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

Rare genetic diseases are a very heterogeneous group of diseases, often undiagnosed, for which patients receive only symptomatic treatment. The development of next-generation massive sequencing technology (Next-Generation-Sequencing; NGS) has made it possible to identify many of the genes responsible for these pathologies, allowing the pathogenic mechanisms to be outlined. The most commonly used methods, such as exome sequencing (Whole-Exome Sequencing; WES) or genome sequencing (Whole-Genome Sequencing; WGS), are based on DNA sequencing and allow to identify pathogenic mutations that cause loss of protein functionality, such as stop mutations, large insertions and deletions and the loss of canonical splicing sites. Conversely, the attribution of pathogenicity to an amino acid substitution variant (missense) or a synonymous variant is often questionable and, consequently, such variants are often categorized as Variants of Unknown Significance (VUS). Furthermore, exome sequencing is unable to identify possible pathogenic variants located in non-coding regions, such as non-canonical splice sites or deep intronic variants.

WGS sequencing, while allowing the identification of variants present in the non-coding regions of a gene, is very expensive and produces an enormous amount of data with low pathogenic information. Algorithms to analyze the possible pathogenicity of intronic variants have little predictive power and the pathogenicity of the identified variants must be validated by lengthy and complex functional analyses. Due to these limitations, approximately 50% of patients with rare genetic diseases remain undiagnosed and cannot benefit from the benefits of personalized medicine. Transcriptome sequencing (RNA-Seq) allows to determine variations in the sequence and levels of RNA, providing complementary information to that obtained with WES and WGS, thus improving the efficiency of genetic diagnosis. Splicing alterations, which are among the main causes of genetic diseases and can reside both in deep intronic regions and in coding regions, can be identified through RNA sequencing. Variants in the regulatory regions of the gene, which are able to influence RNA expression levels, can also be highlighted by RNA-Seq. Since RNA expression, in terms of levels and isoforms produced by alternative splicing, is tissue-specific, the most transcriptionally informative biological material is certainly the affected tissue. However, this is not always easy to achieve and the collection can be too invasive for the patient. In these cases it is possible to resort to the use of alternative tissues, such as whole blood or skin biopsies, which have low invasiveness and a broad RNA expression profile. Fibroblasts obtained from skin biopsies, in particular, have proven to be the most reliable, homogeneous and informative alternative matrix for RNA-Seq analysis, in a wide range of pathologies. It has been previously demonstrated that the information obtained from RNA-Seq from skin fibroblasts was superior to that obtained from blood. Furthermore, the analysis of the transcriptome of skin fibroblasts allowed to identify altered levels of transcripts and splicing abnormalities in neuromuscular and neurodevelopmental diseases. This confirmed the efficacy of the use of skin fibroblasts in the diagnosis of genetic pathologies in WES and WGS negative patients. Last but not least, skin fibroblasts can be reprogrammed into other cell types for possible future analyzes.

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.

Although NGS is essential for the diagnosis of genetic diseases, its success rates range from 30% to 50%. The advent of RNA-Seq increased the chance of diagnosing genetic diseases from 8% to 36%. Through this project the investigators intend to apply the RNA-Seq method, as an integration of the WES analysis, for the diagnosis of rare genetic diseases:

• 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.