meu mais sincero obrigado. Agradeço a todos os professores que deram parte do seu tempo e da sua disponibilidade para enriquecer a minha experiência e conhecimento e que certamente irão fazer de mim um melhor médico. Agradeço por fim à Faculdade de Medicina da Universidade do Porto e à Rühr Universität Bochum por cada passo da minha formação médica.