Chapter Eighteen: Maturity in MH Testing

Chapter 18 Sub-sections

To summarize, contracture testing in muscle strips from the vastus muscle in the thigh is accepted as the Gold Standard and as the putative clinical surrogate. This is because it mimics a direct anesthetic challenge in a human, somewhat similar to the actual challenge in swine. Genetic analysis of DNA further matured MH testing: the polymorphic ryanodine receptor gene has more than 170 missense mutations (Robinson et al, 2006), but just 21 are known to cause MH (Girard et al, 2008). Once DNA analysis detects an MH mutation, blood samples from relatives can be tested for that mutation. If it is found, susceptibility is confirmed; if not, contracture testing on biopsied vastus muscle is desirable to confirm normality (Rueffert et al, 2001; Urwyler et al, 2001; Pollock et al, 2002; Jurkat-Rott et al, 2000; Curran et al, 1999).

The multiple MH mutations are grouped by locus, the location on a chromosome: MHS-1, on chromosome 19q, encodes the ryanodine receptor, the primary MH site. Secondary sites include MHS-2, on chromosome 17q, the voltage dependent sodium channel of skeletal muscle, MHS-3, on chromosome 7q, the dihydropyridine receptor, MHS-4, on chromosome 3q, MHS-5, on chromosome 1q, MHS-6, on chromosome 5p. The equivalent porcine chromosome for MH susceptibility is number 6, and is a single mutation accounting for MH susceptibility in all swine.

Other advances in MH testing that may obviate contracture testing include micro-injection of caffeine or halothane into human quadriceps muscle to measure exaggerated CO2 responses (Anetseder et al, 2002; Schuster et al, 2006, 2007; Bina et al, 2006), and advanced application of leukocyte DNA analysis, since skeletal muscle RYR mutations also appear in leukocytes (Sei et al, 1999; Girard et al, 2001; Kraev et al, 2003; Loke et al, 2003). Micro-injection is explained next.

Adaptation of Isolated Perfused Caudal Pig Preparation to an in Vivo Test

At the invitation of the senior researcher, Dr. Edmund Hartung, I had visited the anesthesia department at the University Hospital in Wurzburg, Germany in 1993, where micro-injection was later developed, and discussed our perfused isolated caudal pig preparation, in which MH could be triggered (Gronert, Milde et al, 1980). This study involved complete section of the body of a pig just below the umbilicus, with perfusion via a cannula in the aorta going to the legs, and another in the inferior vena cava going away from the legs. Dr. Hartung told me in summer 2005 in Stralsund, Germany that my presentation of this technique stimulated his consideration of the possibility of micro-injection of MH triggers, and he began the study. It was further developed after he left Wurzburg. The approach in pigs and humans involves placing a microdialysis catheter into a thigh muscle and adding tiny amounts of MH triggers such as caffeine or halothane. A second catheter is inserted to measure changes in pH, lactate, or CO2 in the region of the injecting catheter. MH susceptible swine or humans safely respond with exaggerated acid production, limited to just that area, i.e., a whole body MH response does not occur. This has developed into a fertile approach toward identification of MH susceptible persons (Anetseder et al, 2002; Schuster et al, 2006, 2007; Bina et al, 2006). Dr. Hartung has been a co-author of two abstracts (Anetseder et al, 2000).

Another minimally invasive non-destructive test is electrical stimulation of a muscle to produce a twitch in susceptible swine that produces a pattern different from that in normal swine. The altered pattern presents an in vivo differentiating test to detect MH susceptibility in swine, but requires sophisticated stimulating equipment. Examination of this approach in susceptible humans was unsuccessful in detecting MH susceptible humans, as their responses were weakened by heterogeneity, e.g., the varied mutations resulted in excessive variability in response to electrical stimulation (Quinlan et al, 1986; Quinlan et al, 1989). Interpretation of human test results and their variations, coordination with clinical findings if a suspected MH episode has occurred, and further MH research are aided by a standardized clinical grading scale, for use in a patient who has had an episode suspicious of MH (Larach et al, 1994). The scale evaluates changes in vital signs and laboratory data and scores them by degree of abnormality. Various case reports of human MH rely upon background information of abnormal physiology, genetics, contracture testing, and altered drug responses (Simmons et al, 1984; Lee et al, 1994; Ogletree et al, 1996; Albrecht et al, 1997; Melton et al, 1999).

Worldwide Human MH Testing

Where is human MH testing in 2008? Major achievements lie with researchers in the European MH Group, South Africa, Japan, China, the United States, and Australia/New Zealand. The United States lags in this area because of our inefficient and fragmentary profit-based health care system. In the U.S., a muscle biopsy and contracture studies costs more than $5000 per patient, and few health care organizations pay for much of that, as opposed to the willingness of medical insurance companies in years past. MH experts are now obtaining Medicare support. Also, there are only about five centers in the US and Canada, and costly travel is involved for testing.

In Europe, where there are more centers, the cost per patient, to the family, is small and the additional amount is subsidized by the government and the university testing center. So, in the U.S., few are tested, while in Europe entire families are routinely tested. Once contracture studies confirm MH susceptibility, genetic analysis can determine the presence of mutations. If a mutation responsible for MH is detected, additional family members can be examined for that specific mutation without need for the invasive muscle biopsy. In time, it may be that relatives who lack the mutation can be considered not susceptible but more patients must be evaluated before making this conclusion.

DNA analysis is usually performed on blood samples. The U.S. MH interests are expanding DNA analyses with in depth analysis of affected families, with modest financial support.

Other Species and MH Susceptibility

Both at UC Davis and Mayo, we analyzed responses of species other than swine that were considered potentially susceptible to MH, particularly wild animals that suffered capture stress or were super athletes. Capture stress occurs in wild animals captured after a prolonged chase or by a net dropped from a helicopter chasing the animal. The clinical picture, as you might expect, resembles an MH episode, rapid pulse rate, trembling in muscles, high temperature, acidosis, and systemic acidosis.

Species sometimes suspected of MH include wild deer, myotonic goats, and greyhound dogs. None demonstrated MH susceptibility, based upon anesthetic exposure and contracture testing (Antognini et al, 1996; Newberg et al, 1983; Cosgrove et al, 1993). Roberts et al have confirmed MH and ryanodine mutations in some dogs, although the canine MH reaction is qualitatively different (Roberts et al, 2001).