La secuenciación masiva del exoma, como estrategia más coste-eficiente en el diagnóstico genético de las enfermedades monogenéticas mendelianas, tiene una limitación impuesta por su propio diseño: no incluye la secuenciación de las regiones intrónicas. Ya es posible realizar la secuenciación masiva de todo el genoma, pero sin embargo, las tecnologías bioinformáticas y la acumulación científica necesaria para procesar la información obtenible por esta técnica aún no han madurado lo suficiente como para que sea utilizada en la práctica clínica diaria. Es por ello que debemos ser conscientes de la posibilidad de que algunas enfermedades puedan ser explicadas por variantes intrónicas profundas no detectadas mediante secuenciación del exoma.
Esto es de interés especial para las enfermedades recesivas, y debe ser tenido en cuenta en caso de detectar una variante patogénica en un alelo, pero no en el otro, en un caso con un fenotipo altamente sugerente de la enfermedad, ante la posibilidad de que exista heterocigosis compuesta.
Splicing.
El mecanismo por el cual las variantes intrónicas tienen consecuencias sobre el fenotipo tiene que ver principalmente con el splicing.
La presencia de intrones en el genoma es un fenómeno biológico característico de las células eucariotas, y la ventaja evolutiva principal que posee es la habilidad para seleccionar diferentes combinaciones de exones, lo cual incrementa la diversidad de expresión génica. El proceso a través del cual, obtenemos una secuencia de ARNm sin intrones, desde una secuencia de pre-ARN, que es copia exacta del ADN, es conocido como splicing. La maquinaria ribonucleoprotéica encargada de dicha función se conoce como spliceosoma.
Existen principalmente 2, el spliceosoma mayor y el spliceosoma menor. Se trata de una maquinaria modulada por múltiples mecanismos, entre ellos los activadores (enhacers) y silenciadores (silencers). La acción de dichos moduladores da lugar a un fenómeno conocido como splicing alternativo, de forma que un mismo gen puede dar lugar a productos proteicos distintos en función de las circustancias. Según las estimaciones actuales, alrededor del 15-50% de las mutaciones puntuales monogénicas causantes de enfermedad afectarían al splicing del pre-ARN, con la mayor parte de ellas afectando a los puntos de unión de splicing (splice junctions).
Mecanismos patogénicos de las mutaciones intrónicas.
- Inclusión de pseudo-exones.
- Competición con los lugares naturales de splicing.
- Disrupción de elementos de regulación de la transcripción.
- Inactivación de ARN no codificantes.
- Reorganizaciones cromosómicas.
Enfermedades con mutaciones intrónicas patogénicas descritas en la literatura.
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