1. Ording H. Incidence of malignant hyperthermia in Denmark. Anesth Analg 1985; 64: 700-4.
2. Larach MG, Brandom BW, Allen GC, Gronert GA, Lehman EB. Malignant hyperthermia deaths related
to inadequate temperature monitoring, 2007-2012: a report from the North American malignant
hyperthermia registry of the malignant hyperthermia association of the United States. Anesth Analg
2014; 119: 1359-66.
3. Maclennan DH, Zvaritch E. Mechanistic models for muscle diseases and disorders originating in the
sarcoplasmic reticulum. Biochim Biophys Acta 2011; 1813: 948-64.
4. Horstick EJ, Linsley JW, Dowling JJ, et al. Stac3 is a component of the excitation-contraction coupling
machinery and mutated in Native American myopathy. Nat Commun 2013; 4: 1952.
5. Robinson R, Carpenter D, Shaw MA, Halsall J, Hopkins P. Mutations in RYR1 in malignant
hyperthermia and central core disease. Hum Mutat 2006; 27: 977-89.
6. Stewart SL, Hogan K, Rosenberg H, Fletcher JE. Identification of the Arg1086His mutation in the alpha
subunit of the voltage-dependent calcium channel (CACNA1S) in a North American family with
malignant hyperthermia. Clin Genet 2001; 59: 178-84.
7. Gillies RL, Bjorksten AR, Du Sart D, Hockey BM. Analysis of the entire ryanodine receptor type 1 and
alpha 1 subunit of the dihydropyridine receptor (CACNA1S) coding regions for variants associated with
malignant hyperthermia in Australian families. Anaesth Intensive Care 2015; 43: 157-66.
8. Oku S, Mukaida K, Nosaka S, et al. Comparison of the in vitro caffeine-halothane contracture test with
the Ca-induced Ca release rate test in patients suspected of having malignant hyperthermia susceptibility.
J Anesth 2000; 14: 6-13
9. Larach MG, Localio AR, Allen GC, et al. A clinical grading scale to predict malignant hyperthermia
susceptibility. Anesthesiology 1994; 80: 771-9.
10. Ibarra M CA, Wu S, Murayama K, et al. Malignant hyperthermia in Japan: mutation screening of the
entire ryanodine receptor type 1 gene coding region by direct sequencing. Anesthesiology 2006; 104:
1146-54.
11. Endo M, Iino M. Measurement of Ca2+ release in skinned fibers from skeletal muscle. Methods Enzymol
1988; 157: 12-26.
12. Kobayashi M, Mukaida K, Migita T, Hamada H, Kawamoto M, Yuge O. Analysis of human cultured
myotubes responses mediated by ryanodine receptor 1. Anaesth Intensive Care 2011; 39: 252-61.
13. Kanda Y. Investigation of the freely available easy-to-use software 'EZR' for medical statistics. Bone
Marrow Transplant 2013; 48: 452-8.
14. Gillies RL, Bjorksten AR, Davis M, Du Sart D. Identification of genetic mutations in Australian
malignant hyperthermia families using sequencing of RYR1 hotspots. Anaesth Intensive Care 2008; 36:
391-403.
15. Miller DM, Daly C, Aboelsaod EM, et al. Genetic epidemiology of malignant hyperthermia in the UK.
11
Br J Anaesth 2018; 121: 944-52.
16. Brandom BW, Bina S, Wong CA, et al. Ryanodine receptor type 1 gene variants in the malignant
hyperthermia-susceptible population of the United States. Anesth Analg 2013; 116: 1078-86.
17. Galli L, Orrico A, Lorenzini S, et al. Frequency and localization of mutations in the 106 exons of the
RYR1 gene in 50 individuals with malignant hyperthermia. Hum Mutat 2006; 27: 830.
18. Kraeva N, Riazi S, Loke J, et al. Ryanodine receptor type 1 gene mutations found in the Canadian
malignant hyperthermia population. Can J Anaesth 2011; 58: 504-13.
19. Broman M, Gehrig A, Islander G, et al. Mutation screening of the RYR1-cDNA from peripheral Blymphocytes in 15 Swedish malignant hyperthermia index cases. Br J Anaesth 2009; 102: 642-9.
20. Carpenter D, Robinson RL, Quinnell RJ, et al. Genetic variation in RYR1 and malignant hyperthermia
phenotypes. Br J Anaesth 2009; 103: 538-48.
21. Ibarra Moreno CA, Hu S, Kraeva N, et al. An assessment of penetrance and clinical expression of
malignant hyperthermia in individuals carrying diagnostic ryanodine receptor 1 gene mutations.
Anesthesiology 2019; 131: 983-91.
22. Schiemann AH, Stowell KM. Comparison of pathogenicity prediction tools on missense variants in
RYR1 and CACNA1S associated with malignant hyperthermia. Br J Anaesth 2016; 117: 124-8.
23. E F Gillard, K Otsu, J Fujii, et al. A substitution of cysteine for arginine 614 in the ryanodine receptor is
potentially causative of human malignant hyperthermia. Genomics 1991; 11: 751-5.
24. Miyoshi H, Yasuda T, Otsuki S, et al. Several Ryanodine Receptor Type 1 Gene Mutations of p. Arg
2508 Are Potential Sources of Malignant Hyperthermia. Aneth Analg: 2015; 121: 994-1000.
25. Danielle Carpenter, Christopher Ringrose, Vincenzo Leo, et al. The role of CACNA1S in predisposition
to malignant hyperthermia. BMC Med Genet 2009; 10: 104.
26. N. Witting. P. Laforêt, N. C. Voermans, et al. Phenotype and genotype of muscle ryanodine receptor
rhabdomyolysis-myalgia syndrome. Acta Neurol Scand 2018; 137:452-461.
27. L Galli, A Orrico, S Cozzolino, et al. Mutations in the RYR1 gene in Italian patients at risk for
malignant hyperthermia: evidence for a cluster of novel mutations in the C-terminal region. Cell
Calcium 2002; 32: 143-51.
28. Sano K, Miura S, Fujiwara R, et al. A novel missense mutation of RYR1 in familial idiopathic hyper
CK-emia. J Neurol Sci. 2015; 356: 142-7.
29. M Snoeck, B G M van Engelen, B Küsters, et al. RYR1- related myopathies: a wide spectrum of
phenotypes throughout life. Eur J Neurol 2015; 22: 1094-112.
30. N Sambuughin, S McWilliams, A de Bantel, et al. Single- amino- acid deletion in the RYR1 gene,
associated with malignant hyperthermia susceptibility and unusual contraction phenotype. Am J Hum
Genet 2001; 69: 204-8.
31.Xia Tian, Wen-Chen Liang, Yanming Feng, et al. Expanding genotype/phenotype of neuromuscular
diseases by comprehensive target capture/NGS. Neurol Genet2015; 1: e14.
12
M80
M68
M35
M60
M21, 25, 27, 69
M43
RYR1 chr19:38934851C>T
RYR1 chr19:38939352G>A
RYR1 chr19:38939352G>C
RYR1 chr19:38946112G>A
RYR1 chr19:38948186G>T
RYR1 chr19:38989863G>A
M23
M78
M38
M60, 84
M51
M74
M42
M40
M82
M51
M3,37
M34
M39
M88
M22
M90
M29
M70
M66
M30
M89
CACNA1S chr1:201061121G>A
RYR1 chr19:38934827C>A
RYR1 chr19:38934892G>T
RYR1 chr19:38935311G>A
RYR1 chr19:38939431G>A
RYR1 chr19:38973997C>T
RYR1 chr19:38987541G>A
RYR1 chr19:38990281G>C
RYR1 chr19:38990282C>A
RYR1 chr19:38990285delGGA
RYR1 chr19:38990344C>G
RYR1 chr19:38993557C>T
RYR1 chr19:39009935A>G
RYR1 chr19:39034034A>G
RYR1 chr19:38934864A>G
RYR1 chr19:38948155T>C
RYR1 chr19:38990320T>C
RYR1 chr19:38990626C>A
RYR1 chr19:38991282C>G
RYR1 chr19:39078017C>G
CACNA1S chr1:201019613T>A
CACNA1S chr1:201028359G>C
CACNA1S chr1:201046197C>T
RYR1 chr19:39071010C>G
RYR1 chr19:39002961G>A
M46
M32, 33
M11, 12, 13, 14, 15(Family8), 16,
17, 19(Family9)
M6, 7(Family5), 24, 75
M44, 71
RYR1 chr19:38991539G>A
No. of
families
Position
Sample
rs763794604
rs901536375
rs377686237
rs118192126
rs193922800
rs193922806
rs1448949048
rs193922797
rs113332073
rs193922777
rs1568436081
rs771058055
rs772226819
rs193922750
rs193922878
rs193922832
rs193922818
rs193922772
rs112563513
rs121918592
rs121918592
rs144336148
rs118192161
rsID
p.F1161L
p.A560T
p.A5025G
p.D1382V
p.D2431E
p.R2454G
p.S604P
p.I2358T
p.N3913D
p.D167G
p.R2625C
p.K3367R
p.E2347del
p.P2366R
p.S2345R
p.V2280I
p.S2345T
p.R367Q
p.P1592L
p.E176D
p.E209K
p.R174W
p.Q155K
p.L4838V
p.E3104K
p.R2508H
p.R614L
p.R2336H
p.G341R
p.G341R
p.R533H
p.R163C
AA change
0.864
0.876
0.824
0.931
0.903
0.898
0.868
0.882
0.91
0.94
0.504
0.711
0.651
0.927
0.641
0.744
0.747
0.88
0.785
0.632
0.924
0.905
EMHG18
EMHG10
EMHG10
EMHG23
EMHG5
EMHG24
EMHG5
EMHG10
EMHG25
Reported10
Reported26
Reported18
Reported17
Reported10
Reported27
Reported28
Reported29
Reported30
Reported5
Reported5
Reported5
Reported32
0.923
0.876
0.605
0.971
0.743
0.893
0.951
0.912
0.959
EMHG10
REVEL
report
26.7
27.7
31.00
28.4
23.1
26.1
28.1
25.1
26.9
25.5
29.1
18.9
21.3
23.7
22.9
25.3
24.7
24.6
27.7
20.9
23.6
29.6
25.7
28.4
26.0
25.7
28.4
25.4
26.0
25.7
24.2
25.7
CADD
0.0001
0.000109
5.46E-05
0.000109
gnomAD
MAF(east
Asia)
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
Likely pathogenic
Pathogenic
Likely pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
Pathogenic
EMHG scoring matrix
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
<1×10-7
gnomAD(East
Asia)㻌 p-value
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
SIFT
MTPred
PP2Pred
0.0003
0.0006
0.0012
ToMMo
MAF
Abbreviations: EMHG, Listed as mutation in EMHG; Reported, reported in other papers, but not listed as mutation in EMHG; N.S., not scored; B, benign; D, disease-causing
shown for reference. The underlines are for the same family.
and MAF with the Tohoku Medical Megabank Organization (ToMMo, https://jmorp.megabank.tohoku.ac.jp/), a genomic study using a large Japanese cohort evaluation is
EMHG scoring matrix and extracted “Pathogeni ”and ”Likely Pathogenic” variants. Evaluation by three algorithms (SIFT; Mutation Taster, MTPred; PolyPhen-2, PP2Pred),
We selected variants with CADD ≥16, REVEL >0.5, and MAF in gnomAD (East Asia) <0.1% in 90 subjects from 77 families. These variants were evaluated using the
Table 1. Variant List
13
Figure Legends
Fig. 1. Percentage of genes associated with pathogenicity in MH patients and families.
Screening results of genes associated with MH pathogenicity in 77 families, including MH
patients and families. Of these, 34 families possessed variants in RYR1 and six families in
CACNA1S. In the remaining 37 families, there were no variants in RYR1 and CACNA1S
(unknown group).
Unknown
48.1%
(37 families)
RYR1
44.2%
(34 families)
CACNA1S
7.8%
(6 families)
Fig.2. Comparison of clinical symptoms for each gene associated with MH pathogenicity.
(A–D) Comparison of clinical symptoms for each gene associated with MH pathogenicity (Max
BT, °C /15 min, Max CK, CGS) is shown. The value of each item is represented by a dot, and
the interquartile range is represented by a line. The Mann–Whitney test was conducted for
groups with RYR and CACNA1S variants. *p < 0.05. Abbreviations: Max BT, maximum body
temperature; ˚C/15 min, elevated body temperature for 15 min; Max CK, maximum creatine
kinase; CGS, Clinical Grading Scale
14
Fig. 3. Comparison of CICR and EC50 for each gene associated with MH pathogenicity and
CICR-˚C/15 min correlation.
Comparison of CICR and EC50 (caffeine, 4-CmC) for each causative gene is shown. CICR was
examined at each of the five Ca concentrations (0, 0.3, 1.0, 3.0, and 10 μM).
(A) Rate of Ca release for each gene associated with MH pathogenicity in Ca 0 μM (Ca-free,
there is no difference related to MH status in Ca 0 μM).
(B) In the other four Ca concentrations, there was a significant difference between the RYR1 and
CACNA1S groups. Ca 3.0 μM is shown as representative.
family
10
11
12
13
14
15
16
17
18
19
20
21
21
No
M1
M2
M3
M4
M5
M6
M7
M8
M9
M10
M11
M12
M13
M14
M15
M16
M17
M18
M19
M20
M21
M22
M23
M24
M25
M26
M27
M28
M29
M30
M31
M32
M33
Sex
Age
(y.o.)
39
65
16
41
73
52
47
72
25
18
77
49
52
27
25
11
44
41
15
13
31
40
39
61
52
18
25
35
23
38
10
Max CK, maximum creatine kinase.
family member
family member
patient
patient
patient
patient
family member
family member
patient
patient
family member
family member
family member
family member
family member
family member
family member
family member
family member
family member
patient
family member
family member
patient
patient
patient
patient
patient
patient
patient
patient
patient
family member
MH or family member
RYR1 p.K3367R
CACNA1S p.R174W
CACNA1S p.R174W
RYR1 p.L4838V
RYR1 p.L4838V
RYR1 p.L4838V
RYR1 p.L4838V
RYR1 p.L4838V
RYR1 p.L4838V
RYR1 p.L4838V
RYR1 p.L4838V
RYR1 p.R614L
RYR1 p.I2358T
RYR1 p.E176D
CACNA1S p.R174W
RYR1 p.R614L
RYR1 p.R614L
RYR1 p.R2454G
CACNA1S p.F1161L
RYR1 p.E3104K
RYR1 p.E3104K
AA change
40.3
41.0
37.6
37.6
38.8
37.4
39.5
38.4
42.5
38.7
43.1
37.4
37.7
38.8
Max BT(ǀC)
Υ/15 (ǀ
C)
1.4
0.5
0.2
0.4
0.18
0.68
0.3
1.9
0.2
2.1
0.6
0.1
0.5
935
2179
907
206
13200
1301
4290
2169
1603
8280
11884
1443
3940
Max CK (IU)
63
58
78
33
43
18
33
18
53
78
45
58
CGS
Abbreviations: AA change, amino acid change; MAX BT, maximum body temperature; °C /15 min, maximum elevated body temperature in 15 min;
listed.
Variants (“Pathogenic” and “Likely pathogenic” based on the EMHG scoring matrix in Table1) and clinical symptoms of 90 people from 77 families are
Supplementary Table 1. Sample List
15
M34
M35
M36
M37
M38
M39
M40
M41
M42
M43
M44
M45
M46
M47
M48
M49
M50
M51
M52
M53
M54
M55
M56
M57
M58
M59
M60
M61
M62
M63
M64
M65
M66
M67
M68
M69
M70
M71
M72
M73
M74
M75
M76
M77
M78
M79
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
60
61
62
63
64
65
66
40
33
34
48
34
31
60
21
47
22
11
14
56
45
40
71
18
89
60
49
15
55
30
20
67
50
42
36
52
33
34
56
52
73
40
37
42
32
37
34
34
20
family member
patient
family member
family member
family member
patient
family member
patient
patient
family member
family member
family member
family member
family member
family member
family member
family member
patient
patient
patient
family member
patient
patient
patient
patient
patient
patient
family member
patient
family member
family member
patient
patient
patient
patient
patient
patient
family member
family member
patient
patient
patient
patient
family member
patient
patient
RYR1 p.N3913D
RYR1 p.G341R
RYR1 p.K3367R
RYR1 p.R367Q
RYR1 p.D167G
RYR1 p.V2346V,E2347del
RYR1 p.S2345R
RYR1 p.R2336H
RYR1 p.Q155K
RYR1 p.R2508H
RYR1 p.R2625C, RYR1 p.V2280I
RYR1 p.R533H, RYR1 p.P1592L
CACNA1S p.D1382V
RYR1 p.G341R
RYR1 p.R614L
RYR1 p.A5025G
RYR1 p.Q155K
RYR1 p.S2345T
CACNA1S p.R174W
RYR1 p.E209K
38.8
38.3
38.4
39.4
40.7
41.3
38.9
37.9
39.5
36.9
41.2
38.0
40.0
39.4
39.0
0.36
1.4
1.2
0.5
0.5
0.5
0.8
0.17
0.9
0.2
71310
10525
14000
2392
1270
40640
386
14066
48000
25024
3304
8000
43
40
68
48
43
53
45
73
48
10
58
30
53
30
40
38
20
16
67
68
69
70
71
72
73
74
75
76
77
44
56
19
55
18
51
62
53
12
15
patient
patient
patient
family member
patient
family member
family member
family member
patient
patient
patient
1) RYR ex34 chr19:38976792-38976812
2) RYR1 ex41 chr19:38987132-38987182
3) RYR1 ex48 chr19:38993338-38993368
4) RYR1 ex74 chr19:39019246-39019273
5) RYR1 ex85 chr19:39034172-39034300
6) RYR1 ex90 chr19:39055699-39056399
7) RYR1 ex99 chr19:39070745-39070768
8) CACNA1S ex43 chr1:201009359-201009506
Supplementary Table 3. Eight areas not covered by AmpliSeq designer
Excel file of AmpliSeq amplicons.
Supplementary Table 2. Primer set designed by AmpliSeq designer
M80
M81
M82
M83
M84
M85
M86
M87
M88
M89
M90
RYR1 p.R163C
RYR1 p.P2366R
RYR1 p.P1592L
RYR1p.R163C
RYR1 p.S604P
CACNA1S p.A560T
RYR1 p.D2431E
40.6
39.6
41.9
40.7
42.0
37.4
40.8
0.9
0.6
0.75
0.8
1.2
0.33
1.6
3332
1803
36258
2194
4984
53
56
58
53
43
78
17
...