Clinical trial NCT06083584: Development of Targeted RNA-Seq for Amyotrophic Lateral Sclerosis Diagnosis
Official title: Development of Targeted RNA-Seq for Amyotrophic Lateral Sclerosis Diagnosis
Study type: OBSERVATIONAL
Overall status: RECRUITING
Conditions: Amyotrophic Lateral Sclerosis
Lead sponsor: Centre Hospitalier Universitaire de Nīmes
Sponsor class: OTHER
Collaborators: name: Association pour la Recherche sur la Sclérose Latérale Amyotrophique et autres maladies du motoneurones
First posted: 2023-10-16
Enrollment: 192
Sex: ALL
Minimum age (years): 18
Standard ages: ADULT
OLDER_ADULT
Accepts healthy volunteers: no
Observational model: COHORT
Time perspective: PROSPECTIVE
Sampling method: NON_PROBABILITY_SAMPLE
Study population: Patients will be recruited during a consultation for a clinical diagnosis of ALS for which a request for a genetic diagnosis has been made in one of the prescribing centers: CHU Lyon, Montpellier University Hospital, Marseille University Hospital, Clermont-Ferrand University Hospital, Bordeaux University Hospital, Toulouse University Hospital.

Brief 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.


Eligibility criteria: Inclusion Criteria:

* Have a prescription for a genetic diagnosis of ALS (or familial hypercholesterolemia for the control cohort)
* Have given their informed consent for the genetic study and the biobank
* The patient must be a member or beneficiary of a health insurance plan

Exclusion Criteria:

* The patient is under safeguard of justice or state guardianship

Primary outcomes:
measure: Concordance between the RNA-Seq results (index text) versus RT-PCR followed by Sanger sequencing (reference technique) on positive plus negative controls
timeFrame: Day 0
Secondary outcomes:
measure: Frequency of splicing anomalies detected in ALS patients in the "exploratory cohort" versus negative controls.
timeFrame: Day 0
Other outcomes:
Locations:
city: Bordeaux
country: France
facility: CHU de Bordeaux
city: Clermont-Ferrand
country: France
facility: CHU de Clermont-Ferrand
city: Lyon
country: France
facility: CHU de Lyon
city: Marseille
country: France
facility: La Timone
city: Montpellier
country: France
facility: CHU de Montpellier
city: Nîmes
country: France
facility: CHU de Nîmes
city: Toulouse
country: France
facility: CHU de Toulouse
Study officials:
affiliation: CHU de Nimes
name: Claire Guissart
role: PRINCIPAL_INVESTIGATOR
References: