Referral Indications for Malignant Hyperthermia Susceptibility Diagnostics in Patients without Adverse Anesthetic Events in the Era of Next-generation Sequencing

This study was performed with approval of the Research Ethics Boards from the Canisius Wilhelmina Hospital, Nijmegen, The Netherlands (registration No. 067-2020, date of approval September 8, 2020); Skane University Hospital, Lund, Sweden (registration No. 2019-03960, date of approval October 9, 2019); Toronto General Hospital, Toronto, Ontario, Canada (registration No. 19-5365, date of approval May 12, 2019); and Antwerp University Hospital/University of Antwerp, Antwerp, Belgium (registration No. 1805016N, date of approval September 28, 2015). Informed consent was waived due to the retrospective nature of the study.

This retrospective multicenter cohort study focusing on the referral indications for MH susceptibility diagnostics consists of two parts in line with the two aims of the study. The primary analysis is a retrospective evaluation of the referral indications and the use of next-generation sequencing during the study period. The secondary analysis is a detailed evaluation of the MH diagnostics performed and the test results of patients referred to the participating MH units without a personal or family history of an adverse anesthetic event suspected to be MH.

The medical records of patients referred to one North American (Toronto, Ontario, Canada) and three European (Antwerp, Belgium; Lund, Sweden; and Nijmegen, The Netherlands) MH investigation units were evaluated. All medical records of patients referred to the participating centers between January 1, 2010, and December 31, 2019, were reviewed. Medical records of the following patients were excluded from the primary analysis to prevent ascertainment bias:

• - Relatives of previously tested patients

• - Patients who underwent MH susceptibility diagnostics before 2010 and were referred again for genetic testing to facilitate MH susceptibility diagnostics by genotyping in family members

Medical records of the following patients were excluded from the secondary analysis:

• - Patients referred because of a personal or family history of adverse anesthetic event suspected to be MH

• - Patients referred because of a referral indication categorized as other (see Referral Indication section)

• - Patients genetically investigated using a targeted technique (e.g., targeted screening for pathogenic RYR1 variants or a hotspot technique), except when referred because of a family history of a diagnosed RYR1-related myopathy or a family history of an RYR1 variant detected in diagnostic testing in the neuromuscular clinic without a specific clinical or histopathologic diagnosis; these patients were parents of neuromuscular patients who were too young to be investigated by a CHCT/in vitro contracture test and were genetically investigated utilizing a targeted technique to identify which family members are carriers of the RYR1 variant(s) identified in the neuromuscular patient.

The study design and the selection process of medical records for the primary and secondary analysis are summarized in figure 1. Some of the medical records included in this study cohort have been described before.^8,11–16  Details of the overlapping medical records are summarized in Supplemental Digital Content 1 (https://links.lww.com/ALN/C833).

The collected data included demographic characteristics (age at referral and sex), referral details (date of referral, indication for referral, and the indication for RYR1, CACNA1S, or STAC3 sequencing), clinical grading scale¹⁷  for the referred probands, resting creatine kinase levels, details on the performed genetic tests (genes analyzed, technique used, and test results), and CHCT/in vitro contracture test results.

Referral indications were categorized into two main groups: those with a personal or family history of adverse anesthetic events suspected to be MH and those without. Since our objective was to improve counseling for patients without a history of adverse anesthetic events, referral indications from the latter category were subcategorized into different groups.

A personal or family history of adverse anesthetic events suspected to be MH was defined as follows:

• 1. A personal history of an adverse anesthetic event suspected to be MH (probands)

• 2. A family history of an adverse anesthetic event suspected to be MH (relatives who were investigated instead of the proband)

Other referral indications without a history of adverse anesthetic events suspected to be MH were defined as follows:

• 3. Personal history of exertional and/or recurrent rhabdomyolysis, defined as a creatine kinase value during the rhabdomyolysis event of more than 10,000 U/l, where recurrent is defined as at least two episodes of rhabdomyolysis

• 4. Personal history of a diagnosed RYR1-related myopathy (central core disease, multiminicore disease, centronuclear myopathy, congenital fiber type disproportion, King–Denborough syndrome, periodic paralysis, and axial myopathy)

• 5. Family history of a diagnosed RYR1-related myopathy

• 6. Personal history of an RYR1 variant detected on diagnostic testing in the neuromuscular clinic for symptoms suggestive of a neuromuscular disorder but without a specific clinical or histopathologic diagnosis and/or fulfilling one of the other referral criteria; these are often coincidentally found RYR1 variants; frequent reasons for RYR1 sequencing in the neuromuscular clinic are myalgia, muscle cramps, and muscle weakness^18,19 

• 7. Family history of an RYR1 variant detected during diagnostic testing in the neuromuscular clinic for symptoms suggestive of a neuromuscular disorder but without a specific clinical or histopathologic diagnosis and/or fulfilling one of the other referral criteria

• 8. Personal history of idiopathically elevated resting creatine kinase values

• 9. Personal history of exertional heat stroke defined as a temperature higher than 40°C/104°F with central nervous system dysfunction

• 10. Other

Based on the available information concerning genetic and CHCT/in vitro contracture test results, all medical records were classified as MH-susceptible, non–MH-susceptible, or unknown. Those who tested positive for both halothane and caffeine, positive for halothane only, or positive for caffeine only by CHCT/in vitro contracture test were classified as MH-susceptible. Those who were tested negative for both halothane and caffeine by CHCT/in vitro contracture test were classified as non–MH-susceptible. Patients with an RYR1/CACNA1S variant diagnostic for MH according to the European Malignant Hyperthermia Group list of diagnostic variants (www.emhg.org/diagnostic-mutations; Lund, Sweden) were classified as MH-susceptible.²⁰ 

In cases in which the medical records did not support any of the above criteria, the records were classified as unknown.⁴  Based on the available information concerning the diagnostic procedures performed, these records could not be classified as MH-susceptible or non–MH-susceptible because the relevant investigations (genetic testing and/or CHCT/in vitro contracture test) were not (yet) performed due to the waiting lists in the participating MH units, patient’s refusal, missing data, or a combination of these reasons.

Individuals referred to the MH unit in Toronto were investigated by CHCT according to the North American MH protocol. The CHCT has a sensitivity of 97% and specificity of 78%.^1,2  Individuals referred to Nijmegen, Lund, and Antwerp were investigated by in vitro contracture test according to the European Malignant Hyperthermia Group protocol. The in vitro contracture test has a sensitivity of 100% and specificity of 94%.^3,4 

To study whether computational evidence is useful when counseling patients without a history of an adverse anesthetic event suspected to be MH, the Rare Exome Variant Ensemble Learner score²¹  was used. During the secondary analysis, the Rare Exome Variant Ensemble Learner score was calculated for the RYR1 missense variants that were not on the European Malignant Hyperthermia Group list of diagnostic variants.²⁰  As the Rare Exome Variant Ensemble Learner score was developed for missense variants, other types of RYR1 variants (e.g., duplication or deletion variants) were excluded from the analysis. When two or more RYR1 missense variants were identified, the variant with the highest Rare Exome Variant Ensemble Learner score was included in the analysis. According to the ClinGen Variant Curation Expert Panel for RYR1 pathogenicity classification recommendations for RYR1 pathogenicity assertions in MH susceptibility,¹⁰  a Rare Exome Variant Ensemble Learner score of 0.5 or lower is evidence against pathogenicity, a Rare Exome Variant Ensemble Learner score of 0.5 to 0.85 neither is evidence against nor supports pathogenicity, and a Rare Exome Variant Ensemble Learner score of 0.85 or higher supports pathogenicity.

The sample size was based on the available data. Therefore, no statistical power analysis was performed. Normality of continuous variables was assessed with the use of histograms. Continuous variables were reported as mean ± SD for normally distributed data and as median and interquartile range for nonnormally distributed data. Categorical variables were reported using frequencies and percentages. The chi-square test was used to compare categorical variables. Records classified as unknown were included in the descriptive statistics regarding MH diagnostic tests used and also in the analysis regarding the referral criteria during the study period. These records were excluded from the statistical analysis comparing the proportion of records classified as MH-susceptible between those with or without personal or family history of adverse anesthetic events suspected to be MH. All statistical analyses were performed using IBM SPSS Statistics for Windows version 24.0 (USA). A two-tailed P value < 0.05 was used as a cutoff for significance. The statistical analysis plan, definition of the subgroups, and outcomes were documented before accessing the data.

In the primary analysis, 520 medical records were included (fig. 1). Median age at referral was 37 yr (interquartile range, 28 to 50), and 265 (51.0%) were male. Based on the information in their MH unit medical records, 180 (34.6%) patients were categorized as MH-susceptible, 160 (30.8%) were categorized as non–MH-susceptible, and 180 (34.6%) were categorized as unknown. In seven (1.8%) medical records, the result of the genetic analysis could not be identified. There were no missing CHCT/in vitro contracture test results. The clinical grading scale¹⁷  was available for 170 probands, of which 109 completed the full diagnostic process of MH susceptibility. Resting creatine kinase values were available in 134 (25.8%) medical records, of which 115 completed the full diagnostic process for MH susceptibility. The demographic characteristics are summarized in table 1.

The most frequent referral indication was a personal history of an anesthetic adverse event suspected to be MH (n = 211; 40.6%). A total of 194 (37.3%) patients referred to the participating MH units did not have a personal or family history of adverse anesthetic events suspected to be MH. Referral indications changed during the study period; 28.4% (61 of 215) of the patients referred between 2010 to 2014 did not have a personal or family history of an adverse anesthetic event suspected to be MH, while this increased to 43.6% (133 of 305) between 2015 to 2019 (P < 0.001). Distribution of the referral criteria for each MH unit is summarized in table 2.

A CHCT/in vitro contracture test was performed in 288 (55.4%) patients, genetic investigation was performed in 399 (76.7%) patients, and 192 (36.9%) were investigated both genetically and by CHCT/in vitro contracture test. Of the 399 genetically investigated patients, 168 (42.1%) were investigated by sequencing of both the RYR1 and CACNA1S genes. The use of next-generation sequencing increased during the study period; 49.3% (106 of 215) of the patients referred between 2010 to 2014 were investigated by next-generation sequencing, while this increased to 68.2% (208 of 305) between 2015 and 2019 (P < 0.001). Referral indication and the use of next-generation sequencing during the study period are summarized in figure 2.

In the subgroup of those without a personal or family history of an adverse anesthetic event suspected to be MH (n = 194), 47 were diagnosed as MH-susceptible, 73 were diagnosed as non–MH-susceptible, and 74 were classified as unknown. Patients with a personal or family history of an anesthetic adverse event suspected to be MH were more frequently diagnosed as MH-susceptible (133 of 220; 60.5%) than those without (47 of 120; 39.2%; P < 0.001). A total of 180 records were excluded from the analysis because of insufficient information, resulting in classification as unknown. Referral indications and details of the diagnostic tests performed are summarized in table 1.

The results of the genetic analysis, overall and for the subgroups (MH-susceptible, non–MH-susceptible, and unknown), are summarized in table 3. We did not identify any STAC3 variants; however, this gene was specifically investigated in only one patient. All identified variants in the RYR1 and CACNA1S genes and in other genes relevant for the neuromuscular clinic are given in Supplemental Digital Content 2 (https://links.lww.com/ALN/C834).

The medical records of 158 patients without a personal or family history of an anesthetic adverse event suspected to be MH were included in the secondary analysis (fig. 1). A total of 42 referred because of a personal history of exertional and/or recurrent rhabdomyolysis were included in the secondary analysis. Based on the information contained in these records, 16 of 42 were diagnosed as MH-susceptible. Only one of six patients referred because of exertional heat stroke was diagnosed as MH-susceptible. However, only two of six were investigated by CHCT/in vitro contracture test. The details of the CHCT/in vitro contracture test and genetic analysis results from patients referred because of a personal history of exertional and/or recurrent rhabdomyolysis and exertional heat stroke are summarized in table 4.

Of 29 patients referred for a RYR1-related myopathy, 5 were diagnosed MH-susceptible. Of 19 patients for whom referral was for a family history of RYR1-related myopathy, 1 was diagnosed as MH-susceptible. A total of 11 of 32 patients were diagnosed as MH-susceptible when the referral indication was an RYR1 variant detected in patients seen in the neuromuscular clinic and tested for symptoms suggestive of a neuromuscular disorder but without a specific clinical or histopathologic diagnosis. Of 20 patients for whom a family history of an RYR1 variant detected in diagnostic testing in the neuromuscular clinic without a specific clinical or histopathologic diagnosis was the referral indication in the medical records, 2 were diagnosed as MH-susceptible. Idiopathically elevated creatine kinase level was the referral indication in the medical records of 10 patients, where 1 was diagnosed as MH-susceptible.

The results of the performed CHCT/in vitro contracture test and genetic analysis of patients referred because of a personal or family history of RYR1-related myopathies, idiopathically elevated creatine kinase values, and a personal or family history of an RYR1 variant detected on diagnostic testing in the neuromuscular clinic are summarized in table 5. All neuromuscular symptoms reported in the medical records of patients with an RYR1 variant detected in the neuromuscular clinic without a specific clinical or histopathologic diagnosis are summarized in Supplemental Digital Content 3 (https://links.lww.com/ALN/C835).

A total of 71 patients without a personal or family of an adverse anesthetic event suspected to be MH had at least one missense RYR1 variant. Most of the Rare Exome Variant Ensemble Learner scores (37 of 71) were between 0.5 and 0.85 and therefore not helpful in RYR1 pathogenicity classification; 9 of 71 RYR1 variants had Rare Exome Variant Ensemble Learner scores of 0.5 or lower, and 25 of 71 RYR1 variants had Rare Exome Variant Ensemble Learner scores of 0.85 or higher.

A total of 34 of 71 RYR1 variants had a Rare Exome Variant Ensemble Learner score of 0.5 or lower or 0.85 or higher, indicating a benign (0.5 or lower) or pathogenic (0.85 or higher) variant. The full diagnostic process of MH susceptibility was completed by 19 of 34. In 10 of 19, the Rare Exome Variant Ensemble Learner scores were discordant with the results of MH susceptibility diagnostics. Rare Exome Variant Ensemble Learner scores and the results of MH susceptibility diagnostics are summarized in figure 3.

This retrospective multicenter cohort study shows that the indications for referral to MH units have changed. An increasing number of patients referred to MH units do not have a personal or family history of an adverse anesthetic event suspected to be MH. This trend coincides with the publication of the European Malignant Hyperthermia Group guideline for investigation of MH susceptibility in 2015.⁴  This guideline recommends referral to an MH unit for patients with exertional and/or recurrent rhabdomyolysis, RYR1-related myopathies, and other RYR1-related phenotypes. This might, at least partly, explain the increasing number of referrals concerning patients without a personal or family history of an adverse anesthetic event. These patients carry RYR1 variants identified during the diagnostic workup for exertional and/or recurrent rhabdomyolysis, exertional heat stroke, RYR1-related myopathies, or an unresolved nonspecific neuromuscular phenotype reflecting the wide spectrum of RYR1-related phenotypes.⁸  Since 39.2% of the patients referred to an MH unit without a personal or family history of an anesthetic adverse event suspected to be MH were diagnosed as MH-susceptible, these patients can be at risk of MH when exposed to triggering anesthetic agents. On the other hand, 60.8% of the patients without a personal or family history of an adverse anesthetic event were diagnosed as non–MH-susceptible, indicating the importance of MH susceptibility diagnostics; a non–MH-susceptible test result enables anesthesiologists to treat carriers of RYR1 variants and their family members without MH precaution measures.^4,22 

In our study, 16 of 42 of the patients with exertional and/or recurrent rhabdomyolysis were diagnosed as MH-susceptible, which compared to previous case studies is less frequent than 11 of 12²³  and 5 of 6²⁴  but more frequent than 2 of 14.¹⁸  This variability can, at least partly, be explained by selection bias concerning some of these study cohorts. Another cohort study reporting 17 MH-susceptible patients who suffered more than two episodes of exertional rhabdomyolysis identified 9 patients with RYR1 variants, including two pathogenic variants for MH.¹¹  Previous studies on MH susceptibility in exertional heat stroke patients reported a positive in vitro contracture test in 12 of 28,²⁵  which is higher than in our study, probably due to the low sample size of the exertional heat stroke cohort and the high number of patients classified as unknown in our study.

There are several case reports reporting MH reactions and studies reporting MH susceptibility in patients with RYR1-related myopathies,^26–29  but we did not identify any cohort studies or large case series on MH susceptibility in patients with RYR1-related myopathies. The same applies to patients with an RYR1 variant detected in diagnostic testing in the neuromuscular clinic without a specific clinical or histopathologic diagnosis.¹⁸ 

As next-generation sequencing has become faster and more cost-effective, it is gradually becoming the first-line diagnostic test for genetically heterogenous disorders (such as congenital myopathies). This has resulted in a rapid increase in the identification of both the number of patients with an RYR1 variant and the number of newly identified RYR1 variants. Only a limited number of these RYR1 variants are classified as pathogenic or benign according to the Variant Curation Expert Panel recommendations for RYR1 pathogenicity classification in MH susceptibility¹⁰  and/or the European Malignant Hyperthermia Group scoring matrix for classification of genetic variants in MH susceptibility. However, only variants classified as (likely) pathogenic or benign can be used for genetic MH susceptibility diagnostics.¹⁰  The increasing number of patients without a personal or family history of an adverse anesthetic event with a variant of unknown significance in RYR1 will be a major challenge for MH units in future.

As our study shows, bioinformatic prediction tools are currently insufficient to classify RYR1 missense variants of unknown significance. In 37 of 71 of the patients with a missense variant of unknown significance in RYR1 who did not have a history of an adverse anesthetic event, the Rare Exome Variant Ensemble Learner scores were between 0.5 and 0.85 and were therefore not helpful.¹⁰  Furthermore, the Rare Exome Variant Ensemble Learner score does not take into consideration the possibility of two or more RYR1 variants interacting in a synergistic manner with regards to their pathogenicity. Rare Exome Variant Ensemble Learner scores of 0.5 or lower and 0.85 or higher may be useful as preliminary guidance but are currently not validated to confirm or rule out MH susceptibility. We identified 10 of 19 cases of discordance between the CHCT/in vitro contracture test result and the Rare Exome Variant Ensemble Learner score (fig. 3). These findings are in line with other in silico predictors of pathogenicity in MH.^30,31 

Our results can be used to improve counseling of patients referred to MH units without a personal or family history of an adverse anesthetic event suspected to be MH. Furthermore, these results can also be useful for geneticists, neurologists, and other specialists investigating patients by RYR1 sequencing or whole-exome sequencing. They need to be aware of and inform patients about the possibility of identifying a variant of unknown significance in RYR1 and the potential subsequent need for a muscle biopsy for CHCT/in vitro contracture test and, if relevant, cascade family testing in all first-degree family members before they perform RYR1 sequencing (either targeted or by next-generation sequencing).

Our study has some limitations. Some referral indications were disproportionally distributed between the participating MH units, probably caused by the close collaboration between the MH units in Toronto and Nijmegen and the university hospital neuromuscular clinic, in contrast to the MH unit in Lund, which does not have any collaborations with the local neurology department. In addition, only a limited number of patients were tested for CACNA1S and STAC3 variants, and several patients referred because of a family history of an RYR1-related myopathy (5 of 19) or a family history of an RYR1 variant detected in diagnostic testing in the neuromuscular clinic without a specific clinical or histopathologic diagnosis (12 of 20) were genetically investigated using a targeted technique. Therefore, we are not sure whether these unaffected family members carried RYR1 variant(s) other than those identified in their relatives with neuromuscular symptoms. Furthermore, due to the low penetrance of MH susceptibility,^10,12,32  currently unresolved modifying factors in the occurrence of MH, and ethical limitations, it is not possible to study which MH-susceptible patients suffer an MH reaction when exposed to triggering agents. Since the CHCT/in vitro contracture test and screening for diagnostic variants are the accepted standard in MH susceptibility diagnostics⁴  and an MH reaction can be life-threatening,^13,33  all patients diagnosed as MH-susceptible should be considered at risk for MH when in need of anesthesia.²² 

It is important to mention that not all patients who suffer a rhabdomyolysis and/or exertional heat stroke episode will be referred to MH units as only a limited number of patients have a genetic background associated with an increased susceptibility to rhabdomyolysis³⁴  or exertional heat stroke.^25,35,36  Neurologists and sport physicians only refer patients to an MH unit with signs of an increased genetic susceptibility to rhabdomyolysis and/or exertional heat stroke, resulting in a selection bias. The same selection bias arises for the RYR1 variants within the study cohort. Patients with RYR1 variants resulting in a loss of function in ryanodine receptor 1 or a high prevalence in control populations are unlikely to cause MH.¹⁰  Carriers of these variants are therefore less likely to be referred to an MH unit.

Last, the number of patients in our cohort who did not complete the full process of MH susceptibility diagnostics might have affected our results but probably also reflect the problem our study addresses. The CHCT/in vitro contracture test is an invasive procedure, and some patients referred for MH susceptibility diagnostics refuse muscle biopsy or are unable to undergo muscle biopsy and consider themselves MH-susceptible without confirmation of the diagnosis. Furthermore, worldwide knowledge and expertise needed to perform a reliable CHCT/in vitro contracture test are limited. In some countries, there are no CHCT/in vitro contracture test laboratories, and the established MH units in other countries have long waiting lists, resulting in a very large number of patients carrying RYR1 variants of unknown significance with limited possibilities for MH susceptibility diagnostics. This is also the case for the MH unit in Toronto; most patients who did not complete the full process of MH susceptibility are on the waiting list to be investigated.

Future strategies for MH susceptibility diagnostics should focus on classification of RYR1, CACNA1S, and STAC3 variants utilizing common databases and functional studies. This should not be limited to suspected pathogenic variants because classification of a variant as benign could prevent unnecessary invasive diagnostic procedures. Other potential fields of interest for future research are identification of new genes of interest as 27.7% of the MH-susceptible–diagnosed patients did not have a variant in RYR1, CACNA1S, or STAC3. As our study demonstrates, currently available bioinformatic models such as Rare Exome Variant Ensemble Learner²¹  are insufficient for MH susceptibility diagnostics, but more useful alternatives may emerge in the future.

The proportion of patients referred to MH units without a personal or family history of adverse anesthetic events suspected to be MH has increased. These patients carry RYR1 variants identified during the diagnostics workup for exertional or recurrent rhabdomyolysis, exertional heat stroke, RYR1-related myopathies, or an unresolved neuromuscular phenotype. Since 39.2% of the patients referred to an MH unit without a personal or family history of an anesthetic adverse event suspected to be MH were diagnosed as MH-susceptible, these patients can be at risk for MH when exposed to MH triggering agents, and the referral of such patients to MH units is therefore indicated.

Several authors of this article are members of the Radboud University Medical Center of Expertise for Neuromuscular Disorders (Nijmegen, The Netherlands), the Netherlands Neuromuscular Center (Abcoude, The Netherlands), and the European Reference Network for Rare Neuromuscular Diseases (Paris, France).

Supported by the Radboud University Medical Center (Nijmegen, The Netherlands) Regional Junior Researcher Grant in collaboration with the Canisius Wilhelmina Hospital (Nijmegen, The Netherlands).

Dr. Heytens is supported by Norgine BV (Amsterdam, The Netherlands). The other authors declare no competing interests.