Those of you who are reading this blog probably know that one of the major accomplishments related to MH in recent years has been the introduction of a DNA test, usually obtained by a blood sample, to diagnose MH susceptibility. Three laboratories offer such testing , two for the general public and one for the military. Many individuals have avoided the more invasive muscle biopsy contracture test hoping that DNA test will replace the test. We are not there yet, but we are progressing along the pathway. A recent study may take us further along the road. Furthermore, a more accurate, less invasive test promises to provide insights into problems that are not currently thought to be related to MH, but in reality may be. For example, sudden death with exercise or exposure to hot environments.
Although the DNA based test is useful for certain situations and families, it is definitely not ready for use as a screening test. For example if we test the DNA of 100 patients from different families with a confirmed diagnosis of MH only about 30% will turn out to have one or more of the DNA changes (mutations) known to be causal for MH. We do know that there are other DNA changes that might or might not lead to susceptibility of MH. However those changes have yet to be characterized as to their role in causing MH. Progress in recognizing the significance of those changes is proceeding apace, but the steps involved are complex and require significant resources. Our colleagues in Europe in the European Malignant Hyperthermia Group (EMHG.org) have several laboratories focused on this effort. The methods for determining the significance of such mutations will require a much longer essay.
To date, the DNA changes associated with MH are associated with two genes, the ryanodine receptor gene(RYR-1) which is the skeletal muscle calcium channel that controls calcium release into muscle and the DHPR gene. The DHPR ( dihydropyridine receptor) is a protein that controls the activity of the ryanodine receptor calcium release channel during muscle excitation. At this point about 150 DNA changes or mutations have been found in the RYR-1 of MH susceptible patients. When the DNA is analyzed by determining the sequence of the nucleic acids in the gene in MH susceptibles, even under the best of circumstances only about 70% will be found to have a DNA change that definitely or may possibly lead to MH susceptibility. (The gene for DHPR is found to be abnormal in only a very few MH susceptibles). If this is the case then other currently unidentified genes are likely to cause MH susceptibility in families that lack mutations in RYR-1 and DHPR genes. Despite many studies in humans only the two genes, the RYR -1 and the DHPR genes have been definitively shown to be linked to MH susceptibility.
But now a sophisticated study in genetically engineered mice gives us a major new avenue for exploration of the genetics of MH. Scientists in Italy and at the University of Rochester,(Reference) have looked into the role of a cellular calcium regulating protein called Calsequestrin in MH.
Calsequestrin is found in the storage site for calcium in muscle cells called the sarcoplasmic reticulum (SR). Calsequestrin (CSQ) is one of several other proteins that influence the amount of calcium ions in the SR and also influences the opening and closing of the calcium release channel of the SR(the RYR-1). Thus calsequestrin might be thought of as a sponge that binds free calcium in the SR storage compartment to maintain the levels in normal range, and also influences the activity of the ryanodine receptor calcium channel.
(For a better understanding of the cellular physiology of MH, please refer to the slide show on the MHAUS web site).
Here is how this came about. Dr. Robert Dirksen, a muscle physiologist, and member of our PAC at the University of Rochester has done a lot of research into the control of cell calcium in normal and MH muscle. Working with colleagues in Italy who were also interested in calcium movements in muscle , they generated a genetically engineered mouse that lacked the cardiac form of the calcium regulatory protein, Calsequestrin(CSQ). Their initial interest was principally on the effect of this protein on normal skeletal muscle contraction, however, the investigators were surprised by the fact the male CSQ deficient mice were dying at a much younger age than wild type mice , and that this typically occurred with the mice were mating . Based on this observation, the investigators hypothesized that other stressors might trigger sudden death in these mice. Therefore, the animals were challenged with at trigger anesthetic for MH. Sure enough, these CSQ deficient animals when exposed to either halothane or heat stress developed all the signs of MH! In addition the syndrome could be prevented by pretreatment with dantrolene! Furthermore many of the cellular changes in muscle isolated from the animals after an episode were similar to those found in MH. They also made another intriguing observation. It is well established that on a statistical basis, more males than females die from MH by a factor of between two and four. Intriguingly, a similar male predominance for MH susceptibility (80% of the genetically engineered male animals died from MH, but only 20% of the females) was observed in SCSQ 1 deficient mice. Furthermore, the genetically engineered male animals died at a higher rate when placed in cages with females for mating purposes. Men, bear in mind that one cannot extrapolate the genetic changes found in an animal to a human.
The environmental temperature that the animals were exposed to was 105 degrees F. During the heat challenge, the core body temperature of the genetically engineered animals rose to a higher level than that observed in the control animals.
The significance of these findings is that we now have a clue to look for DNA changes in the CSQ gene. So, this protein, if abnormal, may account for some or all of the cases of MH that do not have a defect or change in the ryanodine receptor protein. Time will tell.
These studies are important not only for understanding MH, but also for the understanding of the role of cellular proteins in muscle contraction and muscle biochemistry. To date, there is no human disorder of skeletal muscle that is characterized by a deficiency or abnormality of the Calsequestrin protein. However, that may be because no one has looked. But, when the analogous protein is missing in the heart cell, patients and animals are at risk for sudden death from rhythm disturbance.
Not so surprising is that animals and people who have mutations in the heart cell equivalent of the skeletal muscle ryanodine receptor calcium channel are also at risk for sudden death from rhythm disturbances.
We are looking forward to other scientists and MH researchers following this lead and hopefully reveal those other genetic changes leading to MH that might improve the sensitivity of DNA testing for MH.These are exciting times.Reference:Marco Dainese, Marco Quarta, Alla D. Lyfenko, Cecilia Paolini, Marta Canato, Carlo Reggiani, Robert T. Dirksen, and Feliciano Protasi. Anesthetic- and heat-induced sudden death in calsequestrin-1-knockout mice . FASEB J. first published on February 23, 2009 as doi:10.1096/fj.08-121335.