Development of Targeted RNA-Seq for Amyotrophic Lateral Sclerosis Diagnosis - Trial NCT06083584

Timeline & Enrollment

Primary Outcome

Concordance between the RNA-Seq results (index text) versus RT-PCR followed by Sanger sequencing (reference technique) on positive plus negative controls

Summary

Genetic diagnosis of Amyotrophic Lateral Sclerosis (ALS) could identify the origin of the
 disease, potentially allowing the patient to pursue targeted/gene therapy. However, many
 familial forms of ALS are genetically undiagnosed, either because no variant has been
 detected in the genes of interest, or because the detected variant(s) have uncertain
 significance. Currently, molecular diagnosis takes place in two stages: 1) Search for the
 GGGGCC expansion in the C9ORF72 gene by RP-PCR; 2) Analysis of the coding regions by
 high-throughput sequencing of a panel of 30 genes involved in ALS.
 
 Many of these variants of uncertain significance affect splicing. Their impact can be
 predicted using in silico tools, but only an analysis of the patient's RNA can confirm their
 pathogenic nature. Currently, the analysis of transcripts is only done a posteriori, when a
 variant predicted to impact splicing is detected on the patient's DNA. RT-PCR followed by
 Sanger sequencing then verifies the impact of the splice variants. This method confirmed the
 impact of certain splice variants in patients. However, this method is time-consuming and
 requires custom development, and is mutation/gene/patient-dependent. In contrast,
 high-throughput RNA sequencing (RNA-Seq) simultaneously analyzes the splicing of numerous
 genes, with a global approach, applicable to all patients. This approach avoids the custom
 design of primers, which can be biased by the interpretation of splicing predictions, while
 RNA-Seq systematically captures and sequences all the transcripts. Finally, RNA-Seq provides
 additional information compared to DNA sequencing such as the detection of exon skipping,
 intron inclusion, and the creation of fusion transcripts.
 
 In the GTEx project (GTEx Consortium, 2013), expression levels of human genome transcripts
 were quantified by RNA-Seq. Using these results, the study investigators measured expression
 of transcripts of known ALS genes in whole blood. Applying a threshold value of 0.5
 transcripts per million reads (TPM), 25 of the 30 ALS genes currently analyzed by NGS in
 routine diagnostics at Nîmes University Hospital could be eligible for a complete analysis by
 RNA-Seq. None of the French laboratories carrying out genetic analyzes of ALS has yet
 developed RNA-Seq as a routine diagnostic tool. The study laboratory receives more than 600
 requests for genetic diagnosis of ALS patients per year. The aim of this study is therefore
 to develop a global method for analyzing RNA transcripts of ALS genes to categorize the
 mutations to improve the diagnostic management of patients.

ICD-10 Classifications