Genetic screening for malignant hyperthermia and comparison of clinical symptoms in Japan

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subunit of the voltage-dependent calcium channel (CACNA1S) in a North American family with

malignant hyperthermia. Clin Genet 2001; 59: 178-84.

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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.

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the Ca-induced Ca release rate test in patients suspected of having malignant hyperthermia susceptibility.

J Anesth 2000; 14: 6-13

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1146-54.

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391-403.

15. Miller DM, Daly C, Aboelsaod EM, et al. Genetic epidemiology of malignant hyperthermia in the UK.

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Br J Anaesth 2018; 121: 944-52.

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2508 Are Potential Sources of Malignant Hyperthermia. Aneth Analg: 2015; 121: 994-1000.

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to malignant hyperthermia. BMC Med Genet 2009; 10: 104.

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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

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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.

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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

...