METHOD AND KIT FOR DIAGNOSIS OF MALIGNANT HYPERPYREXIA

Abstract: A method for detecting in a human or animal patient the muscle membrane defect leading to the susceptibility of the patient to malignant hyperpyrexia (MH) and/or non-anaesthetic complications, comprises the step of determining the level of inositol 1,4,5-trisphosphate phosphatase (InsP35-ase) or nucleotide sequence encoding same or part thereof in a sample taken from the patient, and comparing the level so determined with the corresponding level in a control sample. A diagnostic kit is also disclosed.

Description: METHOD AND KIT FOR DIAGNOSIS OF MALIGNANT HYPERPYREXIA This invention relates to a simple non-invasive test to screen for the muscle membrane defect which predisposes to the anaesthetic complication malignant hyperpyrexia (MH) , and also non-anaesthetic complications of the muscle membrane defect, such as cot death. Malignant hyperpyrexia (MH) was first described in 1960 as a syndrome of life threatening complications during anaesthesia(1,12) . Research into MH expanded rapidly in 1969 when it was discovered that individuals who are susceptible to this anaesthetic complication have an underlying muscle disorder(2,3) . Three clinical myopathies associated with MH have been defined( ,5) . Since then several non-anaesthetic triggers of MH have been described, which include neuroleptic drugs, severe infections and severe exercise in hot conditions(β) . In 1978 an association was discovered between MH and cot death(7), and this observation has been confirmed by a careful epidemiological study carried out in the United States(8) . Susceptibility to MH is not confined to humans but also occurs in certain breeds of pigs(9). Significantly, MH susceptible (MHS) pigs die suddenly in their pens (the porcine stress syndrome or (PSS) . Meat from these animals is unsuitable for eating (the pale, soft, exudatine pork syndrome (PSEP)) . In countries such as Belgium, PSEP is a major problem in the pig industry as it " can affect up to 15% of the animals. Much of the understanding of MH comes from the use of the pig as an animal model for MH, as the clinical manifestations of MH are similar in both species. As a result of experiments on pigs it is now widely accepted that MH results from increased levels of free Ca 2+ in the myoplasm(lθ) . During an MH episode, elevated yoplasmic Ca 2+ leads to muscle rigidity, severe acidosis and an.explosive and sustained rise in core body- temperature. This knowledge has led to the development of a specific diagnostic test for MH, because there is a direct relationship between myoplasmic Ca 2+ concentration and muscle contraction. Susceptibility to MH can also be identified by skeletal muscle biopsy, as isolated susceptible muscle fibres display hypercontractility to various chemical stimuli, such as 3% halothane, 2mM caffeine and 80mM potassium chloride(ll) . The disadvantage of this test is its invasive nature. The site of the lesion which predisposes to the abnormally high Ca 2+ level in MHS muscle is unknown. However events of excitation-contraction coupling (ECC) involving the mechanism of Ca 2+ release from the terminal cisternae of the sarcoplasmic reticulum (SR) have been implicated. The lesion in man and swine appears to be identical (13) . In many cells inositol 1,4,5,-trisphosphate (InsP 3 ) mobilizes Ca 2+ from non-mitochondrial intracellular stores ( 14,15 ) > Recently, it has been proposed that InsP 3 acts as a second messenger during skeletal muscle ECc' 16) . In response to extracellular stimuli transverse tubule phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P 2 ) is degraded to InsP 3 and diacyglycerol. InsP 3 may then open Ca 2+ channels in the SR and the dihydropyridine-sensitive Ca 2+ channels of the transverse tubule membrane, initiating contraction' 16, * ** - 7 ' . InsP 3 is primarily metabolized by a specific 5'-phosphatase, inositol 1,4,5-trisphosphate phosphatase (InsP 3 5-ase) , to inositol 1,4-bisphosphate (Ins(1,4)P 2 ) (14 ' 15 > . The dephosphorylation of InsP 3 is magnesium dependent and inhibited by 2,3-bisphosphoglycerate and the divalent ion of zinc* 14 ' 16) . In work leading to the present invention, it has been found that an abnormality in InsP 3 metabolism is responsible for the elevated myoplasmic Ca 2+ concentration in MH. In the present investigation, a soluble and SR membrane bound form of InsP 3 5-ase was found in both MH susceptible (MHS) and control porcine muscle. The specific 5'-phosphatases were inhibited by Zn 2+ and 2,3-bisphosphoglycerate and dependent on Mg 2+ for activity. The rate of InsP 3 hydrolysis by the myoplasmic InsP 3 5-ase from MHS and control muscle was similar. However, the dephosphorylation of InsP 3 by the MHS SR membrane bound InsP 3 5-ase was significantly diminished in comparison to controls. InsP 3 5-ase is primarily responsible for the degradation of InsP 3 to Ins(l,4)P 2 and the regulation of intracellular Ca + levels. The reduced SR membrane bound InsP 3 5-ase demonstrated in MHS muscle in the present study explains the high InsP levels in MHS muscle, and this, in turn, explains the high myoplasmic Ca 2+ levels in resting MHS muscle cells. In the present investigation it has also been shown that halothane inhibits InsP 3 -ase, which would lead to a further elevation of myoplasmic InsP 3 Ca 2+ and the initiation of the clinical symptoms of malignant hyperpyrexia. Contracture experiments confirm the biochemical findings. Neomycin and the chemicals phosphatase butanedione monoxamine (BDM) inhibit halothane contractures in MHS muscle. Neomycin inhibits the breakdown of Ptdlns-phosphates and the production of InsP 3 ' 16 18 ' . BDM at concentrations below 10 mmol/1 may depress the development of tension in skeletal muscle by dephosphorylating InsP 3 and inhibiting Ca 2+ release from the SR, rather than solely acting at a myofibrillar level (19) . Inhibition of halothane-induced hypercontractility by neomycin and BDM gives further support to the view that InsP 3 plays a primary role in the aetiology of malignant hyperpyre ia. It has now been shown that the inherited defect in malignant hyperpyrexia is a deficiency of the SR membrane bound InsP 3 5-ase enzyme, which leads to high resting myoplasmic InsP and Ca 2+ levels. Halothane inhibits the myoplasmic InsP 3 5-ase and this may lead to further rises in myoplasmic InsP 3 and Ca 2+ levels and the clinical features of malignant hyperpyrexia. According to the present invention, there is provided a method for detecting in a human or animal patient the muscle membrane defect leading to the susceptibility of the patient to malignant hyperpyrexia (MH) and/or non-anaesthetic complications, which comprises the step of determining the level of inositol 1,4,5-trisphosphate phosphatase (InsP 5-ase) in a sample taken from the patient, and comparing the level so determined with the corresponding level in a control sample. In a particularly preferred embodiment, it is the level of membrane bound, particularly SR membrane or endoplasmic reticulum membrane bound, InsP 3 5-ase which is determined. Preferably, the level of InsP 3 5-ase is determined by measuring the level of inositol 1,4,5-trisphosphate in a cell sample taken from the patient, or by measuring the activity of the enzyme in a cell sample taken from the patient, for example, by measuring the rate of dephosphorylation of inositol 1,4,5-trisphosphate by said enzyme in. vitro. The step of determining the level of InsP 3 5-ase activity may be performed by any suitable procedure. Alternatively, the level of InsP 3 5-ase is determined genetically by assaying genomic extracts from cells taken from the patient for the presence or absence of the genetic information (RNA or DNA) encoding InsP 3 5-ase or determining whether said genetic information has altered so as to give an altered (e.g. reduced activity) enzyme. A deficiency in InsP 3 5-ase activity in the patient sample when compared with the control sample is indicative of suceptibility of the patient to malignant hyperpyrexia and/or non-anaesthetic complications arising from the muscle membrane defect responsible for malignant hyperpyrexia. It may be that the abnormality in enzyme activity in MH muscle will be revealed only after exposure to an anaesthetic such as halothane, and accordingly the method of the present invention may include such an exposure step. The present invention also extends to a diagnostic test kit for detecting in a human or animal patient the muscle membrane defect leading to the susceptibility of the patient to malignant hyperpyrexia (MH) and/or non-anaesthetic complications, comprising means for determining the level of InsP 3 5-ase, particularly InsP 3 5-ase activity, in a sample taken from the patient and means for comparing the level so determined with corresponding level in a control sample. Alternatively, the kit- may contain matrial, such as oligonucleotides, for the determination of the genetic information (RNA or DNA) encoding InsP 3 5-ase in cells taken from the patient to ascertain the presence or absence by said information or altered forms thereof. It should be noted that following the demonstration of abnormal enzyme activity in MH muscle cells, a simple test assaying this activity may be performed in readily available cellular tissue since the relevant biochemical pathways appear to be similar in most cellular systems that have been examined to date. The use of blood cells particularly blood cells other than red blood cells, such as platelets, in this diagnostic test is particularly preferred since samples of such cells are readily available and the resulting test is simple and non-invasive. The present invention does extend, however, to the use of samples of muscle tissue, or any other cellular tissue from the patient which is readily available and in which the abnormal enzyme activity can be demonstrated. The present invention also extends to recombinant InsP 3 5-ase and its derivatives and, in particular, to a nucleotide sequence encoding all or part of InsP 3 5-ase or its derivatives. The nucleotide sequence may comprise RNA or DNA encoding the entire enzyme or a substantial part thereof or may comprise oligonucleotides. By "recombinant InsP 3 5-ase" is meant the glycosylated or unglycosylated enzyme produced by recombinant means such as the ligation of the relevant cDNA molecule encoding InsP 3 5-ase or derivatives thereof into an appropriate expression vector in the correct reading frame relative to the promoter and introducing the resultant recombinant expression vector into a suitable host and growing said host under conditions appropriate for the expression and, if necessary, transportation of the recombinant enzyme or derivative thereof from said host and then, if necessary, purifying same. General techniques for cloning, expressing and " manipulating cDNA and other nucleotide molecules can be found in (25) . In general, a partial or complete N-terminal, C-terminal or internal amino acid sequence of InsP 3 5-ase is determined and one or more oligonucleotide sequences deduced therefrom. The oligonucloetides are then used to screen genomic libraries, either in eukaryotes or prokaryotes, for cells carrying the relevant cDNA. Alternatively, a genomic expression library can be screened by assaying for expression of the enzyme. Recombinant InsP 3 5-ase will be useful in preparing large quantities of enzyme for analysis and for preparing antibodies (monoclonal or polyclonal) against same. Furthermore, the enzyme can be studied in detail to determine its catalytic regions. The level of InsP 3 5-ase can be measured indirectly by assaying for the presence or absence of the genetic information (RNA or DNA) encoding same or any alterations thereof. For example, and not by way of limitation, a cell sample taken from a patient is subjected disruption sufficient to release its genomic extracts. By "genomic extract" is meant, depending on the assay employed, the total DNA and/or mRNA. In one assay, the genomic extracts are subjected to electrophoresis and, by known methods, the presence of any nucleotide sequences in said genomic extract which are complementary to one or more oligonucleotides specific for DNA or RNA encoding InsP 3 5-ase determined. Alternatively, the entire cDNA molecule encoding InsP 3 5-ase or a region of said cDNA molecule can be used. In another method, the presence or absence of InsP 3 5-ase-encoding DNA or RNA in a patient's cell genomic extract can be determined by simple "dot blot". In yet another method, where the InsP 3 5-ase-encoding nucleotide sequence in the genome of a patient is altered by the substitution, addition and/or deletion of one or more nucleotides, then these can be detected using specific oligonucleotide probes designed to detect such alterations in nucleotide sequence. These alterations in nucleotide sequence may result in an enzyme with no activity or with reduced or enhanced activity. Polymerase chain reaction (PCR) techniques can also be employed to enhance detection of the target nucleotide sequences. Accordingly, the present invention extends to a method for detecting in a human or animal patient the muscle membrane defect leading to the susceptibility of the patient to malignant hyperpyrexia (MH) and/or non-anaesthetic complications, which comprises the step of determining the presence, absence or alteration in nucleotide sequence encoding inositol 1,4,5-trisphosphate phosphatase (InsP 3 5-ase) in a genomic extract of a cell sample taken from the patient. In one embodiment, the presence, absence or alteration in nucleotide sequence encoding InsP 3 5-ase is determined by contacting the genomic extract either before or after electrophoresis with a nucleotide sequence designed to hybridize under appropriate conditions to specific nucleotide sequences in said genomic extract which, if present, encode InsP 3 5-ase or part thereof. The present invention also contemplates a kit for the genetic determination of InsP 3 5-ase. It will also be appreciated that although one important feature of the present invention lies in the provision of a test for screening human patients for the detection of MH or SIDS (sudden infant death syndrome - "cot death") susceptible individuals, an equally important area of use of the test lies in the screening of animals, particularly pigs which as described earlier may be susceptible to the porcine stress syndrome (PSS) which is a major problem in the pig industry in some countries. Further features of the present invention will be apparent from the following Example, in association with the accompanying drawings, in which: Figure 1 shows HPLC analysis of the metabolism of [ 3 H]-InsP 3 by malignant hyperpyrexia susceptible (MHS) sarcoplasmic reticulum (SR) membranes; and Figure 2 shows the rate of dephosphorylation of [ 3 H]-InsP 3 by MHS and control InsP 3 5-ase. EXAMPLE A. Methods Biopsy specimens of gracilis muscle were taken under nitrous oxide anaesthesia from 14 MHS pigs and 8 control igs* 2 ^- 5 .. Samples were either used for contracture studies or immediately frozen in liquid nitrogen. Contracture Studies Susceptibility to MH was identified by using the established isolated contracture test (20 ) . Halothane (3%), 2 mmol/1 caffeine and 80 mmol/1 potassium chloride induce hypercontractility in MHS muscle. Some preparations were incubated with neomycin or butanedione monoxime (BDM) before being challenged with halothane. Changes in isometric force were monitored as described previously (20 / . Extraction and measurement of intracellular InsP 3 Frozen muscle samples were ground under liquid nitrogen into a fine powder and 500 mg was suspended in 4 volumes of 15% w/v TCA, 1 mmol/1 CdCl 2 and 10 mmol/1 LiCl 2 . Samples were then sonicated (3x15 seconds bursts) and centrifuged at 3,000 g for 20 minutes. Supernatants were then extracted with 10 volumes of water-saturated diethylether and the pH adjusted to 7.5 with NaHC0 3 . All procedures were performed at 4°C. The concentration of InsP 3 in the supernatant was then measured by using a D-myo-InsP 3 -[ 3 H] assay system (Amersham TRK.1000) . Measurement of InsP 3 5-ase Frozen muscle samples (3-5 g) were ground under liquid nitrogen and suspended in 4 volumes of 0.25 mol/1 sucrose/10 mmol/1 MOPS pH 7.4 buffer. The suspension was homogenized in a Waring blender and filtered. The homogenate was then centrifuged at 105,000 g for 1 hour. The resultant supernatant was called the myoplasmic preparation. All procedures were carried out at 4°C. SR membranes were prepared as described previously' 21) . InsP 3 5-ase activity was measured at 37°C in a solution containing 0.25 mol/1 sucrose/10 mmol/1 MOPS pH 7.4, 1 mmol/1 ATP, lμmol/1 InsP 3 ([ 3 H]-InsP 3 , 0.08, μCi) and various concentrations of MgCl 2 . The reaction was initiated by the addition of myoplasmic or SR membrane protein. Protein concentration was estimated by the method of Peterson' 22) . In some experiments the incubation medium was saturated with halothane (thymol free, ICI, Australia). The concentration of halothane was maintained by continuously bubbling halothane through the incubation medium using a Dragewick vapouriser and carbogen (95% CO /0 2 ) . Reactions were stopped by the addition of an equal volume of an ice cold solution containing 15% (w/v) TCA, 10 mmol/1 LiCI 2 and 1 mmol/1 CdCl 2 . Samples were then centrifuged at 3,000 g for 15 minutes, decanted and the supernatants extracted in water-saturated diethylether. The aqueous phase was adjusted to pH 7.4 with MOPS buffer and analysed by high pressure liquid chromatography (HPLC) . HPLC Analysis SR membranes (2mg/ml) were incubated for 2.5 minutes at 37°C in reaction buffer containing 2 mmol/1 MgCl . Samples were loaded on to a Partisil SAX anion-exchange HPLC column (Whatman) . [ 3 H] inositol phosphates were eluted over 50 minutes using a linear gradient of up to 1.7 mol/1 ammonium formate adjusted to pH 3.7 with orthophosphoric acid* 23) . Fractions were collected every minute and 3 H was ' quantified by scintillation counting. Recovery of radioactivity applied was greater than 90%, and results are expressed as a percentage of the total recovery in all peaks. Peaks 1, 3 and 5 were identified as inositol (Ins), Ins(l,4)P 2 and Ins(l,4,5)P 3 respectively by co-elution of known inositol phosphate standards (Amersham) . The elution of peak 2 corresponds to inositol monophosphate (InsP^) and peak 4 either the Ins(l,5)P 2 or Ins(4,5)P isomer of inositol bisphosphate v 24) Similar elution profiles were found after the dephosphorylation of InsP 3 by SR membranes and myoplasmic samples from MHS and control muscle. Measurement of dephosphorylation rate (a) Myoplasmic and SR (2 mg/ml) proteins were incubated for 2.5 minutes at 37°C in reaction buffer containing 2 mmol/1 Mg Cl 2 . The rate of InsP 3 hydrolysis by MHS (n=5) and control (n=5) myoplasmic samples was similar. However MHS SR membrane bound InsP 3 5-ase activity (n=6) was significantly reduced in comparison to control rates (n=7) (P< 0.001; non paired Student's t-test) . (b) MHS Myoplasmic protein (0.5 mg/ml) was incubated at 37°C for 20 minutes in reaction buffer containing 2,3-bisphosphoglycerate, ZnCl 2 and 2 mmol/1 MgCl 2 . Some samples were incubated only with 2 mmol/1 or 0.25 mmol/1 MgCl 2 . The number of observations ranged from 4-6. Similar results were obtained with control samples. Results are expressed as mean values ± s.e.m. B. Results The level of InsP 3 was significantly elevated in soluble extracts from MHS muscle in comparison to control levels (Table I). The mean level of InsP 3 from MHS extracts was approximately ten fold higher than the mean control value. InsP 3 was dephosphorylated primarily to Ins(l,4)P 2 by both myoplasmic and SR membrane preparations (Fig I) . The rate of hydrolysis of InsP 3 by the myoplasmic forms of MHS and control InsP 3 5-ase was similar (Fig Ila) . However, the rate of dephosporylation by the SR bound InsP 3 5-ase from MHS muscle was significantly diminished in comparison to control SR InsP 3 5-ase (Fig Ila) . The degradation of InsP 3 by InsP 3 5-ase from MHS control muscle was Mg + ion dependent and was inhibited by 2,3-phosρhoglycerate and Zn 2+ ions (Fig lib). Halothane also inhibited the dephosphorylation of InsP 3 by myoplasmic InsP 3 5-ase from MHS and control muscle (Table II) . Halothane inhibition was observed after incubations of 20 minutes (twice) 10 and 5 minutes. Halothane-induced contractures were significantly inhibited in MHS muscle preparations by 6 mmol/1 BDM and 2 mmol/1 neomycin (Table III). Lower concentrations of neomycin (0.5 mmol/1) inhibited halothane contractures after increasing the equilibration time from 20 to 40 minutes (results not shown) . TABLE I - LEVELS OF MYOPLASMIC INOSITOL 1,4,5-TRISPHOSPHATE FROM MALIGNANT HYPERPYREXIA SUSCEPTIBLE AND CONTROL PORCINE SKELETAL MUSCLE InsP^ pmol/soluble extract MHS Control 115 35 110 24 108 14 108 5.4 104 2.7 101 2.5 91 1.8 88 1.0 86 1.0 70 62 X = 94.82 9.71 S.D = - 16.35 ± 11.53 n = 11 Individual estimations of InsP 3 levels are shown. These results were obtained from 7 MHS and 4 control pigs. Mean levels of InsP 3 from MHS samples were significantly higner than control levels, P < 0.001, Student's non-paired t-test. SUBSTITUTE SHEET TABLE II - THE EFFECT OF HALOTHANE ON INOSITOL 1,4,5,-TRISPHOSPHAT , DEPHOSPHORYLATION BY MYOPLASMIC INOSITOL 1,4,5-TRISPHOSPHATE PHOSPHATASE Conditions Incubation time Halothane No halothane (minutes) (% InsP 3 remaining) MHS 5 83.0 68.0 ± 0.93 (2) 10 77.2 58.9 - 0.97 (5) 20 83.7/81 42.2 - 0.87 (10) Control 5 82.4 64.8 i 0.73 (2) 10 80.5 54.7 - 1.20 (6) 20 79.6/77.2 43.7 -± 1.22 (8) Myoplasmic protein (0.5 mg/ml) was incubated in reaction buffer containing 2 mmol/1 gCl2 at 37°C in the presence or absence of halothane. Samples were continuously bubbled with halothane for 5, 10 or 20 minute periods. Results are shown for single observations or expressed as means — s.e.m., with the number of observations in parenthesis. SUBSTITUTE SHEET TABLE III - THE EFFECT OF NEOMYCIN AND BUTANEDIONE MONOXIME ON HALOTHANE CONTRACTURES IN MALIGNANT HYPERPYREXIA MUSCLE Contracture (g) Halothane 3% Halothane 3% + Halothane 3% + neomycin BDM (2 mmol/1) (6 mmol/1) 0.93 -± 0.07 0.17 - 0.17* 0.04 - 0.07* (n = 108) (n = 5) (n = 9) Muscle strips were equilibrated with neomycin (20 minutes) or BDM (45 minutes) and then challenged with halothane. Results are expressed as mean value — s.e.m. The number of fibres tested are shown in parenthesis. Significant differences are indicated P < 0.001 non-paired Student's t-test. SUBSTITUTE SHEET REFERENCES: 1. Denborough, M.A. and Lovell, R.R.H. (1960), Lancet 2 , 45. 2. Denborough, M.A., Forster, J.F.A., Hudson, M.C. , Carter, N.G. and Zapf, P. (1970) Lancet 1- 1137. 3. Denborough, M.A. , Ebeling, P., King, J.O. and Zapf, P. (1970) Lancet 1, 1138. 4. King, J.O., Denborough, M.A. and Zapf, P. (1972) Lancet 1, 365. 5. Denborough, M.A. , Dennett, X. and Anderson R.McD. (1973) British Medical Journal 1 , 272. 6. Denborough, M.A. , Collins, S.P. and Hopkinson, K.C. (1984) British Medical Journal 288, 1878. 7. Denborough, M.A. , Galloway, G.J. and Hopkinson, K.C. (1982) Lancet 2., 1068. 8. Peterson, D.R. and Davis, N. (1986). Anesthesia and Analgesia 65. 209. 9. Hall, L.W., Woolf, N., Bradley, J.W.P. and Jolly, D.W. (1966) British Medical Journal 2 , 1305. 10. Denborough, M.A. , Hird, F.J.R., King, J.O., Marginson, M.A., Mitchelson, K.R. , Rex, M.A. and Condron, R.J. (1971) Proceeding of an International Symposium on Malignant Hyperpyrexia. Eds.Gordon, R.A, Britt, B.A. and Kalow,W. Toronto, Thomas, Illinois, 229. 11. Moulds, R.F.W. and Denborough, M.A. (1974) British Medical Journal 2., 245. 12. Denborough, M.A. (1980) Pharmacology and Therapeutics 5., 357. 13. Okumura, F., Crocker, B.D. and Denborough, M.A. (1980) British Journal of Anaesthesia 52, 377. 14. Abdel-Latif, A.A. (1986) Pharmac Rev: 38: 227. 15. Majerus, P.W. Connolly, T.M., Bansal, VS, et al. (1988) J Biol Chem: 263: 3051. 16. Vergara, J, Tsien, R.Y., Delay, M. (1985) Proc Natl Acad Sci; 82: 6352. 17. Vilven, J., Coronado, R. (1988) Nature: 336: 587. 18. Shute, J.K., Smith, M. (1985) Biochem Pharmac; 14 : 2471. 19. Fryer, M.W. , Gage, P.W., Neering, I.R., Dulhunty, A.F. and Lamb, G.D. (1988) Pflugers Arch-Eur. J.Physiol. 411. 76-79. 20. Okumura, F., Crocker, B.D. and Denborough, M.A. (1979) British Journal of Anaesthesia 51. 171-76. 21. White, M.D., Collins, J.G. Denborough, M.A. (1983) Biochem J; 212: 399 22. Peterson, G.L. (1977) Anal Biochem: 83: 346. 23. Irvine, R.F., Anggard, E.E., Letcher, A., et al. (1985) Biochem J_; 22£: 505 24. Rubiera, C. , Velasco, G. Mitchell, R.H. et al (1988) Biochem _l; 255: 131 25. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982). Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. pp.1-545.

Claims: CLAIMS 1. A method for detecting in a human or animal patient the muscle membrane defect leading to the susceptibility of the patient to malignant hyperpyrexia and/or non-anaesthetic complications, which comprises the step of determining the level of inositol 1,4,5-trisphosphate phosphatase (InsP 3 5-ase) in a sample taken from the patient, and comparing the level so determined with the corresponding level in a control sample. 2. A method according to claim 1, wherein the level of membrane bound InsP 5-ase is determined. 3. A method according to claim 2, wherein the level of sarcoplasmic reticulum membrane bound InsP 3 5-ase is determined. 4. A method according to any one of claims 1 to 3, wherein the level of InsP 3 5-ase is determined by measuring the level of inositol 1,4,5-trisphosphate in the sample, and comparing the level so determined with the corresponding level in a control sample. 5. A method according to any one of claims 1 to 3, wherein the level of InsP 3 5-ase is determined by measuring the activity of the enzyme in the sample taken from the patient, and comparing the activity level so determined with the corresponding activity level in a control sample. 6. A method according to claim 5 wherein said measurement of the activity of the enzyme comprises measurement of the rate of dephosphorylation of inositol 1,4,5-trisphosphate by said enzyme in vitro. 7. A method according to any one of claims 1 to 6 including the further step of exposing the sample taken from the patient to an InsP 3 5-ase inhibitor prior to determining the level of InsP 3 5-ase in said sample. 8. A method according to claim 7, wherein said inhibitor is halothane. 9. A method according to claim 1 wherein the level of InsP 3 5-ase is determined by detecting the presence, absence or alteration of a nucleotide sequence encoding InsP 3 5-ase or part thereof in a genomic extract of a cell sample taken from said patient. 10. A method according to claim 9 wherein the presence, absence or alteration of the nucleotide sequence is determined by contacting said genomic extract with a second nucleotide sequence, said second nucleotide sequence capable of hybridizing to at least part of the first nucleotide sequence encoding InsP 5-ase in the genomic extract, for a time and under conditions sufficient for said hybridization to occur and then detecting same. 11. A method according to claim 10 wherein the second nucleotide sequence is a single stranded cDNA. 12. A method according to claim 10 wherein the second nucleotide sequence is mRNA. 13. A method according to claim 10 wherein the second nucleotide sequence is an oligonucleotide. 14. A method according to claim 10 wherein the genomic extract is first subjected to electrophoresis. 15. A method according to any one of claims 1 to 14, wherein said patient is a human. 16. A method according to any one of claims 1 to 14, wherein said patient is a pig. 17. A method according to any one of claims 1 to 16, wherein said sample is a sample of muscular tissue taken from the patient. 18. A method according to any one of claims 1 to 16, wherein said sample is a sample of blood cells (other than red blood cells) taken from the patient. 19. A diagnosic test kit for detecting in a human or animal patient the muscle membrane defect leading to the susceptibility of the patient to malignant hyperpyrexia and/or non-anaesthetic complications, comprising means for determining the level of InsP 3 5-ase in a sample taken from the patient and means for comparing the level so determined with the corresponding level in a control sample. 20. A kit according to claim 19, wherein said means for determining the level of InsP 3 5-ase comprises means for measuring the level of inositol 1,4,5-trisphosphate in the sample. 21. A kit according to claim 19, wherein said means for determining the level of InsP 5-ase comprises means for measuring the activity of the enzyme in the sample taken from the patient. 22. A kit according to claim 21, wherein said means for measuring the activity of the enzyme comprises means for measuring the rate of dephosphorylation of inositol 1,4,5-trisphosphate by said enzyme iri vitro. 23. A diagnostic test kit for detecting in a human or animal patient the muscle membrane defect leading to the susceptibility of the patient to malignant hyperpyrexia and/or non-anaesthetic complications, comprising means for determining the level of InsP 3 5-ase by detecting the presence, absence or alteration of a nucleotide sequence encoding InsP 3 5-ase or part thereof in a genomic extract in a cell sample taken from the patient and means for comparing the level so determined with the corresponding level in a control sample. 24. A kit according to claim 23 wherein said means for determining the level of InsP 3 5-ase comprises a second nucleotide sequence capable of hybridizing to the first nucleotide sequence and means of detecting occurrence of hybridization. 25. A kit according to claim 24 wherein said second nucleotide sequence is an oligonucleotide probe.

Inventors: DENBOROUGH MICHAEL ANTHONY, FOSTER PAUL STEPHEN, DENBOROUGH, MICHAEL, ANTHONY, FOSTER, PAUL, STEPHEN
Applicants: UNIV AUSTRALIAN, THE AUSTRALIAN NATIONAL UNIVERSITY
Application year: 1989
Main classification: C12Q1
IPC codes: C12Q1