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DNA analysis and malignant hyperthermia susceptibility.

The inclusion, in this issue of Anaesthesia and Intensive Care, of a genetic study on malignant hyperthermia (MH) (Gillies et al, p. 391) is both long overdue and timely, as it highlights a number of issues currently facing anaesthetists, clinical geneticists and research scientists in the field.

The 'gold standard' diagnosis of MH is the in vitro contracture test (IVCT) of excised quadriceps muscle. This test is both invasive and labour intensive, but moreover, it is not 100% specific and it can yield equivocal results (MHE) (1). There is currently no simple (2) biochemical test for MH, a metabolic disorder of calcium homeostasis (2). MH is an autosomal dominant inherited disorder (3,4), so development of a DNA-based assay for a causative mutation should significantly reduce the reliance on in vitro contracture testing. Indeed, DNA-based diagnosis for MH-susceptibility in selected families has been carried out in New Zealand since 19985. This, however, is problematic on a number of counts. First, the underlying genetic cause of MH is heterogeneous. Not only has more than one gene been linked to MH in association studies (2), but over 180 mutations have been identified in the skeletal muscle ryanodine receptor gene (RYR1), the gene most commonly linked to MH (6,7). Second, a mutation must be shown to be causative before DNA-testing can be used for diagnosis of MH susceptibility (MHS) (8). Third, an MH-negative (MHN) diagnosis cannot be made by DNA-testing alone, due to the occasionally observed discordance between genotype and phenotype (i.e. negative DNA test results may be associated with abnormal muscle contractures). This may indicate more than one mutation segregating in an MH family (9-11).

Genotype/phenotype discordance is not universal and it could be argued that in some families a DNA-based MHN diagnosis could be made safely. A clinical geneticist or research scientist may have sufficient pedigree information coupled with extensive genetic analysis demonstrating clear lines of inheritance, as well as absence of the mutation and associated genotypes. This should allow diagnosis of non-susceptibility on the basis of a DNA test. (The exception would be in the case of a de novo causative mutation, but the likelihood of this occurring in an MHS family is identical to that in a non-MH family). But would a DNA-based MHN diagnosis be accepted by an anaesthetist? Probably not. If a patient does not have a familial mutation, does the research scientist then start screening the entire RYR1 gene for a second causative mutation, or is an IVCT immediately indicated? The former is expensive and time consuming and the researcher must weigh up the pros and cons of embarking on what may be a futile exercise. The latter is less expensive but invasive, and we have found that most of the time it will in fact yield an MHN result (unpublished data).

How can these problems be rectified? A simple in vitro functional assay demonstrating altered calcium homeostasis could solve many of these problems. Of the over 180 RYR1 mutations, only a relatively small number (29 or 30 (12,13)) have been shown to be causative and can thus be used in DNA-based diagnosis. A range of methodologies is currently available for demonstrating function. These include use of recombinant DNA technology together with expression in heterologous systems (14-16); immortalised lymphocytes from patients diagnosed MHS (12,17-19); or primary myoblasts isolated from MHS muscle biopsy samples (16,20,21). Depending on the system, a number of different functional assays can be carried out (12,22-24). This is the realm of the research scientist and while the products of such work will eventually lead to the development and implementation of alternative robust diagnostic methods, it requires a significant financial investment by already overstretched grant funding authorities. None of these techniques is trivial. The RYR1 cDNA has over 15,000 base pairs; a formidable challenge for cloning, site-directed mutagenesis and expression (23,25). Establishment of myoblasts and differentiation into myotubes is relatively simple, but requires muscle biopsy tissue and the myoblasts have limited life (26,27). Immortalisation of B lymphocytes is a relatively simple procedure, requires only a blood sample and functional assays can normally be carried out after approximately one month in culture.

An MHN result from in vitro calcium release assays in B lymphocytes, coupled with a mutation negative DNA test could feasibly be used in diagnosis, to replace or augment the IVCT, notwithstanding the contribution of other genes to MH. While considerably less invasive than the IVCT, the costs involved with both cell culture and functional assay are likely to preclude funding by the relevant health providers. They nevertheless remain extremely useful tools for both basic research and confirmation that a specific mutation has a functional effect on calcium homeostasis, the most important criterion for DNA-based diagnosis of malignant hyperthermia susceptibility.

To make matters even more complicated, any mutation screening exercise is likely to reveal novel as well as uncharacterised mutations (not yet shown to be causative of abnormal calcium homeostasis) as in the study by Gillies et al. Haplotype (linked alleles that are transmitted together) analysis adds support to genotype/phenotype relationships and can provide information about founder effects versus common occurrence. Moreover, haplotype analysis can assist acceptance of diagnosis by DNA testing as inheritance of chromosomal regions can be tracked through several generations. As with most good research, the study by Gillies et al has generated more questions to answer than have been answered. The MHE subjects did not exhibit familial mutations. What are the underlying molecular mechanisms of the MHE result? This study also revealed two RYR1 mutations segregating with MHS in the same family. What is the contribution of each mutation to MHS? Are the novel mutations identified actually causative of altered calcium homeostasis? Mutations were identified in about 45% of families studied. This is similar to the detection rate in our New Zealand studies, but considerably less than 70% reported in a United Kingdom study (7). Is this a function of screening only 'hot spot' regions as in the study by Gillies et al, or is a greater percentage of MH in New Zealand and Australia due to defects in genes other than RYR1?

As with many other reports concerning mutation screening for MH, these questions highlight an urgent need for a consolidated research effort to identify and characterise mutations associated with this disorder. Not only will such research effort lead to a confidence in and eventually acceptance of DNA-based diagnosis for MH, but it will also provide a molecular, biochemical and physiological understanding of calcium homeostasis and signaling in skeletal muscle.

REFERENCES

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(13.) European Malignant Hyperthermia Group. From: http://www.emhg.org Accessed February 2008.

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(20.) Lopez JR, Linares N, Pessah IN, Allen PD. Enhanced response to caffeine and 4-CmC in malignant hyperthermia susceptible muscle is related in part to chronically elevated resting [[Ca.sup.2+]]i. Am J Physiol Cell Physiol 2005; 288:606-612.

(21.) Yang T, Esteve E, Pessah IN, Molinski TF, Allen PD, Lopez JR. Elevated resting [[Ca.sup.(2+)]](i) in myotubes expressing malignant hyperthermia RyR1 cDNAs is partially restored by modulation of passive calcium leak from the SR. Am J Physiol Cell Physiol 2007; 292:C1591-1598.

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(24.) Treves S, Larini F, Menegazzi P, Steinberg TH, Koval M, Vilsen B et al. Alteration of intracellular [Ca.sup.2+] transients in COS-7 cells transfected with the cDNA encoding skeletal-muscle ryanodine receptor carrying a mutation associated with malignant hyperthermia. Biochem J 1994; 301:661-665.

(25.) Brini M, Manni S, Pierobon N, Du GG, Sharma P, MacLennan DH et al. [Ca.sup.2+] signaling in HEK-293 and skeletal muscle cells expressing recombinant ryanodine receptors harboring malignant hyperthermia and central core disease mutations. J Biol Chem 2005; 280:15380-15389.

(26.) Wehner M, Rueffert H, Koenig F, Neuhaus J, Olthoff D. Increased sensitivity to 4-chloro-m-cresol and caffeine in primary myotubes from malignant hyperthermia susceptible individuals carrying the ryanodine receptor 1 Thr2206Met (C6617T) mutation. Clin Genet 2002; 62:135-146.

(27.) Wehner M, Rueffert H, Koenig F, Olthoff D. Functional characterization of malignant hyperthermia-associated RyR1 mutations in exon 44, using the human myotube model. Neuromuscul Disord 2004; 14:429-437.

K. M. STOWELL

Institute of Molecular Biosciences,

Massey University,

Palmerston North, New Zealand

N. POLLOCK

Department of Anaesthesia and Intensive Care,

Palmerston North Hospital,

Palmerston North, New Zealand
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Author:Stowell, K.M.; Pollock, N.
Publication:Anaesthesia and Intensive Care
Article Type:Editorial
Geographic Code:8NEWZ
Date:May 1, 2008
Words:2027
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