The project entitled “Unidentified myopathies after negative results for corresponding thematic panels” proposed as part of the France Genome Medicine Plan 2025 is about to begin. Undertaken by Tanya Stojkovic*, and also France Leturcq**, the goal of this project is to integrate high throughput sequencing technologies into the diagnostic strategy for unidentified myopathies in order to tailor the multidisciplinary treatment of the patient and in anticipation of complications, to avoid diagnostic errors and examination redundancy, to have access to reliable genetic consultation, and to allow inclusion in therapeutic trials. In this same context, the project will also act as a pilot for RNAseq (sequencing on RNA), which will make it possible to characterise and quantify genome transcripts from patient biopsies.
What is your project?
Two genome platforms, SeqOIA and AURAGEN, have been created in France, covering different areas of mainland France. These are high throughput sequencing (HTS) platforms for the exome (1) and the genome (2) in rare diseases. For the geographical zone that it covers, SeqOIA will include all the genes that have already been studied on the FILNEMUS 13 thematic panels (3), i.e. a total of 200 genes in one go.
What is the current diagnostic strategy used at the neuromuscular disease referral centres?
When a patient presents clinical signs indicating a muscular disease, the expert clinician at the referral centre, guided by the clinical examination, defines which examinations should be performed, such as muscle biopsy, blood assays (CK, etc.), electromyogram, muscle imaging, etc. These examinations sometimes make it possible to steer the molecular tests towards the suspected gene. Two situations can occur: either the molecular testing will relate to a single gene, with the examinations indicating a single hypothesis (as in the case of Steinert disease or facioscapulohumeral muscular dystrophy), or the clinician defines one or two gene panels to be studied, because the patient’s phenotype could correspond to several genes (for example limb-girdle muscular dystrophies, congenital myopathies, etc.). At the end of this panel testing, either a genetically defined myopathy is diagnosed (for example mutation in the dysferlin gene), or the tests do not identify any abnormality. In the latter case, the myopathy is said to be unidentified.
So it is at this stage that tests using the HTS platform will be proposed to patients?
Indeed, the SeqOIA or AURAGEN platform will make it possible to test, in one go, the 200 genes related to various myopathies. The simultaneous testing of these genes via the exome will make it possible to identify patients whose atypical clinical presentation is associated with myopathy genes that are already “known”. Therefore, the next step is to analyse the exome, or indeed the genome. By screening for all the known myopathy genes, it will be possible to identify a gene that has not been thought of, because it is not usually associated with the phenotype of the myopathy being considered. These tests will help us to identify mutations in an intronic region, or duplications, triplications (copy number variants [CNVs]), or large deletions that cannot be detected using NGS panels.
Since the MyoCapture project (sequencing of 1000 exomes) has been stopped, we no longer have the capability in France to perform this type of analysis systematically and we have had to send samples abroad, thus losing all control over the data. Now, with the SeqOIA and AURAGEN genome platforms, we are keeping the genetic data in France, which, in addition to everything that concerns the patient directly, helps to advance research.
Will genome analysis be limited to the 200 known genes?
This is still being discussed, but we would like to extend the research to all the OMIM genes, i.e. all the genes that are known to produce rare diseases, because we know that there are overlaps and that, for example, a gene that produces a neuropathy could also potentially produce a muscular disease.
Finally, as a third resort, we would like to have access to all the (potentially pathogenic) class 3 Variants of Uncertain Significance [VUS], because our goal would also be to identify new genes. In order to achieve this, in addition to the data analysed by the platform bioinformaticians and geneticists, we would need to have access to the raw genome data in order to be able to re-analyse them using our own methods in our molecular biology laboratories, under the supervision of the neuromuscular disease specialists, and this final point is also under discussion. But in any case, since the exome and the genome are highly complicated to analyse, one needs bioinformaticians, and also molecular biologists, clinicians and geneticists to relate the genetic data found to the pathology phenotypes.
Will all patients with “unidentified” myopathies be eligible?
Initially, all the case files must – and this is a request that comes from the Ministry of Health – necessarily be selected during multidisciplinary team (MDT) meetings, first at the regional level, then, according to what we are seeing emerging at the moment, by a national adult MDT and a national child MDT. These successive MDTs will help us to validate the family case files that can be added to the platform. In particular, one needs to have sufficient family samples taken (patient and his/her parents and/or siblings) and to check that everything possible that can be done at an earlier stage has been done. The idea is that HTS be offered as routine care for these families.
How many case files do you plan to deal with each year?
We plan to deal with 50 case files in total in the first year at the national MDT level. Putting together the case files is fairly burdensome: on the dedicated software system that will allow all the patient data to be entered, we need to code each symptom using HPO (Human Phenotype Ontology) terms, enter the genetic data that have already been collected such as panels, biomedical data, available DNA, we must also recall the patients and all the family members included in the analysis, get them to sign new specific consent forms and take new samples.
When will you start?
We still need to obtain the codes to enter onto the platform. Furthermore, a two-day training course is planned shortly for the geneticists and biologists, organised by our colleagues from the “Mitochondrial diseases of particular severity” project who have already done this work, to train them in this type of analysis, to avoid the stumbling blocks they have already encountered, and to discuss with them the best way to release the results. Indeed, a number of questions arise: what should be contained in the report? How should the results be released? Which data, if any, should be stored and how? What should we do with the incidentalomes (pathogenic genes not searched for and discovered during the analysis)? How should functional tests be performed? Training on the platform is also planned for clinicians. Finally, certain details of the project are still under discussion and the clinicians who will form the MDTs are currently in the process of being recruited, but we believe that the examination of the first case files will begin around March/April 2020.
* Tanya Stojkovic, Neurologist in the Service of Neuro-Myologie at the Institute of Myology, Hôpital La Pitié-Salpêtrière, Paris.
** France Leturcq, Molecular biologist in the Genetics Department and Molecular Biology, Hôpital Cochin, Paris.
(1) Exome: sequencing of all the exons.
(2) Genome: sequencing of the whole genome (WGS), exons and introns.
(3) The thematic panels are made up of a series of genes to be tested that are known and are associated with a myopathy. The 13 panels dedicated to myopathies have been defined by the FILNEMUS Network.
