Establishment And Clinical Application Of The Laboratory Diagnostic Test Of Malignant Hyperthermia

Part 1 Establishment of the laboratory diagnostic test of malignant hyperthermiaObjective To establish laboratory diagnostic test of malignant hyperthermia (MH) -Caffeine and halothane contracture test (CHCT).Methods According to the North America MH Group protocol, the laboratory devices of CHCT were established. Ten Chinese experimental miniature pigs (CEMP) and one pietrain swine were selected for this study to validate the stability of laboratory devices of CHCT. 1.Experiment on the induction of MH in swine: After basal anesthesia with ketamine and diazepam, the pigs were anesthetized with thiopental sodium and tracheal intubation was facilitated with succinylcholine (2mg/kg) intravenously. Mechanical ventilation was applied and the anesthesia was maintained with 2～3% halothane inhalation in oxygen and air (O₂:air=1:1). All the pigs were observed whether the MH episodes were initiated or not. 2.Gene detection: The genome DNA were extracted by classic SDS-proteinase K-hydroxybenzene-chloroform methods from ear tissues of the swines. In order to detect mutations in the RYR1 gene, specific primers were designed and exons were amplified by polymerase chain reaction (PCR) and then subjected to HhalI restriction endonuclease analysis. 3. CHCT: 2～3cm femoral quadriceps' muscle fibers near tendon of the swines were collected and fixed in Krebs-Ringer solution at 37℃constant temperature with 95% oxygen and 5% carbon dioxide. The biopsies were connected to tension sensor and electrical stimulation instrument and then given certain electrical stimulations and the tension changes of the muscles under different concentrations of halothane and/or caffeine were determined.Results 1.Experiment on the induction of MH in swine: The processes of experiments on the induction of MH were stationary and the life signs were stable and clinical manifestations of fulminant MH were not found in all the CEMPs. But the Pietrain swine presented clinical manifestations of MH after anerthesia, such as extremities rigor, increased PaCO₂ and body temperature, elevation of serum CK and serum and urinary myoglobin. 2.Gene detection: The RYR1 genotype of the CEMPs were HALNN and the Pietrain swine's was HALnn, therefore the phenotype of the CEMPs were MH negative and the Pietrain swine's was MH positive. 3. CHCT: All the CEMPs were diagnosed as MH negative (MHN) beaucase caffeine test and halothane test in CHCT were both negative in each animal. But the Pietrain swine were diagnosed as MH susceptibility (MHS) beaucase of both positive results of caffeine test and halothane test in CHCT.Conclusion The results of CHCT were consistent with those of clinical manifestations and genetic screen, demonstrating that the devices of CHCT in present study were overall steady-going and reliable. Part 2 Clinical application of the laboratory diagnostic test of malignant hyperthermiaObjective To apply caffeine and halothane contracture test (CHCT) for the confirmation of clinical diagnosis of malignant hyperthermia (MH).Methods Four cases of MH were summerised. The characteristics of the four cases, including drugs and methods of anesthesia, clinical manifestations, results of lab examination,emergency treatment methods and prognosis were reported. Two cases were analyzed and reported, including clinical manifestations, results of lab examination and prognosis. One case was neuroleptic malignant syndrome(NMS) who was suspicious of MH episode imultaneously and the other case was pheochromocytoma whose symptoms of rhabdomyolysis were suspicious to be caused by domperidone. CHCT was applied for the confirmation of clinical diagnosis of MH by the steps as follow: 2～3cm muscle fibers of femoral quadriceps or rectus abdominis of the patients were collected and fixed in Krebs-Ringer solution at 37℃constant temperature with 95% oxygen and 5% carbon dioxide. The biopsies were connected to tension sensor and electrical stimulation instrument and then given certain electrical stimulations and the tension changes of the muscles under different concentrations of halothane and/or caffeine were determined.Results All the results of caffeine test of the four cases who were clinically diagnosed as MH were positive and then the patients were all diagnosed as MH susceptibility (MHS) according to diagnostic criteria of the North America MH group. The other two cases were both diagnosed as MH negative (MHN) beaucase caffeine test and halothane test in CHCT were both negative in each patient.Conclusion The CHCT in our laboratory can be used in diagnosis and differential diagnosis of MH.Part 3 Molecular genetic study of malignant hyperthermia in Chinese PedigreesObjective To investigate genetically Chinese families who diagnosed as malignant hyperthermia (MH) by caffeine and halothane contracture test (CHCT).Methods Based on abiding strictly by the doctrine of informed consent of patients, this study was performed in four Chinese families(family A, family B, family C and familyD,respectively). Probands of all the family presented as typical clinical manifestations of MH during anesthesia and the diagnosis of MH were confirmed by CHCT. Total genome was extracted conventionally from peripheral leucocytes of the MH patients and other family members'. In order to detect mutations in the RYR1 gene, part exons were amplified by polymerase chain reaction (PCR) and then subjected to automatic DNA sequencing. According to the sequencing results, those who had gene mutation received Fok I restriction analysis to confirm the detected mutation in the index patient and other family members.Results DNA sequencing of PCR-amplified fragments of the index patient in family A revealed c.6724C>T (p.T2206M), a recurrent missense mutation which had been detected in Caucasian MH cases. Restriction analysis of all available family members of family A showed that one daughter and one son of the index patient were heterozygous for the same mutation, indicating that they were MH-susceptible individuals. DNA sequencing of PCR-amplified fragments of all available family members in family B, family C and family D did not reveal mutation in the hot spots of mutation in RYR1 gene.Conclusion Some of the Chinese MH susceptibles carries the similar gene mutation to the Caucasian one, but further studies should be conducted on molecular genetic characteristics of malignant hyperthermia in Chinese pedigrees.