Decifrando os mecanismos moleculares de teratogênese da talidomida : genética, embriologia e biologia de sistemas

A exposição à talidomida no início da gestação levou ao nascimento de milhares de crianças com um conjunto de malformações que foi depois denominado de Embriopatia da Talidomida (TE). Dois aspectos principais que permanecem não elucidados a respeito da teratogênese da talidomida são (1) a susceptibilidade à TE, uma vez que 20-50% dos embriões expostos à talidomida desenvolveram a embriopatia, e (2) a variabilidade interespecífica da TE. Assim, o objetivo dessa tese foi utilizar diferentes abordagens para entender os mecanismos moleculares de teratogenicidade da talidomida em humanos e nos principais modelos animais disponíveis, focando em susceptibilidade genética e de variabilidade fenotípica da TE. Duas variantes regulatórias no gene NOS3, que codifica a proteína eNOS, apresentaram-se em maior frequência em indivíduos com a TE, quando comparados a brasileiros da população geral, sem anomalias congênitas. Essas variantes estão associadas a menor expressão de NOS3, sugerindo-se um efeito pré-transcricional. A análise de polimorfismos foi expandida para outros genes da via de angiogênese, NOS2, PTGS2 e VEGFA, no entanto nenhum efeito de susceptibilidade foi encontrado. Uma revisão de literatura e avaliação de redes de interação proteína-proteína indicou duas proteínas centrais na ação antiangiogênica da talidomida: beta-catenina e eNOS. Outra abordagem visou sequenciar a região de CRBN que codifica a porção de ligação de Cereblon a talidomida. Não foram identificadas variantes exônicas, porém, foram identificadas variantes raras em maior frequência nos afetados pela TE do que nos controles sem anomalias congênitas. Uma associação entre rs1620675, polimorfismo no intron 10, e a presença de anomalias neurológicas foi identificada. Além de CRBN, a análise foi expandida, adicionando-se DDB1 e CUL4A, genes do complexo E3-ubiquitina-ligase (CRL4CRBN) e suas proteínas-alvo, IKZF1 e IKZF3. Após sequenciar esses genes em 35 afetados pela TE, foram encontradas 145 variantes entre os cinco genes, sendo mais de 90% delas em regiões regulatórias. Para entender o impacto dessas variantes, um score de impacto deletério foi desenvolvido e aplicado após predição funcional in silico. 12 Indivíduos com anomalias de membros de padrão pré-axial longitudinal possuíam score mais alto do que os afetados com focomelia. A partir de análise de dados secundários, foi avaliada a expressão gênica diferencial; em células-tronco embrionárias de camundongos a expressão de Crbn, Cul4a e Ddb1 encontrava-se reduzida. Uma experiência de aprendizado em embriologia experimental também foi desenvolvida no presente estudo. Ensaios de exposição com apremilast, análogo anti-inflamatório da talidomida, e diclofenaco foram realizados em embriões de galinha. Não foram encontradas anomalias nos embriões, porém alta taxa de letalidade foi visualizada em altas doses. Ainda em embriões de galinha, tentou-se induzir a TE sem o uso de talidomida, a partir da inserção de esferas nos vasos sanguíneos (tentativa de mimetizar o efeito anti-angiogênico) e pela retirada de porções da crista ectodérmica apical dos brotos dos membros desses embriões. Apesar de pequenas anomalias terem sido visualizadas, a capacidade regenerativa dos membros era alta. Foi então avaliado o padrão de conservação das proteínas afetadas pela talidomida em espécies que desenvolvem a TE versus espécies resistentes à embriopatia típica (ratos e camundongos). O gene CYP2C19, de metabolização, apresenta variação no número de cópias entre as espécies. Também, foram identificadas duas variantes exclusivas dos ratos e camundongos, uma no gene GSTP1 e outra em RECQL4. O último é uma helicase associada à Síndrome de Baller-Gerold, uma condição genética autossômica recessiva caracterizada por malformações de membros similares à TE, sua fenocópia. Ainda, encontrou-se uma correlação entre Recql4 e Crbn em células-tronco embrionárias de camundongos; essa correlação é fortemente alterada na exposição à talidomida. A fim de integrar todas as hipóteses da teratogênese da talidomida, usou-se de ferramentas de biologia de sistemas. Foi identificado que a β-catenina, proteína importante no desenvolvimento de membros, interconecta os principais mecanismos. Avaliando a expressão gênica diferencial a partir de dados secundários, encontrou-se enriquecimento das ontologias gênicas: “ciclo celular”, “replicação de DNA” e “reparo de DNA”. Desses genes com expressão reduzida, destaca-se ESCO2 e SHISA3. Mutações em ESCO2 levam a Síndrome de Roberts, 13 outra condição genética da qual a TE é uma fenocópia. SHISA3 é responsável pela degradação de β-catenina. Por fim, uma análise de passeio aleatório demonstrou que o gene codificante de β-catenina (CTNNB1) e SHISA3 são os mais próximos funcionalmente da susceptibilidade à talidomida. A partir dos resultados dessa tese, surgem novas vertentes de pesquisa para a compreensão da teratogênese da Talidomida, como os mecanismos compartilhados entre TE e as síndromes genéticas (fenocópias), e o papel central da β-catenina na TE. Ambos merecem ser analisados experimentalmente para que possam fornecer maiores evidências. Ainda assim, esse é o primeiro estudo a gerar uma hipótese integradora entre os diferentes mecanismos de teratogênese da talidomida, quem vem sendo discutidos ao longo de quase 60 anos. As perspectivas geradas a partir desse trabalho podem contribuir significativamente no esforço pela obtenção de uma alternativa segura à talidomida e para uso de biomarcadores no uso terapêutico da molécula.Thalidomide exposure in early pregnancy led to the birth of thousands of children with a spectrum of malformations, lated named Thalidomide Embryopathy (TE). Two main aspects regarding thalidomide teratogenesis that remain unknown are (1) genetic susceptibility to TE, once 20-50% of the embryos exposed to thalidomide developed the embryopathy; and (2) the interspecific variability in TE. Hence, the aim of this thesis was to use from different approaches to understand the molecular mechanisms of thalidomide teratogenicity in humans and in the main animal models available, focusing on genetic susceptibility and phenotypic variability in TE. Two regulatory variants in NOS3 gene, that encodes eNOS protein, were present in higher frequency in TE, when compared to Brazilians of the general population, without congenital anomalies. These variants are associated to reduced NOS3 expression, suggesting a pre-transcriptional effect. The analysis of polymorphisms was expanded to other genes of the angiogenesis pathway, NOS2, PTGS2 and VEGFA, however no susceptibility effect was encountered. A literature review and a network avaliation of protein-protein interactions identified two central proteins for thalidomide antiangiogenesis action: beta-catenin and eNOS. Another approach aimed to sequence CRBN gene region that encodes thalidomide binding site. There were none exoniic variants, although rare variants were identified in higher frequency in TE affected in comparison to controls without congenital anomalies. One association between rs1620675, a polymorphism in intron 10, and the presence of neurological anomalies was found. Besides CRBN, we expanded the analysis adding DDB1 and CUL4A, genes of the E3-ubiquitinligase complex (CRL4CRBN) and its target proteins IKZF1 and IKZF3. After sequencing these genes in 35 TE affected, 145 variants were encountered in these five genes; more than 90% were in regulatory regions. To understand the impact of these variants, a score of deleteriousness was developed and applied after in silico functional prediction. Individuals with pre-axial longitunial limb anomalies have a higher score than the ones with phocomelia. Using from secondary data analysis, 15 differential gene expression was evaluated in murine embryonic stem-cells; the expression of Crbn, Cul4a, and Ddb1 was downregulated. A learning experience experimental embryology approach was also developed in the present study. Exposure assays with apremilast, an antiinflammatory analogue of thalidomide, and diclofenac were performed in chicken embryos. Congenital anomalies were not identified in the embryos, although a high rate of lethality was visualized in high doses. Still in chicken embryos, it was tried to induce TE without thalidomide, using from the insertion of beads in the blood vessels (attempt to mimic the antiangiogenic effect) and cutting portions of the apical ectodermal ridge of the limb buds of these embryos. Some anomalies were visualized; however, the regenerative capacity of the limbs was high. The conservation pattern of affected proteins was evaluated, comparing species that develop TE versus the ones resistant to the typical embryopathy (rats and mice). Gene CYP2C19, of metabolization, presents variation in the number of copies between the species. It was also identified two exclusive variants in rats and mice, once in GSTP1 and another in RECQL4. The latter RECQL4 is a helicase associated to Baller-Gerold syndrome, a genetic autosomal recessive condition, characterized by limb malformations similar to TE, its phenocopy. Furthermore, a correlation between Recql4 and Crbn was identified, in murine embryonic stemcells; this correlation is highly affected in thalidomide exposure. To integrate all the hypotheses of thalidomide teratogenesis, we used from systems biology tools. It was identified that beta-catenin, an important protein in limb development, interconnects the main mechanisms. Evaluating the differential gene expression from secondary data, we identified 126 genes with altered expression in human pluripotent stem-cells, after thalidomide exposure. The main gene ontologies affected are “cell cycle”, “DNA replication, and “DNA repair”. Of these genes with downregulated expression, we highlight ESCO2 and SHISA3. Mutations in ESCO2 lead to Roberts syndrome, another genetic condition from which TE is a phenocopy. SHISA3 is responsible for the degradation of beta-catenin. Finally, a random walk analysis demonstrated that beta-catenin gene (CTNNB1) and SHISA3 are the most closely related functionally to thalidomide susceptibility. According to the results of this thesis, we suggest new strands of research for the comprehension of thalidomide teratogenesis, including the shared mechanisms between TE and the genetic syndromes (phenocopies), and the central role of beta-catenin in TE. Both deserve to be evaluated experimentally to provide more evidence. Still, this is the first study to generate an integrative hypothesis between the different teratogenesis mechanisms of thalidomide, that have been discussed in the last 60 years. The perspectives generated from this study can contribute significantly in the effort for the obtention of a safe alternative to thalidomide, and for the use of biomarkers in the therapeutic use of the molecule

Expression profiles of meiotic genes in male vs. female gonads and gametes : insights into fertility issues

Gametes are specialized cells that, at fertilization, give rise to a totipotent zygote capable of generating an entire organism. Female and male germ cells undergo meiosis to produce mature gametes; however, sex-specific events of oogenesis and spermatogenesis contribute to specific roles of gametes in reproductive issues. We investigate the differential gene expression (DGE) of meiosis-related genes in human female and male gonads and gametes in normal and pathological conditions. The transcriptome data for the DGE analysis was obtained through the Gene Expression Omnibus repository, comprising human ovary and testicle samples of the prenatal period and adulthood, additionally to male (nonobstructive azoospermia (NOA) and teratozoospermia), and female (polycystic ovary syndrome (PCOS) and advanced maternal age) reproductive conditions. Gene ontology terms related to meiosis were associated with 678 genes, of which 17 genes in common were differentially expressed between the testicle and ovary during the prenatal period and adulthood. Except for SERPINA5 and SOX9, the 17 meiosis-related genes were downregulated in the testicle during the prenatal period and upregulated in adulthood compared to the ovary. No differences were observed in the oocytes of PCOS patients; however, meiosis-related genes were differentially expressed according to the patient’s age and maturity of the oocyte. In NOA and teratozoospermia, 145 meiosis-related genes were differentially expressed in comparison to the control, including OOEP; despite no recognized role in male reproduction, OOEP was co-expressed with genes related to male fertility. Taking together, these results shed light on potential genes that might be relevant to comprehend human fertility disorders

Comparative Genomics Identifies Putative Interspecies Mechanisms Underlying Crbn-Sall4-Linked Thalidomide Embryopathy

The identification of thalidomide–Cereblon-induced SALL4 degradation has brought new understanding for thalidomide embryopathy (TE) differences across species. Some questions, however, regarding species variability, still remain. The aim of this study was to detect sequence divergences between species, affected or not by TE, and to evaluate the regulated gene co-expression in a murine model. Here, we performed a comparative analysis of proteins experimentally established as affected by thalidomide exposure, evaluating 14 species. The comparative analysis, regarding synteny, neighborhood, and protein conservation, was performed in 42 selected genes. Differential co-expression analysis was performed, using a publicly available assay, GSE61306, which evaluated mouse embryonic stem cells (mESC) exposed to thalidomide. The comparative analyses evidenced 20 genes in the upstream neighborhood of NOS3, which are different between the species who develop, or not, the classic TE phenotype. Considering protein sequence alignments, RECQL4, SALL4, CDH5, KDR, and NOS2 proteins had the biggest number of variants reported in unaffected species. In co-expression analysis, Crbn was a gene identified as a driver of the co-expression of other genes implicated in genetic, non-teratogenic, limb reduction defects (LRD), such as Tbx5, Esco2, Recql4, and Sall4; Crbn and Sall4 were shown to have a moderate co-expression correlation, which is affected after thalidomide exposure. Hence, even though the classic TE phenotype is not identified in mice, a deregulatory Crbn-induced mechanism is suggested in this animal. Functional studies are necessary, especially evaluating the genes responsible for LRD syndromes and their interaction with thalidomide–Cereblon