RESUMEN

Single strand conformational polymorphism analysis was used to screen exons 43 and 44 in the skeletal muscle ryanodine receptor gene from 17 positively diagnosed members of families in which chromosome 19-linked malignant hyperthermia (MH) was segregating. A polymorphism in two unrelated individuals was found to result from the substitution of A for G7297, leading to the substitution of Arg for Gly2433. This mutation is adjacent to a mutation (Arg2434 to His) previously linked to MH and central core disease (Y. Zhang et al., Nature Genet. 1993, 5, 46-50). Subsequent screening showed the presence of the mutation in four of 106 MH families tested and its absence from about 1000 other chromosomes. The mutation was present in all six individuals in four families who had had an MH reaction, in two obligate carriers and in 10 individuals diagnosed as MH susceptible by the caffeine/halothane contracture test (CHCT). The mutation was present in an individual with a normal response to the CHCT and was absent in three individuals with a positive CHCT response. These discrepancies would be consistent with inaccuracies in the CHCT and/or with segregation of a second MH allele within two of the four affected families.

Asunto(s)

RESUMEN

Malignant hyperthermia (MH) is a potentially lethal condition in which sustained muscle contracture, with attendant hypercatabolic reactions and elevation in body temperature, are triggered by commonly used inhalational anaesthetics and skeletal muscle relaxants. In humans, the trait is usually inherited in an autosomal dominant fashion, but in halothane-sensitive pigs with a similar phenotype, inheritance of the disease is autosomal recessive or co-dominant. A simple and accurate non-invasive test for the gene is not available and predisposition to the disease is currently determined through a halothane- and/or caffeine-induced contracture test on a skeletal muscle biopsy. Because Ca2+ is the chief regulator of muscle contraction and metabolism, the primary defect in MH is believed to lie in Ca2+ regulation. Indeed, several studies indicate a defect in the Ca2+ release channel of the sarcoplasmic reticulum, making it a prime candidate for the altered gene product in predisposed individuals. We have recently cloned complementary DNA and genomic DNA encoding the human ryanodine receptor (the Ca2(+)-release channel of the sarcoplasmic reticulum) and mapped the ryanodine receptor gene (RYR) to region q13.1 of human chromosome 19 (ref. 14), in close proximity to genetic markers that have been shown to map near the MH susceptibility locus in humans and the halothane-sensitive gene in pigs. As a more definitive test of whether the RYR gene is a candidate gene for the human MH phenotype, we have carried out a linkage study with MH families to determine whether the MH phenotype segregates with chromosome 19q markers, including markers in the RYR gene. Co-segregation of MH with RYR markers, resulting in a lod score of 4.20 at a linkage distance of zero centimorgans, indicates that MH is likely to be caused by mutations in the RYR gene.

Asunto(s)

RESUMEN

The cytoplasmic concentration of ionised calcium, [Ca2+]i, is believed to be altered by agents that induce a malignant hyperthermia (MH) crisis in susceptible individuals. MH patients were identified by the halothane and halothane/caffeine contracture tests done in isolated muscle biopsy specimens. [Ca2+]i was measured in isolated peripheral blood mononuclear cells from MH patients and controls by means of the fluorescent calcium ion indicator quin2. In the absence of halothane there was no significant difference in [Ca2+]i in cells from normal and MH patients. Addition of halothane (4 microliter/ml) significantly increased [Ca2+]i in cells from MH patients but not in controls. The halothane-induced increase in [Ca2+]i required extracellular calcium ions. This is the first evidence of the mechanism of action of halothane in cells of MH patients; the differential effect of halothane on [Ca2+]i might constitute the basis for a non-invasive screening test for MH.

Asunto(s)

RESUMEN

Dantrolene sodium, a hydantoin analogue, is efficacious in the therapy of malignant hyperthermia (MH). In order to improve our knowledge of the mode of action of dantrolene, we have examined the influence of dantrolene sodium on: (1) twitch and resting tensions, in the absence and the presence of caffeine, of intact skeletal muscle fascicles; and (2) caffeine induced tension rises of single chemically skinned skeletal muscle fascicles. We have found that dantrolene appears to exert its beneficial action on malignant hyperthermia susceptible (MHS) skeletal muscle by an indirect action on the sarcoplasmic reticulum (SR). Thus dantrolene inhibits twitch tensions of skeletal muscle fascicles, probably by indirectly preventing the release of calcium from the SR. To a lesser extent dantrolene inhibits caffeine induced contractures of skeletal muscle fascicles, probably by indirectly accelerating the uptake of calcium into the SR. Because the former effect is greater than the latter in vivo dantrolene sodium is effective only when given prior to total loss of calcium from the SR. Vigilant temperature and EKG monitoring of all patients during anaesthesia is, therefore, essential.

Asunto(s)

RESUMEN

We have compared and contrasted two diagnostic tests for Malignant Hyperthermia (MH) - the Caffeine-Halothane Contracture Test and the Caffeine Skinned Fibre Tension Test. Both tests show a strongly positive relationship both with the occurrence of MH reactions and with each other. The former test is more rapid and requires less skill. The latter test can be performed on much less muscle and permits storage of the muscle over prolonged periods of time.

Asunto(s)

RESUMEN

Quercetin inhibited Ca2+-dependent ATP hydrolysis, ATP-dependent Ca2+ uptake, chelator-induced [ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] Ca2+ release, and ATP synthesis coupled to Ca2+ release in isolated vesicles of sarcoplasmic reticulum. Use of this inhibitor permitted evaluation of whether Ca2+ release from sarcoplasmic reticulum in situ occurs through a reversal of the uptake pathway. Release of Ca2+ from the sarcoplasmic reticulum of skinned muscle fibers can be detected by the measurement of tension in the fiber. If the sarcoplasmic reticulum of these preparations is first allowed to accumulate Ca2+, tension development may be induced by the addition of Ca2+ itself or of caffeine to the bathing medium or by depolarization with Cl-. The presence of quercetin during the loading phase inhibited Ca2+ uptake by sarcoplasmic reticulum in situ. When quercetin was added together with initiators of tension development, however, the rate of tension development was enhanced 4- to 7-fold and the relaxation rate of the fibers was greatly inhibited. These results suggest that quercetin had no effect on Ca2+ release in skinned fiber; its effect on Ca2+ reuptake could account for the apparent enhancement of the release rate and for the prolonged relaxation time. These observations rule out reversal of the Ca2+ pump as the mechanism of Ca2+ release in situ.

Asunto(s)

Asunto(s)

RESUMEN

We have compared the combined effects of halothane, enflurane, isoflurane and methoxyflurane on caffeine-induced contractures of normal and malignant hyperthermia susceptible (MHS) skeletal muscle fascicles. We have found that caffeine contractures without and with the addition of any of these four anaesthetics are higher in MHS than in normal muscle. The differences between the normal and MHS muscle are about the same for all drug combinations. For all four anaesthetics the degree of increase of the contracture is about the same in the normal as in the MHS muscle. For both the MHS and the normal muscle the caffeine contractures are from greatest to least: halothane greater than isoflurane greater than enflurane greater than methoxyflurane. Examination of the relationships among the caffeine specific concentrations in the presence of the various anaesthetics shows significant differences for the comparisons of halothane with the other three anaesthetics but, for the most part, the comparisons among methoxyflurane, enflurane and isoflurane are not meaningful statistically.

Asunto(s)