High-grade instability was present in 60.6% of the tumor samples from index patients. Twenty-three germ-line DNA sequence variants in 24/46 families and four somatic mutations in three tumors were detected in MLH1 and MSH2. Fifteen mutations are novel. None of the newly identified germ-line variants was found in 100 alleles of healthy control individuals. We were able to characterize two intronic variants (MLH1 c.589-10T>A; MSH2 c.367-1G>A) with regard to their effect on mRNA. Both created new splice sites that replaced the regular ones. Germ-line mutations occurred in 44.8% of the families fulfilling the Amsterdam criteria and in 35.3% of the Bethesda patients. The detection of a pathogenic mutation was strongly correlated with microsatellite instability in the tumor DNA (p=0.007). This study is the first comprehensive report of mutations in mismatch repair genes in Austrian patients with HNPCC.

Malignant hyperthermia (MH) is an inherited skeletal muscle disorder triggered by commonly used anesthetics. Mutated ryanodine receptors have been identified as molecular targets. The sensitivity of myotubes from individuals classified by the in vitro contracture test as MH susceptible (MHS), normal (MHN), and equivocal (MHEH) was assessed for the Ca2+-releasing activity of 4-chloro-m-cresol (4-CmC) and caffeine. In this study, we sought to determine whether 4-CmC can differentiate the MH status of an individual on the basis of the release of intracellular Ca2+, particularly in regard to MHEH diagnosis. Intracellular Ca2+ concentration was determined photometrically with Fura2. Regions of the ryanodine receptor 1 harboring most of the described MH mutations were sequenced from MHS and MHEH cells. One MH mutation (Gly2434Arg) was found in one MHS individual. Results of the caffeine-induced Ca2+ release in MHS and MHN cells correlated well with the in vitro contracture test results. MHS cells showed a higher sensitivity against caffeine and, to a lesser extent, against 4-CmC. Cells of MHEH individuals showed low sensitivities against both caffeine and 4-CmC, comparable to those of the MHN group. Therefore, with myotubes, caffeine was able to discriminate between MHS and MHN cells, but both caffeine and 4-CmC failed to detect MHEH cells.

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