Phenotyping Protocol Research Articles

A novel ryanodine receptor mutation and genotype-phenotype correlation in a large malignant hyperthermia New Zealand Maori pedigree.

Malignant hyperthermia (MH) is a pharmacogenetic disorder that predisposes to a sometimes fatal hypermetabolic reaction to halogenated anaesthetics. MH is considered to originate from abnormal regulation of skeletal muscle Ca(2+) release. Current diagnosis of MH susceptibility (MHS) relies on in vitro contracture testing (IVCT) of skeletal muscle. The ryanodine receptor (RYR1) encoding the major Ca(2+) release channel in the skeletal muscle sarcoplasmic reticulum has been shown to be mutated in a number of MH pedigrees. The large Maori pedigree reported here is the largest MHS pedigree investigated to date and comprises five probands who experienced clinical episodes of MH and 130 members diagnosed by the IVCT. Sequencing of the 15 117 bp RYR1 cDNA in a MHS individual from this pedigree identified a novel C14477T transition that results in a Thr4826 to Ile substitution in the C-terminal region/transmembrane loop of the skeletal muscle ryanodine receptor. This is the first mutation in the RyR1 C-terminal region associated solely with MHS. Although linkage analysis showed strong linkage (max LOD, 11.103 at theta = 0.133) between the mutation and MHS in the pedigree using the standardized European IVCT phenotyping protocol, 22 MHS recombinants were observed. The relationship between the IVCT response and genotype was explored and showed that as IVCT diagnostic cut-off points were made increasingly stringent, the number of MHS discordants decreased with complete concordance between the presence or absence of the C14477T mutation and MHS and MH normal phenotypes, respectively, using a cut-off of 1.2 g tension at 2.0 mM caffeine and 1.8 g tension at 2.0% halothane. Many MHS pedigrees investigated have been excluded from linkage to the RYR1 gene on the basis of a small number of recombinants; however, the linkage analysis reported here suggests that other recombinant families excluded from linkage to the RYR1 gene may actually demonstrate linkage as the number of members tested within the pedigrees increases. The high number of discordants observed using the standardized diagnostic cut-off points is likely to reflect the presence of a second MHS susceptibility locus in the pedigree.

Developmental regulation of the rosy locus in Drosophila melanogaster.

The rosy locus of Drosophila melanogaster encodes the enzyme xanthine dehydrogenase (XDH, xanthine: NAD oxidoreductase). The most thoroughly studied gene-enzyme system in higher eukaryotes (Sang, 1984), and long a paradigm for eukaryotic gene organization, the rosy locus has recently become a fertile system for analysis of eukaryotic gene regulation. The major reasons for this are: 1 The locus encodes an enzyme (XDH) that has been isolated and characterized and is easily assayed in extracts of small numbers of whole organisms or isolated tissues (see Sections 3 and 6). 2 The locus has been subject to intensive fine-structure genetic analyses that have defined its limits and organization (see Sections 4 and 7). 3 Effective phenotypic selection and screening protocols are available (see Section 2), taking advantage of conditional lethality at the locus, and resulting in the isolation of a wealth of mutants, including regulatory variants (see Section 7). 4 It is clear that expression of rosy is regulated at both the stage and tissue levels during development (see Section 6); mutants that affect particular stages and tissues have been identified (see Section 7). 5 The locus has been cloned and sequenced. Thus, molecular probes are available for analyses at the DNA and RNA levels (see Sections 4 and 5). 6 The technique of P element-mediated transformation was initiated using the rosy system, and genetic and molecular analyses using transposons are now quite advanced (see Sections 4 and 7). Manipulation of rosy transposons also permits analysis of a range of regulatory phenomena not exclusive to XDH, including euchromatic position effects, heterochromatic position effects, and dosage compensation (see Section 7).