+ MODEL

Journal of the Formosan Medical Association xxx (xxxx) xxx

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: www.jfma-online.com

Original Article

Next-generation sequencing and

bioinformatics to identify genetic causes of
malignant hyperthermia
Huei-Ming Yeh a, Min-Hua Liao b, Chun-Lin Chu c, Yin-Hung Lin d,
Wei-Zen Sun a, Ling-Ping Lai e, Pei-Lung Chen f,g,*

a
Department of Anesthesiology National Taiwan University Hospital, Taipei, Taiwan
b
Scientist, Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan
c
Department of Anesthesiology, National Taiwan University Hospital, Yun-Lin Branch, Yunlin, Taiwan
d
Graduate Institute of Medical Genomics and Proteomics, National Taiwan University College of
Medicine, Taipei, Taiwan
e
Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
f
Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan
University College of Medicine, Taipei, Taiwan
g
Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan

Received 14 January 2020; received in revised form 2 June 2020; accepted 18 August 2020

KEYWORDS Background/purpose: Malignant hyperthermia (MH) is a life-threatening pharmacogenetic dis-

Malignant ease with only two known causative genes, RYR1 and CACNA1S. Both are huge genes containing
hyperthermia; numerous exons, and they reportedly only account for 50e70% of known MH patients. Next-
Genetics; generation sequencing (NGS) technology and bioinformatics could help delineate the genetic
Next-generation diagnosis of MH and several MH-like clinical presentations.
sequencing (NGS); Methods: We established a capture-based targeted NGS sequencing framework to examine the
Bioinformatics; whole genomic regions of RYR1, CACNA1S and the 16.6 Kb mitochondrial genome, as well as 12
RYR1 other genes related to excitation-contraction coupling and/or skeletal muscle calcium homeo-
stasis. We applied bioinformatics analyses to the variants identified in this study and also to the
48 documented RYR1 pathogenic variants.
Results: The causative variants were identified in seven of the eight (87.5%) MH families, but in
none of the 10 individuals classified as either normal controls (N Z 2) or patients displaying
MH-like clinical features later found to be caused by other etiologies (N Z 8). We showed that
RYR1 c.1565A>G (p.Tyr522Cys)(rs118192162) could be a genetic hot spot in the Taiwanese pop-
ulation. Bioinformatics analyses demonstrated low population frequencies and predicted
damaging effects from all known pathogenic RYR1 variants. We estimated that more than
one in 1149 individuals worldwide carry MH pathogenic variants at RYR1.

* Corresponding author. Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan University College
of Medicine, No 1 Jen-Ai Road Section 1, Taipei 100, Taiwan. Fax: þ886 2 23813690.
E-mail address: paylong@ntu.edu.tw (P.-L. Chen).

https://doi.org/10.1016/j.jfma.2020.08.028
0929-6646/Copyright ª 2020, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: Yeh H-M et al., Next-generation sequencing and bioinformatics to identify genetic causes of malignant hy-
perthermia, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.08.028
 + MODEL
2 H.-M. Yeh et al.

Conclusion: NGS and bioinformatics are sensitive and specific tools to examine RYR1 and CAC-
NA1S for the genetic diagnosis of MH. Pathogenic variants in RYR1 can be found in the majority
of MH patients in Taiwan.
Copyright ª 2020, Formosan Medical Association. Published by Elsevier Taiwan LLC. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).

Introduction Comprehensively sequencing by the traditional Sanger

Malignant hyperthermia (MH) is a potentially fatal phar-
macogenetic disorder triggered by certain drugs used for
general anesthesia,1,2 leading to circulatory collapse and
death if not treated quickly.3e5 According to different re-
ports, MH reaction incidence ranges from 1:5000 to
1:50,000e100,000 anesthesias. However, MH susceptibility
(MHS) prevalence was estimated to be as high as one in
839e3000 individuals.2,4e8 MH diagnosis is based on either
clinical presentation or laboratory tests.2e5 However, it is
often difficult to obtain comprehensive clinical data for
definitive MH diagnosis, and it is not possible for prediction.
The in vitro contracture test (IVCT) is the current “gold
standard” for MH diagnosis and can even be used for pre-
diction. However, IVCT cannot be widely used because of
its invasiveness, expensiveness and imperfect clinical
correlation.2,4e7
There are six reported loci of malignant hyperthermia
susceptibility (MHS)2e5 as Table 1. MHS1 was identified as
the RYR1 (ryanodine receptor) gene on chromosome
19q13.1 and MHS5 was identified as the CACNA1S (calcium
channel, voltage-dependent, L type, alpha-1S subunit)
gene on chromosome 1q32.1.2e5 Causative variants in RYR1
account for 50e70% of MH cases.2,5e7,9,10 The current
consensus guidelines for MH genetic diagnosis by the Eu-
ropean Malignant Hyperthermia Group (EMHG) were
described in Urwyler et al.11 and Hopkins and colleagues.12
Recently Merritt and colleagues13 reported 5 RYR1 variants
fulfilling the criteria for MH pathogenicity using the HEK293
functional assays. According to the EMHG official website
(https://emhg.org/genetics/), currently only 48 of the
reported w300 RYR1 variants and 2 reported CACNA1S
variants worldwide have proven pathogenicity according
to those stringent criteria.1,4,5,14,15
RYR1 and CACNA1S are both big genes containing
numerous exons (106 and 44 exons, respectively).
dideoxyNTP termination method is challenging, let alone
other possible genes in the genome.15e19 The ability of
next-generation sequencing (NGS) to offer high throughput
at low cost (per base-read) makes it one of the most
exciting technological advances in biomedical sciences over
the past 10 years.20 NGS for MH diagnosis has been tried by
several teams with various degrees of success and
limitations.14,18,21e23
Here we report our results applying the NGS framework
for genetic testing of MH patients and controls (including
several individuals with MH-like clinical presentations), as
well as a bioinformatics analysis of the documented path-
ogenic RYR1 variants and the hypothetical possible
involvement of mitochondrial genome that incorporates
data from several large-scale sequencing projects
worldwide.
Methods
Ethic statement
This study was approved by the Institutional Review Board
of National Taiwan University Hospital (Ref:
201305099RINB). All participants provided written informed
consent.
Participant enrollment and diagnosis
Suspected MH patients were reported from hospitals across
Taiwan, a single-payer national health insurance country
with 23 million people. All patients were identified by an-
esthesiologists and had typical clinical presentations of MH
including high fever, marked muscle enzyme increase, renal
failure, and hyperkalemia. After enrollment, specimen of
survivals and families of patients who died during operation

Table 1 Six reported loci of malignant hyperthermia susceptibility (MHS).2e5

Locus Gene Genomic region Inheritance Proportion of patients
attributed to the locus
MHS1 RYR1 19q13.2 AD 50e70%
MHS2 unknown 17q11.2-q24 AD Unknown
MHS3 unknown 7q21-q22 AD Unknown
MHS4 unknown 3q13.1 AD Unknown
MHS5 CACNA1S 1q32.1 AD 1%
MHS6 unknown 5p AD Unknown
Abbreviation: AD, autosomal dominant; MHS, malignant hyperthermia susceptibility.

Please cite this article as: Yeh H-M et al., Next-generation sequencing and bioinformatics to identify genetic causes of malignant hy-
perthermia, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.08.028
 + MODEL
Next-generation sequencing for malignant hyperthermia
were referred to genomics laboratory via anesthesiology
department of National Taiwan University Hospital. Clinical
diagnosis was made by three anesthesiologists in our team
based on reported clinical presentation, medical history,
family history, and usage of dantrolene.
MH clinical diagnosis was based on the clinical grading
scale (CGS) according to Larach and colleagues.24 Briefly,
the CGS addresses several indicators, including masseter
spasms or generalized muscle rigidity (process I: rigidity),
maximum serum creatine kinase or maximum serum
myoglobin levels (process II: muscle breakdown), maximum
PaCO2 (process III: respiratory acidosis), maximum tem-
perature (process IV: temperature increase), tachycardia or
ventricular arrhythmia (process V: cardiac involvement),
negative base excess, arterial acidosis, and rapid reversal
of MH signs after intravenous dantrolene (other indicators).
The likelihood of an acute MH crisis is predicted by MH rank
determined by the resulting raw scores: MH rank 1: “almost
never,” rank 2: “somewhat less than likely,” rank 3:
“likely,” rank 4: “somewhat greater than likely,” rank 5:
“very likely,” and rank 6: “almost certain”.
According to the clinical grading scales, patients with MH
rank of 5 or more were enrolled as MH-suspect cases and
those with MH rank of 4 or less were enrolled as MH-like
cases. We also included two normal individuals into our
study as normal control group.
Roche/NimbleGen targeted enrichment panel
We targeted the full-length sequences of the two major MH
genes (RYR1 and CACNA1S ) as well as the entire 16.6 Kb
mitochondrial genome. We also covered 12 additional genes
related to excitation-contraction coupling and/or skeletal
muscle calcium homeostasis, including JSRP1(JP45),
FKBP1B, CASQ1, ASPH, HRC, SYPL2(MG29), CALM1, CALM2,
CALM3, ITPR1, ITPR2 and ITPR3; we included the full-length
sequences the first nine genes, and the coding regions of
the last three genes.
We used the Roche/NimbleGen SeqCap EZ Choice Kit
(http://www.nimblegen.com/seqcapez/) and performed
targeted enrichment following manufacturer’s protocol.
We prepare libraries using the Illumina TruSeq DNA PCR-
Free Sample Preparation Kit (http://www.illumina.com/
products/truseq-dna-pcr-free-sample-prep-kits.ilmn), and
barcodes were added for multiplex NGS experiments. We
applied the final libraries to the Illumina MiSeq NGS
sequencer, and used the MiSeq Reagent Kit v3
(2  300 bp) because of its long reads, high yield, and
reliable quality.
Illumina MiSeq sequencing and bioinformatics
analysis
The bioinformatics analysis was processed using a computer
cluster at the High-Performance Computing (HPC) center at
the National Center for High-Performance Computing at
National Applied Research Laboratories, Taiwan.
We began the analysis using BurrowseWheeler Aligner
(BWA)25 for initial read mapping to the reference sequence
Please cite this article as: Yeh H-M et al., Next-generation sequencing and bioinformatics to identify genetic causes of malignant hy-
perthermia, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.08.028
3
of the GRCh37/hg19 assembly. We used SAMtools26 (version
1.3.1) for data conversion, sorting and/or indexing, then we
used the HaplotypeCaller of Genome Analysis Toolkit
(GATK)27 (version 3.6) for realignment and SNP/indel call-
ing. We applied aggressive filtering to further reduce false
positive results, and Pindel28 or Breakdancer29 to identify
structural variants (such as big deletion, big insertion or
inversion). Then we applied ANNOVAR30 for the annotation
of the genetic variants. SIFT (http://sift.jcvi.org) and
PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) were
used to predict the biological significance of the genetic
variants. We visualized the results using Integrative
Genomics Viewer (IGV).31 If available we used the intra-
familial segregation pattern as an important filter to find
the genuine causative variants. We also took advantage of
population frequency databases, such as genome
Aggregation Database (gnomAD),32 and Taiwan Biobank
(https://taiwanview.twbiobank.org.tw/index),33 to filter
out common variants, which we defined as an allele
frequency greater than 1%. We used Sanger sequencing to
confirm the genotyping calls.
Establishment of a genetic testing framework for
MH, using capture-based target enrichment
followed by NGS
We customized Roche/NimbleGen SeqCap EZ Library probes
(http://www.nimblegen.com/seqcapez/) to set up a target
enrichment gene panel. We targeted the full-length
sequence of 11 genes (including RYR1, CACNA1S,
JSRP1(JP45), FKBP1B, CASQ1, ASPH, HRC, SYPL2(MG29),
CALM1, CALM2 and CALM3), the coding sequences of three
genes (ITPR1, ITPR2 and ITPR3), and the entire 16.6 Kb
mitochondrial genome. Our study design and flow chart is
illustrated in Fig. 1. .
The target regions (excluding the repetitive elements
inside the regions) are listed in Table 2, showing the 14
candidate genes and their position. Across all the target
regions the average read depth was 249 (standard
deviation Z 37), and coverage was 100% (all regions at a
minimum of 50X reads).
Results
Identification of the causative variants in RYR1 in
seven out of the eight MH families (87.5%)
The characteristics and clinical features of the patients are
summarized in Table 3. Eight MH-suspect patients were
reported and two of them died during operation with their
family (MH 02-1, 02-2, 15) enrolled. We identified the RYR1
causative variants in seven of the eight probands (and/or
associated family members) with a definite MH diagnosis
(87.5%) (Fig. 1 and Table 4)
The identified causative RYR1 variants were c.1565A>G
(p.Y522C) (two families), c.6488G>T (p.R2163L),
c.6502G>A (p.V2168M), c.7304G>A (p.R2435H) and
c.7373G>A (p.R2458H) (two families), respectively.
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4 H.-M. Yeh et al.

Figure 1 Study design and flow chart.

No causative variants identified in other related

Table 2 List of the 14 candidate genes and their
positions. candidate genes in MH patients
Chromosome Start End Gene
We included 12 additional genes (JSRP1(JP45), FKBP1B,
chr1 201005639 201086694 CACNA1S CASQ1, ASPH, HRC, SYPL2(MG29), CALM1, CALM2, CALM3,
chr1 110004099 110027764 SYPL2 ITPR1, ITPR2 and ITPR3) known to be related to RYR1and
chr1 160155284 160174676 CASQ1 CACNA1S into the target panel to identify possible novel
chr2 47384220 47408740 CALM2 causative genes, and we also captured the entire 16.6 Kb
chr2 24267583 24289550 FKBP1B mitochondrial genome for sequencing.
chr3 4535031 4889524 ITPR1 We did not identify any additional possibly pathogenic
chr6 33589155 33664348 ITPR3 variants in any of those 12 genes or in the mitochondrial
chr8 62410114 62632199 ASPH genome in our eight confirmed MH families.
chr12 26488284 26986131 ITPR2
chr14 90858326 90877619 CALM1 No false positive results in 10 individuals without
chr19 2249249 2261422 JSRP1 MH
chr19 49651455 49663681 HRC
chr19 38919339 39081204 RYR1
In MH-like group, two cases with osteogenesis imperfecta,
chr19 47099511 47117039 CALM3
one case with neurogenic paralysis, one case with eye
disease, one case with dissecting aortic aneurysm, one case
with tibia fracture, and two cases with severe infection
were enrolled in this study, as all of them displayed some
symptoms and signs similar to MH during anesthesia. We
Representative NGS sequencing results and Sanger confir-
also included two normal individuals without previous
mation (c.7304G>A (p.R2435H), patient id: MH005) are
anesthesia. The NGS-based genetic testing panel detected
shown in Fig. 2.
no potential causative variants in these individuals (Fig. 1
The first two variants (p.Y522C and p.R2163L) had not
and Table 4).
been recorded in any big population genetic database,
were predicted damaging by software packages and
occurred at evolutionarily conserved amino acid residues RYR1 c.1565A>G (p.Tyr522Cys) may be a founder
with previous documented MH pathogenic variants (Table causative variant in Taiwan
4, Fig. 3 and Table S1, listing the 48 documented patho-
genic MH variants, with allele frequencies and pathoge- Two of the eight MH families were identified to carry RYR1
nicity predictions). The other three variants were c.1565A>G (p.Y522C). This variant was also reported pre-
documented MH pathogenic variants listed at the EMHG viously by our team as a MH-causal variant in one out of five
website. unrelated families in another study.16 Therefore, three of

Please cite this article as: Yeh H-M et al., Next-generation sequencing and bioinformatics to identify genetic causes of malignant hy-
perthermia, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.08.028
 + MODEL
Next-generation sequencing for malignant hyperthermia 5

Table 3 Surgical patients (or relatives) with suspected malignant hyperthermia (or malignant hyperthermia-like) events.
Sex Onset age Clinical event Grading score Final diagnosis Operation
MH01 M 42 years Fever, muscle rigidity, 6 MH Laparoscopic
hypercapnia, CK, CK-MB Cholecystectomy
elevation, dantrolene used
MH02-1 M No Father of suspected MH young n/a Father of a MH patient n/a
boy who deceased when having
circumcision
MH02-2 F No Mother of suspected MH young n/a Mother of a MH patient n/a
boy who deceased when having
circumcision
MH03 M 58 years Fever, tachycardia, CO2 6 MH Hepatectomy
elevation, muscle rigidity, CK,
CK-MB elevation, dantrolene
used
MH04 M 71 years Fever, muscle rigidity, CO2, CK, 6 MH Laparoscopic Colectomy
CK-MB elevation, dantrolene
used
MH05 F 3 years Fever, tachycardia, unstable 4 Osteogenesis imperfecta Hip correction
blood pressure, rhabdomyolysis
MH06 M 56 years Fever, tachycardia, CO2, CK, 6 MH Laparoscopic
CK-MB elevation, dantrolene Appendectomy
used
MH07 M 8 years Fever, tachycardia, 5 Brain lesion with paralysis Brain tumor excision
hypercapnia, rhabdomyolysis,
unstable blood pressure
MH08 M 14 years Fever, tachycardia, 5 Osteogenesis imperfecta Bone tumor
hypercapnia, unstable blood
pressure, rhabdomyolysis
MH09 M 32 years Fever, rhabdomyolysis 4 Dissecting aortic aneurysm Aortic repair
MH10 M 10 years Fever, masseter muscle spasm, 5 Appendicitis with rupture Laparoscopic
unstable blood pressure, CK, Appendectomy
CK-MB elevation, dantrolene
used
MH11 M 7 years Fever, tachycardia, CO2 4 Strabismus Strabismus
elevation, unstable blood
pressure, stop surgery
MH12 M 10 years Fever, muscle rigidity, 6 MH Wound debride
tachycardia, hypercapina, CK,
CK-MB elevation, dantrolene
used
MH13 M 13 years Fever, tachycardia and 4 Pterygium Pterygium
hypercapnia, unstable blood
pressure
MH14 M 15 years Fever, tachycardia and muscle Tibia fracture Tibia fracture
rigidity, hypercapina
MH15 M NA Brother of a MH patient who n/a Brother of a MH patient n/a
deceased when having
cosmetic surgery
MH16 F 44 years Fever, tachycardia and 5 MH Stapedectomy with
Hypotension, hypercapnia, prosthesis
dantrolene used
Abbreviation: MH, malignant hyperthermia; n/a, not applicable.

Please cite this article as: Yeh H-M et al., Next-generation sequencing and bioinformatics to identify genetic causes of malignant hy-
perthermia, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.08.028
 + MODEL
6 H.-M. Yeh et al.

Table 4 Next-generation sequencing (NGS)-based genetic test results.

Case Variant PolyPhen2 SIFT gnomAD Taiwan Biobank Pathogenic
MH01 RYR1 c.7373G>A:p.R2458H 0.001 0.999 0.000004 0 Yes
MH02-1 CACNA1S c.5554G>A:p.G1852R 0 0.54 0.0002 0 No
MH02-2 CACNA1S c.5399T>C:p.L1800S 0.99 0.02 0.23 0 No
MH03 RYR1 c.7304G>A:p.R2435H 0.001 0.999 0 0 Yes
MH04 RYR1 c.6502G>A:p.V2168M 1 0 0 0 Yes
MH05 RYR1 c.2750G>A:p.R917K 0.36 1 0 0 No
RYR1 c.9198C>G:p.N3066K 0.004 0.22 0.0004 0 No
MH06 RYR1 c.7373G>A:p.R2458H 0.001 0.999 0.000004 0 Yes
MH07 RYR1 c.11266C>G:p.Q3756E 0 1 0.03 0 No
MH08 no finding n/a n/a n/a n/a n/a
MH09 RYR1 c.11443A>T:p.M3815L 0.15 0.82 0 0 No
RYR1 c.12880A>G:p.T4294A 0.49 0 0 0 No
MH10 CACNA1S c.5399T>C:p.L1800S 0.99 0.02 0.23 0 No
MH11 no finding n/a n/a n/a n/a n/a
MH12 RYR1 c.1565A>G:p.Y522C 1 0 0 0 Yes
MH13 no finding n/a n/a n/a n/a n/a
MH14 no finding n/a n/a n/a n/a n/a
MH15 RYR1 c.1565A>G:p.Y522C 1 0 0 0 Yes
MH16 RYR1 c.6488G>T:p.R2163L 0.99 0 0 0 Yes
Ctl01 No finding n/a n/a n/a n/a n/a
Ctl02 No finding n/a n/a n/a n/a n/a
Abbreviation: MH, malignant hyperthermia; Ctl, control; n/a, not applicable.

the 12 MH families with identified RYR1 causative variants
detected in these two cohorts carried c.1565A>G
(p.Y522C).
The majority of MH patients/families in the
Taiwanese population carry pathogenic variants of
the RYR1 gene
Seven out of the eight MH families (87.5%) in the current
cohort were found to carry pathogenic variants of the RYR1
gene. Combining this cohort with the previous 5-family
cohort (all of the 5 families with identified variants),16 12
out of 13 MH families (92.3%) could be assigned pathogenic
variants of RYR1.
Approximately 0.087% individuals worldwide carry
definitive MH causative variants of RYR1
all the variants, according to gnomAD the total allele fre-
quency is 0.000436, which extrapolates to a population
carrier rate of 0.00087 across the world, assuming
HardyeWeinberg equilibrium.
Bioinformatics analysis demonstrated that the 48
documented RYR1 pathogenic variants are all
located at the highly evolutionarily conserved
residues
Multiple alignment of the RYR1 proteins across human and
several model organisms revealed that all 48 documented
RYR1 pathogenic DNA substitutions (45 amino acid-level
changes) are located at highly evolutionarily conserved
amino acid residues (down to zebrafish, Fig. 3).
Discussion

We analyzed publicly available NGS data from tens of
thousands of individuals from large scale sequencing pro-
jects around the world, including the Exome Sequencing
Project (ESP6500), 1000 Genomes Phase 3 dataset, the
Exome Aggregation Consortium (ExAC),32 the genome Ag-
gregation Database (gnomAD),32 and Taiwan Biobank
(https://taiwanview.twbiobank.org.tw/index) in order to
retrieve the frequencies of the 48 documented causative
variants in RYR1.33 No variant was found at high fre-
quency in the database. To sum up the allele frequencies of
We successfully established a capture-based targeted NGS
sequencing framework to examine the whole genomic re-
gions of RYR1, CACNA1S and the 16.6 Kb mitochondrial
genome, as well as 12 additional genes related to
excitation-contraction coupling and/or skeletal muscle
calcium homeostasis. We identified the causative variants
in seven of eight confirmed MH families (87.5%), and did not
find causative variants in any of the 10 individuals who were
classified as either normal controls (N Z 2) or who were
patients that showed clinical features similar to MH that

Please cite this article as: Yeh H-M et al., Next-generation sequencing and bioinformatics to identify genetic causes of malignant hy-
perthermia, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.08.028
 + MODEL
Next-generation sequencing for malignant hyperthermia 7

Figure 2 Representative genetic testing results. a. NGS sequencing results showed heterozygous c.7304G>A (p.R2435H) path-
ogenic variant. b. Sanger sequencing of this patient (id: MH03) confirmed the variant (upper panel) compared to the normal control
(lower panel). c. Sanger sequencing of his family members detected the same pathogenic variant in another MH case, and also
identified many other individuals carrying this variant.

were later categorized to be caused by other etiologies
(N Z 8).
Genetic testing of a specific disease entity requires
targeted enrichment, which can be achieved through either
capture or amplification. The classical method for
amplification-based target enrichment is the polymerase
chain reaction (PCR), an approach that has been used
previously for MH genetic testing.22 The PCR-based method
is feasible and straightforward, but might suffer from allele
dropout, could miss structural variants (such as big de-
letions) and can be labor-intensive. There were also a
handful of capture-based NGS genetic testing strategies
previously available for MH. Whole-exome sequencing
(WES) for MH18,23 was used primarily in proof-of-concept
experiments, as WES is still considerably more expensive
with a much shallower read depth compared to the MH
specific target panel. WES might also have difficulty iden-
tifying structural variants. Compared to the previous at-
tempts with a MH specific target panel,14,21 the current
study covered all exons (including the 50 - and 30 -untrans-
lated regions) and introns of RYR1 and CACNA1S, had
deeper read depth, and tested more MH families (as well as
controls).
We did not identify any variants convincing enough to be
classified as MH-causing variants other than RYR1 variants.
One possible explanation is that the majority (87.5%) of
cases were caused by the RYR1 gene in our cohort. As we
test more and more MH patients this approach may help to
identify novel MH-causing or severity-modifying genes in
the future.

Please cite this article as: Yeh H-M et al., Next-generation sequencing and bioinformatics to identify genetic causes of malignant hy-
perthermia, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.08.028
 + MODEL
8 H.-M. Yeh et al.

Figure 3 The multiple alignment of the amino acids of RYR1 genes of human, mouse, and zebrafish, and the proven pathogenic
RYR1 variants in MH. The 45 documented MH-causing amino acid changes at the EMHG website are shown as red marks under the
multiple sequence alignment tracts. At any given position, there could be more than one amino acid change that can cause MH. All
of the 48 amino acids map to the positions conserved down to zebrafish. The multiple alignment and figure drawing were performed
using the Geneious software (http://www.geneious.com/) Build 2018-11-06.

We observed strong evolutionary conservation at the
MH-causing amino acid residues of RYR1. It is hard to image
environmental chemicals with similar characteristics to
current anaesthetic agents sufficient to impose selection
pressure during human evolution or even earlier. However,
after analyzing all the documented MH-causing residues
and variants in RYR1 (45 amino acid changes from the 48
DNA variants), we found that all amino acid residues were
conserved down to zebrafish (Fig. 3). We found that all of
the 48 DNA variants of RYR1 had very low allele frequencies
(Table S1). The frequency of the most prevalent variant,
c.1598G>A (p.R533H), was only 0.0001. We suggest to
include “low population allele frequency” and “evolu-
tionary conservation” as additional criteria for the predic-
tion of the pathogenicity of future potential variants.
Although individually rare, the 48 documented MH-
causing RYR1 variants actually sum up to a noticeable
genetic burden in the population. The summed allele
frequency is 0.000436 according to the gnomDB database,
which transforms to a carrier frequency of 0.00087. This
means that as many as one in 1149 individuals are at risk
of MH worldwide, a higher number than previous
estimates.2,4e7 This new estimate represents the lower
limit of the real burden, as other unconfirmed RYR1 var-
iants as well as variants of other MH genes are not
included in the estimation. Given the high probability of
any individual of receiving anesthesia coupled with the
life-threatening outcome of MH, inclusion of the RYR1
genetic information in medical records should be a high
priority.
RYR1 c.1565A>G (p.Tyr522Cys) might be a founder MH-
causing variant in Taiwan. In this study we identified this
variant in two out of the eight MH families, and this variant
was found in one out of five MH families in our previous
report.16 According to the current consensus guidelines by
the EMHG this variant has not yet been definitively assigned
as pathogenic because there was no functional assay.11,12
However, a variant documented to cause MH
(p.Tyr522Ser) is located at the same amino acid residue, a
residue conserved down to zebrafish. This variant was ab-
sent from all public databases, including gnomDB, ExAC,
PSP6500, and Taiwan Biobank (997 individuals), and was
predicted damaging using SIFT and PolyPhen2. A functional
study will be done to fulfill the criteria of pathogenicity.
We achieved high genetic diagnostic rate for MH patients
in Taiwan. We identified the MH-causing variants in 12 out
of 13 families (92.3%), including all the five families re-
ported previously16 combined with seven of the eight new
families in this study. The high diagnostic rate could be
partially attributed to our strict and accurate clinical
diagnosis, as well as the comprehensive sequencing of the
full length of RYR1. However, it might also reflect inter-
population genetic difference across the globe. There
have been no similar genetic studies of comparable scale in
other Asian countries but only a handful of genetic reports
of MH cases.34e36 It is not uncommon to observe inter-

Please cite this article as: Yeh H-M et al., Next-generation sequencing and bioinformatics to identify genetic causes of malignant hy-
perthermia, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.08.028
 + MODEL
Next-generation sequencing for malignant hyperthermia
population genetic/allelic differences in pharmacogenetic
markers.37e39 It is possible that additional MH susceptibility
genes might be identified in the future in other populations
with only moderate genetic diagnostic rates after exam-
ining RYR1. Relative few case numbers in our study is the
major limitation. It is necessary to mention that bio-
informatic prediction result should be treated cautiously
and be used in combination of other available data.40
In conclusion, we demonstrate that NGS and bioinfor-
matics are sensitive and specific tools for genetic diagnosis
of MH. Pathogenic variants in RYR1 can be found in the
majority of MH patients in Taiwan. We estimate that more
than one in 1149 individuals carry MH pathogenic variants in
RYR1 worldwide.
Declaration of Competing Interest
The authors have no conflicts of interest relevant to this
article.
Acknowledgments
We would like to thank National Core Facility for Bio-
pharmaceuticals (NCFB, MOST 106-2319-B-492-002) and
National Center for High-performance Computing (NCHC) of
National Applied Research Laboratories (NARLabs) of
Taiwan for providing computational resources and storage
resources. We are also acknowledged for MH case contrib-
utors: Wei-Zen Sun Ph.D. and Ya-Jung Cheng Ph.D. from
Department of Anesthesiology, National Taiwan University,
Taipei, Taiwan; Ton-Min Chang M.D. from Department of
Pediatrics, Changhua Christian Hospital, Changhua, Taiwan;
Yu-Hua Lin from Department of Anesthesiology, National
Taiwan University Hospital Yun-Lin branch, Yunlin, Taiwan;
Yih-Giun Cherng M.D. from Department of Anesthesiology,
Taipei Medical University Shuang Ho Hospital, Taipei,
Taiwan.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jfma.2020.08.028.
References
1. Stowell KM. Malignant hyperthermia: a pharmacogenetic dis-
order. Pharmacogenomics 2008;9:1657e72.
2. Rosenberg H, Davis M, James D, Pollock N, Stowell K. Malignant
hyperthermia. Orphanet J Rare Dis 2007;2:21.
3. Vladutiu GD, Isackson PJ, Kaufman K, Harley JB, Cobb B,
Christopher-Stine L, et al. Genetic risk for malignant hyper-
thermia in non-anesthesia-induced myopathies. Mol Genet
Metab 2011;104:167e73.
4. Rosenberg H. Continued progress in understanding the molec-
ular genetics of malignant hyperthermia. Can J Anaesth 2011;
58:489e93.
5. Hirshey Dirksen SJ, Larach MG, Rosenberg H, Brandom BW,
Parness J, Lang RS, et al. Special article: future directions in
malignant hyperthermia research and patient care. Anesth
Analg 2011;113:1108e19.
Please cite this article as: Yeh H-M et al., Next-generation sequencing and bioinformatics to identify genetic causes of malignant hy-
perthermia, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.08.028
9
6. Sato K, Pollock N, Stowell KM. Functional studies of RYR1 mu-
tations in the skeletal muscle ryanodine receptor using human
RYR1 complementary DNA. Anesthesiology 2010;112:1350e4.
7. Lanner JT, Georgiou DK, Joshi AD, Hamilton SL. Ryanodine
receptors: structure, expression, molecular details, and func-
tion in calcium release. Cold Spring Harb Perspect Biol 2010;2:
a003996.
8. Mungunsukh O, Deuster P, Muldoon S, O’Connor F,
Sambuughin N. Estimating prevalence of malignant hyper-
thermia susceptibility through population genomics data. Br J
Anaesth 2019;123:e461e3.
9. Hwang JH, Zorzato F, Clarke NF, Treves S. Mapping domains
and mutations on the skeletal muscle ryanodine receptor
channel. Trends Mol Med 2012;18:644e57.
10. Kraeva N, Riazi S, Loke J, Frodis W, Crossan ML, Nolan K, et al.
Ryanodine receptor type 1 gene mutations found in the Ca-
nadian malignant hyperthermia population. Can J Anaesth
2011;58:504e13.
11. Urwyler A, Deufel T, McCarthy T, West S. Guidelines for mo-
lecular genetic detection of susceptibility to malignant hy-
perthermia. Br J Anaesth 2001;86:283e7.
12. Hopkins PM, Ruffert H, Snoeck MM, Girard T, Glahn KP, Ellis FR,
et al. European Malignant Hyperthermia Group guidelines for
investigation of malignant hyperthermia susceptibility. Br J
Anaesth 2015;115:531e9.
13. Merritt A, Booms P, Shaw MA, Miller DM, Daly C, Bilmen JG,
et al. Assessing the pathogenicity of RYR1 variants in malignant
hyperthermia. Br J Anaesth 2017;118:533e43.
14. Schiemann AH, Durholt EM, Pollock N, Stowell KM. Sequence
capture and massively parallel sequencing to detect mutations
associated with malignant hyperthermia. Br J Anaesth 2013;
110:122e7.
15. Carpenter D, Ringrose C, Leo V, Morris A, Robinson RL,
Halsall PJ, et al. The role of CACNA1S in predisposition to
malignant hyperthermia. BMC Med Genet 2009;10:104.
16. Yeh HM, Tsai MC, Su YN, Shen RC, Hwang JJ, Sun WZ, et al.
Denaturing high performance liquid chromatography screening
of ryanodine receptor type 1 gene in patients with malignant
hyperthermia in Taiwan and identification of a novel mutation
(Y522C). Anesth Analg 2005;101:1401e6.
17. Kaufmann A, Kraft B, Michalek-Sauberer A, Weindlmayr M,
Kress HG, Steinboeck F, et al. Novel double and single ryano-
dine receptor 1 variants in two Austrian malignant hyperther-
mia families. Anesth Analg 2012;114:1017e25.
18. Gonsalves SG, Ng D, Johnston JJ, Teer JK, Stenson PD,
Cooper DN, et al. Using exome data to identify malignant hy-
perthermia susceptibility mutations. Anesthesiology 2013;119:
1043e53.
19. Nagele P. Exome sequencing: one small step for malignant
hyperthermia, one giant step for our specialty–why exome
sequencing matters to all of us, not just the experts. Anes-
thesiology 2013;119:1006e8.
20. Lifton RP. Individual genomes on the horizon. N Engl J Med
2010;362:1235e6.
21. Broman M, Kleinschnitz I, Bach JE, Rost S, Islander G,
Muller CR. Next-generation DNA sequencing of a Swedish ma-
lignant hyperthermia cohort. Clin Genet 2015;88:381e5.
22. Fiszer D, Shaw MA, Fisher NA, Carr IM, Gupta PK, Watkins EJ,
et al. Next-generation sequencing of RYR1 and CACNA1S in
malignant hyperthermia and exertional heat illness. Anesthe-
siology 2015;122:1033e46.
23. Kim JH, Jarvik GP, Browning BL, Rajagopalan R, Gordon AS,
Rieder MJ, et al. Exome sequencing reveals novel rare variants in
the ryanodine receptor and calcium channel genes in malignant
hyperthermia families. Anesthesiology 2013;119:1054e65.
24. Larach MG, Localio AR, Allen GC, Denborough MA, Ellis FR,
Gronert GA, et al. A clinical grading scale to predict malignant
hyperthermia susceptibility. Anesthesiology 1994;80:771e9.
 + MODEL
10
25. Li H, Durbin R. Fast and accurate long-read alignment with
Burrows-Wheeler transform. Bioinformatics 2010;26:
589e95.
26. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N,
et al. The sequence alignment/map format and SAMtools.
Bioinformatics 2009;25:2078e9.
27. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K,
Kernytsky A, et al. The Genome Analysis Toolkit: a MapReduce
framework for analyzing next-generation DNA sequencing
data. Genome Res 2010;20:1297e303.
28. Ye K, Schulz MH, Long Q, Apweiler R, Ning Z. Pindel: a pattern
growth approach to detect break points of large deletions and
medium sized insertions from paired-end short reads. Bioin-
formatics 2009;25:2865e71.
29. Chen K, Wallis JW, McLellan MD, Larson DE, Kalicki JM, Pohl CS,
et al. BreakDancer: an algorithm for high-resolution mapping
of genomic structural variation. Nat Methods 2009;6:677e81.
30. Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation
of genetic variants from high-throughput sequencing data.
Nucleic Acids Res 2010;38:e164.
31. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M,
Lander ES, Getz G, et al. Integrative genomics viewer. Nat
Biotechnol 2011;29:24e6.
32. Lek M, Karczewski KJ, Minikel EV, Samocha KE, Banks E,
Fennell T, et al. Analysis of protein-coding genetic variation in
60,706 humans. Nature 2016;536:285e91.
33. Chen CH, Yang JH, Chiang CWK, Hsiung CN, Wu PE,
Chang LC, et al. Population structure of Han Chinese in the
Please cite this article as: Yeh H-M et al., Next-generation sequencing and bioinformatics to identify genetic causes of malignant hy-
perthermia, Journal of the Formosan Medical Association, https://doi.org/10.1016/j.jfma.2020.08.028
H.-M. Yeh et al.
modern Taiwanese population based on 10,000 participants
in the Taiwan Biobank project. Hum Mol Genet 2016;25:
5321e31.
34. Kondo T, Yasuda T, Mukaida K, Otsuki S, Kanzaki R, Miyoshi H,
et al. Genetic and functional analysis of the RYR1 mutation
p.Thr84Met revealed a susceptibility to malignant hyperther-
mia. J Anesth 2018;32:174e81.
35. Li DW, Lai PS, Lee DW, Yong RY, Lee TL. Malignant hyper-
thermia and ryanodine receptor type 1 gene (RyR1) mutation
in a family in Singapore. Ann Acad Med Singapore 2017;46:
455e60.
36. Li W, Zhang L, Liang Y, Tong F, Zhou Y. Sudden death due to
malignant hyperthermia with a mutation of RYR1: autopsy,
morphology and genetic analysis. Forensic Sci Med Pathol
2017;13:444e9.
37. Chung WH, Hung SI, Hong HS, Hsih MS, Yang LC, Ho HC, et al.
Medical genetics: a marker for Stevens-Johnson syndrome.
Nature 2004;428:486.
38. Chen PL, Shih SR, Wang PW, Lin YC, Chu CC, Lin JH, et al.
Genetic determinants of antithyroid drug-induced agranulo-
cytosis by human leukocyte antigen genotyping and genome-
wide association study. Nat Commun 2015;6:7633.
39. Johnson JA, Cavallari LH. Warfarin pharmacogenetics. Trends
Cardiovasc Med 2015;25:33e41.
40. Schiemann AH, Stowell KM. Comparison of pathogenicity pre-
diction tools on missense variants in RYR1 and CACNA1S asso-
ciated with malignant hyperthermia. Br J Anaesth 2016;117:
124e8.