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.

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.

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