ONE characteristic of malignant hyperthermia (MH) is the autosomal-dominant mode of inheritance. This implies that at least one parent of an affected patient should be predisposed to MH. In this report, we describe two families in which MH susceptibility developed spontaneously through a neomutation. These families attracted attention because both parents of an MH-susceptible individual were diagnosed as MH-negative in the in vitro  contracture test (IVCT).

The IVCT was performed with halothane and caffeine according to the protocol of the European MH Group. 1This test may provide the following diagnoses: MH-susceptible (MH-positive), MH-negative, or MH-equivocal (possible MH-positive).

Cluster regions of the skeletal ryanodine receptor gene (RYR1), in which MH-related mutations have been identified, were amplified by polymerase chain reaction and subsequently analyzed by the direct sequencing technique previously described. 2Chromosome 19 microsatellite markers were amplified by polymerase chain reaction using labeled fluorescent-tag primers (D19S228, 19S421, RYR.PCR1, D19S422, D19S223, as recommended by the European MH Group, genetic section §) and were checked by fragment gel analysis in the ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA).

A 12-year-old girl related to the first family was referred to our MH investigation unit because of elevated resting creatine kinase levels (700 U/l) and rapid exhaustion after intensive exercise. The suspected MH disposition was confirmed by an MH-susceptible diagnosis in the in vitro  halothane/caffeine muscle test. MH had been transmitted from the mother, who was found to be MH-susceptible 2 months later. Interestingly, both grandparents showed normal responses in the halothane/caffeine muscle test (MH-negative diagnosis) and had undergone previous general anesthesias with no clinical complications. A familial genetic screening for MH-related mutations in the RYR1 gene revealed an Arg2435His mutation (G7304→A) in all MH-susceptible family members, but not in the MH-negative individuals. This substitution is one of the mutations regarded as causative of MH, according to the guidelines of the European MH Group for molecular genetic detection of MH susceptibility. 3To exclude the possibility of false paternity of the grandfather, we performed haplotyping with chromosome 19 microsatellite markers. The results of this segregation analysis proved that a true relationship between the investigated generations was almost certain (fig. 1).

Fig. 1. The segregation of the RYR1 mutations and chromosome 19.13.1 markers in the two investigated MH families. The presence/absence of the mutation is indicated by a + or − sign. The arrows  indicate the occurrence of a neomutation in the corresponding generation. Regarding IVCT results, the filled circles  indicate MH-susceptible–negative; the open circles with N  indicate MH-negative diagnosis, and the white boxes with question marks  indicate patients whose status has not been determined.
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Fig. 1. The segregation of the RYR1 mutations and chromosome 19.13.1 markers in the two investigated MH families. The presence/absence of the mutation is indicated by a + or − sign. The arrows  indicate the occurrence of a neomutation in the corresponding generation. Regarding IVCT results, the filled circles  indicate MH-susceptible–negative; the open circles with N  indicate MH-negative diagnosis, and the white boxes with question marks  indicate patients whose status has not been determined.

Fig. 1. The segregation of the RYR1 mutations and chromosome 19.13.1 markers in the two investigated MH families. The presence/absence of the mutation is indicated by a + or − sign. The arrows  indicate the occurrence of a neomutation in the corresponding generation. Regarding IVCT results, the filled circles  indicate MH-susceptible–negative; the open circles with N  indicate MH-negative diagnosis, and the white boxes with question marks  indicate patients whose status has not been determined.
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Fig. 1. The segregation of the RYR1 mutations and chromosome 19.13.1 markers in the two investigated MH families. The presence/absence of the mutation is indicated by a + or − sign. The arrows  indicate the occurrence of a neomutation in the corresponding generation. Regarding IVCT results, the filled circles  indicate MH-susceptible–negative; the open circles with N  indicate MH-negative diagnosis, and the white boxes with question marks  indicate patients whose status has not been determined.

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In the second family, a 15-year-old girl developed a tachycardia of 140 beats/min, 45 min after induction of anesthesia. Carbon dioxide rose to 90 mmHg, followed by a metabolic acidosis (pH 7.21) and a maximal serum creatine kinase elevation of 1655 U/l. General anesthesia for an operation on the spinal column was induced with thiopental, alfentanil, and vecuronium, and was maintained with isoflurane. When MH was suspected to be the possible cause of the symptoms, the isoflurane supply was interrupted and the symptoms rapidly declined. Dantrolene was not administered. The susceptibility to MH was confirmed by the IVCT 3 months later, and the histopathologic examination of a muscle biopsy also revealed the presence of central core disease. A RYR1 Ile2453Thr (T7358→C) substitution was identified by DNA testing. The patient’s mother, who also showed pathologic responses in the IVCT (MH-susceptible), carried the same Ile2453Thr mutation, but both grandparents showed a diagnostic constellation similar to that of family 1: both were found to be MH-negative in the IVCT, and neither carried the familial mutation.

The response of the muscle specimens from MH-susceptible and MH-negative individuals in the IVCT is shown in table 1, supplemented by sequencing gel traces of normal and mutated RYR1 alleles in figure 2.

Table 1. Results of the In Vitro Contracture Tests

Table 1. Results of the In Vitro Contracture Tests
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Table 1. Results of the In Vitro Contracture Tests
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Fig. 2. The sequencing gel traces of regions of RYR1 exon 45 (a ) and 46 (b ), in which the G7304→A (Arg2435His) and the T7358→C (Ile2453Thr) were detected.
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Fig. 2. The sequencing gel traces of regions of RYR1 exon 45 (a ) and 46 (b ), in which the G7304→A (Arg2435His) and the T7358→C (Ile2453Thr) were detected.

Fig. 2. The sequencing gel traces of regions of RYR1 exon 45 (a ) and 46 (b ), in which the G7304→A (Arg2435His) and the T7358→C (Ile2453Thr) were detected.
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Fig. 2. The sequencing gel traces of regions of RYR1 exon 45 (a ) and 46 (b ), in which the G7304→A (Arg2435His) and the T7358→C (Ile2453Thr) were detected.

Close modal

It is a known phenomenon of the routine MH diagnosis that in a few MH families, the results of the IVCT do not agree with the theoretical autosomal-dominant mode of inheritance of the disorder. This is sometimes a problem for MH investigators, who must interpret such findings accurately while accounting for the patient’s cultural background and personality.

Various reasons must be considered for those constellations in which both parents of an MH-susceptible patient show a normal response in the IVCT. An incorrect biologic paternity appears to be the most likely reason for the nontransmission of the MH disposition from a direct ancestor (frequencies of up to 10% in the general population were found in a study by the University of Virginia Commonwealth at Richmond). However, MH investigators should deal very carefully with any questions of paternity so as to not to get involved in or initiate a family crisis. In the families presented here, the paternity became plausible by the microsatellite marker segregation.

The second theoretical possibility is an incorrect result of the IVCT. However, the in vitro  halothane/caffeine muscle test is a well-established method, elaborated by MH experts to determine an MH disposition preclinically and to ensure the greatest possible safety for the patients. This is reflected in the high sensitivity (99% and 97%, respectively) of the test according to both available protocols. 1,4–6 

An alternative mode of transmission of the MH disposition, e.g. , recessive forms, may be a third reason for MH-normal parents, but so far this has not been described for MH.

Deduced from the results of the segregation of mutations Arg2435His (G7304→A) and Ile2453Thr (T7358→C), both located in the central MH hot spot region of the RYR1 gene 7as well as of the microsatellite markers, it was highly likely that the MH-susceptibility in the presented families was spontaneously caused by a neomutation. The susceptibility was then transmitted in the known autosomal-dominant manner. Other hitherto described de novo  mutations in the RYR1 gene have occurred exclusively in families with a sporadic central core disease, and were found only in the C-terminal domain (MH/central core disease region 3). 8 

Although both identified mutations also appear to correlate with central core disease (see the genetic European MH Group list for the Arg2435His and case report of the Ile2453Thr mutation carrier), there is some functional evidence concerning their possible causative nature for MH. 9–11 

The molecular genetic findings in these two families demonstrate that sporadic forms of MH are of clinical relevance, and that MH may also develop in the normal population. This must be considered when interpreting unclear IVCT constellations and in forensic matters.

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Tong J, Oyamada H, Demaurex N, Grinstein S, McCarthy TV, MacLennan DH: Caffeine and halothane sensitivity of intracellular Ca2+ release is altered by 15 calcium release channel (ryanodine receptor) mutations associated with malignant hyperthermia and/or central core disease. J Biol Chem 1997; 272: 26332–9
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