2025 update of the National French consensus on gene lists for the diagnosis of muscle diseases using high-throughput sequencing

Abstract

Diagnosing rare muscle diseases can be challenging due to their genetic heterogeneity. The French National Network for Rare Neuromuscular Diseases (FILNEMUS) has previously established a pioneering nationwide strategy based on gene lists organized in 13 phenotype-specific gene panels. We now revise these lists and add recently described genes. Using data collected from all FILNEMUS diagnostic laboratories, we also establish a “Major Muscle Genes” panel that includes genes responsible for the most frequent genetic muscle diseases. The updated diagnostic strategy of the FILNEMUS network will help reduce the turn-around time for genetic results and facilitate rapid access to the French national genome sequencing platforms.

Introduction

Diagnosis of genetically heterogeneous muscle diseases has been greatly improved since the arrival of short-read high-throughput sequencing, now commonly used to analyze a set of genes (or gene panel). However, without clear guidelines for gene selection based on gene-disease validity, the diversity of available gene panels can interfere with efficient genetic diagnosis and lead to inconsistent results between diagnostic laboratories. In order to coordinate the diagnostic process across the laboratories, the French National Network for Rare Neuromuscular Diseases (Filière Nationale des Maladies Rares Neuromusculaires, FILNEMUS) established in 2018 a nationwide diagnostic strategy that was adopted by all diagnostic laboratories of the network. ¹ According to these guidelines, 200 muscle disease genes were organized into 13 Gene Panels grouped by different clinical indications. Each diagnostic laboratory of the FILNEMUS network specialized in the analysis of one or several gene panels, providing the vast majority of genetic tests for muscular diseases in France. If the initial phenotype specific panel did not return a positive result, genetic testing was then pursued by analysis of all genes on the FILNEMUS lists or by sequencing the genome by one of the two national genome sequencing platforms.^2,3 This pioneering work was followed by other efforts to systematically harmonize the diagnostic processes across healthcare systems.^4,5 When the first version of FILNEMUS gene panels was finalized, the international efforts to systematically evaluate gene-disease relationships were in their early steps. Indeed, the Congenital Myopathies and Limb-Girdle Muscular Dystrophies ClinGen Gene Curation Expert Panels (GCEP) ⁶ were launched in 2019 in order to evaluate muscle genes according to ClinGen guidelines. ⁷ The global coalition of gene curation activities, GeneCC, has since further advanced the field. ⁸ Taking into account newly available data on the gene-disease relationships for muscle-disease causing genes as well as recent descriptions of novel genes implicated in muscle diseases, we now update the FILNEMUS gene panels and propose a modified national French consensus strategy for genetic diagnosis of muscle disease. These guidelines were elaborated in concert with all French diagnostic laboratories of the FILNEMUS network, one of the largest of 23 nationwide rare-disease structures, coordinating care of more than 50 000 patients affected with rare neuromuscular disease in France. ⁹ The updated diagnostic strategy will thus ensure uniform genetic testing for muscle-disease patients across different regions of the country.

Materials and methods

Update of the gene-disease relationships

Gene-disease curations for the 200 genes from the initial FILNEMUS gene panels ¹ were downloaded from the GenCC database (https://thegencc.org/, accessed June 16th, 2025). ⁸ The obtained entries were then manually filtered to include only the muscle-related phenotypes. Genes classified as “Moderate”, “Strong” or “Definitive” by at least one GenCC submitter and ranked as “CONVINCING” by Krahn et al. were retained in the new version of the phenotype-specific gene panels. Gene-disease relationships for genes with “Limited/Supporting” classifications, genes with discordant (e.g., “Strong” in GeneCC and “LIMITED” in Krahn et al.) classifications or genes with no classification in GenCC database were examined by an expert group. Genes that were removed from the phenotype-specific gene panels were still included in the Extended Gene Panel.

Updating the gene panels to include additional genes implicated in muscle disease

The annually published Muscle GeneTable ¹⁰ (https://musclegenetable.fr/, version GT_NMD 2025 updated 12/03/2025), a valuable resource listing recently discovered genes implicated in neuromuscular disorders, was examined to identify novel genes responsible for muscle disease (disease groups “Muscular dystrophies”, “Congenital muscular dystrophies”, “Congenital myopathies”, “Distal myopathies”, “Other myopathies”, “Metabolic myopathies”). Additional genes were obtained from the literature, pre-prints, scientific conference presentations, the results of genome analysis as well as following suggestions from FILNEMUS member laboratories. We used the following criteria to select new genes for the updated panels: 1) Genes with disease-causing anomalies detectable by short read high-throughput sequencing (i.e., genes with disease-causing repeat expansions were not included); 2) Genes associated with a muscular phenotype as the main feature. The GeneCC database was then used to examine the disease-associations for these new candidate genes.

Creation of the “Major muscle gene panel”

Diagnostic gene panels proposed by Krahn et al. were implemented in 2017 in all eight diagnostic laboratories of the FILNEMUS network, each specializing in different subtypes of muscle disease. We collected the nationwide data for positive genetic diagnoses of muscle disease obtained by gene panels performed in these laboratories during the period from 2017 to 2022. The details on panels proposed by each laboratory are found at the FILNEMUS website. ⁹ Given the inherent differences between the laboratories due to patient recruitment and panel offer, the numbers of positive diagnoses per gene were combined to obtain the nation-wide data. The thirty most frequently mutated genes were then selected to establish the “Major Muscle Gene Panel”. Even though the DMD gene was not part of the first version of the FILNEMUS gene lists (DMD gene testing is coordinated by a specific network in France), it was now added in the Major Muscle Gene panel due to the frequency of DMD-related muscle disease, bringing the total number of genes on this panel to 31.

Creation of the “Actionable Muscle Gene Panel”

The genes on the initial FILNEMUS list have been recently evaluated using ClinGen Actionability criteria by Vecten et al. ¹¹ Of them, 35 genes with an actionability score of 9 or more were selected to be part of the “Actionable Muscle Gene Panel”. Five additional genes (CPT2, ETFDH, FLAD1, CLCN1 and TK2) were added to this panel based on the decision of the expert group.

Results and discussion

Robust evaluation of gene-disease relationships is critical for diagnosis of rare diseases by gene panel approaches.^5,7,8 The initial version of the FILNEMUS gene panels was established more than six years ago, thus requiring an update using more recently published genetic and molecular data for the genes currently included on the panels. We therefore re-evaluated the gene-disease relationships for the genes on the initial panels and removed the following eight genes from the phenotype-specific gene panels: LIMS2 from “Limb Girdle Muscular Dystrophies”, MYBPC3 from “Congenital Myopathies and Congenital Muscular Dystrophies”, KLHL9 from “Distal and Scapuloperoneal Myopathies”, ZBTB42 from “Fœtal and Neonatal Arthrogryposes”, COL5A3 and COL6A6 from “Retractile Myopathies”, SNAP25 from “Congenital Myasthenias”. These genes were kept on the “Extended Muscle Gene Panel” in order to leave the possibility of identifying new patients with variants in these genes.

A number of novel muscle disease associated genes have been described since the first version of the FILNEMUS gene panels was issued, requiring an update of the initial list of genes. In concert with all FILNEMUS molecular diagnosis laboratories, we carried out a literature search and identified 57 muscle disease genes that were not included in the initial guidelines by Krahn et al. After evaluating the gene-disease relationships, 29 of these genes were added to one or several of the ten phenotype-specific panels. All 57 genes were included in the “Extended Muscle Gene Panel”.

The DMD gene was not initially included in the FILNEMUS gene panels since analysis of this gene was done exclusively by several expert centers as part of a pre-existing network for DMD gene testing in France. We have now included DMD in three FILNEMUS panels (“Limb Girdle Muscular Dystrophies”, “Metabolic Myopathies and Rhabdomyolysis” and “Major Muscle Genes”) in order to reduce unnecessary sequential genetic testing in cases where Duchenne or Becker muscular dystrophy is not evident at initial assessment. Similarly, the SMCHD1 gene has been added to the “Extended Muscle Gene Panel”. The diagnostic laboratories within the FILNEMUS network are encouraged to consult with the reference laboratories for the DMD and SMCHD1 genes in cases when interpretation of an identified variant requires expert analysis.

Several additional modifications of gene panels were made based on the feedback from FILNEMUS laboratories. Four different congenital myopathy and muscular dystrophy gene panels (“Alpha-dystroglycanopathies”, “Congenital muscular dystrophies-except alpha-dystroglycanopathies”, “Congenital myopathies nemaline-positive”, “Congenital myopathies nemaline-negative”) were combined into one (“Congenital myopathies and congenital muscular dystrophies”). The subdivision of gene panels into “core” and “extended” lists was removed to include a single gene list per phenotypic group. The “Metabolic Myopathy” gene panel was renamed “Metabolic Myopathy and Rhabdomyolysis” and extended to include additional genes often associated with hyperCKaemia and exercise intolerance. A new gene panel “Actionable Muscle Genes” was added based on the recent evaluation of the actionability of 200 genes on the initial FILNEMUS list. ¹¹ Finally, using data collected from all FILNEMUS molecular diagnosis laboratories, we established an additional gene list that includes the genes most frequently identified as responsible for muscle diseases in France (“Major Muscle Genes” panel).

Despite the significant drop in sequencing costs, genome and exome analyses remain more expensive and time-consuming than small gene panels, largely due to the many candidate variants identified and the expert interpretation they demand. Based on our data collected during 5 years after the initial FILNEMUS recommendations, the diagnostic yield of most laboratories was around 30–45%, depending on the choice of panels offered and on patient recruitment. It is therefore more cost effective to start genetic analysis by a rapid and targeted gene panel, followed by a broader sequencing analysis if positive diagnosis is not established by this first-tier analysis.

We propose the following updated consensus diagnostic strategy of the FILNEMUS network, as shown in Figure 1. Clinicians will now have a possibility to perform a genetic analysis of the “Major Muscle Gene Panel” (31 most frequent muscle-disease causing genes) for patients with suspected muscle disease, but without clear clinical indication for a particular phenotype-specific gene panel. Introduction of this new gene panel will enable a rapid response to exclude or to confirm the most frequent genetic muscle diseases. The updated diagnostic strategy also provides a possibility to add to the analysis 40 actionable muscle genes in order to quickly identify patients that can benefit from specific treatments or monitoring. In the absence of a positive diagnosis after the analysis of the “ Major Muscle Gene Panel “ or selected “phenotype-specific” gene panels, clinicians will have the option to continue with analysis of 257 genes on the extended list (“Extended Muscle Gene Panel”), analyze exome or to proceed directly to genome analysis by one of the two French national genome sequencing platforms.^2,3 The updated version of the National French consensus gene lists for the diagnosis of muscle diseases will maintain a national standardization of genetic diagnosis for muscle diseases, while helping to avoid unnecessary analysis of variants of uncertain significance, reducing the turn-around time for genetic results and facilitating rapid access to the French national whole-genome sequencing platforms.

Figure 1.

National French consensus strategy for genetic diagnosis of muscle disease. If patient's phenotype is compatible with a particular type of muscle disease, the corresponding phenotype-specific gene panel should be performed as a first-tier genetic analysis. If patient's phenotype is not clear and the referring clinician has difficulties selecting a phenotype-specific gene panel, the updated diagnostic strategy now proposes genetic analysis of 31 genes most often implicated in muscle disease (Major Muscle Gene Panel). Variants with ACMG/AMP classifications of “Variant of uncertain significance” (class 3), “Likely Pathogenic” (class 4) and “Pathogenic” (class 5) will be returned for these first-tier analyses. The clinicians will also have a possibility to analyze 40 actionable muscle genes in order to rapidly establish diagnosis of muscle disease linked to genes with medical value for patient care. Only Likely Pathogenic (class 4) and Pathogenic (class 5) are returned for this panel since the Variants of Uncertain Significance are not actionable in clinical practice. If the initial genetic tests did not establish molecular diagnosis, the Extended Muscle Gene Panel can be proposed. This panel combines all phenotype-specific panels and includes certain genes with less well-established gene-disease relationships. As the number of variants of uncertain significance is expected to increase due to the large number of genes analyzed by this panel, we recommend returning only the variants that will have a clinical impact on patient management (“Likely Pathogenic” (class 4) and “Pathogenic” (class 5)). Close collaboration between clinical and genetic teams is required to identify the variants of uncertain significance that should be further explored in order to be reclassified as Likely Pathogenic or Pathogenic. Alternatively, it is possible to proceed directly to genome sequencing at one of the two French national genome sequencing platforms^2,3 and/or transcriptome analysis etc.

Supplemental Material

sj-docx-1-jnd-10.1177_22143602251400143 - Supplemental material for 2025 update of the National French consensus on gene lists for the diagnosis of muscle diseases using high-throughput sequencing

Supplemental material, sj-docx-1-jnd-10.1177_22143602251400143 for 2025 update of the National French consensus on gene lists for the diagnosis of muscle diseases using high-throughput sequencing by Emmanuelle Pion, Mireille Cossée, Valérie Biancalana, Cécile Acquaviva Bourdain, Céline Bouchet-Seraphin, Julien Fauré, Roseline Froissart, France Leturcq, Rita Menassa, Corinne Metay, Laurence Michel-Calemard, Juliette Nectoux, Francois Petit, John Rendu, Pascale Richard, Damien Sternberg, Sandrine Vuillaumier-Barrot, Charles Van Goethem, Corinne Thèze, Shahram Attarian, Martin Krahn and Svetlana Gorokhova in Journal of Neuromuscular Diseases

Supplemental Material

sj-xlsx-2-jnd-10.1177_22143602251400143 - Supplemental material for 2025 update of the National French consensus on gene lists for the diagnosis of muscle diseases using high-throughput sequencing

Supplemental material, sj-xlsx-2-jnd-10.1177_22143602251400143 for 2025 update of the National French consensus on gene lists for the diagnosis of muscle diseases using high-throughput sequencing by Emmanuelle Pion, Mireille Cossée, Valérie Biancalana, Cécile Acquaviva Bourdain, Céline Bouchet-Seraphin, Julien Fauré, Roseline Froissart, France Leturcq, Rita Menassa, Corinne Metay, Laurence Michel-Calemard, Juliette Nectoux, Francois Petit, John Rendu, Pascale Richard, Damien Sternberg, Sandrine Vuillaumier-Barrot, Charles Van Goethem, Corinne Thèze, Shahram Attarian, Martin Krahn and Svetlana Gorokhova in Journal of Neuromuscular Diseases