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