Developing a Pipeline to Employ RNA-Seq as a Complementary Diagnostic Tool in Rare Diseases

This project aims to identify, through RNA-Seq technology, the genetic alterations underlying undiagnosed rare diseases in pediatric and adult patients with early onset and with negative WES.

* Objective 1: Set up and validate techniques. Set-up and validation of the transcriptome analysis protocol in healthy subjects and in patients with known splicing alterations and/or altered RNA expression. * Objective 2: Diagnostic phase. Study of splicing alterations and RNA levels in cultured fibroblasts obtained from skin biopsies of patients with rare genetic diseases and negative exome.

Exploratory goals

* Compare the RNA expression profile obtained from skin biopsy-derived fibroblasts with the RNA expression profile from blood. The most relevant results will be validated in qRT-PCR. * To analyze the transcriptional and protein profile heterogeneity in skin-derived fibroblasts in enrolled subjects.

To explore the effects of genetic (from WES) and transcriptional (from RNA-seq) alterations in participants' plasma and serum.

Healthy controls Five healthy subjects will be recruited from the staff of the Mario Negri Institute for Pharmacological Research. The coded samples will be used to set up the method of isolation and culture of skin fibroblasts and RNA-Seq.

Validation group For the set-up and validation of the skin fibroblast isolation and RNA-Seq procedure, ten adult patients with known diagnosis and with alterations in RNA levels and/or splicing will be recruited as positive controls.

Patients who meet the requirements described above will be contacted by the doctors of the Daccò Center for an interview explaining the project. Those who agree to participate in the study will be asked to sign the informed consent before proceeding with the experimental part.

"Discovery/Exploration" group The exploration cohort will be composed of 30 symptomatic undiagnosed patients with suspected genetic disease (children and adults with infantile onset) belonging to the Clinical Center of the Mario Negri Institute for Pharmacological Research and for whom WES investigations did not reveal causative genetic alterations.