Fissura de lábio com ou sem fissura de palato não-sindrômica (FL/P NS) é uma malformação craniofacial frequente, com modelo de herança multifatorial, onde fatores de risco genéticos e ambientais atuam na manifestação da doença. Variações nos níveis de expressão gênica têm sido apontadas como um importante mecanismo de susceptibilidade a doenças complexas, e variantes no DNA que regulam esses níveis de expressão (eQTL) têm sido combinadas a estudos de associação para auxiliar no entendimento da etiologia de algumas doenças. No presente trabalho, integramos eQTLs e estudo de associação para 1) verificar se variantes já associadas com FL/P NS possuem um papel regulatório em células-tronco de músculo orbicular do lábio (OOMMSC, um tecido afetado em FL/P NS), e 2) verificar se eQTLs mapeados em OOMMSC teriam associação com a mesma. Para o primeiro objetivo, verificamos a correlação entre os genótipos das variantes rs642961 e rs590223 e os níveis de expressão de IRF6, e também entre rs987525 e os níveis de expressão de MYC. Não encontramos correlação para nenhuma das três variantes testadas. É possível que essas variantes possuam um papel funcional em algum momento específico da embriogênese, ou mesmo que não tenhamos detectado essa correlação devido ao número amostral analisado (N=46). Para o segundo objetivo, realizamos um estudo de associação do tipo caso-controle dos eQTLs rs5011163, rs1505443, rs4793213, rs4793229 e rs1242500. Não encontramos associação entre nenhuma das cinco variantes e FL/P NS. Uma possível explicação para a associação negativa seria a significância marginal dessas variantes como eQTLs em OOMMSC. Além disso, estudos com baixo poder, como o mapeamento de eQTLs em OOMMSC realizado em outro projeto pelo nosso grupo, geralmente detectam os eQTLs de maior efeito, sendo esses frequentemente compartilhados entre tecidos, e, assim, podem não ter relevância para a doença em si. Outros eQTLs de OOMMSC, selecionados por critérios diferentes do presente estudo, estão sendo testados para associação com FL/P NS, o que nos permitirá avaliar a relevância dessa abordagem para detectar variantes de susceptibilidade a FL/P NS / Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a frequent craniofacial malformation, with a multifactorial model of inheritance, in which genetic and environmental risk factors act in disease manifestation. Variation of gene expression has been pointed as an important susceptibility mechanism to complex diseases, and DNA variants that regulate expression levels (eQTLs) have been combined with association studies to help elucidate the etiology of some diseases. In the present work, we integrate eQTL and association studies to 1) verify if variants associated with NSCL/P have a regulatory role in orbicularis oris muscle mesenchymal stem cells (OOMMSC, a tissue affected by NSCL/P); and 2) verify if eQTLs mapped in OOMMSC are associated with the disease. For the first goal, we verified the correlation of the rs642961 and rs590223 genotype variants with IRF6 expression levels, and also between the rs987525 genotype and MYC expression levels. We did not find correlation for any of the three variants tested. Possibly, these variants have a functional role in specific moments of embryogenesis, or sample size (N=46) was insufficient to detect correlation. For the second goal, we did a case-control association study for eQTLs rs5011163, rs1505443, rs4793213, rs4793229 and rs1242500. We did not find association between these variants and NSCL/P. The negative association could be explained by the marginal significance of these variants as eQTLs in OOMMSC. Besides, low-power studies, as the OOMMSC eQTL mapping performed in another project by our group, usually detect eQTLs of larger effect, which are frequently shared among tissues; therefore, they may not be relevant for the disease itself. Other eQTLs, selected under different criteria, are currently being tested for association with NSCL/P, which will enable us to evaluate the relevance of this approach to detect susceptibility variants for NSCL/P

Fissura de lábio com ou sem fissura de palato não-sindrômica (FL/P NS) é uma malformação craniofacial frequente, com modelo de herança multifatorial, onde fatores de risco genéticos e ambientais atuam na manifestação da doença. Variações nos níveis de expressão gênica têm sido apontadas como um importante mecanismo de susceptibilidade a doenças complexas, e variantes no DNA que regulam esses níveis de expressão (eQTL) têm sido combinadas a estudos de associação para auxiliar no entendimento da etiologia de algumas doenças. No presente trabalho, integramos eQTLs e estudo de associação para 1) verificar se variantes já associadas com FL/P NS possuem um papel regulatório em células-tronco de músculo orbicular do lábio (OOMMSC, um tecido afetado em FL/P NS), e 2) verificar se eQTLs mapeados em OOMMSC teriam associação com a mesma. Para o primeiro objetivo, verificamos a correlação entre os genótipos das variantes rs642961 e rs590223 e os níveis de expressão de IRF6, e também entre rs987525 e os níveis de expressão de MYC. Não encontramos correlação para nenhuma das três variantes testadas. É possível que essas variantes possuam um papel funcional em algum momento específico da embriogênese, ou mesmo que não tenhamos detectado essa correlação devido ao número amostral analisado (N=46). Para o segundo objetivo, realizamos um estudo de associação do tipo caso-controle dos eQTLs rs5011163, rs1505443, rs4793213, rs4793229 e rs1242500. Não encontramos associação entre nenhuma das cinco variantes e FL/P NS. Uma possível explicação para a associação negativa seria a significância marginal dessas variantes como eQTLs em OOMMSC. Além disso, estudos com baixo poder, como o mapeamento de eQTLs em OOMMSC realizado em outro projeto pelo nosso grupo, geralmente detectam os eQTLs de maior efeito, sendo esses frequentemente compartilhados entre tecidos, e, assim, podem não ter relevância para a doença em si. Outros eQTLs de OOMMSC, selecionados por critérios diferentes do presente estudo, estão sendo testados para associação com FL/P NS, o que nos permitirá avaliar a relevância dessa abordagem para detectar variantes de susceptibilidade a FL/P NS / Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a frequent craniofacial malformation, with a multifactorial model of inheritance, in which genetic and environmental risk factors act in disease manifestation. Variation of gene expression has been pointed as an important susceptibility mechanism to complex diseases, and DNA variants that regulate expression levels (eQTLs) have been combined with association studies to help elucidate the etiology of some diseases. In the present work, we integrate eQTL and association studies to 1) verify if variants associated with NSCL/P have a regulatory role in orbicularis oris muscle mesenchymal stem cells (OOMMSC, a tissue affected by NSCL/P); and 2) verify if eQTLs mapped in OOMMSC are associated with the disease. For the first goal, we verified the correlation of the rs642961 and rs590223 genotype variants with IRF6 expression levels, and also between the rs987525 genotype and MYC expression levels. We did not find correlation for any of the three variants tested. Possibly, these variants have a functional role in specific moments of embryogenesis, or sample size (N=46) was insufficient to detect correlation. For the second goal, we did a case-control association study for eQTLs rs5011163, rs1505443, rs4793213, rs4793229 and rs1242500. We did not find association between these variants and NSCL/P. The negative association could be explained by the marginal significance of these variants as eQTLs in OOMMSC. Besides, low-power studies, as the OOMMSC eQTL mapping performed in another project by our group, usually detect eQTLs of larger effect, which are frequently shared among tissues; therefore, they may not be relevant for the disease itself. Other eQTLs, selected under different criteria, are currently being tested for association with NSCL/P, which will enable us to evaluate the relevance of this approach to detect susceptibility variants for NSCL/P

Maspin (Mammary Serine Protease Inhibitor) was first reported in 1994 as a serpin with tumor suppressive properties. Maspin was initially isolated through subtractive hybridization and differential display analysis as a 42kDa protein that is expressed in normal mammary epithelial cells but reduced or absent in breast carcinomas. Further research led to maspin's characterization as a class II tumor suppressor based on its ability to inhibit cell invasion, promote apoptosis and inhibit angiogenesis. Since then, efforts have been made to characterize maspin's tumor suppressive mechanisms. In particular, researchers have studied maspin localization, the regulation of maspin expression, and more recently, maspin protein interactions. We employed a maspin-baited yeast two-hybrid system and subsequently identified Interferon Regulatory Factor 6 (IRF6) as a maspin-binding protein. Whereas many of the IRF family members have been well characterized, IRF6 remains poorly understood. In this dissertation, we elucidate some of the complex mechanisms involved in maspin activity, specifically relating to IRF6 regulation and function. We have examined the expression of IRF6 in breast cancer cells and we show that, similar to maspin, IRF6 is reduced or absent in breast carcinomas. We further show that the re-expression of IRF6 in breast cancer cells results in genotypic and phenotypic changes which can be abrogated in the presence of maspin. We identify ERK1/2 as a kinase involved in IRF6 phosphorylation, and we demonstrate a possible role for toll-like receptor signaling in the activation of IRF6. We also evaluate the differential expression of maspin and IRF6 during murine mammary gland development and we show that both maspin and IRF6 are maximally expressed during lactation. These studies have increased our understanding of the complex, pleiotropic nature of maspin and provide an avenue to develop maspin's potential as a diagnostic marker for cancer progression and as a potentially powerful therapeutic agent in the fight against breast cancer.

Association of type and severity of nonsyndromic OROFACIAL CLEFTS with combined genotypes of RFC1 80 GA, MTHFR 677 CT, IRF6 rs642961 and rs2235371 gene polymorphisms in an Indian population Abstract By Chantal Hakim University of the Pacific. Arthur Dugoni School of Dentistry 2020 Introduction. Genetic etiology of nonsyndromic orofacial clefts comprises many genes acting together. However, little is known about their interactions. The purpose of our study was to analyze associations of phenotypic subtypes of nonsyndromic orofacial clefts with combinations of four genotypes involving candidate gene polymorphisms. Materials and Methods. We analyzed a large dataset of cases and controls collected in one location (Karaikal in India) and genotyped for four gene polymorphisms: RFC1 G80A, MTHFR C677T, IRF6 GA rs642961 and IRF6 CT rs2235371 (IRB approval Nr. 17-118 for existing data). The samples were tested for Hardy-Weinberg genetic equilibrium. Combinations of genotypes in cleft subsamples were compared with controls using Odds Ratio and Confidence Interval (95% significance level) calculations. Results. The Hardy-Weinberg equilibrium test showed that all samples were in genetic equilibrium. Some combinations of RFC1 G80A, MTHFR C677T, IRF6 GA rs642961 and IRF6 CT rs2235371 yielded increased or decreased Odds Ratio (OR>1 or OR<1).This means that subtypes of orofacial clefts were differentially determined by genotype combinations of four gene polymorphisms. Conclusions. Our results suggest that combinations of gene polymorphisms may modulate genetic risk in subtypes of nonsyndromic orofacial clefts. Such studies seem to be important for development of a general procedure and how a prevention plan for a specific location needs to be prepared, which data needs to be collected and which analyses need to be performed.

As fissuras orais são as malformações craniofaciais mais freqüentes ao nascimento e apresentam incidências variáveis entre as diversas populações. Essas fissuras são subdivididas em dois grupos principais, as fissuras labiais com ou sem fissura de palato (FL&plusmn;P) e as fissuras palatinas (FP), consideradas entidades distintas do ponto de vista embriológico, epidemiológico e etiológico. Estas malformações podem ser classificadas em sindrômicas ou não sindrômicas (NS) a depender da existência de outras alterações clínicas associadas. Para as FL&plusmn;PNS e FPNS, o padrão de herança multifatorial e o modelo &ldquo;doenças comuns-variantes comuns&rdquo; foram propostos. Diversos estudos têm sido realizados na tentativa de se identificar os fatores genéticos de predisposição a estas malformações, particularmente para as FL&plusmn;PNS. Entre estes fatores, o IRF6, comprovadamente associado à síndrome de Van der Woude (SVW), é um gene candidato associado às fissuras NS. Em algumas situações, a SVW torna-se clinicamente indistinguível das fissuras não sindrômicas, o que pode dificultar o aconselhamento genético destas famílias afetadas por fissuras. A associação entre FL&plusmn;P e variantes comuns no IRF6 tem sido observada em vários estudos em diferentes populações, porém os resultados obtidos não explicam a alta herdabilidade das FL&plusmn;P. Frente a este quadro, foi proposto mais um modelo genético para as fissuras NS, o modelo de &ldquo;doenças comuns-variantes raras&rdquo;. Diante destas questões, o presente trabalho foi realizado para tentar avaliar o impacto fenotípico de variantes no IRF6 em famílias com fissuras NS, através da verificação da ocorrência de mutações patogênicas, do tipo perda de função no IRF6, causativas da SVW entre casos não sindrômicos e da identificação da associação de variantes do IRF6 raras e comuns à predisposição de fissuras NS em casos familiais. Para testarmos estas hipóteses, foi realizado o sequenciamento do IRF6 em 304 pacientes com fissuras orais, não relacionados entre si, e com história familiar positiva para fissuras. Os resultados foram comparados com controles da população brasileira e com bancos de dados internacionais. A avaliação dos resultados sugere uma freqüência de aproximadamente 1% de mutações patogênicas para SVW entre os casos não sindrômicos familiais. Foram também encontradas variantes novas raras, de significado clínico ainda desconhecido, em 3,3% dos casos e observamos um excesso de variantes na região 5\'UTR entre os fissurados sugerindo que estas tenham um efeito funcional na regulação do IRF6 e eventual envolvimento com a ocorrência das fissuras orais NS. Além disso, foram detectadas novas associações com fissuras NS entre as variantes comuns já conhecidas. Além da análise mutacional do IRF6 entre pacientes com FL&plusmn;P e FP, estudamos as FP isoladamente, com o objetivo de verificar a contribuição genética nas fissuras palatinas não sindrômicas, através de um estudo epidemiológico. Entende-se que estes estudos podem fornecer informações relevantes para o delineamento de estudos genéticos e para associações com possíveis fatores ambientais predisponentes à malformação. Entretanto, há um número limitado de estudos epidemiológicos sobre FP na literatura e em particular no Brasil, por isso, objetivamos estimar dados relacionados à consanguinidade, risco de recorrência e herdabilidade das FPNS a partir de uma amostra de 331 casos de FPNS na população brasileira. A frequência de casamentos consanguíneos foi semelhante à da população controle e os riscos de recorrência para irmãos dos probandos (&sim;1,5%) foi menor que o estimado na literatura mundial. Foi estimada uma herdabilidade de 95,81%, o que sugere grande atuação de componentes genéticos na etiologia das FP nesta população. Estes resultados sugerem a validade de maiores estudos na identificação dos fatores genéticos para as FPNS e demonstram a importância de se estudar o efeito dos diversos tipos de variantes no IRF6 em pacientes com fissuras orais / Oral clefts are the most common congenital craniofacial malformation and have different incidences rates among different populations. These oral clefts are subdivided into two main groups, cleft lip with or without palate cleft (CL&plusmn;P) and cleft palate (CP), regarded as separate entities from embryological, epidemiological and etiological perspectives.These malformations are classified into syndromic or nonsyndromic (NS) depending on the existence of other associated clinical changes. For NSCL&plusmn;P and NSCP, the multifactorial inheritance pattern and the &ldquo;common disease-common variants&rdquo; model have been proposed. Several studies have been conducted in an attempt to identify the genetic factors predisposing to these malformations, particularly for NSCL&plusmn;P. Among these factors, the IRF6, demonstrably associated with Van der Woude (SVW) syndrome, is a candidate gene associated to NS clefts. In some situations, SVW becomes clinically indistinguishable from nonsyndromic clefts, which can hamper genetic counseling of cleft affected families. The association between CL&plusmn;P and IRF6 common variants has been observed in several studies in different populations, but the results do not explain the high heritability of CL&plusmn;P. Thus, an additional Another genetic model was proposed for NS clefts, the &ldquo;common disease-rare variant&rdquo; model. Given these issues, the present study tries to assess the phenotypic impact of variants in IRF6 in families with NS clefts by verifying the occurrence of loss-of-function mutations in IRF6, causative of SVW among nonsyndromic cases and also by the indentification of rare and common IRF6 variants predisposing NS clefts in familial cases. To test these hypotheses, the sequencing of IRF6 was performed for 304 unrelated cases of oral clets, with positive family history of clefts. results were compared with Brazilian population controls as well as international databases. The results suggests a prevalence of 1% for pathogenic mutations of SVW among nonsyndromic familial cases. It was also observed new rare variants of still unknown clinical significance in 3.3% of cases and it was observed an excess of variants in the 5\'UTR region among the cleft cases suggesting that they have a functional effect on the regulation of IRF6. Furthermore, new associations with NS clefts were detected among common variants already known. Besides mutational analysis of IRF6 for patients with CL&plusmn;P and CP, the CP cases were studied, in order to verify the genetic contribution in nonsyndromic CP, by means of an epidemiological study. It is understood that these studies may provide relevant information for genetic study guidelines and for possible associations with environmental factors predisposing the malformation. However, there is a limited number of epidemiological studies on CP in the literature and mainly in Brazil, so this study also aims to estimate data related to inbreeding, recurrence risk and heritability of NSCP from a sample of 331 cases from a Brazilian population. The frequency of consanguineous marriages was similar to the control frequency and recurrence risks for siblings of probands (about 1.5%) was lower than estimated in the literature. A heritability of 95.81% was estimated, suggesting a high genetic component interaction in the etiology of CP in this population. Finally these results indicate the relevance of further studies on the identification of genetic factors for NSCP and demonstrate the importance of studying the effect of different types of variants in IRF6 in patients with oral clefts

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2018-06-20T14:04:44Z No. of bitstreams: 2 Dissertação - Betânia Severino da Silva Maranhão - 2016.pdf: 1589818 bytes, checksum: 6614dffb36081f61317f02e41ca0a355 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-06-27T10:35:12Z (GMT) No. of bitstreams: 2 Dissertação - Betânia Severino da Silva Maranhão - 2016.pdf: 1589818 bytes, checksum: 6614dffb36081f61317f02e41ca0a355 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-06-27T10:35:12Z (GMT). No. of bitstreams: 2 Dissertação - Betânia Severino da Silva Maranhão - 2016.pdf: 1589818 bytes, checksum: 6614dffb36081f61317f02e41ca0a355 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-10-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Cleft lip and / or palate are birth defects easily recognizable and widely incidents in the human population. The clefts have a complex etiology involving genetic and environmental factors. They are found in approximately 1 in 700 births and have substantial impact on people's lives. Require always, surgery, dental, speech therapy, psychological and cosmetic. Clefts are recognized as a result of a wide rate of developmental disorders. Approximately 2/3 of the cases are not associated with any other abnormality and are called "non syndromic." The etiology of non syndromic oral clefts remains unknown. Preliminary studies suggest the involvement of a variety of genes and / or loci and environmental factors seem to play an important role in the genesis of such cases. Advances in molecular and quantitative analysis provide new opportunities to identify genes and gene-environment interactions relevant to the etiology of this common defect and representative birth. In this study, we analyzed a set of 80 cases of cleft lip and / or palate of non syndromic cases of patients of Associação de Combate as Deformidades Faciais (REFACE), collecting epidemiological and clinical data and biological sample for DNA extraction. The analysis was performed by Multiplex Ligation-dependent Probe Amplification (MLPA) of IRF6 and GRHL3 genes. This study finds only one individual with a duplication of a genomic region of the gene GRHL3, emphasizing the necessity of a larger sample and different geographical regions. / Fissuras de lábio e/ou palato (FL/P) são defeitos de nascimento facilmente reconhecíveis e amplamente incidentes na população humana. As fissuras têm uma etiologia complexa, envolvendo fatores genéticos e ambientais. São encontradas em aproximadamente 1 em 700 nascimentos e têm substancial impacto na vida das pessoas. As fissuras, de modo geral, acontecem com maior frequência nos homens do que nas mulheres, numa taxa de 2:1. Requerem, sempre, intervenções cirúrgicas, dentárias, fonoaudiológicas, psicológicas e cosméticas. Aproximadamente 2/3 dos casos não estão associadas a qualquer outra anomalia e são chamadas nãosindrômicas (FL/PNS). A etiologia da FL/PNS permanece desconhecida. Estudos preliminares sugerem a participação de uma variedade de genes e/ou loci, bem como fatores ambientais na etiologia desses defeitos congênitos. O gene IRF6 (interferon regulador factor 6) é consistente na sua contribuição como causa das fissuras orais dentre um grande numero de genes candidatos. Mutações nesse gene causam uma forma dominante da síndrome de van der Woude, a qual inclui FL/P, e marcadores polimorficos nesse gene estão fortemente associados a FL/P. Recentemente, o gene GRHL3 foi identificado como um outro gene associado à causa da síndrome de van der Woude. Dessa forma, passou a ser um novo gene candidato as FL/PNS. No presente trabalho, coletamos dados epidemiológicos, ambientais e amostra biológica (sangue periférico) de 80 casos de FL/PNS atendidos na Associação de Combate às Deformidades Faciais (REFACE) da cidade de Goiânia, GO. Investigamos variações nos genes IRF6 e GRHL3, mediante a técnica de Multiplex Ligation-dependent Probe Amplification (MLPA). Os resultados mostraram uma duplicação no exon 4 do gene GRHL3 em quatro indivíduos portadores de fissura de lábio e palato não aparentados. Nenhuma variação foi detectada no gene IRF6. Esse é o primeiro relato de uma microduplicação no gene GRHL3 em indivíduos com FL/PNS. Esses resultados são preliminares e enfatizam a necessidade de aplicação de outros métodos que comprovem esse achado, além de uma maior amostragem e de investigações adicionais no estudo da etiologia das fissuras orais não sindrômicas.

As fissuras orais são as malformações craniofaciais mais freqüentes ao nascimento e apresentam incidências variáveis entre as diversas populações. Essas fissuras são subdivididas em dois grupos principais, as fissuras labiais com ou sem fissura de palato (FL&plusmn;P) e as fissuras palatinas (FP), consideradas entidades distintas do ponto de vista embriológico, epidemiológico e etiológico. Estas malformações podem ser classificadas em sindrômicas ou não sindrômicas (NS) a depender da existência de outras alterações clínicas associadas. Para as FL&plusmn;PNS e FPNS, o padrão de herança multifatorial e o modelo &ldquo;doenças comuns-variantes comuns&rdquo; foram propostos. Diversos estudos têm sido realizados na tentativa de se identificar os fatores genéticos de predisposição a estas malformações, particularmente para as FL&plusmn;PNS. Entre estes fatores, o IRF6, comprovadamente associado à síndrome de Van der Woude (SVW), é um gene candidato associado às fissuras NS. Em algumas situações, a SVW torna-se clinicamente indistinguível das fissuras não sindrômicas, o que pode dificultar o aconselhamento genético destas famílias afetadas por fissuras. A associação entre FL&plusmn;P e variantes comuns no IRF6 tem sido observada em vários estudos em diferentes populações, porém os resultados obtidos não explicam a alta herdabilidade das FL&plusmn;P. Frente a este quadro, foi proposto mais um modelo genético para as fissuras NS, o modelo de &ldquo;doenças comuns-variantes raras&rdquo;. Diante destas questões, o presente trabalho foi realizado para tentar avaliar o impacto fenotípico de variantes no IRF6 em famílias com fissuras NS, através da verificação da ocorrência de mutações patogênicas, do tipo perda de função no IRF6, causativas da SVW entre casos não sindrômicos e da identificação da associação de variantes do IRF6 raras e comuns à predisposição de fissuras NS em casos familiais. Para testarmos estas hipóteses, foi realizado o sequenciamento do IRF6 em 304 pacientes com fissuras orais, não relacionados entre si, e com história familiar positiva para fissuras. Os resultados foram comparados com controles da população brasileira e com bancos de dados internacionais. A avaliação dos resultados sugere uma freqüência de aproximadamente 1% de mutações patogênicas para SVW entre os casos não sindrômicos familiais. Foram também encontradas variantes novas raras, de significado clínico ainda desconhecido, em 3,3% dos casos e observamos um excesso de variantes na região 5\'UTR entre os fissurados sugerindo que estas tenham um efeito funcional na regulação do IRF6 e eventual envolvimento com a ocorrência das fissuras orais NS. Além disso, foram detectadas novas associações com fissuras NS entre as variantes comuns já conhecidas. Além da análise mutacional do IRF6 entre pacientes com FL&plusmn;P e FP, estudamos as FP isoladamente, com o objetivo de verificar a contribuição genética nas fissuras palatinas não sindrômicas, através de um estudo epidemiológico. Entende-se que estes estudos podem fornecer informações relevantes para o delineamento de estudos genéticos e para associações com possíveis fatores ambientais predisponentes à malformação. Entretanto, há um número limitado de estudos epidemiológicos sobre FP na literatura e em particular no Brasil, por isso, objetivamos estimar dados relacionados à consanguinidade, risco de recorrência e herdabilidade das FPNS a partir de uma amostra de 331 casos de FPNS na população brasileira. A frequência de casamentos consanguíneos foi semelhante à da população controle e os riscos de recorrência para irmãos dos probandos (&sim;1,5%) foi menor que o estimado na literatura mundial. Foi estimada uma herdabilidade de 95,81%, o que sugere grande atuação de componentes genéticos na etiologia das FP nesta população. Estes resultados sugerem a validade de maiores estudos na identificação dos fatores genéticos para as FPNS e demonstram a importância de se estudar o efeito dos diversos tipos de variantes no IRF6 em pacientes com fissuras orais / Oral clefts are the most common congenital craniofacial malformation and have different incidences rates among different populations. These oral clefts are subdivided into two main groups, cleft lip with or without palate cleft (CL&plusmn;P) and cleft palate (CP), regarded as separate entities from embryological, epidemiological and etiological perspectives.These malformations are classified into syndromic or nonsyndromic (NS) depending on the existence of other associated clinical changes. For NSCL&plusmn;P and NSCP, the multifactorial inheritance pattern and the &ldquo;common disease-common variants&rdquo; model have been proposed. Several studies have been conducted in an attempt to identify the genetic factors predisposing to these malformations, particularly for NSCL&plusmn;P. Among these factors, the IRF6, demonstrably associated with Van der Woude (SVW) syndrome, is a candidate gene associated to NS clefts. In some situations, SVW becomes clinically indistinguishable from nonsyndromic clefts, which can hamper genetic counseling of cleft affected families. The association between CL&plusmn;P and IRF6 common variants has been observed in several studies in different populations, but the results do not explain the high heritability of CL&plusmn;P. Thus, an additional Another genetic model was proposed for NS clefts, the &ldquo;common disease-rare variant&rdquo; model. Given these issues, the present study tries to assess the phenotypic impact of variants in IRF6 in families with NS clefts by verifying the occurrence of loss-of-function mutations in IRF6, causative of SVW among nonsyndromic cases and also by the indentification of rare and common IRF6 variants predisposing NS clefts in familial cases. To test these hypotheses, the sequencing of IRF6 was performed for 304 unrelated cases of oral clets, with positive family history of clefts. results were compared with Brazilian population controls as well as international databases. The results suggests a prevalence of 1% for pathogenic mutations of SVW among nonsyndromic familial cases. It was also observed new rare variants of still unknown clinical significance in 3.3% of cases and it was observed an excess of variants in the 5\'UTR region among the cleft cases suggesting that they have a functional effect on the regulation of IRF6. Furthermore, new associations with NS clefts were detected among common variants already known. Besides mutational analysis of IRF6 for patients with CL&plusmn;P and CP, the CP cases were studied, in order to verify the genetic contribution in nonsyndromic CP, by means of an epidemiological study. It is understood that these studies may provide relevant information for genetic study guidelines and for possible associations with environmental factors predisposing the malformation. However, there is a limited number of epidemiological studies on CP in the literature and mainly in Brazil, so this study also aims to estimate data related to inbreeding, recurrence risk and heritability of NSCP from a sample of 331 cases from a Brazilian population. The frequency of consanguineous marriages was similar to the control frequency and recurrence risks for siblings of probands (about 1.5%) was lower than estimated in the literature. A heritability of 95.81% was estimated, suggesting a high genetic component interaction in the etiology of CP in this population. Finally these results indicate the relevance of further studies on the identification of genetic factors for NSCP and demonstrate the importance of studying the effect of different types of variants in IRF6 in patients with oral clefts

Cleft lip and/or palate (CL/P) is a group of congenital birth defect caused by the failure of the lip and/or palate to properly fuse during facial development. This defect occurs in approximately 1:700 live births and is the most second most common developmental defect. Twin studies and evaluation of family history reveals that risk for CL/P is influenced by genetics. However, to date less than half of the heritable risk for CL/P has been ascribed to specific genes. To identify new genes involved in CL/P, our colleagues, Dr. Manak and Dr. Murray, screened DNA from CL/P patients for rare copy number variants. A single copy deletion of Isthmin1 (ISM1) was identified in this screen. To test the hypothesis that this deletion contributed to the pathogenesis of CL/P we conducted functional tests of the zebrafish ortholog, isthmin1. The results indicated that Ism1 is necessary for development of the face in zebrafish, supporting the hypothesis. Together with Dr. Bassuk, we applied a similar approach to another structural birth defect, spina bifida. Dr. Manak discovered a de novo single copy deletion encompassing Glypican 5 (GPC5) and part of Glypican 6 (GPC6) in a spina bifida patient. Our functional tests in zebrafish support the notion that mutations in GPC5 cause spina bifida in some patients. To identify additional CL/P loci, we investigated two putative transcriptional targets of Interferon Regulatory Factor 6 (IRF6), a transcription factor encoded by a gene which is mutated in the majority of patients with Van der Woude orofacial clefting syndrome. The Cornell lab found evidence that Irf6 regulates expression of grhl3 and klf4 in zebrafish periderm. Partly in response to our findings, Jeff Murray's group sequenced the coding region GRHL3 gene in Van der Woude patients lacking IRF6 mutations and found 8 different coding mutations in GRHL3. We tested 5 of theses GRHL3 mutations in zebrafish-based functional studies, and found the 5 patient-derived GRHL3 variants had dominant negative effects. We conclude that GRHL3 is indeed a CL/P locus. Because all the patient derived variants were dominant negative (as opposed to null), and because such variants would be expected to block the function of the other copy of GRHL3 as well as other family members (GRHL1 and GRHl2), which are also cleft candidate genes. Multiple KLF4 coding mutations were also detected in patients with CL/P. We tested one of them but did not detect evidence of disruption in protein function. We conclude that this mutation does not seem to affect the function of KLF4, even though it was predicted to be damaging. This enforces the idea that conclusions from in silico studies should be examined in vivo. Finally, the protein structure of Irf6 has been examined to identify important residues within the C-terminus of the protein. Using constructs built by Dr. Mankad, we tested 5 different phosphor-mimetic amino acid substitutions. Of the variety of constructs, only Irf6 S447D in zebrafish Irf6 was able to cause ectopic expression of downstream Irf6 targets. This predicts that phosphorylation at S447 is sufficient to activate Irf6. All of these studies have expanded our understanding of the genetics behind CL/P, either by discovering new loci in human patients and testing them in Danio rerio, or from finding downstream targets of Irf6 in zebrafish, sequencing human patients for mutations in those genes, and then testing the functional changes of those variants. From our work with zebrafish, we can determine additional components of the IRF6 regulatory network.

Non-syndromic orofacial clefting (nsOFC) is among the most common congenital birth defects occurring up to 1 in 800 live births, with genetic and environmental causes. Genome wide association studies (GWAS) have identified several genetic loci that confer risk for nsOFC. However, more than half the heritable risk for nsOFC remains unknown and is considered ‘missing’. Moreover, continued sequencing of nsOFC patient DNA by whole exome sequencing and whole exome sequencing identify hundreds of single nucleotide polymorphism (SNPs). The identification of causal SNPs, however, continues to be a challenge in the OFC community. This is fueled partly by a lack of understanding of: (i) molecular mechanism and, (i) the gene regulatory network (GRN) governing differentiation of the relevant tissue, the embryonic superficial epithelia, also known as the periderm. Research has demonstrated that aberrant differentiation of the periderm, particularly the oral periderm results in pathological adhesions of surfaces within the developing oral cavity resulting in OFC. Further these adhesions can extend to the limbs which is a hallmarks feature in some forms of syndromic OFC (sOFC). In zebrafish, our model system of choice, knock-out of interferon regulatory factor 6 (irf6) ablated periderm marker expression and subsequently induces early embryonic lethality. The ortholog of IRF6 is a major genetic locus of Van der Woude syndrome (VWS) the most common form of sOFC and variants of IRF6 elevate risk for nsOFC. Therefore, we hypothesize that GRN of zebrafish periderm differentiation under the control of irf6 is a tool that can be used to identify novel OFC loci. Supporting this view, we have recently demonstrated that knock-down of an irf6 dependent gene encoding transcription factor Grainy-head like 3 (Grhl3) results in aberrant zebrafish periderm differentiation and GRHL3 was recently discovered as a novel VWS genetic locus. Hence it is likely that orthologs of genes encoding additional members of the periderm GRN harbor mutations in OFC patients. To identify cis–regulatory and transcriptional components in the periderm GRN, we performed: (i) a screen for periderm enhancers through in vivo green fluorescent protein (GFP) reporter assays, and, (ii) irf6 RNA-seq, followed by irf6 ChIP-seq to identify direct targets. From our screen for cis-regulatory elements we have identified a candidate human ZNF750 enhancer that directs GFP reporter expression in the zebrafish periderm. From our screen for irf6 direct targets we have identified several transcription factors including klf17, tfap2a and grhl3, all of which have variants in the human orthologs found in OFC patients. We further resolve the structure of the periderm differentiation GRN in zebrafish by assessing loss of function profiles for klf17, tfap2a and grhl3. Additionally, among the irf6 direct targets is a gene encoding another transcription factor, Zinc finger protein 750 (Znf750). We provide evidence to show that znf750 is expressed weakly in the zebrafish periderm. Further, we sequenced DNA in 500 nsOFC patient samples and identify a novel missense Ser160Pro ZNF750 variant which phenocopies the early embryonic lethality observed in irf6 mutants. Therefore, investigation of the zebrafish periderm GRN structure has facilitated the identification of OFC-associated risk loci.

A Fissura Oral (FO) é uma malformação craniofacial comum na espécie humana e sua etiologia é complexa com aspectos genéticos e ambientais envolvidos na sua formação. Por ser uma malformação de prevalência variável, estudos de associação em populações distintas são necessários, principalmente em populações heterogêneas como no Brasil. A suplementação com ácido fólico está envolvida na redução do risco de recorrência para algumas malformações, mas a natureza da reação entre a ingestão do ácido fólico, a interação entre os genes da rota metabólica e o seu efeito nas concentrações de folato é pouco caracterizada. Além disso, existem poucos estudos envolvendo um grande número de genes e a suplementação com ácido fólico a longo prazo. O objetivo desse trabalho foi estudar o papel dos genes MSX1 e IRF6 e região 8q24 em indivíduos com fissuras orais não sindrômicas de diferentes regiões do Brasil e analisar o efeito da suplementação com ácido fólico e dos polimorfismos nos genes da rota metabólica do folato nos níveis de folato séricos e eritrocitários. Nossos resultados mostram associação positiva entre o alelo 4 do polimorfismo de repetição CA (MSX1) e FO, alelo A da variante rs987525 (8q24) foi associado com FL/P e o haplótipo G/A (rs2235371/rs642961) do gene IRF6 associado com o aumento do risco para FL/P. Dos 23 genes da rota metabólica do ácido fólico estudados, 5 (FPGS, FOLR1, FOLR2, SHMTI e MTHFR) foram relacionados com os níveis de folato sérico e eritrocitário. As variantes rs7033913 (FPGS), rs11235462 (FOLR1) e rs2276048 (FOLR2) foram associadas com os níveis de folato sérico após suplementação. Os polimorfismos rs2168781 e rs2461837 (SHMT1) foram relacionados com os níveis de folato eritrocitário basal e o rs1801131(MTHFR) com os níveis de folato eritrocitário durante a suplementação. Conhecer a etiologia das fissuras orais e entender os efeitos da suplementação e de variantes dos genes da rota do folato nos níveis basais de folato é essencial tanto para auxiliar no manejo clínico através de uma medicina personalizada quanto para aconselhamento genético. / Oral cleft (OC) is a common craniofacial malformation. The etiology is complex and involves genetic and environmental factors. OC have a variable prevalence and association studies are needed in different populations, especially in heterogeneous populations as the Brazilian. Folic acid supplementation reduce the recurrence risk for some malformation, but the reaction between folic acid intake, the interaction between genes of metabolic pathway and effect on folate concentrations is poorly characterized. Furthermore, there are few studies with a large number of genes and long-term folic acid supplementation. The aim was to analyze the role of MSX1 and IRF6 gene and 8q24 region in individuals with non-syndromic oral clefts in different regions of Brazil and to analyze the effect of folic acid supplementation in folate pathway genes and correlate to levels of serum and red blood cell (RBC) folate. Our results have shown a positive association between the CA repeat polymorphism 4 allele (MSX1) and OC, between rs987525 A allele (8q24) and CL/P and the G/A haplotype (rs2235371 / rs642961) of IRF6 associated with increased risk of CL/P. The 23 folate pathway genes studied, 5 (FPGS, FOLR1, FOLR2, SHMTI and MTHFR) were correlated to serum and red blood cell (RBC) folate levels. The variants rs7033913 (FPGS), rs11235462 (FOLR1) and rs2276048 (FOLR2) were associated to serum folate levels after supplementation. Polymorphisms in SHMT1 (rs2168781 and rs2461837) were associated with basal RBC folate while MTHFR (rs1801131) were associated with RBC folate levels during supplementation. Understanding of oral cleft etiology and folate gene pathway will assist clinic management and genetic counseling since folate is involved in important biologic processes.