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Depósito De Tesis 080715.Pdf DEPARTAMENTO DE CIRUGÍA TESIS DOCTORAL Identificación y análisis de nuevos genes y mecanismos moleculares implicados en la Enfermedad de Hirschsprung María del Valle Enguix Riego Sevilla, 2015 DEPARTAMENTO DE CIRUGÍA Memoria realizada en la Unidad de Gestión Clínica de Genética, Reproducción y Medicina Fetal del Hospital Universitario Virgen del Rocío bajo la dirección de Da. Salud Borrego López, Doctora en Medicina y Cirugía, y la tutoría de D. Guillermo Antiñolo Gil, Doctor en Medicina y Cirugía, y Profesor Titular del Departamento de Cirugía de la Universidad de Sevilla, que presenta la Licenciada en Farmacia María del Valle Enguix Riego para optar al grado de Doctor. La Directora, El Tutor, Fdo. Salud Borrego López Fdo. Guillermo Antiñolo Gil Doctora en Medicina y Cirugía Doctor en Medicina y Cirugía Hospital Universitario Virgen del Rocío Profesor Titular Universidad de Sevilla La Doctoranda, Fdo. María del Valle Enguix Riego Índice ÍNDICE Índice Índice de Contenido Índice de Contenido ............................................................................................................i Índice de Figuras .......................................................................................................... iv Índice de Tablas .......................................................................................................... vii ABREVIATURAS .............................................................................................................. xiii 1. INTRODUCCIÓN ...................................................................................................... xvi 1.1 El Sistema Nervioso Entérico ................................................................................. 1 1.1.1 Principales rutas de señalización durante el desarrollo del SNE .................... 5 1.1.2 Factores de transcripción implicados en el desarrollo del SNE ..................... 10 1.1.3 Regulación epigenética de las células de la cresta neural y su posible implicación en el desarrollo del SNE ....................................................................... 18 1.2 Neurocristopatías................................................................................................. 22 1.2.1 La enfermedad de Hirschsprung .................................................................... 23 1.2.2 Síndrome de Waardenburg ............................................................................ 34 2. HIPÓTESIS DE TRABAJO Y OBJETIVOS ....................................................................... 37 2.1 Hipótesis de trabajo ............................................................................................. 39 2.2 Objetivos del estudio ........................................................................................... 40 3. METODOLOGÍA .......................................................................................................... 41 3.1 Pacientes y Controles ........................................................................................... 43 3.1.1 Pacientes HSCR ............................................................................................... 43 3.1.2 Individuos Control .......................................................................................... 44 3. 2 Animales de Experimentación ............................................................................ 44 3.2.1 Ratones .......................................................................................................... 44 3.2.2 Pez Cebra ........................................................................................................ 44 3.3 Métodos ............................................................................................................... 47 MV. Enguix-Riego -i Índice 3.3.1 Técnicas generales utilizadas para estudios moleculares de ADN genómico 47 3.3.2 Extracción de precursores neurales crecidos como NLBs a partir del SNE humano y de ratón .................................................................................................. 55 3.3.3 Estudios en Pez-Cebra .................................................................................... 68 3.3.4 Análisis estadístico ......................................................................................... 71 3.3.5 Herramientas Bioinformáticas ....................................................................... 73 4. RESULTADOS .............................................................................................................. 75 4.1 Identificación de variantes patogénicas en dos pacientes con Síndrome de Waandenburg Tipo IV. ............................................................................................... 77 4.1.1 Historia clínica y eventos moleculares presentes en el Paciente 1. .............. 77 4.1.2 Historia clínica y eventos moleculares presentes en el Paciente 2. .............. 78 4.2 Implicación del gen DNMT3B en la patogénesis de la enfermedad de Hirschsprung .............................................................................................................. 81 4.2.1 Análisis de la expresión diferencial de genes de pluripotencia en precursores neurales de SNE de pacientes HSCR versus controles ............................................ 81 4.2.2 Expresión de la proteína DNMT3B en muestras de precursores neurales de SNE de pacientes HSCR versus controles. ............................................................... 86 4.2.3 Análisis de los niveles de metilación en precursores entéricos de pacientes HSCR versus controles. ............................................................................................ 87 4.2.4 Rastreo mutacional de la secuencia codificante de DNMT3B en la cohorte HSCR ........................................................................................................................ 88 4.2.5 Estudio de DNMT3B en Pez-cebra ................................................................. 92 4.3 Implicación del gen PAX6 en la patogénesis de la enfermedad de Hirschsprung ................................................................................................................................. 101 4.3.1 Análisis de la expresión diferencial de factores de transcripción en precursores neurales de SNE de pacientes HSCR versus controles. ..................... 101 4.3.2 Expresión de la proteína PAX6 en muestras de precursores neurales de SNE de pacientes HSCR versus controles. .................................................................... 104 4.3.3 Estudio de expresión de las dos isoformas mayoritarias de PAX6 .............. 105 ii - MV. Enguix-Riego Índice 4.3.4 Rastreo mutacional de la secuencia codificante del factor de transcripción PAX6 y análisis del SRT (AC)m(AG)n localizado en el promotor P1. ..................... 107 4.3.5 Evaluación de PAX6 como diana de p300. ................................................... 111 4.3.6 Evaluación de PAX6 como diana de la metiltransferasa DNMT3B. ............. 113 5. DISCUSIÓN ................................................................................................................ 117 5.1 Identificación de dos variantes patogénica en Síndrome de Waardenburg Tipo IV. ............................................................................................................................. 119 5.2 Identificación de genes de pluripotencia que presentaban niveles de expresión diferencial en precursores neurales de SNE de pacientes HSCR versus controles .. 124 5.2.1 Disminución de la expresión de DNMT3B en muestras de precursores neurales de SNE de pacientes HSCR versus controles .......................................... 128 5.2.2 Niveles de metilación global anormalmente disminuidos en precursores entéricos de pacientes HSCR en comparación con controles. .............................. 129 5.2.3 Identificación de variantes dentro de la secuencia de DNMT3B en la cohorte de pacientes HSCR ................................................................................................. 130 5.2.4 Estudio de DNMT3B en Pez-cebra ............................................................... 132 5.3. Identificación de factores de transcripción con diferencias en los niveles de expresión entre precursores neurales de SNE de pacientes HSCR versus controles. ................................................................................................................................. 135 5.3.1 El balance en la expresión de las dos isoformas mayoritarias de PAX6 no parece ser necesario a nivel de precursor entérico .............................................. 138 5.3.2 Diferente distribución alélica y genotípica del SRT (AC)m(AG)n localizado en el promotor P1 de PAX6, en pacientes HSCR versus controles. ........................... 139 5.3.3 PAX6 es una diana de la metiltransferasa DNMT3B. ................................... 141 6. CONCLUSIONES ........................................................................................................ 143 7. BIBLIOGRAFÍA ........................................................................................................... 147 8. ANEXO ...................................................................................................................... 175 9. PUBLICACIONES........................................................................................................ 179 MV. Enguix-Riego - iii Índice Índice de Figuras Figura 1: Sección esquemática del tracto gastrointestinal (GI) adulto ...........................
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