DNA QUANTIFICATION TECHNIQUE AS A INTERPRETATION TOOL IN MITOCHONDRIAL DISEASES

IMPLEMENTING A SINGLE MUSCLE FIBER DNA QUANTIFICATION TECHNIQUE AS A INTERPRETATION TOOL FOR THE VARIANTS OF UNKNOWN SERVICE IN MITOCHONDRIAL DISEASES

2622/5000 Mitochondrial diseases (MM) are the most common metabolic diseases. Since these pathologies are very heterogeneous in clinical terms, only the identification of mutations in nuclear genes or mitochondrial DNA confirms the diagnosis.

The full-scale study of mtDNA by high-throughput sequencing (NGS) is a first step in the diagnostic approach. The recent introduction of this revolutionary new technology has greatly increased the efficiency of mutation identification. However, in addition to known pathogenic mutations, NGS reveals numerous variants whose significance is currently unknown. A major challenge to obtain a reliable diagnosis is therefore the interpretation of the clinical impact of these new rare variants which proves to be very difficult.

Pathogenicity criteria allow the classification of variants from benign to pathogenic. One of the major pathogenicity criteria is a good correlation of heteroplasmic level with tissue or cellular involvement. Indeed, mtDNA mutations are generally heteroplasmic, which corresponds to the coexistence of normal and mutated molecules in the same cell or tissue, the most affected tissues having a high rate of mutation. On a muscle biopsy of an affected patient, the fibers often present an enzyme deficiency in cytochrome c oxidase (COX-negative) which can be demonstrated in immunohistochemistry. The single fiber study allows to isolate the deficient fibers and to quantify the heteroplasmic rate of a variant. The presence of a high level of heteroplasm in the COX-negative fibers, unlike fibers without deficit, is a strong argument in favor of the pathogenicity of this variant. Currently, this technique is not used routinely in diagnostic laboratories but only occasionally in a research framework in some laboratories. It is a heavy technique that consists of a first stage of laser microdissection of the various muscle fibers followed by a second step of quantification of the variant from each fiber. This second step requires a specific focus for each identified variant.

The aim of this pilot study is to develop a new technique for quantification of single-fiber heteroplasmics isolated by NGS laser microdissection. This, independent of the type of variant, will avoid the long and costly adjustments required for each new variant identified and thus facilitate its use

Study Overview

• Status: Completed
• Conditions: Mitochondrial Diseases
• Intervention / Treatment: Biological: samples

• Study Type: Interventional
• Enrollment (Actual): 10
• Phase: Not Applicable

Contacts and Locations

Study Locations

• France
  • Nice, France, 06200
    • CHU de Nice

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child; Adult; Older Adult
• Accepts Healthy Volunteers: No

Description

Inclusion Criteria:

• general criteria: major or minor patients, sporadic or isolated cases
• criteria related to pathology:
• Suspected mitochondrial pathology which will be evaluated according to the following criteria (expertise of the clinician of the MM reference center):

• Clinical picture suggestive of a mitochondrial pathology ("illegitimate association" of symptoms, specific syndrome of MELAS type for example, muscular deficit, ptosis ...) AND / OR

  • Metabolic assessment suggestive of respiratory tract involvement AND / OR
  • Identification of a deficiency involving one or more complexes of the respiratory chain from a muscular specimen
• Presence on the histological analysis of the muscle biopsy of COX-negative fibers
• signing of informed consent for minor patients signed by at least one of the parents or the representative of the parental authority

Exclusion Criteria:

• Persons deprived of their liberty by a judicial or administrative decision;
• Persons hospitalized without consent;
• Persons admitted to a health or social institution for purposes other than research;
• Persons of legal age who are under protection or who are unable to express their consent. Inability to co-operate.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: N/A
• Interventional Model: Single Group Assignment
• Masking: None (Open Label)

Number of Arms

1

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Other: biological samples; biological samples on patients with MITOCHONDRIAL DISEASES	Biological: samples; Blood samples, oral smear and urine

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Time Frame
number of amplification with the NGS technique	36 months

Secondary Outcome Measures

Outcome Measure	Time Frame
comparaison of the number of amplification between NGS technique and the PCR-RFLP technique	36 months

Collaborators and Investigators

• Sponsor: Centre Hospitalier Universitaire de Nice

Study record dates

Study Major Dates

• Study Start (Actual): February 2, 2018
• Primary Completion (Actual): September 30, 2020
• Study Completion (Actual): September 30, 2020

Study Registration Dates

• First Submitted: July 7, 2017
• First Submitted That Met QC Criteria: July 11, 2017
• First Posted (Actual): July 13, 2017

Study Record Updates

• Last Update Posted (Actual): November 15, 2023
• Last Update Submitted That Met QC Criteria: November 14, 2023
• Last Verified: November 1, 2023

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Metabolic Diseases; Mitochondrial Diseases
• Other Study ID Numbers: 16-AOIP-04

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