Mary Lehane

Identification of Novel Mutations in the Ryanodine-Receptor Gene (RYR1) in Malignant Hyperthermia: Genotype-Phenotype Correlation

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle that is triggered in genetically predisposed individuals by common anesthetics and muscle relaxants. The ryanodine receptor (RYR1) is mutated in a number of MH pedigrees, some members of which also have central core disease (CCD), an inherited myopathy closely associated with MH. Mutation screening of 6 kb of the RYR1 gene has identified four adjacent novel mutations, C6487T, G6488A, G6502A, and C6617T, which result in the amino acid alterations Arg2163Cys, Arg2163His, Val2168Met, and Thr2206Met, respectively. Collectively, these mutations account for 11% of MH cases and identify the gene segment 6400-6700 as a mutation hot spot. Correlation analysis of the in vitro contracture-test data available for pedigrees bearing these and other RYR1 mutations showed an exceptionally good correlation between caffeine threshold and tension values, whereas no correlation was observed between halothane threshold and tension values. This finding has important ramifications for assignment of the MH-susceptible phenotype, in genotyping studies, and indicates that assessment of recombinant individuals on the basis of caffeine response is justified, whereas assessment on the basis of halothane response may be problematic. Interestingly, the data suggest a link between the caffeine threshold and tension values and the MH/CCD phenotype.

A multicenter study of 4-chloro-m-cresol for diagnosing malignant hyperthermia susceptibility.

Standardization of the in vitro contracture test (IVCT) for malignant hyperthermia (MH) susceptibility has resulted in very rare false negative tests. However, false positive results stigmatizing the patient seem to be more frequent than false negative results and make supplementary tests desirable. This multicenter approach studied the usefulness of an IVCT with 4-chloro-m-cresol (4-CmC), a ryanodine receptor-specific agonist for a better definition of MH susceptibility. Diagnosis made by the standard IVCT was compared with the results of this 4-CmC test on muscle specimens of 202 individuals from 6 European MH centers. In the 4-CmC test, the results of the MH susceptible group differed significantly from both the MH normal and the MH equivocal group. 4-CmC revealed a qualitatively dose response-curve similar to caffeine. A correlation index of r = 0.79 for the concentration thresholds underlined the strong concordance of the caffeine and the 4-CmC effects. The optimal threshold concentration was determined to be 75 &mgr;M in the pooled data of all centers and is much lower than that of caffeine (2 mM), suggesting a more than 25-fold higher affinity of 4-CmC. The predictive value of 4-CmC is as high as that of caffeine and consequently higher than that of halothane. 4-CmC seems to be a suitable drug to refine diagnosis of MH susceptibility and could be used as an additional test substance. Implications Although in vitro contracture testing for malignant hyperthermia diagnosis is well standardized, with a relatively high sensitivity and specificity, false test results cannot be excluded and may be associated with serious disabilities for the concerned individuals. In this multicenter study, 4-chloro-m-cresol was evaluated as a new test substance for the in vitro contracture testing. Its use improves the accuracy of in vitro diagnosis of malignant hyperthermia susceptibility.

Detection of a novel mutation in the ryanodine receptor gene in an Irish malignant hyperthermia pedigree: correlation of the IVCT response with the affected and unaffected haplotypes.

Defects in the ryanodine receptor (RYR1) gene are associated with malignant hyperthermia (MH), an autosomal dominant disorder of skeletal muscle and one of the main causes of death resulting from anaesthesia. Susceptibility to MH (MHS) is determined by the level of tension generated in an in vitro muscle contracture test (IVCT) in response to caffeine and halothane. To date, mutation screening of the RYR1 gene in MH families has led to the identification of eight mutations. We describe here the identification of a novel mutation, Arg552Trp, in the RYR1 gene, which is clearly linked to the MHS phenotype in a large, well characterised Irish pedigree. Considering that the RYR1 protein functions as a tetramer, correlation of the IVCT with the affected and unaffected haplotypes was performed on the pedigree to investigate if the normal RYR1 allele in affected subjects contributes to the variation in the IVCT. The results show that the normal RYR1 allele is unlikely to play a role in IVCT variation.

Diagnosis of susceptibility to malignant hyperthermia with flanking DNA markers.

OBJECTIVE--To define the region on human chromosome 19 carrying the gene for malignant hyperthermia susceptibility and to evaluate the use of flanking DNA markers in diagnosing susceptibility. DESIGN--Prospective molecular genetic linkage studies in a large malignant hyperthermia pedigree. SETTING--Irish malignant hyperthermia testing centre. SUBJECTS--A large Irish malignant hyperthermia pedigree. MAIN OUTCOME MEASURES--Routine diagnosis of susceptibility to malignant hyperthermia with in vitro contracture test on muscle biopsy specimens and genetic linkage between susceptibility and polymorphic DNA markers in a malignant hyperthermia family. RESULTS--Genetic typing of polymorphic DNA markers in a large Irish malignant hyperthermia pedigree generated a lod score of greater than 3 for the marker D19S9 and showed that the gene for susceptibility is flanked by the markers D19S9 and D19S16. These tightly linked flanking markers allowed non-invasive presymptomatic diagnosis of susceptibility in five untested subjects in the large pedigree with an accuracy of greater than 99.7%. CONCLUSIONS--DNA markers flanking the gene for susceptibility to malignant hyperthermia can be used with high accuracy to diagnose susceptibility in subjects in large known malignant hyperthermia pedigrees and may replace the previous in vitro contracture test for diagnosing this inherited disorder in large families with malignant hyperthermia.

Diagnosis of malignant hyperthermia: a comparison of the in vitro contracture test with the molecular genetic diagnosis in a large pedigree.

Malignant hyperthermia (MH) is an inherited skeletal muscle disorder and is one of the major causes of death resulting from anaesthesia. MH is currently diagnosed by the in vitro contracture test performed on a muscle biopsy. Genetic linkage analysis on an Irish MH pedigree showed that when the thresholds for the standardised European protocol for MHS diagnosis was applied, linkage between the MHS phenotype and the RYR1 locus was excluded. When we raised the threshold values for assignment of MHS status and assumed MHN diagnosis in subjects where this threshold was not attained, tight linkage between MHS and RYR1 markers was observed, suggesting that MHS is linked to the RYR1 locus in this pedigree. Confirmation of these results was borne out by the fact that all of the MHS patients in the pedigree exceeding the raised threshold carried the known MHS Gly341Arg RYR1 mutation. The results obtained could be explained (1) by false positive diagnosis of MHS in the recombinant subjects, (2) by the presence of a mutation in a predisposing gene other than RYR1, or (3) by the presence of mild subclinical myopathies. The implications of these results for heterogeneity studies is discussed.

Genetics in Anesthesiology

Other, vital, facts are simply missing. There is no real discussion of population genetics. One looks in vain to see real figures for just how prevalent in the population such genes might be today and therefore just what fast breeders the sad, mad, bad, and the stupid might have to be in order to trigger the explosion of the gene bomb which Dr Comings so fears. There is a scrappy two page illustration of the results of using one of the equations that calculates the rate at which the frequency of a gene with such a selective advantage will increase over succeeding generations. The equation itself is not given, nor is there any discussion of what factors might limit the spread of the gene. In 1938, the late great J B S Haldane showed in his little book Heredity and Politics that eugenic programmes in the USA in the 1920s and 1930s for sterilising mental defectives would have had a negligible effect on the average IQ of the population. Dr Comings presents no figures on how many of the sad, mad, bad, and stupid would have to refrain from reproduction (voluntarily) before the streets of Harlem or inner city Los Angeles would once again be safe for decent, educated people to walk down. With errors and omissions of fact go astonishing assumptions about moral and ethical values. Dr Comings tells us that our IQ is like the core of our essence. Really? A Christian might say that the foundation of our being is our relationship to a loving God, whereas a modern secular philosopher might make membership of the moral community the defining characteristic of humanity. Neither of these characteristics necessarily correlates with IQ and the kingdom ofheaven certainly is not a paradise of the intellectual. The point here is not who is right or wrong but that Dr Comings initial assumption is dubious, and so much of what follows is also dubious. The fundamental problem is that we have all been here before, many times. In the 1 920s and 1930s the concern in the USA was not inner city Blacks and Hispanics but the white rural poor. Statistics put forward then by geneticists at least as respectable as Dr Comings, and with better worked out population genetics, showed irrefutably that the rural poor were significantly stupider and more fecund than the urban middle classes. But, strange to relate, the USA did not decline into a rural slum populated by village idiots. Instead, it became the worlds most technologically advanced nation while the children and the grandchildren of the white trash of the 1 920s turned into very capable electronic engineers and rocket scientists enabling NASA to put the first man on the moon. Now thats what I call a reality check.

Diagnosis of Susceptibility to Malignant Hyperthermia with Flanking DNA Markers

OBJECTIVEnTo define the region on human chromosome 19 carrying the gene for malignant hyperthermia susceptibility and to evaluate the use of flanking DNA markers in diagnosing susceptibility.nnnDESIGNnProspective molecular genetic linkage studies in a large malignant hyperthermia pedigree.nnnSETTINGnIrish malignant hyperthermia testing centre.nnnSUBJECTSnA large Irish malignant hyperthermia pedigree.nnnMAIN OUTCOME MEASURESnRoutine diagnosis of susceptibility to malignant hyperthermia with in vitro contracture test on muscle biopsy specimens and genetic linkage between susceptibility and polymorphic DNA markers in a malignant hyperthermia family.nnnRESULTSnGenetic typing of polymorphic DNA markers in a large Irish malignant hyperthermia pedigree generated a lod score of greater than 3 for the marker D19S9 and showed that the gene for susceptibility is flanked by the markers D19S9 and D19S16. These tightly linked flanking markers allowed non-invasive presymptomatic diagnosis of susceptibility in five untested subjects in the large pedigree with an accuracy of greater than 99.7%.nnnCONCLUSIONSnDNA markers flanking the gene for susceptibility to malignant hyperthermia can be used with high accuracy to diagnose susceptibility in subjects in large known malignant hyperthermia pedigrees and may replace the previous in vitro contracture test for diagnosing this inherited disorder in large families with malignant hyperthermia.