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by Dr. Barbara Brandom (Children’s Hospital) with Dr. Sheila Muldoon (Uniformed Services University of the Health Sciences). Both Dr. Brandom and Dr. Muldoon are members of the Professional Advisory Council of the North American Malignant Hyperthermia Registry of the (MHAUS).

Antwerp, Belgium, May 16-18, 2002 – The 21st Annual Meeting of the European Malignant Hyperthermia Group (EMHG) was hosted by EMHG Chairman Professor Albert Urwyler; Dr. Luc Heytens; and the head of the Department of Intensive Care, University Hospital Antwerp, Professor L. Bossaert, who welcomed attendees from all parts of Europe, and two from the United States. More than 20 reports of original research studies were presented, most of which investigated aspects of the pharmacology of malignant hyperthermia (MH). There were also several review lectures on cell biology, genetics, and central core syndromes. In this overview, we report on selected presentations that we consider particularly relevant to the North American Malignant Hyperthermia Registry (NAMHR).

Noteworthy discussion included implementation of standard and alternative contracture tests within the EMHG. Halothane 2% and caffeine 2 mM are still recognized as the best tests of MH susceptibility. Children should be at least 10 years old before undergoing contracture testing. Larger bath volumes may be preferable. Dr. Barbara Brandom gave an update on the NAMHR programs in the past year, describing the number of biopsies entered, improvements planned for the system, and progress made by the biopsy centers in genotyping patients. To date, 105 patients have been genotyped. The detection rate for RYR1 mutations using our screening strategy was 24%. Dr. Renee Krivosic-Horber, from Lille Center in France, presented two patients with positive IVCT tests, who experienced myopathic symptoms after receiving ‘statin’ drugs. These results supported the argument that patients with chronically elevated creatine kinase (CK) levels should undergo contracture (IVCT) testing to evaluate MH susceptibility. The sibling of one of these index patients was also found to be MHS by contracture testing, although this relative had never been treated with any cholesterol-lowering drugs.

Alternative and new diagnostic techniques:

Dr. Susan Treves, from Basel, discussed the use of B-lymphocytes to study calcium flux after transfection with normal and mutated ryanodine receptors (RYR1). In cells transfected with the RYR1 mutation (V2168M), the most common MH mutation in Switzerland, the EC-50 for 4-chloro-m-cresol- (4CmC)-induced calcium release decreased approximately two-fold. This indicates that the sensitivity of the mutated RYR1 receptor must be considerably increased in MHS cells, in comparison to normal cells.

A similar but greater effect was observed in B-lymphocytes transfected with mutated RYR1 from central core disease (CCD) patients. These cells were characterized by smaller intracellular Ca++ stores than control cells carrying a wild type RYR1 or the MH linked mutation Val2168Met. Furthermore, B-lymphocytes from CCD patients exhibited release of Ca++ even in the absence of pharmacological activators of the RYR1. Such unstimulated calcium transients were never seen in cell lines from control individuals or from individuals with the Val 2168Met mutation, indicating a severely enhanced Ca++ leakage in CCD. Dantrolene pretreatment restored normal Ca++ control in these CCD cells.

Dr. Marcos Wehner, from Leipzig, used primary cultures of myotubes to show differential changes in EC50 for 4CmC-induced calcium release as a function of mutation site. For example, in myotubes with IL2182Phe mutation, the EC50 for all standard RYR1 triggers was reduced approximately 50%. However, in myotubes with GLU2375Ala, there was no decrease in 4CmC EC50 and the halothane /caffeine EC50 reduction was attenuated. He concludes that the different mutations can have differing functional consequences.

Investigators from Wuerzburg presented two different in-vivo studies of muscle that could discriminate between MHS, MHN, and normal patients. Dr. Martin Anetseder placed a fiberoptic pCO2 probe in the rectus femoris, and 500 µl of 80 mM caffeine was injected. He found that local intramuscular administration of caffeine caused a greater increase in pCO2 at a faster rate in MHS than in MHN or control subjects. Local CK levels increased only in the MHS subjects.

Dr. Andreas Hoyer studied the kinetics of the contractile response of the adductor pollicis muscles at different stimulation frequencies. He found that at 2Hz stimulation, the contraction velocity at 60% maximum amplitude was 27% faster in the MHS subjects than in MHN subjects. This is consistent with increased calcium release for a given stimulus. He also observed a more intense fatigue of MHS muscle. If further studies confirm the reliability of this promising and minimally invasive test, it could be developed to identify MH susceptible individuals who are unable to undergo contracture testing.

Several investigators discussed the three areas on the RYR1 where mutations are frequently found. The relationships between each mutation, MH status, and potentially related illness, such as the genetically heterogeneous CCD, are being investigated. The general conclusion of this ongoing research, as well as that of the retrospective study of Dr. Rainer Muller and others from Wuerzburg, is that contracture testing must be done as the first step in describing the MH status of individuals and their family members. Muller reviewed 213 patients who had undergone muscle biopsy and contracture testing between 1989 and 2001. Of the 55% who had experienced an MH event during anesthesia, only 13% had a family history of similar problems, and only 2% had chronically elevated CK. Contracture testing found 60% of these patients NOT to be MH susceptible. Twenty-five percent of the patients with myopathic symptoms had family members with similar complaints. Sixty-nine percent had chronically elevated CK. They concluded that 70% of these patients did not have MH-susceptible contracture test results, and therefore family history, serum CK levels, and histopathologic findings hardly help to detect a patient at risk for MH. The contracture test is the sole diagnostic tool that can lead to a definite diagnosis of MH susceptibility.

One session was devoted to papers relating to central core disease (CCD) and multi-minicore disease (MmD). There were eight presentations from seven centers. CCD is a congenital myopathy clinically characterized by generalized muscle weakness and morphologically defined by the presence of cores on staining for oxidative enzymes. These cores extend throughout the length of the fiber. Autosomal dominant inheritance is clearly predominant. Linkage to RYR1 on chromosome 19q was demonstrated 10 years ago. Several RYR1 mutations that co-segregate with clinical and/or histologic findings of CCD have been identified. Sporadic cases do occur. Recessive inheritance has not been reported.

In contrast, MmD is an autosomal recessive congenital myopathy and is morphologically defined by the presence of multiple, very small zones of sarcomeric disorganization and lack of oxidative activity in both type 1 and type 2 muscle fibers. Most patients share a recognizable phenotype marked by the predominance of axial muscle weakness, severe scoliosis, and respiratory insufficiency. Dr. Joel Lunardi, from France, presented an update on RYR1 mutations in CCD patients. Analyzing a panel of 34 families recruited on the basis of both clinical and morphologically expressed CCD, he identified 12 different mutations in the C-terminal domain of RYR1 in 16 unrelated families. The presence of neomutations in the RYR1 gene was identified in four families, indicating that neomutations in the RYR1 gene are not rare events and must be considered in genetic studies of families that present with myopathies.

Dr. Clemens Mueller, of Wuerzburg, also presented a series of CCD patients screened for mutations in the C terminal of the RYR1 gene. To date, a cohort of 65 unrelated CCD cases had been screened and mutations identified in 27 patients (41.5%). One of three mutations occurred in 63% of the cases. In 12 cases, the disease appeared familial with at least two affected persons. Fifteen cases were sporadic with no family history of CCD. In six of the sporadic cases, it was possible to study both unaffected parents. In five patients the mutation had arisen de novo, while in the sixth family both parents were heterozygous. In summary, all 13 mutations clustered in exons 101 or 102 of the RYR1 gene.

Dr. V. Tegazzin, and colleagues from the departments of Anesthesiology and Neurology in Padua, Italy, compared contractures in response to caffeine and halothane in muscle from four groups: MH patients, others histologically diagnosed as CCD, MCD, and core-like myopathies. All 85 patients underwent IVCT, RYR1 screening for 16 mutations, and histologic studies. The IVCT responses identified 35 MHS, 10 MHE, and 40 MHN. The average muscle tension was greater in the CCD and MCD groups than the other groups. CCD and MCD muscle also had a greater sensitivity to halothane and caffeine. The most interesting finding in this study was that the mutations found in four CCD and four MCD patients were not confined to the C-terminal region. Instead, they were distributed throughout the RYR1 gene.

Dr. Ana Ferreiro and colleagues identified a novel homozygous mutation in a poorly studied region of RYR1 associated with a recessive form of central core disease transiently presenting as MCD.

Implementation of a Diagnostic Screening Service

Dr Jane Halsall described the implementation of a diagnostic screening service for MH susceptibility based on EMHG guidelines. Patients who are diagnosed as MHS by the IVCT are tested for 15 causative mutations, as documented in the British Journal of Anaesthesia (2001:86:283-2870).

When this group began, they had over 500 families in their database, with DNA research information available on approximately 250. From this data, they knew that around 25% of families could benefit from DNA testing. Mutation frequency was used to guide the screening strategy.

Their first priority was to check all research data through an accredited DNA laboratory, which included getting blood samples from all the MHS-diagnosed patients. When a mutation is found they rebleed all IVCT-tested individuals in the family in order to keep track of discordancy problems. Once they have established that they do not have discordancy in the family, DNA screening is offered to the remaining untested individuals.

DNA screening has led to enormous administrative changes. These include alterations to the database, filing, and record keeping, and a system of counseling patients with their DNA results.

Session 7: Concordance/discordance – IVCT versus molecular genetics

This session included six presentations from five MH centers. Dr. Albert Urwyler, president of the EMHG, established a European database to determine the frequency of RYR1 mutations in Europe and to determine the mutations causative for MH. The structure of the data bank was discussed and agreed upon at the last meeting. Data was collected from 10 European centers, and 11 of the 15 causative mutations were identified. In 110 of 245 subjects, the Arg614Cys was identified. However, this data did not include patients from the United Kingdom.

Dr. Thierry Girard, from Basel, presented “Genotype – Phenotype Comparison of the MHEH population.” RYR1 mutations in 24 IVCT-positive families were studied. Sixteen of the 24 families had the Swiss V2168M mutation. Three families had R614C, three families had G2434R, one family had G341R, and one family had 2458C. Out of the MHEH families, 10 had a mutation, 30 did not. The contractures were higher in individuals with the RYR1 mutation than in individuals without it.

Dr. Rachael Robinson of Leeds sent a questionnaire to centers in Europe and collected data on more than 800 families. Centers participating were from Belgium, France, Germany, Switzerland, Italy, and the UK. The questionnaire focused on the 15 causative mutations clustered in two regions of the RYR1, the N-terminal region with amino acids from 35 to 614, and the central region from amino acids 2163 to 2458. Not all families were screened for all 15 mutations.

Based on the data collected:

• Mutation prevalence varies across Europe.
• DNA testing of the 15 current mutations may benefit up to 30% of patients.
• Discordance was observed for the most prevalent mutations.
• Discordance is associated with weaker contractures.
• About 2.5% of families may have a false positive diagnosis based on genetic testing.
• Genetic testing guidelines will eliminate the risk of a false negative diagnosis.

Robinson concluded:

“Our objective has been to develop an effective RYR1 mutation testing service. The UK population frequencies of 15 RYR1 mutations were established by screening 300 unrelated MH-susceptible and 200 unrelated normal control individuals. Data indicated that DNA testing will benefit 25% of patients when MH status has been confirmed by a positive IVCT result in the family. Mutation frequency was used to devise a testing strategy in which the most frequent mutations were screened first, followed by the less frequent ones, and so on.”

Because of space and time limitations, we have not been able to discuss all the interesting presentations made at the meeting. It was an extremely informative and stimulating meeting, and we urge you to attend next year’s International MH Meeting, which will precede the EMHG meeting. The International Workshop and the annual meeting of the European group will both be held at Brunnen, in Switzerland, June 11-14, 2003. The details will be given on the EMHG Website.