Development of Targeted RNA-Seq for Amyotrophic Lateral Sclerosis Diagnosis (ROSA)

May 15, 2025 updated by: Centre Hospitalier Universitaire de Nīmes

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.

Study Overview

Status

Recruiting

Conditions

Intervention / Treatment

Study Type

Observational

Enrollment (Estimated)

192

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Contact

Study Locations

  • France
      • Bordeaux, France
        • Recruiting
        • CHU de Bordeaux
        • Contact:
        • Principal Investigator:
          • Gwendal Le Masson
        • Sub-Investigator:
          • Stephane Mathis
      • Clermont-Ferrand, France
        • Recruiting
        • CHU de Clermont-Ferrand
        • Contact:
        • Principal Investigator:
          • Nathalie Guy
        • Sub-Investigator:
          • Annaick Desmaison
      • Lyon, France
      • Marseille, France
        • Recruiting
        • La Timone
        • Contact:
        • Principal Investigator:
          • Annie Verschueren
        • Sub-Investigator:
          • Aude-Marie Grapperon
      • Montpellier, France
        • Recruiting
        • CHU de Montpellier
        • Contact:
        • Principal Investigator:
          • Elisa De la Cruz
        • Sub-Investigator:
          • Florence Esselin
        • Sub-Investigator:
          • Alix Durand
      • Nîmes, France
        • Recruiting
        • CHU de Nîmes
        • Contact:
        • Principal Investigator:
          • Claire Guissart
      • Toulouse, France
        • Recruiting
        • CHU de Toulouse
        • Contact:
        • Principal Investigator:
          • Blandine Acket

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

  • Adult
  • Older Adult

Accepts Healthy Volunteers

No

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.

Description

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

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

Cohorts and Interventions

Group / Cohort
Intervention / Treatment
Positive controls
6 patients already in database. The 6 confirmed splicing mutations are: DCTN1 (NM_004082.5): c.3209G>T, OPTN (NM_001008211.1) : c.1613-7T>G, FUS (NM_004960.4) : c.764+8T>A, GRN (NM_002087.4): c.835+1G>A, GRN (NM_002087.4): c.709-3C>G, SPG11 (NM_025137.4): c.3039-5T>G
Other: RNA sequencing
RNA-Seq (Sureselect XT HS2 RNA) from patient blood sample
Negative controls
30 patients with familial hypercholesterolemia. The absence of splicing anomalies in the SLA genes after confirmation by RT-PCR followed by Sanger sequencing of the absence of anomalies for the 6 variants listed above for each of the 30 individuals.
Other: RNA sequencing
RNA-Seq (Sureselect XT HS2 RNA) from patient blood sample
Exploratory cohort
156 ALS: 20 ALS patients with splice variants predicted to be deleterious by in silico prediction software; 136 panel-analysis-negative ALS patients (priority will be given to familial ALS)
Other: RNA sequencing
RNA-Seq (Sureselect XT HS2 RNA) from patient blood sample

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Concordance between the RNA-Seq results (index text) versus RT-PCR followed by Sanger sequencing (reference technique) on positive plus negative controls
Time Frame: Day 0
Sashimi Plot visualized in Integrative Genomics Viewer
Day 0

Secondary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Frequency of splicing anomalies detected in ALS patients in the "exploratory cohort" versus negative controls.
Time Frame: Day 0
Frequency
Day 0

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

Centre Hospitalier Universitaire de Nīmes

Collaborators

Association pour la Recherche sur la Sclérose Latérale Amyotrophique et autres maladies du motoneurones

Investigators

  • Principal Investigator: Claire Guissart, CHU de Nîmes

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

November 22, 2023

Primary Completion (Estimated)

May 1, 2027

Study Completion (Estimated)

May 1, 2027

Study Registration Dates

First Submitted

October 9, 2023

First Submitted That Met QC Criteria

October 9, 2023

First Posted (Actual)

October 16, 2023

Study Record Updates

Last Update Posted (Estimated)

May 20, 2025

Last Update Submitted That Met QC Criteria

May 15, 2025

Last Verified

May 1, 2025

More Information

Terms related to this study

Additional Relevant MeSH Terms

Other Study ID Numbers

  • NIMAO/LOCAL/2023/CG-01

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

NO

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

product manufactured in and exported from the U.S.

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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