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Master's Dissertation
DOI
10.11606/D.61.2013.tde-12112013-150520
Document
Author
Full name
Ana Laís Bignotto da Rocha
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Bauru, 2013
Supervisor
Committee
Bicudo, Lucilene Arilho Ribeiro (President)
Conte, Agnes Cristina Fett
Maximino, Luciana Paula
Title in Portuguese
Sequenciamento direto dos genes SIX3, SHH, TGIF1, ZIC2 e array-CGH no estudo de pacientes com holoprosencefalia
Keywords in Portuguese
CGH-array
Holoprosencefalia
sequenciamento direto
Abstract in Portuguese
Objetivos: Analisar por meio da técnica de sequenciamento direto a presença de alterações moleculares nos genes SHH, SIX3, ZIC2 e TGIF1 em indivíduos com diagnóstico clínico de HPE. Analisar por meio da técnica de CGH-array a presença de alterações moleculares em indivíduos com diagnóstico clínico de HPE previamente submetidos à análise por sequenciamento direto. Local: Laboratório de Genética e Citogenética Humana HRAC/USP, Bauru-SP. Casuística e metodologia: Foram selecionados 50 indivíduos, de ambos os sexos com idades entre 03 meses a 50 anos com diagnóstico clínico para HPE. Todos foram analisados por meio da técnica de sequenciamento direto para os genes SHH e TGIF1 completamente e para os genes ZIC2 e SIX3 parcialmente. Dentre os indivíduos que não apresentaram alterações na técnica de sequenciamento oito indivíduos com fenótipo mais grave foram selecionados para a análise por CGH-array. Resultados e discussão: Foram analisados 50 indivíduos por meio da técnica de sequenciamento direto dos gene SHH e TGIF1, foram encontradas duas variantes patogênicas na análise do gene SHH, no caso 1 a variante p.24G>P foi identificada, e no caso 2 foi identificada a variante c.1031del C. No gene TGIF1 foram encontrados cinco polimorfismos já descritos na literatura. Foi identificada uma nova variante silenciosa no éxon 1 do gene ZIC2 p.Q46Q (c. 431 G>A) e um polimorfismo já descrito na literatura em dois indivíduos no gene SIX3. A análise por CGH-array revelou a presença de uma microdeleção no caso 37, de 1,5Mb no cromossomo 17p12 entre as posições genômicas 14,052,279-15,102,307. A mesma deleção foi encontrada na mãe, sendo que esta região nunca foi associada a HPE. Conclusão: A técnica de sequenciamento direto é uma ferramenta muito importante no diagnóstico molecular da HPE, a padronização do sequenciamento direto para os genes ZIC2 e SIX3 poderá auxiliar em diagnósticos mais precisos em estudos futuros dentro do HRAC/USP. O emprego de novas técnicas como CGH-array pode indicar novas relações entre regiões cromossômicas e os múltiplos fatores envolvidos na formação da HPE.
Title in English
Direct sequencing of the genes SIX3, SHH, TGIF1, ZIC2 and array-CGH on the study of patients with holoprosencephaly
Keywords in English
Array-CGH
direct sequencing
Holoprosencephaly
Abstract in English
Objective: Analyze through direct sequencing technique the presence of molecular changes on the genes SHH, SIX3, ZIC2 and TGIF1 on individuals with clinical diagnosis of HPE. Analyze through array-CGH technique the presence of molecular changes on individuals with clinical diagnosis of HPE previously submitted to the direct sequencing analyzes. Local: Genetics and Human Cytogenetics Laboratory, HRAC/USP, Bauru-SP. Methods: Were selected 50 individuals from both genders with ages between 03 months and 50 years clinically diagnosed with HPE. Everyone was analyzed through the direct sequencing technique for the genes SHH and TGIF1 completely and for the genes ZIC2 and SIX3 partially. From those individuals which did not have shown changes on the direct sequencing technique, eight individuals with more severe phenotype were selected to the analysis through array-CGH. Results an Discussion: Were analyzed 50 individuals through the technique of direct sequencing of the genes SHH and TGIF1, were found two pathogenic variants in the analysis of SHH gene, in the case 1, the variant p.G24P was identified, and in the case 2 was identified the variant c.1031delC. On the TGIF1 gene were found five polymorphisms already described on the literature. Was identified a new silent variant on the exon 1 of the ZIC2 gene p. Q46Q(c.431G>A) and a polymorphism already described in the literature in two individuals on the gene SIX3. The analysis through array-CGH revealed the presence of one microdeletion in the case 37, of 1,5 Mb on the region 17p12 between the genomic positions 14,052,279-15,102,307. The same deletion was detected in the mother, though this region was never associated to the HPE. Conclusion: The direct sequencing technique is a very important tool for the molecular diagnosis of the HPE, and the direct sequencing standardization for the genes ZIC2 and SIX3 might help in more precise diagnostics on HRAC/USP future studies. The employ of new techniques such as array-CGH may indicate new relations between chromosomal regions and the multiple hit involved in the development of HPE.
 
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DissAnaLaisRocha.pdf (1.45 Mbytes)
Publishing Date
2013-11-13
 
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