O desenvolvimento embrionário dos membros é um processo complexo e dinâmico. É controlado por diversos genes e diversos mecanismos morfogenéticos, muitos dos quais ainda não estão completamente elucidados. O objetivo deste estudo é identificar as alterações genéticas responsáveis por defeitos de membros em quatro famílias brasileiras. A família 1 apresenta três indivíduos com um espectro extremamente variável de displasia ectodérmica, ectrodactilia e fenda labiopalatina, características da síndrome EEC. O gene TP63 foi analisado por sequenciamento de Sanger e foi descoberta uma variante nunca descrita anteriormente, c.1037C>A (p.Ala346Gly), em heterozigose, segregando com o fenótipo. Mutações no gene TP63 já foram associadas com casos da síndrome EEC. Concluímos que a variante no gene TP63 é a responsável pelo quadro clínico de EEC na família. A família 2 incluiu cinco indivíduos afetados por polegar trifalângico. O fenótipo de um dos indivíduos é mais grave e se caracteriza por, além do polegar trifalângico, pela presença de braço direito grosseiramente curto e deformado com dedos hipoplásicos, pé direito malformado com metatarsos hipoplásicos e orelha direita pequena e protuberante. Estudos de mapeamento gênico com arrays de SNPs revelaram Lod scores sugestivos de ligação com 8 regiões cromossômicas distintas. O sequenciamento massivo em paralelo do exoma com a amostra de um dos afetados, seguida da seleção das variantes presentes nas regiões com Lod scores sugestivos, revelou a presença da variante c.410dupG (p.Gly138Argfs∗43) no gene SALL4, a qual nunca havia sido descrita. Mutações no SALL4 que alteram a matriz de leitura já foram associadas com casos da síndrome de Okihiro, a qual é caracterizada por defeitos radiais de membros e anomalia oftalmológica de Duane. A segregação da variante entre os indivíduos afetados foi confirmada por sequenciamento de Sanger. Concluimos que esta variante é responsável pelo quadro clínico da família, a qual apresenta a síndrome de Okihiro, porém sem a anomalia de Duane. A análise de todos os casos descritos com variantes no gene SALL4 sugere que há uma correlação entre defeitos nos membros inferiores e o tamanho reduzido da proteína truncada SALL4. A família 3 apresenta três indivíduos afetados por sindactilia completa com polidactilia pré-axial nas mãos, classificada como sindactilia do tipo IV. O sequenciamento massivo em paralelo do exoma com as amostras de três indivíduos afetados e dois normais não revelou a presença de qualquer alteração genética candidata a explicar o quadro clínico. O estudo do array-CGH revelou uma duplicação de aproximadamente 97 kb no gene LMBR1 abrangendo a região de enhancer (ZRS) do gene SHH. Duplicações no ZRS já foram identificadas em casos de sindactilia do tipo IV. Concluímos que a duplicação no ZRS é responsável pelo quadro. A revisão da literatura sobre os casos com duplicação dessa região sugere uma correlação entre duplicações com tamanho ao redor de 97 kb e o fenótipo clássico de sindactilia do tipo IV. A família 4 apresenta seis indivíduos afetados por uma síndrome relativamente nova descrita por nossa equipe em 2008, a síndrome de Santos. Essa síndrome é caracterizada por aplasia/hipoplasia fibular e anoniquia/hipoplasia ungueal dentre outros defeitos de membros. O estudo de ligação com array de SNP e marcadores de microssatélites, com posterior cálculo de Lod score, indicou duas regiões no cromossomo 3 como candidatas a conter a alteração genética responsável pelo fenótipo. Análise dos genes presentes nessas regiões indicou o gene WNT7A como o principal candidato. Alterações neste gene já foram associadas com a síndrome de Fuhrmann e a AARRS (Al-Awadi⁄Raas-Rothschild syndrome), de herança recessiva, as quais apresentam fenótipos correlatos aos presentes na síndrome de Santos. O sequenciamento de Sanger deste gene revelou a presença da variante c.934G>A (p.Gly312Ser), não descrita, em homozigose em cinco dos seis indivíduos afetados da família. O sexto indivíduo afetado é heterozigoto e apresenta fenótipo muito menos grave. Não apresenta qualquer defeito fibular e ungueal, característicos da síndrome. Esse é o primeiro relato de um caso de defeitos de membros como resultado da presença de uma variante em heterozigose no gene WNT7A. O sequenciamento massivo em paralelo do exoma foi realizado com as amostras deste indivíduo e de um dos indivíduos afetados. Contudo, nenhuma outra variante se mostrou candidata a explicar o quadro clínico. Concluímos que a síndrome de Santos é o resultado desta mutação no gene WNT7A. Estudos funcionais in situ baseados na atividade da Luciferase estão em andamento para comprovar o efeito deletério desta variante / Limb development is a complex and dynamic process driven by many different genes and morphogenetic mechanisms. Many of them have not been properly clarified yet. The aim of this study is to identify genetic alterations responsible for limb defects in four Brazilian families. Family 1 includes three individuals affected by an extremely variable phenotype of ectrodactyly, ectodermal dysplasia and cleft lip/palate, and other clinical signs of EEC syndrome. The TP63 gene, associated with this condition, was analyzed through Sanger sequencing and a novel variant, c.1037C>A (p.Ala346Gly), was found; the variant segregated in heterozygous state with the EEC phenotype. Mutations in TP63 gene are already known to be associated with EEC syndrome. Due to the extremely variable phenotype presented by the individuals in the family, the possibility of the mutation c.1037C>G being related to acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome and SHFM cannot be ruled out. Family 2 presents five individuals affected by preaxial polydactyly (triphalangeal thumbs). The phenotype of the proband is more severe, being characterized by triphalangeal thumb, with the following manifestations: grossly shortened and deformed forearm, markedly hypoplastic and appendicular thumb and malformed right foot with hypoplastic metatarsals. Linkage studies by SNP-array pointed to suggestive Lod score values in 8 distinct chromosomal regions. Whole exome sequencing with the sample from one affected individual revealed a novel frameshift variant, c.410dupG (p.Gly138Argfs∗43) in the SALL4 gene. Frameshift mutations in the SALL4 were already associated to Okihiro syndrome, which is characterized by radial limb defects and Duane anomaly. The segregation of the variant among the affected individuals was confirmed by Sanger sequencing. We concluded that this variant is the cause of the clinical signs in the family, which was classified as presenting Okihiro syndrome without Duane anomaly. The review of all clinical cases reported with SALL4 variants indicates a possible correlation between the foot malformation and the reduced size of the SALL4 protein. Family 3 has affected individuals with complete hand syndactyly associated to pre-axial polydactyly (syndactyly type IV). Whole exome sequencing was performed with the samples collected from the three affected individuals and two non-affected ones and no pathogenic variant was detected in genes related to limb development segregating with the phenotype. Through array-CGH, nevertheless, a duplication of about 97 kb including the enhancer (ZRS) of the SHH gene was detected. Duplications in ZRS region were already reported in Syndactyly type IV. We concluded that the 97-kb duplication is the cause of the syndactyly type IV in the family. A review of reported cases (including ours) suggested a possible correlation between the duplication with 97 kb size and the classic phenotype of syndactyly type IV. Family 4 has six individuals affected by a new syndrome (Santos syndrome) described by members of our group in 2008, a condition characterized by fibular agenesis/hipoplasia and ungual hypoplasia⁄anonychia, among other limb defects. Linkage study by SNP-array and microsatellite markers was performed and Lod score calculation pointed to two candidate regions on chromosome 3. Search for candidate genes revealed the WNT7A as the best candidate. Mutations in WNT7A gene, in homozygous state, are known to cause two other limb defect syndromes, Fuhrmann syndrome and AARRS (Al-Awadi/Raas-Rothschild syndrome), both presenting similar phenotypes to Santos syndrome. Sanger sequencing showed a novel variant, in homozygous state, c.934G>A (p.Gly312Ser) in the WNT7A gene in five out of six affected individuals. One affected individual, much less severely affected than his relatives, carries the mutation in heterozygous state. He does not present fibular or ungual malformations, characteristic signals of the syndrome; instead, he is the carrier of a complex polydactyly in his left hand. This is the first report about a heterozygous variant in WNT7A gene resulting in limb defect. Whole exome sequencing was performed with the samples from two affected individuals. However, no other variant in candidate gene to explain the limb defects was detected. We concluded that Santos syndrome is caused by a mutation in the WNT7A gene. Functional assays, based on Luciferase activity, are in execution to test the deleterious effect of the c.934G>A variant in the WNT7A gene

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Here, we validate novel protein interactors of TCF7L1 (also known as TCF3), a downstream transcription factor in the Wnt/β-catenin signaling pathway, from an initial protein interaction screen that utilized the BioID system in mouse embryonic stem cells. The BioID-TCF7L1 screen identified multiple proteins including several transcription factors and numerous epigenetic regulators. Notably, SALL4, a key embryonic stem cell factor belonging to the SPALT family of transcription factors was validated to interact with TCF7L1 through Proximity Ligation Assay (PLA), and Co-Immunopreciptation (Co-IP). Analysing mRNA transcriptomic signatures of TCF7L1-null mEScs and SALL4 overexpressing mESCs, we observed similarly increased output of the pluripotency-gene, Tbx3, suggesting a transcriptionally opposing function between TCF7L1 and SALL4. Furthermore, we identified that SALL4 also interacted with TCF7, suggesting that SALL4 may interact with all four members of the TCF/LEF transcription factor family to regulate Wnt targets. This work further validates the utility and effectiveness of screening transcription factor interactors through the BioID system and provides important insights into SALL4 mediated Wnt regulation through the TCF/LEFs. / Thesis / Master of Science (MSc) / The biology of cells is highly complex. The genes within are under tight regulation to promote balance that is critical to the growth and status of the cell. Cells communicate with one another to support this balance through molecule secretion signaling which dictates biology. Understanding the complex biology within cells is critical, and therefore here we study one of many signaling pathways known as the Wnt Signaling Pathway. This work contributes to the knowledge of Wnt signaling by validating the interaction of proteins that dictate the onset or offset of important genes in mouse embryonic stem cells.