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32 Payne, A. N., Garland, L. G., Lees, 1. W. and Salmon, J. A, (1988) Br. 1. Pharmocol. 94,W

33 Higgs, G. A.. Follenfant, R. L. and Gadand, L. G. (1988) 3~. 1. Fha~ac~l. 94.547-551

34 R&ndeau, D., Falgueyret, J-P., Guay, J., l&da, N. and Yamamoto, S. (1991) Biochem. I. 274.287-292

35 Rouzer, C. A., Riendeau. D., Falgueyret, J-P.. Lau, C K. and Gresser, M. J. (1991) &hem. P~a~a~l. 41,136S-1373

36 Bird, T. G. C. et al. (1991) J. Med. Ckem. 34,2176-2186

37 McMilian, R. M. et d. (1991) Agents Actions 34.110-112

38 McMiUan, R. M., Girodeau, J-M. and Fester, S. J. (1990) Br. J_ Pku~acal. 101, 501-5@3

39 Crawley, G. C. et ul. 1. Med. Chent. (in p-3 -

40 McMiUan, R. M., Spruce, K. E., Crawley, G. C. Walker, E. R. H. and Foster, S. J.

41 Coutts, S. M. ei cii. (i9Ss5) in Prosta- glandins, Leuk&ienes and Lipoxins ;zz J. M., sld.), pp. 627-637, Plenum

42 Musser, J. H. and Kreft, A. F. (1990) Drugs Fut. 15,73-80

43 Kreft, A. F., Musser, J., Marsha& L. and Gromes. D. 11990) DruPs ht. 15.805-807

44 Proudman, ‘K. E’., MGores, S.. M. and M&i&m, R. M. (1991) Br. I. Pharmacol. 102,364P.

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47 AnciIl, R. J.. Takahashi, Y.. Kibune, Y., Cambell, R. and Smith, R. J. (1990) f. lnt. Med. Res. 18.75-88

48 McMilian, R. M., Millest, A. J., Proudman, K. E. and Taylor, K. B. (1986) Br. J. Pharmacol. 87,53P

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50 Rubin, P. et al. (1991) in Progress in Inflammation Research and Therapy (Ackerman, N., Bonney, R. and Docherty, N., eds), pp. 103-112, Birkhauser

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The genetic basis of malignant hypothermia David H. Maclennan

Anaesthzsia can induce skeletal muscle rigidity, hypermetabolism and high feuw in humans genetically predisposed to malignant hyperthermia. If not immediately reversed, such episodes can lead to tissue damage and death. In swine with the ~o~e~~on~ng condition, stress can induce death or lead to valued meat p~ducts. Since muscle contraction is controlled by sarco- plasmic Cd’, the abnormality, as reviewed here by David H. MacLennan, could reside in the skeletal muscle Ca2+ -release channel gene, RYRl. Several observations support the view that a single RYRl mutation is causal of mulignant hyperthennia in all breeds of pigs and in at least some human families: the subs~~tton of Cys for Arg625 as the sole deduced amino acid sequence change in a comparison of malignant hyperthermia and normal porcine RYRl cDNAs; the linkage of this mutation to malignant hyperthermia in over450 pigs in six breeds, including 338 meioses; and the appearance of the corresponding mutation, Cys for Argfil4, across a species barrier, in a few human families, where it also cosegregates with malignant hype~hermia. Linkage of mali~ant hype~he~ia to RYRl is, however, not observed in all human families with malignant hyperthermia. Accordingly, other abnormal genes that may cause the condition are being sought.

Malignant hyperthermia is an in- herited condition in which there is skeIetal muscle contracture with attendant hypermetabolic and hyperthermic reactions. In humans heterozygous for the abnormality, it is most often triggered by a combination of potent inhalational anaesthetics and depolarizing skeletal muscle relaxantsl. There are also indications that physical or emotional stress, excitement or

I). H. Mactennnn is f. W. Biiies Professor of Me&Cal Research, Bunting and Best Depart- ment of Medical Research, University of Toronto, Charles H. Best Institute, 112 College Street, Toronto, Ontario, Canada M5G lL6.

sudden changes in environmental temperature can bring on the symptoms. In swine homozygous for the abnormality, stress is the major triggering factor, hence the name ‘porcine stress syndrome”. The human syndrome was charac- terized in the mid 195Qs, following the extensive anaesthetic use of halothane and suxamethonium. In certain families, the combination was fatal to genetically predisposed individuals3. In humans, the syn- drome appears in about 1 in 15 000 administrations of anaesthetic in children, and 1 in 50000 adult anaesthetics. In swine the inci-

dence is variable, but at least IO-15% of commercial animals are heterozygous for the genetic defect and l-Z% are homozygous.

The aetiopathogenesis of the disease has become apparent from a wide range of studies of both the human and porcine syn~me1,2. In muscle, contraction and gly- colysis both require cytoplasmic Ca2+, which is normally closely regulated by the sarcoplasmic reticulum, together with a series of pumps, channels and exchangers in the muscle cell membrane. Ab- normalities in the Ca’“- release channel of the sarcoplasmic reticu- lum or, potentially, in other Ca*+ regulatory molecules in the cell, manifested under conditions of anaesthesia or stress, would flood muscle cells with excess Ca’+, The resulting sustained muscle con- traction and enhanced glycolytic and aerobic metabolism would use up ATP, glucose and oxygen, produce excess COa, lactic acid and heat, and generate temper- atures as high as 47°C (Fig. 1).

Early clinical symptoms of a malignant hyperthermia episode include skeletal muscle rigidity, elevated end-tidal C02, tachyp- noea, tachycardia, fever, cyan- osis, hypercapnia, lactic acidosis, hyperkalaemia, unstable blood pressure and arrhythmia. If ther- apy is not initiated immediately, death can occur within minutes from ventricular fib~llation, within hours from pulmonary oedema or coagulopathy, or with- in days from neurological damage or obstructive renal failure. Thus

TiPS - August 1992 [Vol. 131 331

normal malignant hyperthemtia

Cap+

glycogen I /I ‘I

glucose-l-P \ III

lack acid

Fig. 1. A proposed mechanism for induction of malignant hyperthem?ia due &J aQnormatities in the Ca*+-retease channel of streletat muscle sarcoplasmic reticulum. Muscle contraction and giycolysis are regulated by cytoptasmic Ca2+ concentrations. In a nomu/ muscle relaxation-contraction cycte, Caps is pumped into the sarcoptasmic reticuium by a Ca2+-transporting ATPase to initiate retaxation, stored within the lumen in association with calsequestrin and releas& through a Ca”-release channel to initiate contmct/on. t3iycotyttc and aerobic metabolism proceed onty rapidfy enough to maintain the energy balance of the ceil. The Ca2+-retease channel can be regulated by Ca*+ itsell ATP, Mg2+ and calmoduiin and, even when stimulated, has a relativefy short open time. In nW#nmt hyparrla, the abnormal Ca2+-release channel is hypersensitive to stimulators of opening and does not close read//y. Hatothane and suxamathonium, as well as other agents inducing malignant hyperthennia, act either directty or fndirectty to hotd the abnormal channel in a hmfler open state, fiooding the cell with Ca*+ und overpowering the Cap+ pump in its attempts to lower oytopfasmic Cap+. Sustained muscte uWactton accounts for rigidity, while sustained glycolytic and aerobic meta&o/ism account for the generation of heat and tactic aeM Tertiary imbalances of ion transport and damage to cell membranes account for the Iife4hreatening systemic problems that a#ear during the progression of a ma/ignant hyperthermia episode. Adapted from Meclennen, D. Il. and Phillips, M. S. (1982) Science 256,78%7W copyright 1992 by the AAAS.

management of the disease re- quires early detection, coupled with measures to reverse its course. In the case of human malignant hyperthermia, itifusion of dantrolene4 to lower intra- cellular Ca2+ concentration has been proven to reverse the pro- gress of the disease. The death rate has been lowered from over 80% to less than 10% through the use of appropriate monitoring for early detection and dantrolene therapy.

A major goal of malignant hy- perthermia research has been to identify individuals susceptible to malignant hyperthermia in ad- vance of anaesthesia. To this end, in vitro caffeine/halothane con- tracture tests were developed, in which fibres from muscle biopsies are attached to a force-displace- ment transducer and then exposed to incremental doses of caffeine, to single or incremental doses of halothane, or to incremental doses of caffeine in the presence of halo-

thane5s6. Under North American’ and European8 protocols, the tests differ both in procedure and in criteria for defining malignant hyperthermia susceptibility. In particular, the North American group defines those individuals whose in vitro tests are positive for either caffeine or halothane as ‘malignant hyperthermia suscep- tible’. The European group de- fines such results as ‘malignant hyperthermia equivocal’ and re- quires positive reactions with both halothane and caffeine for a diagnosis of malignant hyper- thermia susceptibility. From the clinical point of view, however, malignant hyperthermia equivocal individuals are treated as suscep- tible. The tests are invasive, ex- pensive and, because of the potentially disastrous results of a false-negative diagnosis, tend to err on the side of the false- positive.

Malignant hyperthermia in swine is associated with lean,

heavily-muscled breeds where the malignant hyperthermia gene appears to contribute 2-3% to dressed carcass weight9. Malig- nant hyperthermia is a serious economic problem in the pork in- dustry because it leads to sudden, stress-induced deaths and to pale, soft, exudative meat, probably resulting from contracture, hyper- metabolism and hyperthermia in the carcass itseW”.

Swine can be tested for malig nant hyperthermia susceptibility through a halothane challenge test, which will induce signs typical of a malignant hyper- thermia reaction such as muscle rigidity, tachycardia, ventricular arrhythmia, mottled cyanosis and fever in most live homozygous pigs but not in most heterozygous or normal pigs”. Accordingly, heterozygotes escape detection. Analysis of inheritance of the linked markers glucose phosphate isomerase (GPI) and &phospho- gluconate dehydrogenase (PGD),

332 TiPS - August 1992 [Vol. 131

lying in the malignant hyper- thermia (HAL) linkage group, leads to an indirect test for the heterozygote that is accurate within the limits of recombination frequency, but is expensive”. Thus there has been a need for an inexpensive, accurate, non- invasive test for both human and porcine malignant hyperthermia (see Box).

The Caz+-release channel The muscle rigidity associated

with malignant hyperthermia most likely results from abnor- malities in Ca2+ regulation in skeletal muscle. Thus, attention was drawn very early to potential abnormalities in Ca2+ release or reuptake in malignant hyper- thermia skeletal muscle5. Develop- ment of suitable assays for the function of the Ca*+-release chan- nel permitted evaluation of its function in skeletal muscle sarco- plasmic reticulum from malignant hyperthermia and normal indi- viduals.

The rate and extent of Ca*+ release from malignant hyper- thermia sarcoplasmic reticulum exceeded the rate of Ca2+ release from normal sarcoplasmic ret- iculum and the threshold of ac- tivation of Ca2+ release by such agents as caffeine, ATE’ and even Ca2+ itself was lowered in mahg- nant hyperthermia sarcoplasmic reticulum1*16. Moreover, Ca2+- induced channel closing was found to be altered in the porcine mali

Pn ant hyperthermia chan-

nel’ , but not in human malignant hyperthermia channels’s. The tryptic digestion pattern for the malignant hyperthermia channel was also found to be altered, con- sistent with loss of a single tryptic digestion site in the malignant hypertherrnic pig”. Dantrolene, the clinical antidote for malignant hyperthermia in the operating room4, has been reported to in- hibit halothane-induced Ca2+ release14 and Ca2+-induced Ca2+ release” from isolated sarco- plaamic reticulum.

The Ca2+-release channe! was identified and isolated through its high-affinity binding of the modulator ryanodine, hence the name ryanodine receptoP. It is a homotetrameric complex made from identical subunits of 565kDa. Its function is regulated by Ca2+, ATI’, Mg2+ and calmodulin. Full-

Genetic testing for malignant hyperthermia in important question emerging from the studies outlined here is whether, now or in the future, genetic testing can provide a diagnosis for individuals susceptible to malignant hyperthennia. The answer is very clear for malignant hyperthermia in swine, but less so for humans. In all breeds of swine studied to date, the presence of the single Cys-for-Arg615 substitution, tightly linked to malignant hyperthermia, is diagnostic of homozygous or heterozygous malignant hyperthermia-susceptible ani- mals. This test can be performed accurately and rapidly on as little as 50 ul of whole blood. It is anticipated that this test will be used worldwide either to eliminate the gene from swine populations or, if it is truly beneficial in pork production, to establish homozygous malignant hyperthermla boar and homozygous normal sow lines for the production of heterozygous slaughter animals. The economic benefit of such programs could approach a value of several hundred million dollars per year worldwide.

For humans, where there is no selection for or against the malignant hyperthermia gene, it appears that a variety of different mutations in the RYRZ gene will be among those found to be causative of malignant hyperthermia. As each of these mutations is identified and satisfactorily linked to malignant hyperthermia, their subsequent identification within other malignant hyperthermia families, using a simple blood test, will provide reliable information on the malignant hyperthermia status of members of that family. At present, this is true only for the Cys-for-Arg614 substitution which is present in no more than 1 in 35 malignant hyperthennia families. For all other families, diagnosis by a direct genetic test is not yet possible. Indirect genetic tests, based on linkage of benign polymorphisms in the RYRZ gene with malignant hyperthermia, can predict inheritance of malignant hyperthennia mutations within families, provided statistically significant linkage has already been established within that family.

Even after exhaustive studies to identify all RYRZ mutations involved in malignant hyperthermia, it will still not be possible to predict every case of malignant hyperthermia susceptibility. This is because malignant hyper- thermia may also arise in association with muscle-cell deficiencies induced by other neuromuscular diseases. There is also evidence that genes other than RYRl might be causative of malignant hyperthermia susceptibility and potential mutations in alternative genes must also be sought. Accordingly, there is little possibility that a universal genetic test for malignant hyperthermia susceptibility in humans will be easily developed.

length cDNAs encoding rabbit, human and porcine skeletal muscle ryanodine receptors have been cloned and sequenced22-24. The cDNAs are over 15000 base pairs long, encoding proteins of some 5035 amino acids, and the human gene has over 200000 base pairs (Phillips, M. S. et al., unpublished).

Ryanodine receptor/malignant hyperthermia linkage

The RYRl gene encoding the skeletal muscle Ca*+-release channel is localized on human chromosome 13q13.1, in a linkage group containing human GJJP. A linkage group for the porcine HAL gene, localized near the centromere of pig chromosome 6 (Ref. 26), includes GPI and PGD2’, suggesting that parts of these regions of human chromosome 19 and pig chromosome 6 are hom- ologous. In a study of linkage be- tween inheritance of malignant

hyperthermia and one or more of several restriction fragment length polymorphisms found in the human RYRl gene, MacLennan et al.” found co-segregation in 23 meioses in nine families. Using a series of chromosome 19q markers, McCarthy et al. 24 also established linkage of the malignant hyper- thermia locus to the region of human chromosome 19q where RYRl and the malignant hyper- thermia gene had been co-local- ized. Thus RYRl is a candidate gene for malignant hyperthermia in both human and pigs.

Linkage between RYRl and malignant hyperthermia made it imperative to initiate a search for sequence differences in the RYRZ gene between malignant hyper- thermia and normal individuals. In a comparison of the RYRl cDNA sequences of malignant hyperthermia (Pietrain) and nor- mal (Yorkshire) pigs, only a single

TiPS - August 1992 [Vol. 131

deduced amino acid sequence change was found”: substitution of T for C at nucleotide 1843 leads to the substitution of Cys for Arg615 in the deduced amino acid sequence. Association between inheritance of this mutation and malignant hyperthermia was shown in some 80 animals from five different breeds. In analysis of linkage in 376 British Landrace swine, including 338 representing informative meioses, co-segre- gation of the malignant hyper- thermia phenotype with the Cys-for-Arg615 substitution was complete30.

The appearance of an identical mutation in five lean, heaviljr- muscled pig breeds suggested that the mutation in all breeds arose in a founder animal. Haplotype and genotype analysis using three markers covering about 150 kb within the RYRl gene showed the inheritance of the same haplotype in every homozygous malignant hyperthermia animal examined and the potential for the inheri- tance of the chromosome with this haplotype in every heterozygous animal”. Association of the same haplotype with the malignant hyperthermia phenotype suggests that the abnormality originated in a founder animal and was selected in breeding stock.

The selection of a disease gene in five breeds of pigs is consistent with evidence that the malignant hyperthermia gene contributes 2-30/n to dressed carcass weight’. It is conceivable that a hypersen- sitive Ca2+-release channel could give rise to spontaneous muscle contraction and the resulting re- petitive toning of the muscle could lead to muscle hypertrophy. The use of ATP for spontaneous contraction would limit the de- position of fat. Alternatively, the malignant hyperthermia mutation could be very closely linked to gene(s) responsible for the desir- able carcass traits and maintained by disequilibrium in the linkage between the genes.

In a search for the correspond- ing mutation in 35 human malig- nant hyperthermia families, the equivalent mutation was found in a single family of five members in which the mutation segregated with malignant hyperthermia31. Co-segregation of the mutation with malignant hyperthermia in swine, combined with the ap

pearance of the corresponding mutation across a species barrier between swine and humans, strongly supports the proposal that this mutation is causative of malignant hyperthermia suscepti- bility in most pigs and in at least some human families.

Lack of ryanodine receptor/ malignant hyperthermia linkage

Linkage between RYRl and human malignant hyperthermia has not been found in all human families studied32-34. This is not surprising since episodes of malignant hyperthermia could, presumably, also arise under con- ditions where a normal Ca2’ release channel is triggered to open as a result of poor Ca2+ regulation. There could be a rise in muscle cell Ca2+ concentration, for example, because of defects in other proteins such as Ca” pumps, channels or exchangers. Alternatively, there might be increased cell membrane per- meability to Ca2+ due to secondary causes. As examples, individuals with such myopathies as central core disease, King-Denborough syndrome, myoadenylate deamin- ase deficiency, periodic paralysis, myotonia car_genita or Duchenne muscular dystrophy have had malignant hyperthermia reactions or responded positively in caf- feine/halothane contracture tests35.

It is of interest that individuals with central core disease are malignant hyperthermia suscep- tible35 and that linkage between RYRl and central core disease has been established with a high lod score (the logarithm of the odds that genetic linkage exists)36. In this syndrome, a potential abnor- mality in RYRZ may give rise to multiple physiological abnor- malities, leading to both malignant hyperthermia and muscle myo- pathy.

In a study of a large French- Canadian family, no linkage was found if criteria for diagnosis of malignant hyperthermia in the halothane/caffeine contracture tests were rigorously applied=. On the other hand, by using caf- feine/halothane contracture test limits below those currently recommended, co-segregation of RYRZ and malignant hyper- thermia genes was demonstrated in this family. Either the criteria for the normal responses were set

333

too conservatively, leading to false-positive diagnoses, or the genetic model of co-inheritance of malignant hyperthermia and an RYRZ allele was incorrect.

In other studies where linkage could not be established32J3, diagnosis appeared to be accurate and no evidence could be ob- tained for an alternative my- opathy. In at least some of these cases, both false-positive and false-negative diagnoses would have had to have been invoked to prove linkage. Thus abnormalities in proteins other than the Ca*+- release channel, leading to poor Ca2+ regulation within the celI, may eventually be shown to give rise to other forms of malignant hyperthermia susceptibility in those families in which malignant hyperthermia cannot be Iinked to RYRl.

0 0 cl

Future studies must be directed in two areas. First, the search must continue for additional mutations in the RYRZ gene that are likely to be causative of malignant hyper- thermia. It is not inconceivable, given +he large size of the gene and its complexity, that many RYRZ mutations will be uncovered that can be associated with malig- nant hyperthermia. At the same time, there must be concern about those families in which there is no linkage between malignant hyper- thermia and RYRZ and where there is no evidence for other neuromuscular disease causing the syndrome. Either the pheno- typic diagnosis is in error, leading to false-positive or false-negative diagnoses for individuals in the family, or the defective gene does not lie on chromosome 19q13.1.

It is reasonable, given the avail- ability of DNA probes for dif- ferent chromosomal regions, to consider searching for linkage of inheritance of malignant hyper- thermia with inheritance of poly- morphisms in other regions of the human genome. On the other hand, if the diagnosis is faulty, linkage will not be established in any part of the genome. Thus the elucidation of all aspects of malig- nant hyperthermia is dependent on accurate phenotypic diagnosis as well as on the ability of geneti- cists to find each of the possible defects leading to the condition.

334 TiPS - Augusf 1992 [Vol. 131

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Acknowledgements Contributions from many collab-

orators, including the illustration by Mr Michael Phillips, are grate- fully acknowledged. Parts of the original research described in this review were supported by grants to D.H.M. from the Canadian MRC and the Muscular Dystrophy Association of Canada.

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A clinical tale: Gingko bitoba goes west Gingko biloba Extract (EGb 761): Pharmacological Activities and Clinical Applications

by F. V. DeFeudis, Elsevier, 2992. FFr250.00 in France, FFr280.00 else- where (xii + 287 pages) ISBN 2 906077 24 0 kbk, 2 906077 21 6 pbk

Anything that survives 4000 years must have a few tricks up its leaves. The Gingko biloba tree has a life span of between 2000 and 4000 years and is regarded as a living fossil, since this species has been in existence for between 150 and 200 million years. Chinese herbal pharmacopoeias, both ancient and modern, contain references to the extract of Gingko biloba leaves as being ‘good for the heart and lung’.

In this monograph, DeFeudis describes more recent investi- gations of the basic and clinical pharmacology of the extract of

Gingko biloba leaves (EGb 761) that has been in use in Europe since 1965. Specific indications include stroke, vasospastic and ischaemic disorders and primary degenerative or vascular demen- tia. My particular interest in this book derives from the fact that Gingko biloba extract contains active amounts of gingkolides A, B and C. Gingkolide B, also known as BN52021, is a relatively potent platelet-activating factor (PAF) receptor antagonist.

The prologue suggests that the monograph has been written for both the ‘average’ person and the health specialist - an ambitious undertaking and one that I do not believe has been achieved in the pages that follow. The language and detail of the treatise are clearly directed at a scientifi~y literate and clinical readership and consequently should prove of most value to prescribers of Gingko biloba extract and to those interested in researching further the fascinating activities of the ex- tract. Those with an interest in the

pharmacology of PAF and PAF antagonists would also find the monograph of value.

The first chapter provides a brief and interesting history of the use of Gingko biloba extract, initially in Chinese herbal medi- cine followed by some detail of its first use in Western medicine. The chemical composition, though not yet fully elucidated, is described and much attention is focused on standardization of the content of the extract to specific percentages of flavonoids and terpenoids, the former possessing antioxidant ac- tivity, the latter comprising some PAF antagonists, There is, how- ever, no description of the chemi- cal stability of this complex mix- ture. The limited amount of pharmacokinetic data is reviewed, but given the number of poten- tially active substances in the extract, any systematic pharmaco- kinetic evaluation would be im- practical. Subsequent chapters deal with in vitro, in viva and clinical studies, with a further four chapters covering clinical perspec- tives, safety, proposed mechan- isms of action and general con- clusions. The monograph is well organized with a number of useful diagrams. Extensive reproduction of published data in both tabular