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.