SHORT REPORT ABSTRACT: In this study we present 3 families with malignant hyperther-mia (MH), all of Indian subcontinent descent. One individual from each ofthese families was fully sequenced for RYR1 and presented with the non-synonymous change c.11315G>A/p.R3772Q. When present in the homo-zygous state c.11315*A is associated with myopathic symptoms.

Muscle Nerve 40: 633–639, 2009

A RYR1 MUTATION ASSOCIATED WITHRECESSIVE CONGENITAL MYOPATHY ANDDOMINANT MALIGNANT HYPERTHERMIA INASIAN FAMILIES

DANIELLE CARPENTER, PhD,1 AZZAM ISMAIL, FRCPath,2

RACHEL L. ROBINSON, PhD,1 CHRISTOPHER RINGROSE, BSc,1

PATRICK BOOMS, PhD,3 DAVID E. ILES, PhD,3 P. JANE HALSALL, MBChB,1

DEREK STEELE, PhD,4 MARIE-ANNE SHAW, PhD,3

and PHILIP M. HOPKINS, MD, FRCA1

1MH Investigation Unit, Academic Unit Anaesthesia, St. James’s University Hospital,Leeds LS9 7TF, UK

2Department of Histopathology, Leeds General Infirmary, Leeds, UK3 Institute of Integrative and Comparative Biology, Faculty of Biological Sciences,University of Leeds, Leeds, UK

4 Institute of Membrane and Systems Biology, Faculty of Biological Sciences, Universityof Leeds, Leeds, UK

Accepted 31 March 2009

Malignant hyperthermia (MH) is a potentially life-threatening pharmacogenetic disorder. Susceptibleindividuals have a defect in skeletal muscle thatmanifests as an increased release of calcium intothe myoplasm under certain conditions. Undernormal physiological conditions this can be com-pensated for by increased rates of calcium seques-tration. However, inhalational anesthetics and themuscle relaxant suxamethonium can trigger anMH crisis during which a massive concentration ofcalcium accumulates within the cells. This leads toacceleration of muscle metabolism and contractileactivity. This generates heat and leads to hypoxe-mia, metabolic acidosis, rhabdomyolysis, and a

rapid rise in body temperature, which can be fatalif it is not recognized and treated promptly.

Laboratory confirmation of suspected MH casesrequires open skeletal muscle biopsy and a subse-quent in vitro contracture test (IVCT), for whichthere is a standardized and validated protocol. TheIVCT involves exposure of freshly excised musclebiopsy specimens to incremental concentrations ofhalothane or caffeine, and the extent of musclecontracture is measured. The phenotypes of MHsusceptible (MHS), MH normal (MHN), or MHequivocal (MHE) are assigned according to the Eu-ropean MH Group protocol (www.emhg.org),where the MHE phenotype represents increasedsensitivity to one of the stimulants only.

MH exhibits an autosomal-dominant mode ofinheritance. The gene that encodes the skeletalmuscle ryanodine receptor (RYR1 on chromosome19q13.1) has been shown to play a major role. Itaccounts for more than 70% of MH cases in theUK,1 and recently a total of 178 non-synonymouschanges were described across RYR1.1 Mutations inthis gene have also been described in connectionwith patients suffering from congenital myopa-thies,1–3 including central core disease (CCD; 53

Abbreviations: ATPase, adenosine triphosphatase; CCD, central coredisease; CK, creatine kinase; IVCT, in vitro contracture test; MH, malig-nant hyperthermia; MHE, MH equivocal; MHN, MH normal; MHS, MH sus-ceptible; MmD, multiminicore disease; NADH, nicotinamide adeninedinucleotide; PAS, periodic acid–Schiff; SDH, succinate dehydrogenase;SNP, single-nucleotide polymorphism

Correspondence to: D. Carpenter; e-mail: danielle.carpenter@nottingham.ac.uk

VC 2009 Wiley Periodicals, Inc.Published online 30 July 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mus.21397

Key words: Indian subcontinent; King–Denborough syndrome; malignanthyperthermia (MH); myopathy; RYR1

Short Reports MUSCLE & NERVE October 2009 633

reported RYR1 mutations), multiminicore disease(MmD; 17 reported RYR1 mutations), and an iso-lated case of a congenital myopathy characterizedon histology by cores and rods.1,2 Patients with my-opathy are often susceptible to MH, although thesegregation of MH and myopathy is not always con-cordant within families.4

Herein we describe a non-synonymous changein RYR1 (p.R3772Q) that we believe to be of partic-ular interest. It has been detected exclusively inpatients of Indian subcontinent descent, and, dueto the presence of consanguineous marriages, ispresent in both the heterozygous and homozygousstate. Interestingly, this change has previously beendescribed in two other unrelated consanguineousfamilies, including one from Asia.5 The clinicalphenotypes and muscle histology observed with thismissense change in 3 families, show for the firsttime that, in the homozygous state, it appears tooccur with both myopathy, MH phenotypes, and insome instances as an overlap with King–Denbor-ough syndrome. This pattern is characteristic of arecessive mode of inheritance, whereas in the heter-ozygous form only the MH phenotype is observed.

METHODS

MH Phenotyping. The MH phenotype of each indi-vidual in this study has been determined using theIVCT according to the European MH group proto-col (www.emhg.org).

Muscle Histopathology. Fresh samples from theopen muscle biopsies were fixed and stained usingthe following:

1. Hematoxylin and eosin (H&E), for general mus-cle fiber morphology.

2. Gomori trichrome, for general muscle fibermorphology and visualization of nemaline rods.

3. Nicotinamide adenine dinucleotide (NADH), astain for oxidative enzymes and the principalstain for visualizing cores.

4. Succinate dehydrogenase (SDH), a stain for mi-tochondrial pathology.

5. Periodic acid–Schiff (PAS), which stains glyco-gen stores.

6. Fiber typing, using pH-specific adenosine tri-phosphatase (ATPase).

Screening of c.11315G>A/p.R3772Q. The RYR1mutation c.11315G>A/p.R3772Q in exon 79 was

identified in 3 apparently unrelated samples froma cohort of 96 independent MHS individuals, onwhich full RYR1 cDNA sequencing was performedand has previously been described.1 Subsequentscreening of a further 374 independent UK MHpatients was done using an assay developed in-house as follows: forward 50-GCA GGA GTG AGAATG TGA GG-30, and reverse 50-AGG AGG AGTGAG GTT AGG AT-30 primers generating anamplicon of 236 bp in length. In the normal situa-tion (c.11315*G) this is cleaved by MspI to pro-duce 2 fragments of 131 bp and 105 bp. Cleavagefails to occur in the presence of the mutated allele,c.11315*A. When mutations were detected,extended pedigrees were investigated, wheneverpossible, to assess the concordance between clini-cal, histological, and IVCT phenotypes andc.11315G>A genotype. The c.11315G>A mutationwas not detected on screening the 374 independ-ent UK MH patients nor on screening 200 inde-pendent normal controls.

RESULTS

Screening of 470 independent UK MH patientsidentified 3 families carrying the mutationc.11315G>A in concordance with the MH IVCTphenotype. These families were exclusively of In-dian subcontinent descent and account for 43% (3of 7) of all families in the UK National MH regis-ter of this descent. This mutation was not observedin families of any other descent in the UK cohort.Interestingly, 4 patients were further observed tocarry two copies of the c.11315*A allele, likely as aconsequence of consanguineous marriages, andthis led to a more complex myopathic phenotype.

Family 1. Family 1 is a two-generation family (Fig.1). The MH proband (I:i) presented with spasm ofthe jaw muscles after exposure to suxamethonium,followed by tachycardia, mixed respiratory andmetabolic acidosis, and a temperature of 38.5�Cduring maintenance of anesthesia. Postoperatively,he developed myoglobinuria, a creatine kinase(CK) level of 300,000 IU/L, and severe musclestiffness for 4–5 days. This individual had no clini-cal features of myopathy, and muscle histology wasnormal. Unfortunately, no DNA was available formolecular analysis. Following referral of I:i, weestablished that 2 other family members, II:ii andII:vi, had developed problems during anesthesia.

Individual II:vi, the product of a consanguine-ous marriage in generation I, has a congenital my-opathy that presented in infancy with muscle

634 Short Reports MUSCLE & NERVE October 2009

flaccidity and weakness, Achilles tendon contrac-tures, and eventration of the diaphragm. She hadher first general anesthetic at age 3 months for pli-cation of the diaphragm, with a myotonic responseto the suxamethonium noted. A muscle biopsytaken during that procedure was reported to show‘‘mild non-specific myopathic changes.’’ At 5 yearsof age she had a reaction during anesthesia forrelease of Achilles tendon contractures. She devel-oped tachycardia, tachypnea, a temperatureincrease, and mixed respiratory and metabolic aci-dosis. Postoperatively, serum CK concentration was7360 IU/L. She was first investigated for MH suscep-tibility when she was age 14 years. She was thin andof short stature with mild weakness of the limbs; alltendon reflexes were reduced, with the legs being

more affected than the arms. The serum CK concen-tration, measured from a blood sample taken priorto the muscle biopsy, was 1298 IU/L. The results ofthe IVCT indicated an MHS phenotype, while thehistology was also abnormal. There were markednon-specific myopathic features with necrosis and fi-brosis of the muscle, internal nuclei, and cores lack-ing oxidative enzymes. Screening for c.11315G>A/p.R3772Q confirmed that II:vi was homozygous forthe non-synonymous change c.11315*A in RYR1.Full cDNA sequencing of RYR1 revealed no othernon-synonymous change in the gene.

The cousin of II:vi, II:ii, was also confirmed tobe homozygous for c.11315*A through DNAscreening. He presented for investigation of MHsusceptibility at 25 years of age. He had a history

FIGURE 1. Family pedigree information. Each pedigree is shown in duplicate with the inheritance of MH and myopathy drawn separately, in

combination with the genotypic information (GG/GA/AA), and individual identifiers. Disease status is shown by the shading. Solid black

symbols represent the phenotypes of either MHS or myopathy, black and white symbols represent MHE, and white symbols represent a nor-

mal phenotype. Individuals with a ‘‘?’’ have an unknown phenotype. A double bar between parents represents a consanguineous marriage.

Short Reports MUSCLE & NERVE October 2009 635

of muscle weakness, principally in the legs. At 12years of age, a tonsillectomy procedure was aban-doned because his muscles ‘‘went tight’’ afterinduction of anesthesia. On examination there wasobvious wasting of the peroneal and calf muscles.The preoperative serum CK concentration was2857 IU/L. The results of the IVCT indicated anMHS phenotype, while the histology had featuresin common with that of II:vi. There was markedvariation in fiber size with small atrophic type 1and 2 fibers, hypertrophic type 1 fibers with manyinternally located nuclei, and necrotic and regen-erative fibers (Fig. 2). With NADH staining therewere distinct peripheral cores, mainly in the type Imuscle fibers (Fig. 3).

The four siblings to the proband, I:ii, I:iii, I:iv,and I:v, the two nephews, II:i and II:v, and theniece, II:viii, are all clinically and histologically nor-mal, but they are heterozygous for mutation

c.11315G>A. Three of the siblings (I:iii, I:iv, andI:v), and the niece (II:viii) have the abnormalIVCT phenotype, MHE, with increased sensitivityto halothane but not caffeine, whereas the othersibling (I:i) and the nephews (II:i and II:v) havethe MHS IVCT phenotype. Individuals II:iii, II:iv,and II:vii have the MHN (normal) IVCT pheno-type and normal muscle histology consistent withthe genotype of wild-type homozygote c.11315*G.

Family 2. Family 2 is a two-generation family inwhich there is a suspected consanguineous mar-riage. Individual II:i was found to be homozygousfor c.11315*A through DNA screening. The familywas referred for MH testing, as the clinical featuresof II:i were consistent with a diagnosis of King–Denborough syndrome, an illness associated withMH. The child (II:i) presented with scoliosis, dys-morphic features (ptosis, widely spaced eyes),

FIGURE 2. Histological samples (H&E stain; original magnifica-

tion �100) from individual II:ii in family 1 (A) and individual I:I in

family 1 (B). Histological slide (A) shows features of myopathy.

There is marked variation in fiber size, many small angulated

and atrophic fibers (square), fibers with internal nuclei (rounded

rectangle), and one necrotic fiber (circle). Histological slide (B)

shows a normal muscle phenotype and no evidence of myopa-

thy. [Color figure can be viewed in the online issue, which is

available at www.interscience.wiley.com.]

FIGURE 3. Histological studies (NADH stain) from individual II:ii

in family 1 (A) and individual I:i in family 1 (B). Histological slide

(A) (original magnification �200) shows evidence for areas

within the muscle fibers that are deficient in oxidative enzyme

activity (cores), at the periphery of the sarcoplasm (rectangles).

Histological slide (B) (original magnification �100) shows a nor-

mal muscle phenotype with no evidence of cores. [Color figure

can be viewed in the online issue, which is available at

www.interscience.wiley.com.]

636 Short Reports MUSCLE & NERVE October 2009

kyphosis, lumbar lordosis, and undescended testes;serum CK concentration was slightly raised at 351IU/L. A muscle biopsy performed at 2 years ofage during the course of an orchidopexy proce-dure showed marked variation in fiber size, loss offiber-type differentiation, and ‘‘marked moth-eatenchange’’ on NADH stain. An anesthetic regimenthat avoided MH-triggering agents was uneventful.As II:i was too young for IVCT testing, his father,and subsequently his siblings, were tested for MHsusceptibility.

The father (I:ii) had a normal clinical pheno-type, but his CK was slightly raised at 301 IU/L.His IVCT phenotype was MHS, but he had normalmuscle histology and was heterozygous for themutation c.11315G>A. The siblings of the pro-band were clinically normal, and both had abnor-mal IVCT responses. II:ii had the MHE phenotype(increased sensitivity to halothane only), and II:iiihad the MHS phenotype. Both had normal musclehistology but, interestingly, II:ii had a slightlyraised CK of 258 IU/L and was homozygous forc.11315*G. II:iii had a normal CK of 153 IU/L butwas heterozygous for the mutation c.11315G>A.Individual II:iii has been fully sequenced for RYR1and is known to possess no other non-synonymouschange in this gene. The mother of the probandhas not been tested for MH susceptibility, but sheis heterozygous for the mutation c.11315G>A.

Family 3. The MH proband of family 3 (I:i) ishomozygous for c.11315*A, which has been con-firmed through DNA screening. He has been fullycDNA sequenced for RYR1 and is known to possessno other non-synonymous changes. This individualpresented with persistent tachycardia, hyperventila-tion, and temperature increase under general an-esthesia for an orchidopexy procedure. He hadmuscle stiffness for 1 week postoperatively. He wassubsequently referred to a clinical geneticist whosuspected King–Denborough syndrome because ofdelayed motor milestones, dysmorphic features,kyphosis, lumbar lordosis, scoliosis, and unde-scended testes. There was muscle weakness in thelegs and arms, and a history of cramps in thecalves. Serial blood samples revealed a slightly ele-vated serum CK in the range 270–370 IU/L. Hewas evaluated for MH susceptibility at the age of11 years. The results of testing revealed the MHSIVCT phenotype. Histological examination showednon-specific myopathic changes with minimal varia-tion in fiber size and occasional angulated andatrophic muscle fibers, but no evidence of cores

(Fig. 4). The parents are cousins, neither of whomhave any clinical features of myopathy. Their MHstatus and genotypes are unknown, and they havenot had a muscle biopsy.

As full RYR1 cDNA sequencing was carried outon a single member of each of these families, 36potentially informative exonic single-nucleotidepolymorphism (SNP) loci were identified acrossthe 158-kb RYR1 gene, as previously described.6

Thirty were located before and 6 after the c.11315site (Fig. 5). A detailed study from the RYR1 cDNAsequencing profile of 2 of the c.11315*A homozy-gous individuals, II:vi of family 1 and I:i of family3, showed homozygosity at all 36 exonic SNP loci,and identical genotyping for both individuals. Thissuggests a single common founder haplotype. TheRYR1 cDNA sequencing profile for the heterozy-gous individual who presented with MH only, II:iiiof family 2, identified a single copy of the haplo-type surrounding the c.11315 site. This is identicalto the haplotype homozygous in the other 2 indi-viduals, but it was broken down 23 kb distal to thec.11315 site in the genomic DNA, prior to the SNPp.Pro3062 (Fig. 5).

DISCUSSION

Observations from these 3 families suggest that car-riage of allele c.11315*A can result in susceptibilityto MH, and that carriage of two copies of this al-lele leads to the MH phenotype along with a com-plex muscle phenotype. Each family expressed themutation in the homozygous state, and all 4affected individuals had an apparently non-pro-gressive mild congenital myopathy. Two also hadfeatures of King–Denborough syndrome. Many of

FIGURE 4. Histological sample (H&E stain; original magnifica-

tion �100) from individual I:i in family 3, shows evidence of mild

myopathic changes and marked variation in muscle fibers with

some pale fibers. [Color figure can be viewed in the online

issue, which is available at www.interscience.wiley.com.]

Short Reports MUSCLE & NERVE October 2009 637

the histological changes in the muscle of these 4individuals with confirmed homozygous status wereof a non-specific nature, but cores were identifiedin the two homozygous members of family 1. Inter-estingly, a previous observation with this mutationdemonstrated multiple cores in the homozygousstate.5

The presence of a dominantly inherited myopa-thy associated with malignant hyperthermia wasfirst reported by King and colleagues.7 The 2 casesreported herein in families 2 and 3 represent thefirst examples in which the underlying geneticdefect has been demonstrated in the homozygousstate. The fact that not all homozygous carriers ofthis mutation have sufficient clinical features to bedefined as having the syndrome, and indeed thespectrum of the myopathic features of this geno-type, suggests a potential for modifier effects. Thepresence of MH susceptibility in individual II:ii offamily 2 in association with the homozygousc.11315*G (wild-type) genotype suggests (an) addi-tional genetic factor(s) in operation, at least inthis family. It should be further noted that, due tothe presence of consanguineous marriages, thepatients presenting as homozygous at the RYR1locus are probably homozygous at other, poten-tially deleterious, loci. These other loci couldpotentially be modifier loci, which may have a sig-nificant influence on the resulting phenotype, par-ticularly in the manifestation of King–Denboroughsyndrome. Further analysis of candidate genes inMH and myopathy would be of interest.

Homozygosity of RYR1 mutations associatedwith human MH has been reported rarely, andreports from murine studies have suggested thathomozygosity of certain RYR1 mutations can bedevelopmentally deleterious, causing death inutero or immediately postnatally.6,8 However, por-cine MH is associated exclusively with recessive in-heritance of p.R615C.9 Several RYR1 mutationshave been observed previously in the homozygousstate in association with recessive CCD10 andMmD,5,11 but their overlap with the MH phenotypeis unclear. A functional study of some previouslydescribed recessive mutations suggested a small butsignificant effect on resting Ca2þ concentrations inthe homozygous state, but these mutations may notcause MH in the heterozygous state.12

Considering both the phenotypic and geno-typic data presented herein, carriage of allelec.11315*A can result in an MH phenotype,whereas in the homozygous state both MH andclinical myopathy are observed. It would be ofinterest to conduct an independent functionalanalysis of the c.11315G>A to support theseobservations.

This study was supported by a grant through the Department ofHealth Pharmacogenetics Research Grant Programme.

REFERENCES

1. Robinson RL, Carpenter D, Shaw M-A, Halsall J, HopkinsPM. Mutations in RYR1 in malignant hyperthermia andcentral core disease. Hum Mutat 2006;27:977–989.

FIGURE 5. SNP breakdown for the 3 individuals, 1 from each family, that were fully sequenced for the coding region of RYR1 (family

1, individual II:vi, coded as F1 II:vi; family 2, individual II:iii, coded as F2 II:iii; family 3, individual I:i, coded as F3 I:i). Each exonic SNP

is given in consecutive order along RYR1, with exonic position, rs number, where ascribed, and distance between adjacent SNPs pre-

sented. The identified mutation p.R3772Q is shown in bold.

638 Short Reports MUSCLE & NERVE October 2009

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