Variants in ASPH cause exertional heat illness and are associated with malignant hyperthermia susceptibility

Exertional heat illness (EHI) and malignant hyperthermia (MH) are life threatening conditions associated with muscle breakdown in the setting of triggering factors including volatile anesthetics, exercise, and high environmental temperature. To identify new genetic variants that predispose to EHI and/or MH, we performed genomic sequencing on a cohort with EHI/MH and/or abnormal caffeine-halothane contracture test. In five individuals, we identified rare, pathogenic heterozygous variants in ASPH, a gene encoding junctin, a regulator of excitation-contraction coupling. We validated the pathogenicity of these variants using orthogonal pre-clinical models, CRISPR-edited C2C12 myotubes and transgenic zebrafish. In total, we demonstrate that ASPH variants represent a new cause of EHI and MH susceptibility.


Editorial assessment and review synthesis Editor's summary and assessment
Exertional heat illness (EHI) and malignant hyperthermia (MH) are related conditions caused by stimulus-driven skeletal muscle breakdown. Here, the authors performed whole exome or whole genome sequencing on a cohort of 103 individuals with EHI/MH and/or abnormal caffeine-halothane contracture test. They identified heterozygous pathogenic variants in the junctin isoform of the ASPH gene that might underlie EHI or MH susceptibility in five of these individuals, and used two orthogonal models (transgenic zebrafish and CRISPR-edited C2C12 myoblasts) to validate the pathogenicity of these variants. Altogether, they demonstrate that ASPH variants represent a new cause of EHI and MHS.
The editors jointly decided to send this manuscript out to review based on the integration of genetic testing and preliminary validation in multiple systems. However, the degree of advance provided and the preliminary nature of these functional experiments in zebrafish and C2C12 cells prevented further consideration by Nature Genetics.

Editorial synthesis of reviewer reports
The reviewers largely find the study to be well-designedl, but provide several comments on potential discrepancies in the datasets and figures that should be discussed in a revision. Reviewer #1 also comments on the validity of the normalization approach for swimming distance, considering some of the variability in controls on a daily basis. The reviewers also commented on the framing of the manuscript, which should be improved to promote readability and appropriate context for these results.
While Nature Genetics is unable to offer a revision, Nature Communications would be interested in considering a revision that comprehensively addresses reviewer concerns about your experimental controls and setup. Communications Biology would also be interested in considering a manuscript that addresses all discussion points raised by the reviewers.

Nature Genetics
Revision not invited The degree of advance provided or the breadth of potential interest to researchers has not matched the criteria for further consideration at Nature Genetics.

Major revisions
A revision for Nature Communications would require you to fully address the concerns from Reviewer #1 regarding the variability of swimming distances in your controls; please provide additional experimental work, controls and replicates as appropriate. Your revision would also be expected to address the comments from Reviewer #3 about the promoter that you used in your assays. Please address all the other comments raised by the three Reviewers as well.

Communications Biology
Minor revisions A revision for Communications Biology would only have to justify the normalization approach for swimming distances (noted by Reviewer #1) and address other discussion points raised by the reviewers. We believe these points could be addressed textually, without additional experimental evidence.

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Annotated reviewer reports
The editors have included some additional comments on specific points raised by the reviewers below, to clarify requirements for publication in the recommended journal(s). However, please note that all points should be addressed in a revision, even if an editor has not specifically commented on them.

Reviewer #1 information Expertise
This reviewer has expertise in zebrafish models and muscle physiology.

Editor's comments
This reviewer provided a positive assessment of the manuscript, but highlighted the need to justify the normalization approach for swimming distance datasets.

Section Annotated Reviewer Comments
Remarks to the Author: Overall significance This manuscript identified a candidate gene for exertional heat illness/malignant hyperthermia from two different patient cohorts. All variations in ASPH were heterozygous, suggesting a dominant effect. This and the potential impact of these variants on swimming behavior with/without heat and caffeine challenge were next investigated. The experiments using transgenic/RNA -expressing zebrafish are well done and clearly show that pathogenic variants in ASPH impact swimming behavior and muscle structure in the zebrafish model. The mechanism may be partially through excess ROS, as an antioxidant blunts the damage. Similar experiments in C2C12 cells led to similar, though less striking, results. Biochemical experiments suggest that Junctin interactions with RyR and CASQ1 may mediate Juntin's impacts on calcium homeostasis, muscle structure, and function. The impact of this manuscript is elucidating new genetic causes of EHI/MH, and identifying that the zebrafish model recapitulates important aspects of the phenotype and shows similar drug responsiveness -thus this is an excellent vertebrate model for these important conditions. Signed: Clarissa Henry

Remarks to the Author: Strength of the claims
The data are rigorous and well described. The authors very clearly state in which experiments they used established transgenic lines and which involved mRNA injections -showing similar results from both types of experiments bolsters their data. I only had one "major" comment -although not particularly major.

Major:
1. The variation in swimming distance is somewhat high -with controls swimming different distances (up to 25% difference) in different experiments. This is not alarming, the authors are to be lauded for showing their data clearly, but does raise the question of whether it makes sense to normalize data compared to controls on any given day in a given experiment?
It would be necessary to fully address this concern with further experimental work, controls and replicates as appropriate for further consideration at Nature Communications. Please ensure that your approach controls for heterogeneity across different days and experiments adequately.
It would only be necessary to either justify the current analysis or reanalyze the data to account for day-to-day variability among controls for further consideration at Communications Biology.

Minor:
1. First bit of results section -relies on reader having read introduction but it's potentially worth quickly reiterating that excitation-contraction coupling (ECC) genes are strong candidates…. Reviewer #2 also comments on the need for additional context, for the sake of readability.

Remarks to the Author: Reproducibil ity
Manuscript is well written and described.

Reviewer #2 information Expertise
This reviewer has expertise in cardiovascular and muscle physiology.

Editor's comments
This reviewer also provided positive feedback on the study, but introduced several important discussion points that should be addressed in a revision. They also echo some feedback from Reviewer #1 regarding the study's context, to improve readability.

Remarks to the Author: Overall significance
In the manuscript entitled "Variants in ASPH cause exertional heat illness and are associated with malignant hyperthermia susceptibility.", by Endo et al, the Authors described a novel function of junctin, a variant of the aspartate beta-hydroxylase (ASPH) gene, on the treatment of exertional heat illness (EHI), and malignant hyperthermia susceptibility (MHS) muscle diseases. In the well-written and clear manuscript, the Authors showed that specific mutations found in human muscle samples on junctin (K88T and V54A) are relevant to keep the stability of ryanodine receptors during the excitation-contraction coupling, especially during high temperature and under the effect of halothane. The interesting approach taken by the Authors to understand these mutations was to transfer the mutated gene into two different and wide-used pre-clinical muscle models: zebrafish and C2C12 cell line. The findings in this manuscript are clinically relevant for the EHI and MHS patients, and for finding new alternative approaches for those diseases. There are nevertheless, minor comments that the Authors should address before a final decision.
1) The Authors should give the (in extenso) name of the gene abbreviation.
2) At the end of the introduction, the Authors only mentioned junction as one candidate for EHI/ MHS, without describing how this gene was selected, the function of the protein, and to whom it interacts. These pieces of information are necessary to understand the reason for the work, especially for those readers who are not familiar with the topic. Part of the explanation was found in the results, but still should be present in the introduction for better comprehension of the manuscript. This point (and point #3) echoes similar feedback from Reviewer #1 regarding the context of the manuscript (Minor Point #1).
3) From the introduction, it is difficult to clearly see the aim of the work. The Authors should elaborate a sentence defining the aim(s) and the pieces of evidence to support the aim(s).
4) The Authors should describe in the results the concentration of the drugs used in the study. Moreover, it is lacking on figures 2 and 3 a description of the statistical analyses of whether the pharmacological treatment did or did not produce a significant result different from the control condition.
For the sake of reproducibility, please carefully elaborate on the statistical analyses for these results.

5)
In-text for figure 2, the Authors state ", and identified no obvious differences in baseline motor behavior, muscle ultrastructure, or survival ( Figure 2B)." Although there are no differences, the Authors only showed muscle ultrastructure data, without the other parameters. Maybe just a re-adjust of the position of the label ( Figure 2B) will correct the imprecision of the text. It would be necessary to elaborate on this point for future consideration at Nature Communications and Communications Biology.
6) The Authors should clarify in the results whether the experiments using C2C12 cells were done in myoblast or myotubes, and if so, the passage, confluency, and days after differentiation. The Methods should include sufficient detail for others to repeat these experiments. Please note that we do not have a word limit for the Methods section. Figure 4e, the truncated data is represented with a red bar, instead of the orange bar from the other experiments. Is there a specific reason for the change of color? 8) In the paragraph of the discussion, starting with "Our data expand the knowledge… and positive CHCT in his sibling" the Authors should provide the missing citations.

Remarks to the Author: Impact
The current and the following works on the function of junctin will provide important and relevant pre-clinical and clinical information on the new therapies for EHI and MHS.

Remarks to the Author: Strength of the claims
The data presented in the current manuscript are enough to convince for further studies of junctin related to EHI and MHS.

Remarks to the Author: Reproducibility
The Authors took care of the statistical analyses, giving enough information for the continuity work from independent groups.

Reviewer #3 information Expertise
This reviewer has expertise in zebrafish models of cardiovascular disease.

Editor's comments
While we recognize this review is brief, the reviewer noted some important discrepancies in the manuscript that should be discussed in a revision.

Remarks to the Author: Overall significance
In this manuscript, the authors integrated human genetics, zebrafish genetics and cell culture models to discover causative genes for exertional heat illness (EHI) and malignant hyperthermia (MH). They performed genomic sequencing on a cohort with EHI/MH and identified rare, pathogenic variants in ASPH. They then generated transgenic zebrafish for two of the variants, which recapitulated the corresponding phenotypes in human. They went on to generate knock-in alleles in C2C12 cell and also obtained promising data to prove causality. The logical flow is clear, and data and their conclusions are convincing, and the presentation is excellent.

Remarks to the Author: Impact
Successful modeling of the disease in the efficient zebrafish model is interesting, which shall have great potential for testing additional variants that will be identified in the future. Thus, the impact could be high. 3. Fig. 3A and 4B. Are the units the same? If so, please choose one. It seems data in fish and cell are different. Why? Please clarify the units between these figures, for the sake of consistency.

Remarks to the Author: Reproducibility
Statistical analysis is adequate.

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