Q3143457 (Q3143457): Difference between revisions
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(Created claim: summary (P836): MAIN OBJECTIVES: 1) Functionally analyse the mutations of the candidate genes GPATCH3, LRP2 and GUCA1C, previously identified by our group in the exome study of 26 patients with severe primary congenital glaucoma. 2) Study in zebrafish larvae the ocular phenotypes resulting from reducing or increasing the expression of orthologist genes of the 3 genes mentioned above. 3) Analyse the presence of mutations of candidate LRP2 and GUCA1C genes in 90...) |
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MAIN OBJECTIVES: 1) Functionally analyse the mutations of the candidate genes GPATCH3, LRP2 and GUCA1C, previously identified by our group in the exome study of 26 patients with severe primary congenital glaucoma. 2) Study in zebrafish larvae the ocular phenotypes resulting from reducing or increasing the expression of orthologist genes of the 3 genes mentioned above. 3) Analyse the presence of mutations of candidate LRP2 and GUCA1C genes in 90 patients with congenital glaucoma without known mutations. 4) Identify new genes involved in the disease by reanalysis of 23 of the exomas already sequenced, following a threesome analysis strategy formed by the tester and his healthy parents. METHODOLOGY: Objective 1) The mutations identified will be generated by targeted mutagenesis. Subsequently, they shall be expressed in HEK-293T cells and their stability, subcellular location and transactivation shall be analysed using western blot, immunocytochemistry and luciferase assays, respectively. Objective 2) The expression of orthologist genes of the 3 candidate genes will be studied in zebrafish larvae, using fluorescent in situ hybridisation and confocal microscopy. The ocular phenotype will also be analysed by confocal microscopy after inhibiting the expression of these ortologist genes (knockdown with morfolins) and transiently overexpressing them (mRNA microinjection). Objective 3) The tracing in 90 patients of mutations of the LRP2 and GUCA1C genes will be carried out by mass sequencing (targeted sequencing). Objective 4) For the study of the 23 exomas in threesomes, the parent exome sequence will be determined by mass sequencing. Different filters will be applied to identify the candidate variants and functional studies will be carried out to confirm their pathogenicity. (English) | |||||||||||||||
| Property / summary: MAIN OBJECTIVES: 1) Functionally analyse the mutations of the candidate genes GPATCH3, LRP2 and GUCA1C, previously identified by our group in the exome study of 26 patients with severe primary congenital glaucoma. 2) Study in zebrafish larvae the ocular phenotypes resulting from reducing or increasing the expression of orthologist genes of the 3 genes mentioned above. 3) Analyse the presence of mutations of candidate LRP2 and GUCA1C genes in 90 patients with congenital glaucoma without known mutations. 4) Identify new genes involved in the disease by reanalysis of 23 of the exomas already sequenced, following a threesome analysis strategy formed by the tester and his healthy parents. METHODOLOGY: Objective 1) The mutations identified will be generated by targeted mutagenesis. Subsequently, they shall be expressed in HEK-293T cells and their stability, subcellular location and transactivation shall be analysed using western blot, immunocytochemistry and luciferase assays, respectively. Objective 2) The expression of orthologist genes of the 3 candidate genes will be studied in zebrafish larvae, using fluorescent in situ hybridisation and confocal microscopy. The ocular phenotype will also be analysed by confocal microscopy after inhibiting the expression of these ortologist genes (knockdown with morfolins) and transiently overexpressing them (mRNA microinjection). Objective 3) The tracing in 90 patients of mutations of the LRP2 and GUCA1C genes will be carried out by mass sequencing (targeted sequencing). Objective 4) For the study of the 23 exomas in threesomes, the parent exome sequence will be determined by mass sequencing. Different filters will be applied to identify the candidate variants and functional studies will be carried out to confirm their pathogenicity. (English) / rank | |||||||||||||||
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| Property / summary: MAIN OBJECTIVES: 1) Functionally analyse the mutations of the candidate genes GPATCH3, LRP2 and GUCA1C, previously identified by our group in the exome study of 26 patients with severe primary congenital glaucoma. 2) Study in zebrafish larvae the ocular phenotypes resulting from reducing or increasing the expression of orthologist genes of the 3 genes mentioned above. 3) Analyse the presence of mutations of candidate LRP2 and GUCA1C genes in 90 patients with congenital glaucoma without known mutations. 4) Identify new genes involved in the disease by reanalysis of 23 of the exomas already sequenced, following a threesome analysis strategy formed by the tester and his healthy parents. METHODOLOGY: Objective 1) The mutations identified will be generated by targeted mutagenesis. Subsequently, they shall be expressed in HEK-293T cells and their stability, subcellular location and transactivation shall be analysed using western blot, immunocytochemistry and luciferase assays, respectively. Objective 2) The expression of orthologist genes of the 3 candidate genes will be studied in zebrafish larvae, using fluorescent in situ hybridisation and confocal microscopy. The ocular phenotype will also be analysed by confocal microscopy after inhibiting the expression of these ortologist genes (knockdown with morfolins) and transiently overexpressing them (mRNA microinjection). Objective 3) The tracing in 90 patients of mutations of the LRP2 and GUCA1C genes will be carried out by mass sequencing (targeted sequencing). Objective 4) For the study of the 23 exomas in threesomes, the parent exome sequence will be determined by mass sequencing. Different filters will be applied to identify the candidate variants and functional studies will be carried out to confirm their pathogenicity. (English) / qualifier | |||||||||||||||
point in time: 12 October 2021
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Revision as of 13:29, 12 October 2021
Project Q3143457 in Spain
| Language | Label | Description | Also known as |
|---|---|---|---|
| English | No label defined |
Project Q3143457 in Spain |
Statements
83,600.0 Euro
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104,500.0 Euro
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80.0 percent
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1 January 2016
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31 March 2020
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UNIVERSIDAD DE CASTILLA-LA MANCHA
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38°59'42.32"N, 1°51'21.31"W
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02003
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OBJETIVOS PRINCIPALES: 1) Analizar funcionalmente las mutaciones de los genes candidato GPATCH3, LRP2 y GUCA1C, identificadas previamente por nuestro grupo en el estudio del exoma de 26 pacientes con glaucoma congénito primario grave. 2) Estudiar en larvas de pez cebra los fenotipos oculares resultantes de reducir o incrementar la expresión de los genes ortólogos de los 3 genes mencionados anteriormente. 3) Analizar la presencia de mutaciones de los genes candidato LRP2 y GUCA1C en 90 pacientes con glaucoma congénito sin mutaciones conocidas. 4) Identificar nuevos genes implicados en la enfermedad mediante reanálisis de 23 de los exomas ya secuenciados, siguiendo una estrategia de análisis de tríos formados por el probando y sus progenitores sanos. METODOLOGÍA: Objetivo 1) Las mutaciones identificadas se generarán mediante mutagénesis dirigida. Posteriormente serán expresadas en células HEK-293T y se analizará su estabilidad, localización subcelular y transactivación, mediante western blot, inmunocitoquímica y ensayos con luciferasa, respectivamente. Objetivo 2) Se estudiará en larvas de pez cebra la expresión de los genes ortólogos de los 3 genes candidato, utilizando hibridación in situ fluorescente y microscopía confocal. También se analizará mediante microscopía confocal el fenotipo ocular después de inhibir la expresión de estos genes ortólogos (knockdown con morfolinos) y de sobreexpresarlos transitoriamente (microinyección de ARNm). Objetivo 3) El rastreo en 90 pacientes de mutaciones de los genes LRP2 y GUCA1C se realizará mediante secuenciación masiva (targeted sequencing). Objetivo 4) Para el estudio de los 23 exomas en tríos se determinará la secuencia del exoma de los progenitores mediante secuenciación masiva. Se aplicarán distintos filtros para identificar las variantes candidatas y se realizarán estudios funcionales para confirmar su patogenicidad. (Spanish)
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MAIN OBJECTIVES: 1) Functionally analyse the mutations of the candidate genes GPATCH3, LRP2 and GUCA1C, previously identified by our group in the exome study of 26 patients with severe primary congenital glaucoma. 2) Study in zebrafish larvae the ocular phenotypes resulting from reducing or increasing the expression of orthologist genes of the 3 genes mentioned above. 3) Analyse the presence of mutations of candidate LRP2 and GUCA1C genes in 90 patients with congenital glaucoma without known mutations. 4) Identify new genes involved in the disease by reanalysis of 23 of the exomas already sequenced, following a threesome analysis strategy formed by the tester and his healthy parents. METHODOLOGY: Objective 1) The mutations identified will be generated by targeted mutagenesis. Subsequently, they shall be expressed in HEK-293T cells and their stability, subcellular location and transactivation shall be analysed using western blot, immunocytochemistry and luciferase assays, respectively. Objective 2) The expression of orthologist genes of the 3 candidate genes will be studied in zebrafish larvae, using fluorescent in situ hybridisation and confocal microscopy. The ocular phenotype will also be analysed by confocal microscopy after inhibiting the expression of these ortologist genes (knockdown with morfolins) and transiently overexpressing them (mRNA microinjection). Objective 3) The tracing in 90 patients of mutations of the LRP2 and GUCA1C genes will be carried out by mass sequencing (targeted sequencing). Objective 4) For the study of the 23 exomas in threesomes, the parent exome sequence will be determined by mass sequencing. Different filters will be applied to identify the candidate variants and functional studies will be carried out to confirm their pathogenicity. (English)
12 October 2021
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Albacete
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Identifiers
PI15_01193
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