Catherine Duff

A substitution of cysteine for arginine 614 in the ryanodine receptor is potentially causative of human malignant hyperthermia

Malignant hyperthermia (MH) is a devastating, potentially lethal response to anesthetics that occurs in genetically predisposed individuals. The skeletal muscle ryanodine receptor (RYR1) gene has been linked to porcine and human MH. Furthermore, a Cys for Arg substitution tightly linked to, and potentially causative of, porcine MH has been identified in the ryanodine receptor. Analysis of 35 human families predisposed to malignant hyperthermia has revealed the presence, and cosegregation with phenotype, of the corresponding substitution in a single family. This substitution, by analogy to the findings in pig, may be causal for predisposition to MH in this family.

Polymorphisms and deduced amino acid substitutions in the coding sequence of the ryanodine receptor (RYR1) gene in individuals with malignant hyperthermia.

Twenty-one polymorphic sequence variants of the RYR1 gene, including 13 restriction fragment length polymorphisms (RFLPs), were identified by sequence analysis of human ryanodine receptor (RYR1) cDNAs from three individuals predisposed to malignant hyperthermia (MH). All RFLPs were detectable in PCR-amplified products, and their segregation was consistent with our initial finding of linkage to MH in the nine families previously informative for one or more intragenic markers (MacLennan et al., 1990, Nature 343:559-561). Four amino acid substitutions were identified in the study: Arg for Gly248, Cys for Arg470, Leu for Pro1785, and Cys for Gly2059. Of 45 families tested, a single family presented the Arg for Gly248 substitution where it segregated with malignant hyperthermia, making it a candidate mutation for predisposition to MH in man. The other three polymorphic substitutions failed to segregate with malignant hyperthermia in those families in which they occurred, implying that they represent polymorphisms with little or no effect on the function of the RYR1 gene.

Chromosome stability in CHO cells

The established cell line derived many years ago from Chinese hamster ovary (CHO cells) has been studied for the extent of chromosomal variation. Because this cell line is used extensively for genetic studies, the contribution of chromosome variability to genetic variability has also been examined. The quasidiploid CHO cells were found to have a banded karyotype somewhat altered from that of the Chinese hamster from which the line was derived. However, most of the genome could be accounted for among the rearranged marker chromosomes. In addition, the CHO line was found to have a relatively stable karyotype, the same basic karyotype being found in a majority of the uncloned cells, as well as in most cells of several but not all independent clones. Many, but not all, mutant cell lines derived from CHO also showed the same basic karyotype. Quasitetraploid cells, derived either spontaneously or by Sendai-virus-induced fusion, showed considerably more variation resulting in loss or gain of whole chromosomes, rearrangement of chromosomes, and appearance of new “marker” chromosomes.

Protein-tyrosine phosphatase SH-PTP2 (PTPN11) is localized to 12q24.1-24.3.

A 2.1-kb cDNA probe encoding the human SH2-domain containing protein-tyrosine phosphatase SH-PTP2 (PTPN11) was hybridized to human metaphase chromosomes in three independent experiments. In each instance, hybridization was maximal to chromosome 12q24.1–q24.3. The presence of SH-PTP2 cDNA crosshybridizing sequences located on a number of other chromosomes suggested that SH-PTP2-related genes or pseudogenes are present in the human genome.

Marker segregation without chromosome loss at the emt locus in Chinese hamster cell hybrids.

Segregation, defined as the reexpression of the recessive phenotype, has been examined in Chinese hamster cell hybrids heterozygous at the recessive emtr locus. Segregants were selected in emetine, and the role of chromosome loss in the segregation process was evaluated by detailed karyotype comparison of segregants with their hybrid parent. In emtr CHO × emt+ CHO hybrids (CHO is thought to be hemizygous at the emt locus), segregants were obtained at high frequency, and no consistent chromosome loss was found in the segregants. In hybrids made with Emtr CHO and wild-type lines other than CHO (CHW,CHL,V-79), where two wild-type alleles are thought to be present in the hybrid, segregants were obtained at much lower frequency, consistent with a two-step segregation process. These segregants revealed consistent loss of one chromosome 2 or deletion of a part of the long arm of a chromosome 2. Thus, one step in segregation seems to be chromosome loss while the other step must have a different mechanism, possibly the same mechanism that operates in the CHO × CHO hybrids. Two major conclusions can be drawn: (1) the emt gene maps to a hemizygous region of the long arm of a chromosome 2 in Chinese hamster, and (2) a segregation mechanism other than chromosome loss appears to operate with high efficiency in intraspecific hybrids.