Objective Evaluation of Clinical Actionability for Genes Involved in Myopathies: 63 Genes with a Medical Value for Patient Care

The implementation of high-throughput diagnostic sequencing has led to the generation of large amounts of mutational data, making their interpretation more complex and responsible for long delays. It has been important to prioritize certain analyses, particularly those of “actionable” genes in diagnostic situations, involving specific treatment and/or management. In our project, we carried out an objective assessment of the clinical actionability of genes involved in myopathies, for which only few data obtained methodologically exist to date. Using the ClinGen Actionability criteria, we scored the clinical actionability of all 199 genes implicated in myopathies published by FILNEMUS for the “National French consensus on gene Lists for the diagnosis of myopathies using next generation sequencing”. We objectified that 63 myopathy genes were actionable with the currently available data. Among the 36 myopathy genes with the highest actionability scores, only 8 had been scored to date by ClinGen. The data obtained through these methodological tools are an important resource for strategic choices in diagnostic approaches and the management of genetic myopathies. The clinical actionability of genes has to be considered as an evolving concept, in relation to progresses in disease knowledge and therapeutic approaches.


Results
We used the semi-quantitative actionability metric established by ClinGen [3,4] to score the clinical actionability of 199 genes implicated in 223 myopathies published by FILNEMUS [5]. We choose to offer an overall score for each gene-associated disorder, to mark the most severe phenotype and not each symptom (cardiac involvement, muscle damage, etc.). The four indicators, severity of disease, penetrance/likelihood of disease, effectiveness of specific intervention, and nature of intervention, were each scored from 0 to 3, from a low level to a high level of actionability [3]. For example, the score severity is 0 if the disease has minimal health impact or no morbidity and 3 if there is sudden death or inevitable death; the score penetrance is 0 if <1% chance or unknown, 3 if >40% chance; effectiveness of specific intervention is 0 if ineffective/no intervention, 3 if highly effective; nature of intervention is 0 if high risk/poorly acceptable/intensive or no intervention, and 3 if low risk/medically acceptable/low intensity intervention [3,4]. All scores have been reviewed by the French FILNEMUS expert-network (clinicians and geneticists).
The 223 myopathy genes/disorders were scored from 1 to 12 (Supplemental Material S1). More than 45% of the scores were distributed between 4 and 6, while the scores for genes/disorders of the ClinGen AWG genes list were between 5 and 12, with >50% of them greater than or equal to 9 (Figure 1a).  The four indicators of actionability were severity of disease, penetrance/likelihood of disease, effectiveness of specific intervention, and nature of intervention. These indicators were each scored from 0 to 3, from a low level to a high level of actionability [3]. a/Total scores for the 223 myopathy genes/disorders (from the 199 genes reported by FILNEMUS [5]) and for the 213 ClinGen AWG genes/outcome-intervention pairs (from the 127 genes scored by ClinGen AWG [4]). b/Distribution of the four indicators for the 223 myopathy genes/disorders. c/Distribution of the four indicators for the 213 ClinGen AWG genes/outcome-intervention pairs. d/Distribution of the four indicators for the 43 myopathy genes/disorders (36 genes) with a global score ≥ 9. e/Distribution of the four indicators for the 125 ClinGen AWG genes/outcome-intervention pairs (111 genes) with a global score ≥ 9.
We evaluated the scores distribution of the four indicators for the myopathy genes/ disorders (Figure 1b). Concerning the severity indicator: 43% had very high mortality rates (score 3), 29% had moderate severity (score 2), and 28% low severity (score 1). For the disease penetrance indicator (likelihood): the information was available for only 27% of the myopathy genes/disorders, leading to an artificial score of 0 for 73% of them. For the effectiveness of intervention: 36% were highly or moderate effective (score ≥ 2). The nature of intervention was evenly distributed between the different scores, with a great or high risk (scores 0 and 1) in 44% of cases.
By filtering the two care indicators, effectiveness of specific intervention with a score ≥ 2 and nature of intervention ≥ 1, we retained 63 myopathy genes corresponding to 78 disorders that could be actionable on their management (Table 1). Final scores of actionability of these genes/disorders were between 5 and 12, with the majority ≥ 9 (43/78, 55%). The intervention included either the availability of drugs or a specific cardiac management (defibrillator/pacemaker). Table 1. The 63 myopathy genes with a medical value for patient care. The 63 genes of myopathies were selected based on effectiveness of the treatment with a score ≥ 2 and nature of the intervention ≥ 1. The global score indicated also takes into account the scores of severity and penetrance/likelihood. Genes with a global score ≥ 9 are highlighted in grey. LGMD old/new names according to the LGMD nomenclature are indicated.   We also evaluated the distribution of actionability scores within the 43 pairs genes/ disorders (36 myopathy genes) with higher actionability global scores (≥9) (Figure 1c), of which only 8 had already been scored by ClinGen (Table 1). For the severity indicator: 77% had very high mortality rates, 14% had moderate severity, and 9% low severity. For the disease penetrance indicator: the information was available for 95% of the genes and mostly with full penetrance (81%). The effectiveness of intervention was moderate (score 2) or high (score 3) for all genes. The nature of intervention was moderate or low risk (scores 2 and 3) for 97% of the genes. We then compared these actionability scores with those of ClinGen AWG genes with a global score ≥ 9 (111 genes corresponding to 78 disorders and 125 outcome-intervention pairs) (Figure 1d). We found that the distribution of scores was different between both groups concerning the outcome-related domains (severity and likelihood), with higher scores for myopathy genes (p < 0.05). Results for both interventionrelated indicators were similar in the two categories of genes.

Discussion
We applied here, in an exhaustive way for the first time in the field of myopathies, the ClinGen AWG scoring methodological approach.
We encountered a lack of information on the penetrance data of myopathy genes in the literature (for 74% of genes), which was not the case for genes from the ClinGen AWG list genes (Figure 1e) [4]. This lack of information on penetrance led to a "loss" of 1 to 3 final scoring points, that could account for the lower final average actionability score of myopathy genes (score 4-5) compared to the genes scored by the ClinGen AWG (9-10). Noteworthy, an important proportion of myopathies is of autosomal recessive inheritance, with expected complete penetrance. The use of the data collected by the French National Rare Diseases Data Bank (BAMARA) will possibly make it easier in the upcoming years to access data on penetrance for rare myopathies.
Thanks to our scoring work, we have identified 63 myopathy genes (corresponding to 78 disorders) that could be actionable on their management (intervention moderately or highly effective, without a high risk), including 43 genes/disorders with actionability scores ≥ 9. However, only 12 of these myopathy genes were previously scored by Clin-Gen. Of the 31 genes not scored by ClinGen AWG, 21 are part of congenital myasthenic syndromes (Table 1), for which there are specific treatments, reflecting the value of this scoring work [7]. Some of the 63 myopathy genes/disorders have a lower score of actionability (<9) but are considered to have medical value for patient care. The CLCN1 gene is a good example in the recessive or dominant myotonia congenita ( Table 1). The low global score (7) is due to the modest morbidity (severity scored as 1), but the treatment is highly effective (scored as 3) and acceptable (scored as 3). For some genes associated with several diseases, the increase in the global score for actionability is due to an increase in the severity score. For example, the SCN4A gene is associated with an actionability score of 9 in the hyperkalemic periodic paralysis (severity score at 1), compared to a score of 11 in the congenital myasthenic syndrome type 16 (severity score at 3) [8].
Our results will allow the integration of the notion of actionable genes into strategic choices for molecular diagnostic strategies and management of patients with genetic myopathies. We recommend that these 63 genes be analyzed as a priority in the sequencing of gene panels, exomes, or genomes, in order to offer a rapid diagnosis to patients and optimize patient care (treatabolome database in preparation). Furthermore, these genes (at least the 36 with global score ≥ 9) should be considered for a possible addition to the ACMG actionable gene list, in order to assess them as secondary data of medical value for patient care. Of course, patients' phenotyping remains essential, and the absence of a mutation in this panel of genes does not exclude an intronic mutation in one of these genes. DMD, DM1, and DM2 genes were not on the list of FILENEMUS genes analyzed by NGS [5]. However, they should also be considered actionable, given their important consequences on patient management, as well as the SMN1 gene.
The clinical actionability of genes must be considered as an evolving concept, in relation to progresses in therapeutic approaches, or diagnosis. In recent years, new therapeutic options such as gene therapies have been developed and offer hope for patients with myopathies. It will be important to take these new therapeutic approaches into account in the coming years as several additional myopathic genes will then be considered actionable.

Materials and Methods
We compared the scores obtained for the myopathy genes/disorders with those of the ClinGen AWG (127 genes corresponding to 78 disorders with 213 outcome-intervention pairs) [4], using the nonparametric Kruskal-Wallis test. Thereafter, we analyzed the distribution of scores of the four indicators for the ClinGen AWG and myopathy genes using the chi-square method that admits differences in group size.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.