GENOMICS

13,

1247-1254

(1992)

Polymorphisms and Deduced Amino Acid Substitutions in the Coding Sequence of the Ryanodine Receptor (RYRI) Gene in Individuals with Malignant Hyperthermia ELIZABETH F. GILLARD,“,’ KINYA OTSu, ts2 JUNICHI FuJII, ts3 CATHERINE DUFF, * STELLA DE LEON, t V. K. KHANNA,t BEVERLEYA. BRITT,* RONALD G. WORTON,* AND DAVID H. MACLENNANt’4 *Department of Genetics, Hospital for Sick Children and Department of Molecular and Medical Genetics, University of Toronto, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8; tBanting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, 172 College Street, Toronto, Ontario, Canada M5G 1L6; and *Departments of Anaesthesia and Pharmacology, University of Toronto, CCRW-2-834, Toronto General Hospital, 200 Elizabeth Street, Toronto, Ontario, Canada M5G 2C4 Received

December

10, 1991;

Twenty-one polymorphic sequence variants of the RYRl gene, including 13 restriction fragment length polymorphisms (RFLPs), were identified by sequence analysis of human ryanodine receptor (RYRl) cDNAs from three individuals predisposed to malignant hyperthermia (MH). All RFLPs were detectable in PCR-amplified products, and their segregation was consistent with our initial finding of linkage to MH in the nine families previously informative for one or more intragenie markers (MacLennan et al., 1990, Nature 343:559-561). Four amino acid substitutions were identified in the study: Arg for G~Y~~*, Cys for Arg470, Leu for Pro17*6, and Cys for G1y206’. Of 45 families tested, a single family presented the Arg for Glyz4’ substitution where it segregated with malignant hyperthermia, making it a candidate mutation for predisposition to MH in man. The other three polymorphic substitutions failed to segregate with malignant hyperthermia in those families in which they occurred, implying that they represent polymorphisms with little or no effect on the function of the RYRl gene. o ISW Academic press, I~C.

INTRODUCTION

Malignant hyperthermia (MH) is a dominantly inherited predisposition to uncontrolled muscle contracture and accompanying hypercatabolic reactions, triggered in man by inhalational anesthetics such as haiothane and muscle relaxants such as succinylcholine. Halothane sensitivity commonly occurs in swine, al-

’ Present address: Somatic Cell Genetics Laboratory, Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WC2 3PX, UK. ’ Present address: Department of Pathophysiology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka Suita, Osaka 565, Japan. 3Present address: Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka Suita, Osaka 565, Japan. 4 To whom correspondence should be addressed. 1247

revised

Aph

13, 1992

though the condition in homozygously affected pigs may be triggered by stress as well as by anesthesia (Britt, 1991; O’Brien, 1987; Nelson, 1988; Harrison, 1988). A malignant hyperthermia crisis is potentially fatal, with multiple complications. The fact that Ca”+ plays an essential role in muscle contraction would associate defective Cazf release or Ca2+ reuptake with muscle contracture, as well as with the hypermetabolic activities of the contracted muscle (Brostrom et al., 1971). No evidence for a defect in the Ca2+ pump, which removes Ca2+ from muscle, has yet been obtained (Nelson, 1988). MH episodes may be averted by early intervention with the drug dantrolene (Steward and O’Connor, 1987), known to prevent calcium release into the muscle, either directly or by dissociating early steps in the excitation-contraction coupling process (Harrison, 1988). These observations point to the calcium release channel of skeletal muscle, spanning the gap between the functional face of the sarcoplasmic reticulum and the transverse tubules (Lai and Meissner, 1989), as a potential site for defects causative of MH. Because this channel binds and is modulated by the plant alkaloid ryanodine, it is also referred to as the ryanodine receptor. Further evidence associating defects in the skeletal muscle ryanodine receptor (RYRl) gene with MH susceptibility includes tissue-specific expression of the gene in fast- and slow-twitch skeletal muscle (Otsu et al., 1990), the tissue most affected by the MH reaction. In swine the MH phenotype is accompanied by alterations in the tryptic digestion pattern of the ryanodine receptor (Knudson et al., 1990), alterations in the Ca2+ dependence of ryanodine binding (Mickelson et al., 1988), and facilitation of channel opening and inhibition of channel closing (Ohnishi et al., 1983; Fill et al., 1990). Genetic studies also support RYRl as a candidate gene for MH in both humans and pigs. In humans the RYRl gene is localized to chromosome 19q13.1 (MacKenzie et al., 1990), while in pigs it is localized to a ho0888-7543/92

$5.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

1248

GILLARD

mologous region on chromosome 6 (Harbitz et al., 1990). In both swine (Archibald and Imlah, 1985) and humans (McCarthy et aZ., 1990) genetic markers close to the RYRl gene were found to segregate with the MH phenotype. In studies of the RYRl gene itself (MacLennan et al., 1990), cosegregation of intragenic polymorphisms with the MH phenotype was found in nine human families. In studies of the pig, we discovered that the alteration of c1843to T in the porcine RYRl gene, leading to substitution of Cys for Arg615, segregated with the MH phenotype in over 450 animals, including 338 informative meioses, strongly implicating this genetic alteration as causative of halothane sensitivity (Fujii et al., 1991; Otsu et al., 1991). We also found that the corresponding mutation occurs across a species barrier in humans where it was linked to MH in a single family (Gillard et al., 1991). Despite this powerful evidence for a causative role for the RYRl gene in porcine and human MH, there is evidence for genetic heterogeneity, since lack of linkage between RYRl polymorphisms and MH has now been observed in a number of families (Levitt et al., 1991; Deufel et al., 1992). To define additional candidate MH mutations in the RYRl gene and to detect additional polymorphisms that will be useful for further linkage studies, we undertook systematic sequencing of RYRl cDNA obtained from muscle mRNA from three individuals predisposed to MH. We identified 21 sequence variations in the three patients, 13 of which resulted in loss or gain of a useful restriction enzyme site. These restriction fragment length polymorphisms (RFLPs) were then utilized for linkage analysis in our MH families. Four of the 21 sequence variants observed resulted in amino acid substitutions. One of these, an Arg for G1yz4’ substitution, segregated with MH, making it a candidate mutation potentially causative of MH. MATERIALS

Amplification

AND

of Genomic DNA

METHODS

and cDNA

Genomic DNA was isolated from blood of MH-sensitive (MHS) and normal (MHN) individuals (Miller et al., 1988). RNA was prepared (Chomczynski and Sacchi, 1987) from muscle biopsy samples of three MHS individuals, HJ, TJ, and BT, and subsequent cDNA preparation used random hexamers in a modification of the method of Wang et al. (1989), as described in Bulman et al. (1991). Amplification of the cDNA (2-3 ~1) from approximately 0.5 mg total mRNA by the polymerase chain reaction (Saiki et al., 1988) was followed either by direct sequencing or by subcloning and sequencing. Amplification for restriction endonuclease analysis utilized 200-500 ng of genomic DNA. Each reaction was performed with 100 ng of each primer and 1.5 units of 7’uq polymerase in a DNA thermal cycler (Perkin-Elmer/Cetus) using the buffer described by Kogan et al. (1987). Conditions for amplification of either first-strand cDNA or genomic DNA for most of the primer pairs involved denaturation at 94°C for 30 s, annealing at 58°C for 90 s, extension at 72’C for 150 s, for 35 cycles (with ramp times of 30 s), and then a ‘I-min extension at 72°C before returning the sample to 4°C. Conditions for amplification of primer pairs PI/P2, P3/P4, and P7/P8 involved denaturation at 94°C for 30 s, annealing at 55’C for 30 s, extension at 72°C for 60 s, for 35 cycles (with ramp times of

ET

AL.

15s), and then to 4°C.

a 7-min

extension

at 72°C

before

returning

agarose

according

the sample

Sequencing of PCR Products The

PCR

to Heery et HJ and BT as described by Winship (1989), and cloning of the PCR product and sequencing of multiple clones were carried out for patient TJ. The detection efficiency for the mutant allele should be close to 100% for either strategy as long as the transcript from the mutant allele is represented equally with that from the normal allele in the total mRNA of the patient muscle. For direct sequencing of PCR products, PCR primers were chosen at intervals of approximately 550 bp along the cDNA to give 28 overlapping segments with about 50 bp of overlap at each end. In practice, for the direct sequencing approach, it was found that sequence near the primers was difficult to obtain so that gaps in the data remain at several of the overlap regions. The sequence is 90% complete for BT and 93% complete for HJ. For cloning and sequencing of DNA from patient TJ, PCR products were purified from polyacrylamide gels, and the DNA was then endfilled using the Klenow fragment, digested with XbaI, and cloned into the XbaI site of the Bluescript vector (Stratagene). Sequence analysis (Sanger et al., 1977) utilized the T7 sequencing kit (Pharmacia). Whenever possible, eight clones were sequenced, since this number gives a 99.6% probability that the mutant allele will be present in at least one clone and a 96% probability of being represented more than once. For 15% of the PCR products, only five to seven clones were available for analysis (detection probability of 81-94%) such that the overall detection efhciency is estimated to be 95%. Sequence variations were considered significant only if present in more than one clone, since PCR artifacts are only rarely repeated in multiple clones.

al. (1990),

products

were purified

from

direct sequencingwas performed on patients

Restriction

Fragment

Length Polymorphism

Analysis

PCR primers that amplified each of the 13 RFLPs identified in the sequence analysis were chosen. The amplified products were restriction endonuclease digested and separated on 8-20% polyacrylamide gels. The primer names are indicated by a number followed by F for a forward (coding strand) primer, R for a reverse (antisense) primer, e for an exon-specific primer, and i for an intron-specific primer. “Ex” primers are derived from the tentative number assigned to the exon immediately following. The sequence of the primer pairs used to amplify genomic DNA for restriction endonuclease analysis, ordered 5’to 3’ in the RYRl gene, is as follows: 1Fi 5’.C AAT CGT CTC TGA CTG CCG CA, 2Re 5’CTC TTC GCA GCG GGA GCA GAT, 15Fe 5’-TGA GAT CAG TAC GGG GAG TCA, 16Re 5’.CTT CTT GAG CAC GCC GAGA CCG, HMHlFe 5’-ACT TCT AGA AGC CTG GAC AGC TTC AGC (+9 bp 5’Xbal site), 80Re 5’.ACT TCT AGA CTC CTC CTG GAA GAG GCT CTG (+9 bp 5 Xbal site), Ex8Fe T-ACT TCT AGA TGC TGA CAG TGA TGA CCA GCG CAG (+9 bp 5’ XbaI site), Ex9Re 5’.ACT TCT AGA CTG ATT CTC AGT GGC TCC AGC CTC (+9 bp 5’ Xbcd site), 27Fe 5’-ACT TCT AGA AAC TTG GAC TGG CTG GTC AGC AAG, 63Re 5’-GCG AGG CAG CAA GTT CTC AGT AAT, 93Fe 5’.CAG CGC TGG CAC TTG GGC AGT, 32Re 5’-ATC TCT AGA GAG GAC CTC GCC ATT GAG GGT GAA (+9 bp 5’ XbuI site), 39Fe Z-ACT TCT AGA ATC AGC ATC CAC CTC (+9 bp 5’ XbuI site), Ex33Re 5’.AGG CAC AAA CTG GAA CTC CAC GGA, 7Fi 5’.GGG ATC TCA GAC CCT CAT TC, 8Re 5’-AC AGT GTG CCA GCA GGT CTT, 76Fe S-ACT TCT AGA TTC AGC TAG ATG GAG AGG AGG (+9 bp 5’ XbaI site), 77Re 5’-ACT TCT AGA GGT TTG CTC TCC TCT GCT GAC (+9 bp 5’ XbaI site), 98Fe 5’-ATG GGG CTC TGG TGC AGC CAA, 99Re 5’CAC GTC CAG CAC GTG CAG CAA, 3Fe 5’-TCC TCA CCA ACC ACT ATG AGC, 4Re 5’-TG GGC CAG AGA GTC AAA GATG, 9Fe 5’.AAA TAC GAC CCG GAG CTG TAC, 1ORi 5’-CAG GGT CAG CAG ATG TTG GAG, 50Re 5’.ATC TCT AGA TTC TGC CAG TTG TTC TGC CAT GGC (+9 bp 5’ Xbul site), 51Fe 5’-ATC TCT AGA GAT CCT CGA GAA GGC TAC AAC CCT (+9 bp 5’ XbuI site). 5Fi

RYRl

IN

INDIVIDUALS

WITH

!Y-ATG GTG TCT GAT GTA TTG CGG, 6Re 5'-AGC GTG ATC TCG ATG ACA TGC.

Certainprimershave9-bp XbaI sites at their 5’ ends that are not complementary to the RYRl cDNA, but were added to facilitate cloning. They are not essential for RFLP analysis by PCR amplification and restriction endonuclease digestion. The sequences of PCR primers used in sequencing of the cDNA may be obtained from the corresponding author.

Patient

MALIGNANT

listed. Four of the 21 observedchangesresulted in amino acid substitutions in the protein. Fourteen of the ob-

servedchangesresultedin the lossor gainof a restriction endonuclease site and all but one of these had fragment sizes that were easily detectable and therefore useful for linkage studies. Analysis of Four Anino

Characterization

The three individuals chosen for this study had been referred to the Malignant Hyperthermia Unit of the Toronto General Hospital for assessment of MH susceptibility. Halothane, caffeine, and halothane plus caffeine contracture tests were carried out on muscle biopsies of these individuals and some family members. Contractures resulting in less than 0.3 g tension in 1% halothane indicate normality (N) and those greater than 0.3 g tension indicate susceptibility (H). Recorded doses of caffeine (CSC) are those required to raise the resting muscle tension by 1 g. Doses of greater than 4 mM indicate normality, while doses lower than 4 mM indicate susceptibility (C). In the presence of 1% halothane doses of caffeine (HCSC) greater than 0.5 mM indicate normality, doses of less than 0.35 mA4 indicate susceptibility (K), and those between 0.35 and 0.5 mMcaffeine are considered indeterminate. BT case history. BT was an 18-year-old male of mixed German and Phillipino (Chinese) parentage, who at the age of 18 underwent extraction of wisdom teeth under succinylcholine and halothane. The halothane was switched to enflurane part way through the procedure. He developed generalized muscle rigidity and arrhythmia. Postoperatively, he complained of severe bilateral chest pain that radiated to the back of his neck. His creatine kinase rose to 5500 IU. A junctional rhythm appeared on the EKG. A skeletal muscle biopsy was performed subsequently, the results of which are listed in Table 2. No family members have had reactions. HJ case history. HJ fractured his left femur in a car accident and during the resulting surgery succinylcholine and halothane were administered. This resulted in premature ventricular contraction, sweating, and a fever of 38.5”C. Blood gas levels were measured at P, 0, of 70 mm Hg and Pa CO* of 72 mm Hg. The pH was 7.145 and the base deficit was -7.5 meq/liter. These signs were reversed with dantrolene, sodium bicarbonate, propranolol, and cooling. The creatine kinase level measured in postoperative intensive care was 4410 IU. Skeletal muscle biopsy results are listed in Table 2.

TJ case history. While undergoing a tonsillectomy, patient TJ was administered succinylcholine and halothane. Muscle rigidity, increased respiratory and heart rates, and a fever of 37.8”C resulted. Blood gas levels with hyperventilation were measured at Pa 0, of 65.6 mm Hg and Pa CO, of 53.2 mm Hg. The pH was 7.23 and the base deficit was -6.4 meq/liter. Successful treatment included 40 mg dantrolene and cooling. The creatine kinase level was measured at 712 IU. Skeletal muscle biopsy results are listed in Table 2. The patient was known to suffer from muscle cramps.

RESULTS PCR Sequence Analysis of cDNA To obtain the 15.3-kb RYRl gene coding sequence from three MHS individuals, mRNA was isolated from muscle from each patient and first-strand cDNA was subjected to PCR amplification. The overall probability of detecting any sequence variant present in 50% of the mRNA was estimated to be >95%, as discussed under Materials and Methods. Twenty-one sequence variations identified in the course of this study are presented in Table 1. To eliminate PCR artifacts, only those variants detected or confirmed by direct sequencing are

1249

HYPERTHERMIA

Acid Substitutions

The four amino acid substitutions were all observed in TJ. To determine the significance of these alterations in relation to MH, DNA from MH family members was studied for the presence or absence of the putative mutations. For three substitutions, DNA was analyzed by PCR amplification followed by restriction endonuclease digestion and polyacrylamide gel electrophoresis, since in all three cases a restriction site had been lost or gained (Table 1). In the case of the Arg for G1y248substitution, for which there was no altered restriction site, all DNA samples were examined by PCR amplification, followed by direct sequencing. The substitution of Arg for G1y248was observed in only one of the the 45 families tested-family 39 (Fig. 1). The substitution was observed in the proband, TJ, her brother, SJ, and her mother, CJ. TJ and SJ are both considered to be MHS on the basis of halothane and caffeine contracture tests (Table 2). They share a maternal, but not a paternal, haplotype based on other informative RYRl RFLPs (Fig. 2). Unfortunately, because the mother of TJ and SJ has not been biopsied and repeated efforts to obtain a biopsy from her have failed, her MH status is unknown. These results would be consistent with inheritance of R YRl as an MHS gene from the mother, since she has the amino acid substitution associated with MH in her two children. The corresponding nucleotide substitution is, therefore, a candidate mutation for predisposition to MH. The substitution of Cys for Arg470 results in the loss of a HinPI (or HhaI) site. This substitution was observed only in family 39, the family of TJ (lanes 7-11, Fig. 2). The Cys for Arg470 substitution was seen in TJ and her father but was not observed in her brother or mother. Its segregation is thus consistent with an amino acid polymorphism rather than a candidate mutation. The substitution of Leu for Pro’785 is associated with the loss of a PuuII site. Leu for Pro’785 also failed to segregate with MH in the family of TJ, since it was inherited from her father and was not observed in her brother (Fig. 3). This substitution was also observed in family 10, where it was present only in the normal parent and was not seen in a child whose identical twin died from an MH reaction, nor was it found in his MHS father. Thus, this substitution is consistent with an amino acid polymorphism rather than a candidate mutation. The Cys for G1y205gsubstitution is associated with the gain of a DraIII site (Fig. 4). Patient TJ is homozygous for this substitution and the same substitution was also observed in two other families. In none of these families was it associated with predisposition to MH. This sub-

1250

GILLARD

TABLE

ET

AL.

1

RYRZ cDNA SEQUENCESUBSTITUTIONS' Amino

acid

Substitution

AA change’

RE change

Primers

CTG

> CTA

None

AlwNI

GGG

> AGG

Gly > Arg

None

GCC

> GCT

None

AciI

CGC

> TGC

Arg

TCA

> TCG

None

None

ACA ATC

> ACG > ATT

None None

None TuqI Loss

CCA

> CTA

Pro

AAA

> AAG

None

GGC

> TGC

Gly

GCG

> GCA

None

D&II gain FokI loss

GTG

> GTA

None

None

CAC

> CAT

None

Not

CGC

> CGT

None

HinpI

loss

ACG

> ACA

None

BsuJI

loss

ATT

> ATC

None

None

GAT

> GAC

None

FokI

1Fi 2Re Ex8Fe Ex9Re 15Fe 16Re HMHl 80Re 27Fe 63Re None 93Fe 32Re 39Fe Ex33R 7Fi 8Re 76Fe 77Re 98Fe 99Re 98Fe 99Re 94Fe 4Re 94Fe 4Re 3Fe 4Re 9Fe 1ORi 9Fe 1ORi

GAG AGT

> GAA > AGC

None None

None HinPI

CCA

> CCG

None

CTG

> CTA

PUUII loss, MspI gain Sty1 gain

> Cys

> Leu

HinPI

PUUII FokI

> Cys

loss

loss loss

loss gain

useful

loss

gain

None 51Fe 50Re None None 5Fi 6Re

Fragment 51+39/90,37

sizes

(bp)

common

91f18/110 154/110+44,60

common

72+48/120 226+169/395 156/77+69 160/52+105 33+27+50/33+77

37+25/62 46+86/136

Allele

het.

0.65/0.35, n = 92” Observed family 0.91, 0.09, n= 116 Observed family

freq. 0.52 in one only 0.14 in one only

0.55/0.45, 0.52 n = 42 0.96, 0.04, 0.08 rz = 50 0.92/0.08, 0.15 n = 90 0.90/0.10, 0.12 n = 66 Not done

0.86/0.14, 0.19 n = 42 0.73, 0.27, 0.21 n = 108

38+106/149, 91 common (double digest with Ban11 to give 56+31 bp fragments)

0.75/0.25, n = 92

0.2

205/50+153

0.74/0.26, n = 102

0.37

223/130+89

0.89/0.11, n = 94

0.21

n Only variants confirmed by direct sequencing are listed. In addition, constant differences were observed within four codons in all three patients and also in a normal control and are thus assumed to represent errors in the sequence originally reported by Zorzato et al. (1990). They are now corrected as follows: LeuTgl CTG + CTT; Lys + Asn23*3 AAG + AAC; Ala + Argzs8 GCG + CGG; and Ala + Arg13’9 GCC + CGC. b AA, amino acid; RE, restriction endonuclease site; allele het freq., allele and heterozygote frequencies. The frequency of the more common allele is listed first, corresponding to the more common allele listed first under “fragment sizes.” Allele and heterozygote frequencies were determined using samples from unrelated, mostly Caucasian individuals, selected from families including individuals predisposed to malignant hyperthermia. ’ Number of chromosomes analyzed.

stitution must also be presumed to be a polymorphism rather than a candidate mutation, but occurs more frequently than the other amino acid substitutions described above. Analysis of Restriction RYRl Gene

Site Polymorphisms

in the

The 14 single base changes that result in the loss or gain of a restriction endonuclease site are listed in Table 1. To evaluate these RFLPs for family studies, oligonu-

cleotide primers were designed for PCR amplification of the region surrounding each sequence variant. The primer names are listed in Table 1 and their full sequences are provided under Materials and Methods. Primers were not designed for the Pro3”j” sequence change since this PuuII polymorphism had already been detected by conventional Southern blot analysis and used in MH family linkage studies (MacLennan et al., 1990). The His262o substitution resulted in an altered restriction site for several frequently cutting endonucleases but the cleaved fragments were too small and too

RYRI

IN

INDIVIDUALS

WITH

MALIGNANT

TABLE Caffeine Relationship

Individual HJ BT TJ SJ Family I-l I-2 II-1 II-2 Family I-l I-2 11-l II-2

Patient Patient Patient Brother

Halothane

2

Contracture CSC

1% H k)

of TJ

1251

HYPERTHERMIA

Test

Results HCSC

(mM)

Diagnosis”

(m&f)

1.25 0.4 3.5 1.1

1.12 3.9 1.15 2.4

0.05 0.24 0.06 0.15

RHCK RHCK RHCK HCK

10 Father Mother Brother Patient

of II-2 of II-2 of II-2

0 0 0

3.05 7.75 7

0.47 0.57 0.1

CK N K R

Father Mother Patient Brother

of II-1 of II-1

0.65 0 0 0.2

3.3 8.6 2.5 2.9

0.14 0.48 0.29 0.3

HCK

2

’ In this column: all three tests.

R, MH

of II-l

reaction;

H, positive

halothane

test;

C, positive

similar in size to be useful for genetic testing. The remaining 12 RFLPs were tested to determine allele frequencies and segregation patterns in MH families (Table 1). Allele and heterozygote frequencies were determined using samples from unrelated, mostly Caucasian individuals selected from MH families. The three most useful RFLPs are the AZwNI polymorphism at Let?, the TuqI polymorphism at Ile1151, and the HinPI polymorphism at Serzs6’, with heterozygote frequencies of 0.4-0.5. Our earlier linkage study of RYRl gene polymorphisms in MH families identified nine informative families (MacLennan et al., 1990). One or more of the new

caffeine

test;

K, positive

halothane

N RCK CK

+ caffeine

test;

and N, normal

for

polymorphisms were informative in each of these families and, as expected, the new markers segregated with the previously identified RFLPs and with the MH gene. DISCUSSION Malignant hyperthermia is a complex phenotype expressed in humans in response to a challenge with certain anesthetics. Once an individual has been labeled MH-sensitive by virtue of an MH reaction, other family members are often tested for susceptibility to halothaneor caffeine- induced contractures in muscle biopsies. These invasive tests are known for their difficulty in

Glycine to Arginine 248 Family 39 I

HCK

HCK

AC

G T

A

CG

T

A

C

G

T

ACGT

ACGT 3’ C T C -T/C

5 G A G G/A G

1 E z C

c

G

T C G 5’

A G C 3

FIG. 1. Direct sequencing of amplified cDNA from the region containing the Arg for Gly’@ substitution using reverse primer 80Re. The C to T substitution (G to A in the coding strand) is indicated by an arrow and is present in approximately half of the mRNA of the proband (TJ), her brother, and her mother. H, positive halothane test; C, positive caffeine test; K, positive halothane + caffeine test. Filled symbols indicate MH susceptibility: open symbols indicate no diagnosis.

1252

GILLARD

Ser 2962 Hinpl RFLP

Arg 470 Hinpl RFLP

FIG. 2. Polyacrylamide gel of HinPI restriction endonuclease-digested PCR products from family 39. The C to T substitution at SerZ8s2 results in a HinPI gain (lanes 1 through 5). TJ’s father and brother are heterozygous for the Serz8@ polymorphism while the rest of the family are homozygous for the new allele (153-bp band), demonstrating that TJ and her brother have inherited different paternal alleles. The substitution of Cys for Arg470 results in the loss of a HinPI site (lanes 7 through 11). TJ and her father are heterozygous for the Arg470 polymorphism while her mother and brother are homozygous for the wildtype llO-bp allele, indicating that this sequence substitution is not segregating with MH.

both execution and interpretation, undoubtedly resulting in some cases of misdiagnosis. The development of genetic tests for MH requires that the gene and the mutations causing MH be identified unequivocally. The identification of the ryanodine receptor gene and its linkage to the MH phenotype in family studies (MacLennan et al., 1990) supported, but did not prove, that the RYRl gene is mutated to cause MH in humans. The recent reports of families in which MH (Levitt et fails to segregate with RYRl polymorphisms al., 1991; Deufel et al., 1992) suggest additional caution in the interpretation of the earlier studies and, at the very least, point to genetic heterogeneity among MH families. Many of the data implicating the RYRl gene in defects leading to MH sensitivity come from studies in the pig. We recently identified a candidate mutation, substitution of Cys for Arg615 in the pig genome, and showed that this genetic alteration segregated consistently with Pro 1795 Pvull RFLP Family 39

Family 10

FIG. 3. Polyacrylamide gel of PuuII restriction endonuclease-digested PCR products from families 10 and 39 showing the loss of a PuuII site associated with the substitution of Leu for Prolrffl. Leu”85 (represented by the 395bp band) is not associated with predisposition to MH in either family.

ET

AL.

Family 10

Family 2

FIG. 4. Polyacrylamide gel of &a111 restriction endonuclease-digested PCR products of the region surrounding the Cys for Gly2”9 substitution. Patient TJ (lane 11 was homozygous for the substitution associated with the gain of a D&II restriction site (bands at 112 and 48 bp). In families 2 and 10 this substitution fails to segregate with predisposition to MH. In family 10 individual II-2 and his identical twin both experienced MH reactions; individual II-1 gave conflicting results on his biopsy test (Table 2) and is shaded to indicate his uncertain status. In family 2 individual II-1 experienced an MH reaction. The MH status of other family members was determined by caffeine halothane muscle biopsy contracture tests (Table 2).

the MH phenotype in over 450 animals examined, including 338 meioses. The corresponding mutation in humans, Cys for Arg614, has also been found in one MH family, where it segregates with the phenotype in three meioses, giving support to the hypothesis that certain mutations in RYRl are causative of MH (Gillard et al., 1991). In the present study, we report the identification of several new polymorphisms that can be applied to linkage studies in MH families and we report a second candidate mutation, adding to the evidence implicating the RYRl gene in MH. The RFLPs from the coding region of the RYRl gene, described in this paper, have proven to be informative in most of our MH families, including all of those informative for the earlier markers. Use of these markers was rapid and simple, confirming our earlier findings of linkage between MH and RYRl genes. The eight substitutions that failed to alter a restriction endonuclease site might also be applied to linkage studies and family analysis by means of single strand conformation polymorphisms or SSCPs (Orita et al., 1989). The availability of some 32 polymorphisms in the RYRl gene, 24 of which are detected as RFLPs, will make analysis of inheritance of specific RYRl haplotypes informative in most MH families. Our results exclude three of the four amino acid substitutions that we observed as candidate mutations for predisposition to MH and suggest that the fourth, Arg for G~Y’~~,may predispose to MH in the single family in which it has been observed. This candidate mutation was observed in only the family of TJ, consistent with the RYRl haplotype analysis (Table 3), which demonstrated that TJ did not have the relatively common MH haplotype shared by the majority of our MH patients, including BT. The MH haplotype in TJ’s family closely

RYRZ

TABLE RYRl

Haplotype

IN

INDIVIDUALS

Leuig7 Gl 248 A& Arg470 Ilen5i Pro’785 LYs”“‘z G1yzo5’ Asp= Ser286* Leu3”’

Site/AA change

REFERENCES

of Probands

Allele

AlwNI Gly > Arg AciI Arg > Cys TaqI Pro > Leu FokI Gly > Cys FokI + Ban11 HinPI Sty1

39=1 Gly=l 91=1 Arg=l

72=1 Pro=1 156=1 Gly=l 105=1 205=1 223=1

1253

HYPERTHERMIA

3 BT, HJ, and TJ

BT Amino acid

MALIGNANT

WITH

HJ

Archibald, A. L., and Imlah, P. (1985). and its linkage relationships. Anim.

TJ

N

M

N

M

N

0 1 2 1 1 1 1 1 1 1 1

0 1 2 1 1 1 1 1 1 1 1

0 1 2 1 1 1 2 1 2 2 2

0 1 2 1 1 1 2 1 2 2 2

2

1 1 1 2 2 2 2 2 1 2 1

Note. Partial RYRl haplotypes for each individual sequenced. The more common allele for each polymorphism is represented as a 1, the less common allele as a 2, and the loci heterozygous in a given individual for which phase could not be determined are represented by a 0. Family members of HJ were not available for analysis of phase.

resembles that of the normal maternal chromosome in family 1, differing only at the G1y248locus. HJ, for whom family members are unavailable, has a unique genotype among our MH family members. The association of the Arg248 mutation with MH will require linkage analysis in larger families or direct biochemical or physiological demonstration that this mutation appropriately alters Ca2+-release channel function. Amino acid substitutions have not yet been observed in either HJ or BT. Approximately 10% of the RYRl coding sequence of patients HJ and BT, mostly within 20 bp adjacent to each of the primers used in direct sequencing, remains to be sequenced. Candidate mutations causative for MH in these patients may lie within these regions. The R YRl coding sequence of patient TJ has been completely analyzed by cloning and sequencing with a minimum of five clones covering each region analyzed. This technique avoided the gaps left in the direct sequencing method, but introduced the potential for error due to PCR artifacts or to the lack of amplification of the mutant haplotype in the clones that were analyzed. Thus, there is a slight chance (~4%) that a candidate mutation may have been overlooked by this technique. Accordingly, we cannot state categorically that Arg for G1y248 is the sole candidate mutation in the RYRl gene for predisposition to MH in the family of TJ. Confirmation of Arg for G1yz4* as a mutation causative for predisposition to MH will await its identification and correct segregation in additional families or functional studies on the resultant ryanodine receptor. ACKNOWLEDGMENTS This research was supported by grants to D.H.M. and R.G.W. from the Muscular Dystrophy Association of Canada (MDAC) and by Grant MT-3399 from the Medical Research Council of Canada to D.H.M. J.F. was a fellow of the MDAC.

sensitivity Biochem.

locus Genet.

16:253-263.

M

2 2 1 2 1 2 2 1 2 1

The halothane Blood Groups

Britt, B. A. (1989). The North American caffeine ture test. In “Malignant Hyperthermia: Current Nalda Felipe, S. Gottmann, and H. J. Khambatta, Normed Verlag, Madrid.

halothane contracConcepts” (M. A. Eds.), pp. 53-69,

Britt, B. A. (1991). Malignant hyperthermia: A review. In “Thermoregulation: Pathology, Pharmacology and Therapy” (E. Schonbaum, and P. Lomax, Eds.) pp. 179-292, Pergamon, New York. Brostrom, C. O., Hunkeler, tion of skeletal muscle 246: 1961-1967.

F. L., and Krebs, E. G. (1971). The regulaphosphorylase kinase by Ca2+. Biol. Chem.

Bulman, D. E., Gangopadhyay, S. B., Bebchuck, K. G., Worton, R. G., and Ray, P. N. (1991). Point mutations in the human dystrophin gene: Identification through Western blot analysis. Genomics 10:

457-460. Chomczynski, isolation traction. Deufel, T., hauer, D., K. J., and neity of Genet., in

P., and Sacchi, N. (1987). Single-step method by acid guanidinium thiocyanate-phenol-chloroform Anal. Biochem. 162: 156-159. Golla, A., Iles, D., Meindl, A., Meitinger, T., DeVries, A., Pongratz, D., MacLennan, D. H., Lehmann-Horn, F. (1992). Evidence for genetic malignant hyperthermia susceptibility. Am. press.

Fill, M., Coronado, B. A., and Louis, nels in malignant

R., Mickelson, C. F. (1990). hyperthermia.

of RNA exSchindelJohnson, heterogeJ. Hum.

J. R., Vilven, J., Ma, J., Jacobson, Abnormal ryanodine receptor chanBiophys. J. 50: 471-475.

Fujii, J., Otsu, K., Zorzato, F., De Leon, S., Khanna, V. K. Weiler, J., O’Brien, P. J., and MacLennan, D. H. (1991). Identification of a mutation in porcine ryanodine receptor associated with malignant hyperthermia. Science 253: 448-451. Gillard, mezi, D. H. odine thermia.

E. F., Otsu, K., Fujii, J., Khanna, V. K., De Leon, S., DerdeJ., Britt, B. A., Duff, C. L., Worton, R. G., and MacLennan, (1991). A substitution of cysteine for arginine 614 in the ryanreceptor is potentially causative of human malignant hyperGenomics. 11: 751-755.

Harbitz, I., Chowdhary, B., Thomsen, P., Davies, W., Kaufman, U., Kran, S., Gustavsson, I., Christensen, K., and Hauge, J. (1990). Assignment of the porcine calcium release channel gene, a candidate for the malignant hyperthermia locus, to the 6pll-q21 segment of chromosome 6. Genomics 8: 243-248. Harrison, G. G. (1987). Porcine of the hot pig. In “Malignant 103-136, Nijhoff, Boston. Harrison, aesth.

G. G. (1988). 60: 279-286.

malignant Hyperthermia”

Dantrolene-dynamics

hyperthermia-The (B. A. Britt, and kinetics.

Ed.),

saga pp.

Br. J. An-

Heery, D. M., Gannon, F., and Powell, R. (1990). A simple method for subcloning DNA fragments from gel slices. Trends Genet. 6: 173. Knudson, C. M., Mickelson, J. R., Louis, C. F., and Campbell, K. P. (1990). Distinct immunopeptide maps of the sarcoplasmic reticulum Ca2+ release channel in malignant hyperthermia. J. Biol. Chem.

265:2421-2424. Kogan, S. C., Doherty, M., and Gitschier, J. (1987). An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. N. Engl. J. Med. 317: 985-990. Lai, F. A., and Meissner, G. (1989). The muscle ryanodine receptor and its intrinsic Ca’+ activity. J. Bioenerg. Biomembr. 2 1: 227-246. Levitt, R. C., Nouri, N., Jedlicka, A. E., McKusick, V. A., Marks, A. R., Shutack, J. G., Fletcher, J. E., Rosenberg, H., and Meyers, D. A. (1991). Evidence for genetic heterogeneity in malignant hyperthermia susceptibility. Genomics 11: 543-547. MacKenzie, A. E., Korneluk, R. G., Zorzato, F., Fujii, J., Phillips, M., Iles, D., Wieringa, B., Le Blond, S., Bailly, J., Willard, H. F., Duff, C., Worton, R. G., and MacLennan, D. H. (1990). The human ryan-

1254

GILLARD

odine receptor gene, its mapping to 19q13.1, placement in a chromosome 19 linkage group, and exclusion as the gene causing myotonic dystrophy. Am. J. Hum. Genet. 46: 1082-1089. MacLennan, D. H., Duff, luk, R. G., Frodis, W., odine receptor gene is hyperthermia. Nature

C., Zorzato, F., Fujii, J., Phillips, M., KorneBritt, B. A., and Worton, R. G. (1990). Ryana candidate for predisposition to malignant 343: 559-561.

McCarthy, T. V., Healy, J. M. S., Heffron, J. J. A., Lehane, M., Deufel, T., Lehmann-Horn, F., Farrall, M., and Johnson, K. (1990). Localization of the malignant hyperthermia susceptibility locus to human chromosome 19q12-13.2 Nature 343: 559-561. Mickelson, J. R., Gallant, pel, W. E., and Louis, lum ryanodine receptor 263: 9310-9315.

E. M., Litterer, L. A., Johnson, K. M., RemC. F. (1988). Abnormal sarcoplasmic reticuin malignant hyperthermia. J. Biol. Chem.

Miller, S. A., Dykes, D. D., and Polesky, H. F., (1988). A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16: 1215. Nelson, T. E. (1988). Calcium 9: 257-265. O’Brien, P. d. (1987). mia: Hypersensitive coplamic reticulum.

SR

function

in malignant

hyperthermia.

Etiopathogenetic defect of malignant calcium release channel of skeletal Vet. Res. Commun. 11: 527-559.

Cell

hyperthermuscle sar-

Ohnishi, S. T., Taylor, S., and Gronert, G. A. (1983). Calcium-induced Ca*+ release from sarcoplasmic reticulum of pigs susceptible to malignant hyperthermia. FEBS I&t. 161: 1033107. Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K. (1989). Rapid sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5: 875-879. Otsu, K., Willard, H. F., Khanna, V. K., Zorzato, MacLennan, D. H. (1990). Molecular cloning

and

F., Green, N. M., and of cDNA encoding the

ET

AL.

Ca2+ release sarcoplasmic

channel (ryanodine reticulum. J. Biol.

receptor) of rabbit cardiac Chem. 265: 13,472-13,483.

Otsu, K., Khanna, V. K., Archibald, A. L., and MacLennan, (1991). Cosegregation of porcine malignant hyperthermia probable causal mutation in the skeletal muscle ryanodine gene, in backcross families. Genomics 11: 744-750.

muscle D. H. and a receptor

Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-494. Sanger, F., Nicklen, with chain-terminating 5463-5467.

S., and Coulson, inhibitors.

A. R. (1977). DNA sequencing Proc. N&l. Acad. Sci. USA 74:

Schonk, D., Coerwinkel-Dreisen, M., Van Dalen, I., Oerlemans, F., Smeets, B., Schepens, d., Hulsebos, T., Cockburn, D., Boyd, Y., Davis, M., Rettig, W., Shaw, D., Roses, A., Ropers, H., and Wieringa, B. (1989). Definition of subchromosomal intervals around the myotonic dystrophy gene region at 19q. Genomics 4: 384-396. Steward, D. J., and O’Connor, G. A. R. (1987). Malignant hyperthermia-The acute crisis. In “Malignant Hyperthermia” (B. A. Britt, Ed.), pp. l-9, Nijhoff, Boston. Wang, A. M., Doyle, M. V., and Mark, D. F. (1989). Quantitation of mRNA by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86: 9719-9721. Winship, P. R. (1989). An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide. Nucleic Acids Res. 17: 1266. Zorzato, F., Fujii, J., Otsu, K., Phillips, M., Green, N. M., Lai, F. A., Meissner, G., and MacLennan, D. H. (1990). Molecular cloning of cDNA encoding human and rabbit forms of the Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 265: 2244-2256.

Polymorphisms and deduced amino acid substitutions in the coding sequence of the ryanodine receptor (RYR1) gene in individuals with malignant hyperthermia.

Twenty-one polymorphic sequence variants of the RYR1 gene, including 13 restriction fragment length polymorphisms (RFLPs), were identified by sequence...
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