HUMAN MUTATION Mutation in Brief #913 (2006) Online
MUTATION IN BRIEF
© 2006 WILEY-LISS, INC.
Received 23 January 2006; accepted revised manuscript 18 April 2006.
Frequency and Localization of Mutations in the 106
Exons of the RYR1 Gene in 50 Individuals With
Lucia Galli1, Alfredo Orrico1, Stefania Lorenzini1, Stefano Censini2, Michela Falciani1, Antonello
Covacci2, Vincenzo Tegazzin1, 3, and Vincenzo Sorrentino1*
1Molecular Medicine Section, Department of Neuroscience, University of Siena and Azienda Ospedaliera
Universitaria Senese, Siena, Italy;, 2IRIS, Chiron Vaccines, Siena, Italy; 3Department of Anaesthesia, S. Antonio
University Hospital, Padova, Italy
*Correspondence to: Vincenzo Sorrentino, Molecular Medicine Section, Department of Neuroscience, University
of Siena, Siena, Italy; E-mail: email@example.com
Grant sponsor: Telethon (n° GGP02168); European Union (HPRN-CT-2002-00331); MIUR/ FIRB 2001.
Communicated by Maria Rita Passos-Bueno
Malignant hyperthermia (MH) is a dominantly inherited pharmacogenetic condition that
manifests as a life-threatening hypermetabolic reaction when a susceptible individual is
exposed to common volatile anesthetics and depolarizing muscle relaxants. Although MH
appears to be genetically heterogeneous, RYR1 is the main candidate for MH susceptibility.
However, since molecular analysis is generally limited to exons where mutations are more
frequently detected, these are routinely found only in 30-50% of susceptible subjects. In this
study the entire RYR1 coding region was analyzed in a cohort of 50 Italian MH susceptible
(MHS) subjects. Thirty-one mutations, 16 of which were novel, were found in 43 individuals
with a mutation detection rate of 86%, the highest reported for RYR1 in MH so far. These
data provide clear evidence that mutations in the RYR1 gene are the predominant cause of
MH. © 2006 Wiley-Liss, Inc.
KEY WORDS: RYR1; malignant hyperthermia; muscle contraction; calcium release; ryanodine receptor
Linkage analysis and mutation screening have identified the RYR1 gene (MIM# 180901) on chromosome
19q13.1, as the gene most frequently involved in the pathogenesis of MH (MIM# 145600) [Benkusky et al., 2004;
Hamilton, 2005]). Rare cases of mutations in the α1 subunit of the DHPR gene (CACNA1S: MIM# 114208) have
been reported in MH patients [Monnier et al, 1997; Stewart et al., 2001]. Additional loci have also been linked to
MH, but causative genes have still to be identified [Levitt et al., 1992; Iles et al., 1994; Sudbrak et al., 1995;
Robinson et al., 1998]. In recent years, more than 60 mutations in RYR1 have been identified in MH/CCD families
[Hamilton, 2005]. Mutations in MHS individuals are detected in three regions (exons 2-17, exons 39-46 and exons
90-104) of the RYR1 gene. However, since the large size of the RYR1 gene (106 exons) makes genetic analysis
very laborious and expensive, mutation screening is preferentially performed only in those exons where known
mutations are clustered [Barone et al., 1999; Brandt et al., 1999; Lynch et al., 1999; Galli et al., 2002; Robinson et
al., 2003; Tilgen et al., 2003; Tammaro et al., 2003; Sambuughin et al., 2001]. This approach has resulted in the
2 Galli et al.
identification of mutations in the RYR1 gene in approximately 30-50% of the probands identified as MHS at the in
vitro contraction test (IVCT) [European Malignant hyperpyrexia Group, 1984; Robinson et al., 2003], while
analysis of the entire RYR1 coding sequence is only rarely performed [Sambuughin et al., 2005; Monnier et al.,
2005]. Overall, this may generate a bias that favors extensive identification of mutations in specific areas of the
RYR1 gene while limiting the identification of additional regions where mutations may also occur. Furthermore, it
may also limit the precise assessment of RYR1 mutation frequency in MHS individuals, which is estimated to be
around 50 % of MHS cases. The present study was designed to assess a reliable estimate of the frequency and
localization of RYR1 mutations following the screening of the entire coding region of the RYR1 gene in 50 Italian
MATERIALS AND METHODS
50 unrelated MHS subjects, 19 females and 31 males, with a clinical history including an MH event during
anesthesia, family history of MH, or chronically unexplained elevated serum levels of creatinine kinase (CK) were
enrolled in this study. All individuals had undergone an in vitro contraction test (IVCT) to determine their MH
susceptibility status according to the protocol of the European Malignant Hyperthermia Group [European
Malignant hyperpyrexia Group, 1984]. This test is based on the contractile response of skeletal muscle bundles
taken from diagnostic muscle biopsies, exposed to different concentrations of halothane and caffeine. Individuals
are considered MH-susceptible (MHS) if positive for both halothane and caffeine stimulation, MH-equivocal
(MHE) when positive to only one of the triggering agents, or MH-negative (MHN).
Genomic DNA was extracted from peripheral blood leucocytes by standard procedures. The genomic structure
and intron boundary sequences were deduced from the Homo sapiens chromosome 19 genomic contig NT_011109
and the cDNA sequence NM_000540.1. PCR primers were designed for each exon with the Primer-3 software
(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Primer sequences and conditions are available on
request (firstname.lastname@example.org). Denaturing high-performance liquid chromatography (DHPLC)- analysis was
performed using a WAVE 3500HTsystem (Transgenomic, Cheshire, UK. http://www.transgenomic.com). In brief,
a mixture of 4-6 µl of each amplicon from patient DNA and from a normal individual was heated for 5 min at
95oC, and then cooled to room temperature. A volume of 8-12 µl of each mixture (sample) was analyzed, using at
least two partially denaturing temperatures predicted as the optimal melting temperatures for each amplicon by the
Wavemaker 4.1 software. In addition, mutated samples previously detected by SSCP [Barone et al., 1999; Galli et.,
al., 2002] were used as control of sensitivity, allowing to modify, when necessary, the DHPLC conditions
predicted by the software. DNA fragments with an abnormal chromatogram profile, compared to those obtained
from normal controls, were directly sequenced to confirm putative sequence variations. Individuals who did not
show an abnormal chromatogram profile at the DHPLC analysis were also analyzed by direct sequencing of all
RYR1 exons. In addition, the nucleotide sequence of exon 25 of the CACNA1S gene (Homo sapiens chromosome 1
genomic contig NT_004487 and cDNA sequence NM_000069) was also determined.
RESULTS AND DISCUSSION
Identification of RYR1 mutations
Screening by DHPLC analysis of the 106 exons of the RYR1 gene identified 31 different mutations in 43 out of
50 MHS patients tested, giving a mutation detection rate of 86%, see Table 1. Although rare cases of compound
heterozygotes and homozygotes for RYR1 mutations have been reported, all patients reported were heterozygous
for RYR1 mutations, [Lynch et al., 1997; Rueffert et al., 2001; Jungbluth et al., 2002]. Seven mutations, detected
in subjects 10, 11, 13, 18, 20, 27, 32, 33, 35, 40, 47, 50 have been previously reported in association with MH and
have been demonstrated to be pathogenic mutations on the basis of functional studies (www.emhg.org). These 7
mutations were detected in 12 patients (27.90%), i.e. p.Arg163Cys (n=1), p.Arg614Cys (n=1), p.Arg2163His
(n=1), p.Thr2206Met (n=3), p.Gly2434Arg (n=3), p.Arg2435His (n=1), p.Arg2458Cys (n=2), see Table 1. Eight
further mutations detected in subjects 4, 14, 15, 21, 30, 31, 34, 39, 43 and 45 have been previously detected in MH
probands and found to segregate in affected family members. These 8 mutations were found in 10 subjects
(23.25%) i.e. p.Arg177Cys (n=1), p.Ala2200Val (n=1), p.Val2280Ile (n=1), p.Arg2435Leu (n=1), p.Arg4136Ser
(n=1), p.Val4234Leu (n=1), p.Phe4684Ser (n=1) and p.Arg4737Trp (n=3), see Table 1.
RYR1 Mutations in MH 3
Table 1. RYR1 mutations in the MHS population screened
Amino acid change
aNumbering of nucleotides and amino acids based on the RYR1 cDNA sequence (NM_000540.1 and NP_000531, respectively)
with +1 as the A of the ATG initiation codon, and this ATG codon as codon +1.
Quane et al. 1993
Monnier et al. 2005
Gillard et al. 1991
Manning et al. 1998
Sambuughin et al. 2005
Manning et al. 1998
Manning et al. 1998
Manning et al. 1998
Galli et al. 2002
Keating et al. 1994
Keating et al. 1994
Keating et al. 1994
Zhang et al. 1993
Barone et al. 1999
Manning et al. 1998
Manning et al. 1998
Galli et al. 2002
Galli et al. 2002
Monnier et al. 2005
Galli et al. 2002
Galli et al. 2002
Galli et al. 2002
4 Galli et al.
Sixteen novel missense mutations (48.83%) were detected in 21 subjects (1, 3, 5, 6, 7, 9, 17, 19, 22, 24, 25, 26,
36, 37, 38, 41, 42, 44, 46, 48, 49), i.e. p.Arg44His (n=1), p.Ser71Tyr (n=2), p.Arg156Lys (n=1), p.Arg367Gln
(n=1), p.Val2212Asp (n=1), p.Arg2336Gln (n=2), p.Glu2362Gly (n=1), p.Ala2436Val (n=1), p.Glu2439Asp
(n=1), p.Arg2508His (n=1), p.Arg2591Gly (n=2), p.Val2627Leu (n=1), p.Glu2764Lys (n=1), p.Leu2867Gln
(n=1), p.Arg3903Gln (n=3) and p.Arg4041Trp (n=1), see Table 1.
All the nucleotide variants resulting in predicted amino acid changes were not found in >100 alleles in the
general population. These mutations resulted in changes in amino acids highly conserved in RYR1 throughout
evolution and that were also conserved across the 3 RYR known genes, RYR1, RYR2, and RYR3 (data not shown).
Altogether, the mutations detected in more than one proband were 8, i.e. p.Ser71Tyr, p.Thr2206Met,
p.Arg2336Gln, p.Gly2434Arg, p.Arg2458Cys, p.Arg2591Gly, p.Arg3903Gln and p.Arg4737Trp, accounting for
46.51 % of mutated subjects (20 out of 43 subjects). Mutations p.Thr2206Met, p.Gly2434Arg, p.Arg3903Gln and
p.Arg4737Trp were each detected in 3 different probands, accounting together for 27.9%, of the mutated subjects.
In individuals 2, 8, 12, 16, 23, 28 and 29, no variants were identified by DHPLC screening. To exclude that lack of
variant detection could be due to the limits in the sensitivity of the DHPLC technique, the nucleotide sequence of
all 106 exons of the RYR1 gene of these subjects was determined. This analysis confirmed the absence of
mutations in the RYR1 gene in these 7 subjects, but also indicated that DHPLC analysis represents a highly
efficient methodology for RYR1 mutation detection. Mutations in exon 25 of the CACNA1S gene (p.Arg1086His
and p.Arg1086Cys) have been reported in two MH probands [Monnier et al., 1997; Stewart et al., 2001]. Thus,
exon 25 of the CACNA1S gene of individuals 2, 8, 12, 16, 23, 28 and 29 was analyzed by direct nucleotide
sequencing. Also, this analysis did not identify sequence variation, thus excluding defects in exon 25 of the
CACNA1S gene as possible cause of the MHS status in these subjects.
Segregation of mutations within families of probands
Genetic analysis was extended, when possible, to relatives of the 43 probands with a mutation in the RYR1
gene. A total of 100 first-degree relatives, within 30 independent families, were analyzed. Among the family
members of 11 probands (subjects 10, 11, 13, 18, 20, 27, 32, 35, 40, 47, 50) in which a mutation considered to be
causative according to EMHG criteria was found, the mutations showed a complete co-segregation with the
MHS/MHE phenotypes. In this group, out of 32 first-degree relatives examined, mutations were found in 13/13
MHS/MHE. As it concerns family members of the 10 subjects carrying mutations reported in the literature as
associated with the MHS status, but not functionally characterized in vitro, we analyzed 34 family members of 7
probands (subjects 4, 15, 21, 31, 34, 43, 45). We detected mutations in 10/10 MHS-MHE subjects. The only case
of apparent discrepancy was detected in the family of individual 4, who carries the p.Arg4136Ser mutation, where
the mutation was inherited from the MHN father. Critical evaluation of IVCT data in this family confirmed the
results of the contracture test.
Of the 16 missense mutations reported herein for the first time 4. i.e. p.Ser71Tyr; p.Arg2336Gln;
p.Arg2591Gly; p.Arg3903Gln were detected in more than one proband (subjects 3 and 19; 6 and 41; 7 and 26; 9,
37 and 48, respectively) and in their MHS/MHE relatives. Mutations p.Arg44His p.Glu2362Gly and p.Arg4041Trp
were detected only in one proband, however were found to segregate with the MHS status within their families. In
contrast, for variants p.Arg156Lys, p.Arg367Gln, p.Val2212Asp, p.Arg2508His, p.Val2627Leu, p.Glu2764Lys
and p.Leu2867Gln, there were no family members of probands 22, 24, 25, 38, 42, 46, 49, available for laboratory
investigation. One case of discordance between genetic results and the IVCT phenotypes was observed in the
family of individual 26, who carried the p.Arg2591Gly mutation, that appears to have been inherited from the
MHN father. Critical evaluation of IVCT data in this family confirmed the results of the contraction test. This
study also identified several single nucleotide polymorphisms (SNPs) in the coding sequence of RYR1 gene. Most
of these silent genetic variants have been already reported in the dbSNP database at the National Center for
Biotechnology Information [Aerts et al., 2002]. Out of a total of 29 SNPs identified, eight (27.58%) were novel,
see Table 2.
RYR1 Mutations in MH 5
Table 2. Synonymous SNPs detected in the MHS population screened
SNP identification (ID) refers to the Reference Sequence identifier in the SNP database (dbSNP):
http://www.ncbi.nlm.nih.gov/SNP. Numbering of nucleotides: (a) +1= A of ATG in the cDNA sequence
(NM_000540.1); (b) nucleotide numbers as from the human chromosome 19 contig NT_011109. Minor allele
frequencies were estimated from analysis of 100 chromosomes.
c. 2712 G>A
Localization of mutations in the RYR1 protein
In 33 out of the 43 subjects with mutations in RYR1 (76.74%), mutations were detected in one of three domains
of RYR1 where mutations are more frequently detected (MH domains 1, 2 and 3), while in the remaining ten
subjects, the mutations were found in regions usually not explored. Analysis of exons 1-17, which include the MH
domain 1, identified 7 mutations accounting for 8 subjects (8/43=18.60%). Namely, mutations found in the MH
region 1 were p.Arg44His in exon 2, p.Ser71Tyr in exon 3, p.Arg156Lys, p.Arg163Cys and p.Arg177Cys in exon
6, p.Arg367Gln in exon 11 and p.Arg614Cys in exon 17. Of these mutations p.Arg44His, p.Ser71Tyr,
p.Arg156Lys and p.Arg367Gln were novel, although a different mutation for residue Arg44 (p.Arg44Cys) had
been reported (Tammaro et al., 2003). Interestingly, the p.Ser71Tyr mutation, detected in two independent
subjects, is located in exon 3, an exon not routinely included in analysis of the MH domain 1 of RYR1.
6 Galli et al.
Analysis of exons from 18 to 38, which span the region between MH domains 1 and 2, did not result in the
identification of mutations although three synonymous polymorphisms were detected in this area of the RYR1
Screening of exons 39-46 which cover the MH domain 2 identified 19 subjects (44.18%) with mutations. In this
group the mutations found were p.Arg2163His in exon 39, p.Ala2200Val, p.Thr2206Met, and p.Val2212Asp in
exon 40, p.Val2280Ile in exon 42, p.Arg2336Gln in exon 43, p.Glu2362Gly in exon 44, p.Gly2434Arg,
p.Arg2435His, p.Arg2435Leu, p.Ala2436Val and p.Glu2439Asp in exon 45, p.Arg2458Cys in the exon 46. The
mutations p.Val2212Asp, p.Arg2336Gln, p.Glu2362Gly, p.Ala2436Val and p.Glu2439Asp are novel. The
p.Arg2336Gln Mutation, detected to in two independent subjects, is located in exon 43 that is not usually included
in the screening of the MH domain 2.
Analysis of the region between exons 47 and 89 (that includes exons located between the MH domain 2 and
MH domain 3 and covers 1612 codons that account for 32% of the RYR1 coding sequence) identified 7 mutations:
p.Arg2508His in exon 47, p.Arg2591Gly, p.Arg2591Gly in exon 48, p.Val2627Leu in exon 49, p.Glu2764Lys in
exon 52, p.Leu2867Gln in exon 55, p.Arg3903Gln in exon 85 and p.Arg4041Trp in exon 89. All these mutations
were novel and account for 10 mutated subjects (23.26%) as two of them were found to recur in more than one
individual: p.Arg2591Gly (n=2), p.Arg3903Gln in probands (n=3). Thus, this region, which is generally not
examined in routine investigations, covers 23.26% of the mutated subjects examined in this study. At a closer look
it is possible to note that of the seven mutations detected by analysis of exons 47-85, 5 occur in exons 47, 48, 49,
52 and 55, which may be considered as an extension of the MH domain 2, and the remaining 2 are found in exons
85 and 89 that precede the MH domain 3. We did not find mutations in exons from 56 to 84. Indeed, considering
the current data in the literature [Hamilton 2005, Sambuughin et al., 2005, Monnier et al., 2005], only two
mutations (p.Arg3348His in exon 67 and p.Pro3527Ser in exon 71) have been so far detected in the region encoded
by exons 56 to 84 that is located between MH domain 2 and 3.
Finally, 6 out of 43 subjects (13.95%) presented mutations in the region between exons 90 and 106, which
cover the MH domain 3 and corresponds to the COOH-terminal of RYR1. The mutations found in the MH domain
3 are p.Arg4136Ser in exon 90, p.Val4234Leu in exon 91, p.Phe4684Ser in exon 96, p.Arg4737Trp in exon 98.
Altogether, these data confirmed that the distribution of mutations within the RYR1 gene is very selective. In
fact, 24 of 31 mutations (77.42 %) were localized in the three MH domains that account for 37.9% of the RYR1
coding sequence. Namely, 7 of 31 mutations (22.58 %), were localized in the MH domain 1 that accounts for the
12.4% of the coding sequence, 13 of 31 mutations (41.93 %) were localized in the MH domain 2 that accounts for
7.24% of the coding sequence, while 4 of 31 mutations (12.90 %) were localized in the MH domain 3. It is also
noteworthy that the results obtained indicate that all mutations found in this study were localized in 23 out of the
106 exons of RYR1. This evidence may contribute to select exons to preferentially screen for genetic test of MH.
Critical evaluation of MHS individuals negative for RYR1 mutations
Out of the 50 MHS subjects characterized in this study, 7 were negative for mutations in the 106 exons of the
RYR1 gene and in exon 25 of the CACNA1S gene. From a genetics point of view either changes outside of the
coding region of the RYR1 gene or mutations different from the nucleotide changes in exon 25 of the DHPR α1s
subunit gene or the involvement of an additional unidentified gene may explain the MHS phenotype of these seven
patients. On the other hand, although the IVCT with caffeine and halothane represents the gold standard for the
diagnosis of MH, it should be considered that the possibility exists that a small number of normal subjects may
score positive at the test [Ording et al., 1997; Urwyler et al., 2001]. In order to explore this second possibility, the
clinical and IVCT records of patients 8, 12, 16, 23, 28 and 29 were critically re-evaluated (the original IVCT
records of patient 2 were not available). It is worth noting that no one of these individuals had reported an MH
crisis, but all were referred on the basis of increased levels of CK value. Furthermore, only individual 2 had a
relative who scored MHS, while the remaining six subjects represent sporadic cases, with no clinical history of
MH and without family members available for molecular and/or IVCT investigations. Survey of IVCT data
indicated that only for individual 16 all the four muscle strips (2 for halothane and 2 for caffeine tests) were
positive as observed in almost 90% of individuals who present an MHS response at the IVCT analysis. In the
remaining 5 individuals (8, 12, 23, 28 and 29) a positive response at the cut off point of 2 % halothane or 2 mM
caffeine was observed only in one of the two muscle strips analyzed for each agonist. IVCT records from subject
29 were barely positive at cut-off points for both halothane and caffeine. In addition, for all of them, it was not
RYR1 Mutations in MH 7
possible to observe a clear dose-response dependent contractile response. Therefore, although the IVCT data were
compatible with a diagnosis of MHS for all patients according with the EMHG protocol, we find that for subjects
8, 12, 23, 28 and 29, the IVCT results were not very strong and certainly differed from the more pronounced and
reproducible contractile response presented by other MHS subjects analyzed in this study and in whom mutations
in RYR1 gene were identified.
In conclusion, the data reported here allow for a more precise estimate of the frequency of RYR1 mutations in
the MHS Italian population, where we found 31 different RYR1 mutations, 16 of which were novel, in 43/50 MHS
individuals that identifies 86% of RYR1 mutations in MHS individuals. The mutation rate can rise as high as
100%, if the 7 individuals who scored negative for mutations are not considered since they were referred to IVCT
test because of high CK value, which may be due to a condition different from MH, hence the poor performance at
the IVCT. In both cases, these results represent the highest rate of mutations in the RYR1 gene in MHS subjects
and strongly suggest that RYR1 mutations are the predominant cause of MH in the vast majority of cases. Taking
into account that for almost all MH family studied mutations in RYR1 confirmed the diagnosis, these results
indicate that search for mutations in the RYR1 gene may provide confirmation of the diagnosis for MH probands.
Furthermore, it may allow genetic counseling and preventive molecular test for family members. It is interesting to
note that RYR1 mutations were found either within the three MH domains or in exons close to MH domain 2 and
3. This is in agreement with data in the literature that outside of the three MH domains, i.e. in exons from 18 to 38
and from 56 to 84, mutations are either not detected or are very rare. Additional analysis of all 106 RYR1 exons in
MHS subjects will certainly help in further understanding the distribution of mutations in this gene and to better
understand the relationship between structure and function in the RYR1 channels.
This work was supported by grants from Telethon (n° GGP02168), European Union (HPRN-CT-2002-00331)
and from MIUR/ FIRB 2001 to Vincenzo Sorrentino.
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