Multigenerational Brazilian family with malignant hyperthermia and a novel mutation in the RYR1 gene.
ABSTRACT Malignant hyperthermia (MH) is a pharmacogenetic disease triggered in susceptible individuals by the administration of volatile halogenated anesthetics and/or succinylcholine, leading to the development of a hypermetabolic crisis, which is caused by abnormal release of Ca2+ from the sarcoplasmic reticulum, through the Ca2+ release channel ryanodine receptor 1 (RyR1). Mutations in the RYR1 gene are associated with MH in the majority of susceptible families. Genetic screening of a 5-generation Brazilian family with a history of MH-related deaths and a previous MH diagnosis by the caffeine halothane contracture test (CHCT) in some individuals was performed using restriction and sequencing analysis. A novel missense mutation, Gly4935Ser, was found in an important functional and conserved locus of this gene, the transmembrane region of RyR1. In this family, 2 MH-susceptible individuals previously diagnosed with CHCT carry this novel mutation and another 24 not previously diagnosed members also carry it. However, this same mutation was not found in another MH-susceptible individual whose CHCT was positive to the test with caffeine but not to the test with halothane. None of the 5 MH normal individuals of the family, previously diagnosed by CHCT, carry this mutation, nor do 100 controls from control Brazilian and USA populations. The Gly4932Ser variant is a candidate mutation for MH, based on its co-segregation with disease phenotype, absence among controls and its location within the protein.
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ABSTRACT: Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder that is manifested on exposure of susceptible individuals to halogenated anesthetics or succinylcholine. Since MH is associated primarily with mutations in the ryanodine receptor type 1 (RYR1) gene, the purpose of this study was to determine the distribution and frequency of MH causative RyR1 mutations in the Canadian MH susceptible (MHS) population. In this study, we screened a representative cohort of 36 unrelated Canadian MHS individuals for RYR1 mutations by sequencing complete RYR1 transcripts and selected regions of CACNA1S transcripts. We then analyzed the correlation between caffeine-halothane contracture test (CHCT) results and RYR1 genotypes within MH families. Eighty-six percent of patients had at least one RyR1 mutation (31 out of 36), five of which were unrelated individuals who were double-variant carriers. Fifteen of the 27 mutations identified in RYR1 were novel. Eight novel mutations, involving highly conserved amino acid residues, were predicted to be causal. Two of the mutations co-segregated with the MHS phenotype within two large independent families (a total of 79 individuals). Fourteen percent of MHS individuals (five out of 36) carried neither RYR1 nor known CACNA1S mutations. The distribution and frequency of MH causative RyR1 mutations in the Canadian MHS population are close to those of European MHS populations. Novel mutations described in this study will contribute to the worldwide pool of MH-associated mutations in the RYR1 gene, ultimately increasing the value of MH genetic diagnostic testing.Canadian Anaesthetists? Society Journal 03/2011; 58(6):504-13. · 2.31 Impact Factor
Faculdade de Medicina
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The Brazilian Journal of Medical and Biological Research is partially financed by
Braz J Med Biol Res, December 2009, Volume 42(12) 1218-1224
Multigenerational Brazilian family with malignant hyperthermia
and a novel mutation in the RYR1 gene
A.R. Matos, N. Sambuughin, F.D. Rumjanek, N.D. Amoedo, L.B.P. Cunha, G. Zapata-Sudo and
Volume 42 (12) 1119-1247 December 2009
Brazilian Journal of Medical and Biological Research (2009) 42: 1218-1224
Multigenerational Brazilian family with
malignant hyperthermia and a novel
mutation in the RYR1 gene
A.R. Matos1, N. Sambuughin4, F.D. Rumjanek2, N.D. Amoedo2,
L.B.P. Cunha3, G. Zapata-Sudo1 and R.T. Sudo1
1Programa de Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas,
2Instituto de Bioquímica Médica, and 3Serviço de Anestesiologia, Faculdade de Medicina,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil
4Uniformed Services, University of the Health Sciences, Bethesda, MD, USA
Malignant hyperthermia (MH) is a pharmacogenetic disease triggered in susceptible individuals by the administration of volatile
halogenated anesthetics and/or succinylcholine, leading to the development of a hypermetabolic crisis, which is caused by
abnormal release of Ca2+ from the sarcoplasmic reticulum, through the Ca2+ release channel ryanodine receptor 1 (RyR1).
Mutations in the RYR1 gene are associated with MH in the majority of susceptible families. Genetic screening of a 5-generation
Brazilian family with a history of MH-related deaths and a previous MH diagnosis by the caffeine halothane contracture test
(CHCT) in some individuals was performed using restriction and sequencing analysis. A novel missense mutation, Gly4935Ser,
was found in an important functional and conserved locus of this gene, the transmembrane region of RyR1. In this family, 2
MH-susceptible individuals previously diagnosed with CHCT carry this novel mutation and another 24 not previously diagnosed
members also carry it. However, this same mutation was not found in another MH-susceptible individual whose CHCT was
positive to the test with caffeine but not to the test with halothane. None of the 5 MH normal individuals of the family, previously
diagnosed by CHCT, carry this mutation, nor do 100 controls from control Brazilian and USA populations. The Gly4935Ser
variant is a candidate mutation for MH, based on its co-segregation with disease phenotype, absence among controls and its
location within the protein.
Key words: Malignant hyperthermia; Mutation; Ryanodine receptor; Calcium channel
Braz J Med Biol Res 42(12) 2009www.bjournal.com.br
Correspondence: R.T. Sudo, Programa de Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas, CCS, UFRJ, Bloco J,
sala 14, 21941-590 Rio de Janeiro, RJ, Brasil. Fax: +55-21-2562-6505. E-mail: firstname.lastname@example.org
Received August 14, 2009. Accepted October 28, 2009. Available online November 13, 2009. Published December 4, 2009.
Malignant hyperthermia (MH) is an autosomal dominant
pharmacogenetic disease (1,2). Individuals who are suscep-
tible to MH develop a hypermetabolic crisis after the adminis-
tration of volatile general anesthetics and/or succinylcholine,
with signs of tachycardia, arrhythmia, muscle rigidity, and
hyperthermia. This life-threatening condition is caused by the
increased release of Ca2+ from the sarcoplasmic reticulum to
the cytoplasm by the ryanodine receptor 1 (RyR1) (3).
MH susceptibility has been linked to several loci in the
genome (4-8), but most cases are associated with the RYR1
gene located on chromosome 19 (9,10), one of the largest and
most complex genes in the human genome, comprising 106
exons and transcribing a 15,117 nucleotide-long RNA (11),
which encodes a 563-kD homotetrameric protein. Worldwide,
about 70-80% of MH-susceptible (MHS) individuals have
mutations in RYR1 (12-15).
More than 180 different mutations (16) and many poly-
morphisms (14,15) have been described in RYR1 among
MHS families. These mutations are frequently clustered in
the hot-spot domains of the protein, which are the N-terminal
region (amino acids 34-614), the central region (amino
acids 2163-2458) and the C-terminal region (amino acids
Currently, MH is diagnosed by the contracture test,
which is the diagnostic model adopted at the Brazilian MH
Center. The caffeine-halothane contracture test (CHCT)
is applied according to the North American protocol (17).
However, because of its invasiveness, this test presents
Novel mutation for malignant hyperthermia
Braz J Med Biol Res 42(12) 2009
some disadvantages and, especially in Brazil, which has
only one center for MH diagnosis, patients frequently also
need to travel long distances and to spend much time and
effort in order to undergo this test. All of these circumstances
create great difficulties in the access to the diagnosis of MH
Molecular tests based on genetic analysis are desirable
because they are less invasive and more informative for pa-
tients, especially when familial mutation is known. However,
the complexity of this gene and the increasing number of
reported mutations limit routine screening of RYR1 in MHS
families. To this end, the development of a genetic test is
underway in several countries, as well as the elaboration of
guidelines applicable to molecular genetics (18,19).
A previous study of a Brazilian MH family revealed a
new mutation in RYR1, Arg2355Cys (20), which permitted
a genetic diagnosis according to the guidelines proposed in
other members of the family that could not undergo CHCT.
Genetic screening for RYR1 was reported thereafter in
other Brazilian families (21,22). The Brazilian MH Center
has registered many other families who are affected by this
disorder but have not been investigated genetically.
In the present investigation, we studied a large multi-
generational Brazilian family, in which several deaths and
cases of hypermetabolic crisis under general anesthesia
had occurred and were associated with MH. First, an MHS
member of the family diagnosed by CHCT was screened for
mutations in the RYR1 gene. Then, the genetic diagnosis
test was applied to the rest of the family.
Subjects and Methods
Blood samples from 101 individuals of a Brazilian fam-
ily were sent to the Brazilian MH Center of Universidade
Federal do Rio de Janeiro (UFRJ) for analysis. Eight of
these individuals had already been diagnosed by CHCT
according to the North American protocol (17) at the Brazil-
ian MH Center of UFRJ. Three of them (IV-37, IV-41 and
IV-42) were diagnosed as MHS and 5 (III-5, III-7, III-11,
IV-30, and IV-31) were diagnosed as MH normal. It should
be noted that IV-42 had a positive result for the test with
caffeine but a negative result for the test with halothane,
being diagnosed as MHS. The clinical history of the family
describes death that could be associated with MH in II-1
and III-1 and with a hypermetabolic crisis after general
anesthesia in IV-54.
Genomic DNA was extracted from whole blood samples
using the phenol-chloroform method (23). All the exons
within the three mutational hot spots were sequenced
using the Big DyeTerminator™ Cycle Sequencing kit and
ABI 3100 Genetic Analyzer (Perkin Elmer, USA). A novel
variant was found in exon 102 of RYR1. Primers 5’-CCTCC
CTCAGTGTTACCTGTTT-3’ and 5’-GCCCAAGGTCTCAC
AGTAGC-3’ were used for PCR amplification of a 296-bp
fragment that includes the locus of the mutation in exon
102. In some members of the family investigated, this
mutation was also detected by restriction analysis. DNA
sequence analysis revealed that the mutation created the
restriction site GGATC for the enzyme HinfI. DNA fragments
were then digested with HinfI (Invitrogen, USA) according
to the manufacturer protocol, at 37°C for 1 h. Restriction
fragments were then fractionated by agarose gel electro-
phoresis and visualized by staining with ethidium bromide
(GE Health Sciences, USA). Next, the fragments of exon
102 from 19 members of the family chosen at random were
sequenced in both directions at Macrogen Inc. (Korea) to
confirm the results of restriction analysis. DNA sequenc-
ing was analyzed using the Contig and Align from Vector
program (available at www.invitrogen.com). In addition, 100
control subjects from Brazilian and USA populations were
screened for this variant.
CHCT data for the MHS individuals of the family are
presented in Table 1, including individual IV-42, who had a
positive contracture response to the test with caffeine (3.2
g - threshold: 0.2 g), but not for the test with halothane (0.5
g - threshold: 0.7 g). The clinical data of the family revealed
an MH reaction under general anesthesia for individual IV-
54 and an unexplained sudden death associated with MH
for individuals II-1 and III-1. The complete pedigree for six
generations of this family is shown in Figure 1.
After screening for the hot spots of the RYR1 gene, a
novel variant was found in exon 102, producing the missense
mutation Gly4935Ser, which corresponds to a G14803A
change at the DNA level. Furthermore, this alteration creates
a new restriction site for the enzyme HinfI, which was used
Table 1. Caffeine halothane contracture test data of some of the
malignant hyperthermia-susceptible members of the family.
SubjectCaffeine (g)Halothane (g)
The maximal contracture response of the muscle fragment from
each individual in the test of exposure to caffeine (2.0 mM) and
halothane (3%) is shown. The threshold contracture value for the
test to be considered positive is 0.2 g for caffeine and 0.7 g for
halothane. Note that all individuals had positive results for the
tests with caffeine and halothane, except for individual IV-42, who
was positive to caffeine but not to halothane.
A.R. Matos et al.
Braz J Med Biol Res 42(12) 2009
Figure 1. Pedigree of the Brazilian family of this study. The green symbols indicate individuals diagnosed as malignant hyperthermia
(MH)-susceptible by the caffeine halothane contracture test (CHCT). The light blue symbols indicate individuals diagnosed as MH
normal by CHCT. The red symbols indicate individuals who carry the Gly4935Ser mutation. The dark blue symbols indicate individuals
who do not carry this mutation. The open symbols indicate individuals who were not studied by either the DNA test or CHCT. Note that
6 children descending from the first individual (I-1) generated the rest of the pedigree (II-1 to II-6).
Novel mutation for malignant hyperthermia
Braz J Med Biol Res 42(12) 2009
to investigate all the members of the family. After restriction
analysis of the DNA fragment containing exon 102, the
occurrence of the missense mutation was detected by the
presence of additional fragments of 166 and 130 bp, and the
original fragment of 296 bp, visualized by agarose gel elec-
trophoresis (Figure 2). To confirm these results, 19 samples
chosen at random were sequenced in both directions as
described. This mutation was found in 26 members of this
family. The electropherograms from individuals displaying
or not the mutation are shown in Figure 3.
This mutation was found in individuals IV-37 and IV-41,
who had been previously diagnosed as MHS by CHCT, but
not in individual IV-42, who was confirmed by experiments
of DNA restriction and sequencing analysis, also diagnosed
as MHS by CHCT. The results in Table 1 show that the
CHCT results for individuals IV-37 and IV-41 were positive
for the test with caffeine and halothane, while the results for
individual IV-42 were only positive for the test with caffeine.
Individuals III-13 and IV-43 DNAs were not available.
One hundred and one individuals from the family were
screened for the Gly4935Ser variant, which was found in
26, including 2 MHS individuals previously diagnosed by
CHCT. This variant was not found in any of the 5 individu-
als who had been previously diagnosed by CHCT as MH
normal (III-5, III-7, III-11, IV-30, and IV-31). Screening for
this variant in 100 control subjects, including Brazilian and
USA populations, showed negative results.
We studied a large Brazilian family distributed along six
generations with a history of MH. Some of the members
were suspected to have died due to MH and others were
diagnosed as MHS by CHCT. A novel amino acid variant,
Gly4935Ser, was identified by direct sequencing. This
variant was found in 26 members of the family, including
Figure 2. Photographs of 1.2% agarose gels stained with ethidium bromide and visualized under UV light. A, PCR amplification; note
that the arrow shows the 296-bp PCR fragment resulting from the individuals indicated above the panel. B, Restriction analysis of the
DNA fragments with the enzyme HinfI, which cuts the fragments that carry the mutation. Note that the DNA fragments from individuals
IV-46, IV-47, IV-51, IV-52, and IV-39 do not have their resulting band cut by the enzyme because no additional band was observed,
indicating absence of the mutation. Individuals IV-53, IV-54, IV-55, IV-56, and IV-38 had their resulting band cut by Hinf because they
all have two additional bands (166 and 130 bp) and the original band fragment (296 bp), indicating the presence of the mutation in one
allele of the gene. 100 bp - Molecular weight marker.
A.R. Matos et al.
Braz J Med Biol Res 42(12) 2009
two previously diagnosed MHS, individuals IV-37 and IV-
41. The CHCT of these individuals were both positive to
halothane and caffeine (Table 1), showing agreement of
the phenotype with the genotype results.
In the other MHS individual diagnosed, IV-42, this muta-
tion was not found, as confirmed by experiments of DNA
restriction and sequencing analysis. This individual was
sensitive to caffeine but not to halothane during the CHCT,
a fact that raised the possibility of a false-positive test for
this subject. According to the North American protocol, a
person is diagnosed as MHS if he shows a positive result to
one of the substances of the test (17). However, according
to the European MH Group (EMHG) protocol, a person is
diagnosed as equivocal for MH when only one of the tests
shows a positive result (24).
It should be considered that CHCT has a high sensitivity
of 99%, but a lower specificity of 78% for the North American
protocol (25). Discordance between CHCT and genetic stud-
ies has already been reported (26-30), indicating that some
familial mutations are not found in some MHS members of
the family. It is also possible that this discordant individual
has another mutation in another part of RYR1 not analyzed
or in another gene related to MH (4-7). Some families that
display more than one causative mutation leading to MH
have been reported (16,31).
The missense mutation Gly4935Ser is located in exon
102, inside the last hot spot of the gene. This region has
been reported to be less frequently mutated than the others
(32). The position Gly4935 is evolutionarily conserved and
also conserved between the other two isoforms of RyR and
the IP3R protein (Figure 4) and its mutation was absent
among the 100 control subjects studied here.
The C-terminus of the protein is a hydrophobic region
that contains the conduction pore of the Ca2+ release chan-
nel (33), the transmembrane segments (34,35), one of the
binding sites for Ca2+ (36), and the binding site for ryanodine
(37). According to a proposed model (38), the position of the
Gly4935 is at the end of the last transmembrane domain of
RyR1, very close to the C-terminus of the protein.
We conclude that the Gly4935Ser variant is a candi-
date mutation for MHS in this Brazilian family, based on its
co-segregation with disease phenotype, absence among
controls and its location within the protein.
The CHCT is not easily available in Brazil. Its limited
availability and invasiveness prevented its use in high-risk
family members. Our study is an example of the possible
use of a molecular genetic test for the diagnosis and preven-
tion of MH. A genetic test for MH is especially important for
large families in which many members, particularly children,
can benefit without undergoing an invasive contracture
test. This is even more important because, as a result of
the speed and ease with which the analysis is carried out,
a more comprehensive profile can be achieved.
This study was supported in part by FAPERJ, CNPq,
CAPES, Cristália Produtos Químicos e Farmacêuticos
Ltda., FUJB, and by CNPq fellowships (A.R. Matos, G.
Zapata-Sudo, R.T. Sudo).
Figure 3. DNA sequencing of the fragment generated by PCR,
which contains a region of exon 102 of the RYR1 gene including
the locus of the mutation. The Gly4935Ser mutation corresponds
to a G14803A change at the DNA level. A, Electropherogram
from an individual without this RYR1 mutation. The arrow shows
a single peak corresponding to a G at nucleotide position 14803.
B, Electropherogram from an individual carrying the RYR1 mu-
tation. In contrast, there are two superposed peaks, which cor-
respond to a G and an A at nucleotide position 14803, where
the program shows the letter N (arrow). This indicates that this
person is heterozygous for this mutation.
Figure 4. Comparison of the amino acid sequences of the re-
gion of the mutation in RyR1 found in the family. Note that this
is a conserved domain among different species and isoforms of
RyR, as well as in the IP3R protein. Letters in bold and under-
lined correspond to the site of the mutation. Normal letters are
the conserved amino acids. Letters in italics correspond to non-
conserved amino acids in the sequence.
Novel mutation for malignant hyperthermia
Braz J Med Biol Res 42(12) 2009
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