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

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ClinicalTrials.gov Identifier: NCT06083584
Recruitment Status : Recruiting
First Posted : October 16, 2023
Last Update Posted : January 31, 2024
Sponsor:
Collaborator:
Association pour la Recherche sur la Sclérose Latérale Amyotrophique et autres maladies du motoneurones
Information provided by (Responsible Party):
Centre Hospitalier Universitaire de Nīmes

Study Description
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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.


Condition or disease Intervention/treatment
Amyotrophic Lateral Sclerosis Other: RNA sequencing

Study Design
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Study Type : Observational
Estimated Enrollment : 192 participants
Observational Model: Cohort
Time Perspective: Prospective
Official Title: Development of Targeted RNA-Seq for Amyotrophic Lateral Sclerosis Diagnosis
Actual Study Start Date : November 22, 2023
Estimated Primary Completion Date : May 2025
Estimated Study Completion Date : May 2025

Resource links provided by the National Library of Medicine


Groups and Cohorts
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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


Outcome Measures
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Primary Outcome Measures :
  1. 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


Secondary Outcome Measures :
  1. Frequency of splicing anomalies detected in ALS patients in the "exploratory cohort" versus negative controls. [ Time Frame: Day 0 ]
    Frequency


Eligibility Criteria
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Ages Eligible for Study:   18 Years and older   (Adult, Older Adult)
Sexes Eligible for Study:   All
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.
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
Contacts and Locations
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Information from the National Library of Medicine

To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.

Please refer to this study by its ClinicalTrials.gov identifier (NCT number): NCT06083584


Contacts
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Contact: Claire Guissart 04 66 68 32 07 claire.guissart@chu-nimes.fr

Locations
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France
CHU de Bordeaux Recruiting
Bordeaux, France
Contact: Gwendal Le Masson       gwendal.le-masson@chu-bordeaux.fr   
Principal Investigator: Gwendal Le Masson         
Sub-Investigator: Stephane Mathis         
CHU de Clermont-Ferrand Recruiting
Clermont-Ferrand, France
Contact: Nathalie Guy       nguy@chu-clermontferrand.fr   
Principal Investigator: Nathalie Guy         
Sub-Investigator: Annaick Desmaison         
CHU de Lyon Recruiting
Lyon, France
Contact: Emilien Bernard       emilien.bernard@chu-lyon.fr   
Principal Investigator: Emilien Bernard         
La Timone Recruiting
Marseille, France
Contact: Annie Verschueren       annie.verschueren@ap-hm.fr   
Principal Investigator: Annie Verschueren         
Sub-Investigator: Aude-Marie Grapperon         
CHU de Montpellier Recruiting
Montpellier, France
Contact: Elisa De la Cruz       e-delacruz@chu-montpellier.fr   
Principal Investigator: Elisa De la Cruz         
Sub-Investigator: Florence Esselin         
Sub-Investigator: Alix Durand         
CHU de Nîmes Recruiting
Nîmes, France
Contact: Anissa Megzari    04.66.68.42.36    drc@chu-nimes.fr   
Principal Investigator: Claire Guissart         
CHU de Toulouse Recruiting
Toulouse, France
Contact: Blandine Acket       acket.b@chu-toulouse.fr   
Principal Investigator: Blandine Acket         
Sponsors and Collaborators
Centre Hospitalier Universitaire de Nīmes
Association pour la Recherche sur la Sclérose Latérale Amyotrophique et autres maladies du motoneurones
Investigators
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Principal Investigator: Claire Guissart CHU de Nimes
More Information
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Responsible Party: Centre Hospitalier Universitaire de Nīmes
ClinicalTrials.gov Identifier: NCT06083584    
Other Study ID Numbers: NIMAO/LOCAL/2023/CG-01
First Posted: October 16, 2023    Key Record Dates
Last Update Posted: January 31, 2024
Last Verified: January 2024

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Studies a U.S. FDA-regulated Drug Product: No
Studies a U.S. FDA-regulated Device Product: No
Additional relevant MeSH terms:
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Motor Neuron Disease
Amyotrophic Lateral Sclerosis
Sclerosis
Pathologic Processes
Neurodegenerative Diseases
Nervous System Diseases
 Neuromuscular Diseases
Spinal Cord Diseases
Central Nervous System Diseases
TDP-43 Proteinopathies
Proteostasis Deficiencies
Metabolic Diseases