Functional analysis of RYR1 variants in patients with confirmed susceptibility to malignant hyperthermia.

BACKGROUND: A major bottleneck to the introduction of noninvasive presymptomatic diagnostic tests for the pharmacogenetic disorder malignant hyperthermia is the lack of functional data for associated variants. METHODS: We screened 50 genes having a potential role in skeletal muscle calcium homeostasis using the HaloPlex™ (Agilent Technologies, Santa Clara, CA, USA) target enrichment system and next-generation sequencing. Twenty-one patients with a history of a clinical malignant hyperthermia reaction together with a positive in vitro contracture test were included. Eight variants in RYR1 were subsequently introduced into the cDNA for the human ryanodine receptor gene and tested in cultured human embryonic kidney (HEK293) cells for their effect on calcium release from intracellular stores in response to the ryanodine receptor-1 agonist 4-chloro-m-cresol using fura-2 as calcium indicator. Each variant was subjected to in silico curation using the European Malignant Hyperthermia Group scoring matrix and ClinGen RYR1 variant curation expert panel guidelines. RESULTS: Potentially causative RYR1 variants were identified in 15 patients. Of these, two families carried two RYR1 variants, five variants had been previously reported as 'pathogenic', two variants had been previously reported as 'likely benign', and eight were of 'uncertain significance'. Of these eight variants, four showed hypersensitivity to 4-chloro-m-cresol. Three variants were reclassified as either 'pathogenic' or 'likely pathogenic'. Two were classified as 'benign', whilst three remained of 'uncertain significance'. CONCLUSIONS: Three (p.Tyr1711Cys, p.Val2280Ile, and p.Arg4737Gln) additional variants can be added to the list of RYR1 disease-associated variants managed by the European Malignant Hyperthermia Group. These can therefore be used diagnostically in the future. Three variants (p.Glu2348Gly, p.Asn2634Lys, and p.Arg3629Trp) that remained classified as of uncertain significance require further family studies or a different functional test to determine clinical relevance in malignant hyperthermia.

A CpG mutational hotspot in a ONECUT binding site accounts for the prevalent variant of hemophilia B Leyden.

Hemophilia B, or the "royal disease," arises from mutations in coagulation factor IX (F9). Mutations within the F9 promoter are associated with a remarkable hemophilia B subtype, termed hemophilia B Leyden, in which symptoms ameliorate after puberty. Mutations at the -5/-6 site (nucleotides -5 and -6 relative to the transcription start site, designated +1) account for the majority of Leyden cases and have been postulated to disrupt the binding of a transcriptional activator, the identity of which has remained elusive for more than 20 years. Here, we show that ONECUT transcription factors (ONECUT1 and ONECUT2) bind to the -5/-6 site. The various hemophilia B Leyden mutations that have been reported in this site inhibit ONECUT binding to varying degrees, which correlate well with their associated clinical severities. In addition, expression of F9 is crucially dependent on ONECUT factors in vivo, and as such, mice deficient in ONECUT1, ONECUT2, or both exhibit depleted levels of F9. Taken together, our findings establish ONECUT transcription factors as the missing hemophilia B Leyden regulators that operate through the -5/-6 site.

DNA testing for malignant hyperthermia: the reality and the dream.

The advent of the polymerase chain reaction and the availability of data from various global human genome projects should make it possible, using a DNA sample isolated from white blood cells, to diagnose rapidly and accurately almost any monogenic condition resulting from single nucleotide changes. DNA-based diagnosis for malignant hyperthermia (MH) is an attractive proposition, because it could replace the invasive and morbid caffeine-halothane/in vitro contracture tests of skeletal muscle biopsy tissue. Moreover, MH is preventable if an accurate diagnosis of susceptibility can be made before general anesthesia, the most common trigger of an MH episode. Diagnosis of MH using DNA was suggested as early as 1990 when the skeletal muscle ryanodine receptor gene (RYR1), and a single point mutation therein, was linked to MH susceptibility. In 1994, a single point mutation in the α 1 subunit of the dihydropyridine receptor gene (CACNA1S) was identified and also subsequently shown to be causative of MH. In the succeeding years, the number of identified mutations in RYR1 has grown, as has the number of potential susceptibility loci, although no other gene has yet been definitively associated with MH. In addition, it has become clear that MH is associated with either of these 2 genes (RYR1 and CACNA1S) in only 50% to 70% of affected families. While DNA testing for MH susceptibility has now become widespread, it still does not replace the in vitro contracture tests. Whole exome sequence analysis makes it potentially possible to identify all variants within human coding regions, but the complexity of the genome, the heterogeneity of MH, the limitations of bioinformatic tools, and the lack of precise genotype/phenotype correlations are all confounding factors. In addition, the requirement for demonstration of causality, by in vitro functional analysis, of any familial mutation currently precludes DNA-based diagnosis as the sole test for MH susceptibility. Nevertheless, familial DNA testing for MH susceptibility is now widespread although limited to a positive diagnosis and to those few mutations that have been functionally characterized. Identification of new susceptibility genes remains elusive. When new genes are identified, it will be the role of the biochemists, physiologists, and biophysicists to devise functional assays in appropriate systems. This will remain the bottleneck unless high throughput platforms can be designed for functional work. Analysis of entire genomes from several individuals simultaneously is a reality. DNA testing for MH, based on current criteria, remains the dream.

Functional characterization of 2 known ryanodine receptor mutations causing malignant hyperthermia.

BACKGROUND: Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic disorder. More than 300 variants in the ryanodine receptor 1 (RYR1) have been associated with MH; however, only 31 have been identified as causative. To confirm a mutation in RYR1 as being causative for MH, segregation of the potential mutation in at least 2 unrelated families with MH susceptibility must be demonstrated and functional assays must show abnormal calcium release compared with wild-type RYR1. METHODS: We used "Hot-spot" DNA screening to identify mutations in RYR1 in 3 New Zealand families. B-lymphoblastoid cells were used to compare the amount of calcium released on stimulation with 4-chloro-m-cresol between wild-type RYR1 cells and cells carrying the new variants in RYR1. RESULTS: We identified a known RYR1 mutation (R2355W) in 2 families and another more recently identified (V2354M) mutation in another family. Both mutations segregated with MH susceptibility in the respective families. Cell lines carrying a mutation in RYR1 showed increased sensitivity to 4-chloro-m-cresol. CONCLUSIONS: We propose that R2355W is confirmed as being an MH-causative mutation and suggest that V2354M is a RYR1 mutation likely to cause MH.

Skeletal muscle ryanodine receptor mutations associated with malignant hyperthermia showed enhanced intensity and sensitivity to triggering drugs when expressed in human embryonic kidney cells.

BACKGROUND: Mutations within the gene encoding the skeletal muscle calcium channel ryanodine receptor can result in malignant hyperthermia. Although it is important to characterize the functional effects of candidate mutations to establish a genetic test for diagnosis, ex vivo methods are limited because of the low incidence of the disorder and sample unavailability. More than 250 candidate mutations have been identified, but only a few mutations have been functionally characterized. METHODS: The human skeletal muscle ryanodine receptor complementary DNA was cloned with or without a disease-related variant. Wild-type and mutant calcium channel proteins were transiently expressed in human embryonic kidney-293 cells expressing the large T-antigen of simian virus 40, and functional analysis was carried out using calcium imaging with fura-2 AM. Six human malignant hyperthermia-related mutants such as R44C, R163C, R401C, R533C, R533H, and H4833Y were analyzed. Cells were stimulated with a specific ryanodine receptor agonist 4-chloro-m-cresol, and intracellular calcium mobility was analyzed to determine the functional aspects of mutant channels. RESULTS: Mutant proteins that contained a variant linked to malignant hyperthermia showed higher sensitivity to the agonist. Compared with the wild type (EC50=453.2 µM, n=18), all six mutants showed a lower EC50 (21.2-170.4 µM, n=12-23), indicating susceptibility against triggering agents. CONCLUSIONS: These six mutations cause functional abnormality of the calcium channel, leading to higher sensitivity to a specific agonist, and therefore could be considered potentially causative of malignant hyperthermia reactions.

Metabolite profiling and quantification of phytochemicals in potato extracts using ultra-high-performance liquid chromatography-mass spectrometry.

BACKGROUND: Potatoes contain a diverse range of phytochemicals which have been suggested to have health benefits. Metabolite profiling and quantification were conducted on plant extracts made from a white potato cultivar and 'Urenika', a purple potato cultivar traditionally consumed by New Zealand Maori. There is limited published information regarding the metabolite profile of Solanum tuberosum cultivar 'Urenika'. RESULTS: Using ultra-high- performance liquid chromatography-mass spectrometry (UHPLC-MS), a total of 31 compounds were identified and quantified in the potato extracts. The majority of the compounds were identified for the first time in 'Urenika'. These compounds include several types of anthocyanins, hydroxycinnamic acid (HCA) derivatives, and hydroxycinnamic amides (HCAA). Six classes of compounds, namely organic acids, amino acids, HCA, HCAA, flavonols and glycoalkaloids, were present in both extracts but quantities varied between the two extracts. CONCLUSIONS: The unknown plant metabolites in both potato extracts were assigned with molecular formulae and identified with high confidence. Quantification of the metabolites was achieved using a number of appropriate standards. High-resolution mass spectrometry data critical for accurate identification of unknown phytochemicals were achieved and could be added to potato or plant metabolomic database.

Expression and role in glycolysis of human ADP-dependent glucokinase.

A novel murine enzyme, ADP-dependent glucokinase (ADPGK), has been shown to catalyse glucose phosphorylation using ADP as phosphoryl donor. The ancestral ADPGK gene appears to have been laterally transferred from Archaea early in metazoan evolution, but its biological role has not been established. Here, we undertake an initial investigation of the functional properties of human ADPGK in human tumour cell lines and specifically test the hypothesis that ADPGK might prime glycolysis using ADP under stress conditions such as hypoxia. Recombinant human ADPGK was confirmed to catalyse ADP-dependent glucose phosphorylation in vitro, with an apparent K (M) for glucose of 0.29 mM. Expression databases and western blotting of surgical samples demonstrated high expression in many human tissues, including tumours. Unlike hexokinase-2 (HK2), RNAi studies with exon arrays showed that ADPGK is not a transcriptional target of hypoxia inducible factor-1. Consistent with this, ADPGK protein was not upregulated by hypoxia or anoxia. Surprisingly, stable fivefold overexpression of ADPGK in H460 or HCT116 cells had no apparent effect on proliferation or glycolysis, and did not rescue clonogenicity or glycolysis when HK2 was suppressed by siRNA. Furthermore, suppression of ADPGK by siRNA did not cause detectable inhibition of glycolysis or cell killing by anoxia, although it did induce a statistically significant decrease in plating efficiency of H460 cells under aerobic conditions. Thus, human ADPGK catalyses ADP-dependent phosphorylation of glucose in vitro, but despite its high expression in human tumour cell lines it appears not to make a quantifiable contribution to glycolysis under the conditions evaluated.

Treatment with the PARP-inhibitor PJ34 causes enhanced doxorubicin-mediated cell death in HeLa cells.

Adjuvant therapies can incorporate a number of different drugs to minimize the cardiotoxicity of cancer chemotherapy, decrease the development of drug resistance and increase the overall efficacy of the treatment regime. Topoisomerase IIα is a major target of many commonly used anticancer drugs, where cell death is brought about by an accumulation of double-strand DNA breaks. Poly (ADP-ribose) polymerase (PARP)-1 has been extensively studied for its role in the repair of double-strand DNA breaks, but its ability to add highly negative biopolymers (ribosylation) to target proteins provides a vast number of pathways where it can also be important in mediating cell death. In this study, we combine the classical topoisomerase IIα poison doxorubicin with the PARP inhibitor PJ34 to investigate the potentiation of chemotherapeutic efficiency in HeLa cells. We demonstrate that PJ34 treatment has the capacity to increase endogenous topoisomerase IIα protein by about 20%, and by combining doxorubicin treatment with PJ34, we observed a 50% improvement in doxorubicin-mediated cell death in HeLa cells. These results were correlated with the ribosylation of transcription factor specificity factor 1 after doxorubicin treatment, thereby altering its affinity for binding to known regulatory elements within the human topoisomerase IIα promoter. Taken together, these results highlight the synergistic potential of combining PARP inhibitors with classical topoisomerase IIα-targeting drugs.

Functional studies of RYR1 mutations in the skeletal muscle ryanodine receptor using human RYR1 complementary DNA.

BACKGROUND: Malignant hyperthermia is associated with mutations within the gene encoding the skeletal muscle ryanodine receptor, the calcium channel that releases Ca from sarcoplasmic reticulum stores triggering muscle contraction, and other metabolic activities. More than 200 variants have been identified in the ryanodine receptor, but only some of these have been shown to functionally affect the calcium channel. To implement genetic testing for malignant hyperthermia, variants must be shown to alter the function of the channel. A number of different ex vivo methods can be used to demonstrate functionality, as long as cells from human patients can be obtained and cultured from at least two unrelated families. Because malignant hyperthermia is an uncommon disorder and many variants seem to be private, including the newly identified H4833Y mutation, these approaches are limited. METHODS: The authors cloned the human skeletal muscle ryanodine receptor complementary DNA and expressed both normal and mutated forms in HEK-293 cells and carried out functional analysis using ryanodine binding assays in the presence of a specific agonist, 4-chloro-m-cresol, and the antagonist Mg. RESULTS: Transiently expressed human ryanodine receptor proteins colocalized with an endoplasmic reticulum marker in HEK-293 cells. Ryanodine binding assays confirmed that mutations causing malignant hyperthermia resulted in a hypersensitive channel, while those causing central core disease resulted in a hyposensitive channel. CONCLUSIONS: The functional assays validate recombinant human skeletal muscle ryanodine receptor for analysis of variants and add an additional mutation (H4833Y) to the repertoire of mutations that can be used for the genetic diagnosis of malignant hyperthermia.

Identification and Functional Analysis of RYR1 Variants in a Family with a Suspected Myopathy and Associated Malignant Hyperthermia.

BACKGROUND: The ryanodine receptor 1 (RyR1) is a major skeletal muscle calcium release channel located in the sarcoplasmic reticulum and involved in excitation-contraction coupling. Variants in the gene encoding RyR1 have been linked to a range of neuromuscular disorders including myopathies and malignant hyperthermia (MH). OBJECTIVE: We have identified three RYR1 variants (c.1983 G>A, p.Trp661*; c.7025A>G, p.Asn2342Ser and c.2447 C>T, p.Pro816Leu) in a family with a suspected myopathy and associated malignant hyperthermia susceptibility. We used calcium release assays to functionally characterise these variants in a recombinant system. METHODS: Site-directed mutagenesis was used to introduce each variant separately into the human RYR1 cDNA. HEK293-T cells were transfected with the recombinant constructs and calcium release assays were carried out using 4-chloro-m-cresol (4-CmC) as the RyR1 agonist to investigate the functional consequences of each variant. RESULTS: RYR1 c.1983 G>A, p.Trp661* resulted in a non-functional channel, c.7025A>G, p.Asn2342Ser in a hypersensitive channel and c.2447 C>T, p.Pro816Leu in a hypersensitive channel at higher concentrations of 4-CmC. CONCLUSIONS: The p.Trp661* RYR1 variant should be considered as a risk factor for myopathies. The p.Asn2342Ser RYR1 variant, when expressed as a compound heterozygote with a nonsense mutation on the second allele, is likely to result in MH-susceptibility. The role of the p.Pro816Leu variant in MH remains unclear.

Masseter muscle rigidity and the role of DNA analysis to confirm malignant hyperthermia susceptibility.

Malignant hyperthermia (MH) is an uncommon, autosomal dominant disorder of skeletal muscle, triggered by inhalational anaesthetics or depolarizing muscle relaxants. Masseter muscle rigidity (MMR) can be regarded as potentially a preceding sign for an MH reaction. Susceptibility to MH can be determined by the in vitro contracture test (IVCT) or DNA analysis where a familial variant is known. Our aims were to review patients with MMR, where IVCT and DNA analysis had been undertaken, to determine if DNA analysis could be used as an initial screening tool for MH susceptibility, and, by reviewing standard monitored variables (SMVs), to determine if any clinical characteristics could be used to differentiate between MMR patients who are MH susceptible (MHS) and those who are not. Patients with MMR were identified from the Palmerston North Hospital MH Reactions Database. IVCT and DNA analysis results were documented. DNA testing was performed retrospectively in the majority of patients as many patients had presented before DNA analysis was available. Forty-one patients were analysed. Fourteen were DNA positive/IVCT positive and six DNA positive only (48% in total), seven were IVCT positive/DNA negative and 14 were IVCT normal. Increased creatine kinase (>18,000 units/L) was consistent with MH susceptibility. Severity of MMR was not linked to MH susceptibility. This study confirmed that DNA analysis can be used as a first-line test for MH susceptibility in patients presenting with MMR (consistent with European MH Group recommendations). Creatine kinase was the only SMV that was significantly different between MHS and MH normal individuals.

Identification of ryanodine receptor 1 single-nucleotide polymorphisms by high-resolution melting using the LightCycler 480 System.

High-resolution melting (HRM) allows single-nucleotide polymorphism (SNP) detection/typing using inexpensive generic heteroduplex-detecting double-stranded DNA (dsDNA) binding dyes. Until recently HRM has been a post-PCR process. With the LightCycler 480 System, however, the entire mutation screening process, including post-PCR analysis, can be performed using a single instrument. HRM assays were developed to allow screening of the ryanodine receptor gene (RYR1) for potential mutations causing malignant hyperthermia (MH) and/or central core disease (CCD) using the LightCycler 480 System. The assays were validated using engineered plasmids and/or genomic DNA samples that are either homozygous wild type or heterozygous for one of three SNPs that lead to the RyR1 amino acid substitutions T4826I, H4833Y, and/or R4861H. The HRM analyses were conducted using two different heteroduplex-detecting dsDNA binding dyes: LightCycler 480 HRM dye and LCGreen Plus. Heterozygous samples for each of the HRM assays were readily distinguished from homozygous samples with both dyes. By using engineered plasmids, it was shown that even homozygous sequence variations can be identified by using either small amplicons or the addition of exogenous DNA after PCR. Thus, the LightCycler 480 System provides a novel, integrated, real-time PCR/HRM platform that allows high throughput, inexpensive SNP detection, and genotyping based on high-resolution amplicon melting.

Identification and biochemical characterization of a novel ryanodine receptor gene mutation associated with malignant hyperthermia.

BACKGROUND: Mutations in the skeletal muscle ryanodine receptor gene may result in altered calcium release from sarcoplasmic reticulum stores, giving rise to malignant hyperthermia (MH). MH is a pharmacogenetic skeletal muscle disorder triggered by volatile anesthetics and depolarizing muscle relaxants. Diagnosis of MH is by in vitro contracture testing of quadriceps muscle. DNA analysis of causative mutations is limited by the large number of mutations that cosegregate with MH and the relatively few that have been biochemically characterized. METHODS: DNA sequence analysis was used to screen the skeletal muscle ryanodine receptor gene in MH-susceptible individuals. A diagnostic test using real-time polymerase chain reaction was developed to detect the mutation in individuals diagnosed as MH susceptible by in vitro contracture testing. The functional relevance of this mutation was examined in Epstein-Barr virus-immortalized B-lymphoblastoid cells. RESULTS: A novel ryanodine receptor mutation (cytosine 14997 thymine resulting in a histidine 4833 tyrosine substitution) was identified in pathology specimens from two patients with fatal MH reactions. B lymphocytes from patients with this mutation were approximately twofold more sensitive than MH-negative cells to activation with 4-chloro-m-cresol. The amount of Ca released from B lymphocytes of MH-susceptible patients was significantly greater than that released from cells of family members without this mutation. Haplotype analysis suggests that both families had a common ancestor. CONCLUSIONS: DNA analysis to detect mutations which cosegregate with MH as well as biochemical assays on cultured lymphocytes obtained from blood can serve as useful diagnostic tools for MH susceptibility and genotype-phenotype correlations.

GcSTUA, an APSES transcription factor, is required for generation of appressorial turgor pressure and full pathogenicity of Glomerella cingulata.

Glomerella cingulata, which infects a number of different hosts, gains entry to the plant tissue by means of an appressorium. Turgor pressure generated within the appressorium forces a penetration peg through the plant cuticle. A visible lesion forms as the fungus continues to grow within the host. A G. cingulata homolog (GcSTUA) of the genes encoding Asm1, Phd1, Sok2, Efg1, and StuA transcription factors in Magnaporthe grisea and other fungi was cloned and shown to be required for infection of intact apple fruit and penetration of onion epidermal cells. Mobilization of glycogen and triacylglycerol during formation of appressoria by the GcSTUA deletion mutant appeared normal and melanization of the maturing appressoria was also indistinguishable from that of the wild type. However, GcSTUA was essential for the generation of normal turgor pressure within the appressorium. As is the case for its homologs in other fungi, GcSTUA also was required for the formation of aerial hyphae, efficient conidiation, and the formation of perithecia (sexual reproductive structures).

Down-regulation of human topoisomerase IIalpha expression correlates with relative amounts of specificity factors Sp1 and Sp3 bound at proximal and distal promoter regions.

BACKGROUND: Topoisomerase IIalpha has been shown to be down-regulated in doxorubicin-resistant cell lines. The specificity proteins Sp1 and Sp3 have been implicated in regulation of topoisomerase IIalpha transcription, although the mechanism by which they regulate expression is not fully understood. Sp1 has been shown to bind specifically to both proximal and distal GC elements of the human topoisomerase IIalpha promoter in vitro, while Sp3 binds only to the distal GC element unless additional flanking sequences are included. While Sp1 is thought to be an activator of human topoisomerase IIalpha, the functional significance of Sp3 binding is not known. Therefore, we sought to determine the functional relationship between Sp1 and Sp3 binding to the topoisomerase IIalpha promoter in vivo. We investigated endogenous levels of Sp1, Sp3 and topoisomerase IIalpha as well as binding of both Sp1 and Sp3 to the GC boxes of the topoisomerase IIalpha promoter in breast cancer cell lines in vivo after short term doxorubicin exposure. RESULTS: Functional effects of Sp1 and Sp3 were studied using transient cotransfection assays using a topoisomerase IIalpha promoter reporter construct. The in vivo interactions of Sp1 and Sp3 with the GC elements of the topoisomerase IIalpha promoter were studied in doxorubicin-treated breast cancer cell lines using chromatin immunoprecipitation assays. Relative amounts of endogenous proteins were measured using immunoblotting. In vivo DNA looping mediated by proteins bound at the GC1 and GC2 elements was studied using the chromatin conformation capture assay. Both Sp1 and Sp3 bound to the GC1 and GC2 regions. Sp1 and Sp3 were transcriptional activators and repressors respectively, with Sp3 repression being dominant over Sp1-mediated activation. The GC1 and GC2 elements are linked in vivo to form a loop, thus bringing distal regulatory elements and their cognate transcription factors into close proximity with the transcription start site. CONCLUSION: These observations provide a mechanistic explanation for the modulation of topoisomerase IIalpha and concomitant down-regulation that can be mediated by topoisomerase II poisons. Competition between Sp1 and Sp3 for the same cognate DNA would result in activation or repression depending on absolute amounts of each transcription factor in cells treated with doxorubicin.

Functional Characterization of C-terminal Ryanodine Receptor 1 Variants Associated with Central Core Disease or Malignant Hyperthermia.

BACKGROUND: Central core disease and malignant hyperthermia are human disorders of skeletal muscle resulting from aberrant Ca2+ handling. Most malignant hyperthermia and central core disease cases are associated with amino acid changes in the type 1 ryanodine receptor (RyR1), the skeletal muscle Ca2+-release channel. Malignant hyperthermia exhibits a gain-of-function phenotype, and central core disease results from loss of channel function. For a variant to be classified as pathogenic, functional studies must demonstrate a correlation with the pathophysiology of malignant hyperthermia or central core disease. OBJECTIVE: We assessed the pathogenicity of four C-terminal variants of the ryanodine receptor using functional analysis. The variants were identified in families affected by either malignant hyperthermia or central core disease. METHODS: Four variants were introduced separately into human cDNA encoding the skeletal muscle ryanodine receptor. Following transient expression in HEK-293T cells, functional studies were carried out using calcium release assays in response to an agonist. Two previously characterized variants and wild-type skeletal muscle ryanodine receptor were used as controls. RESULTS: The p.Met4640Ile variant associated with central core disease showed no difference in calcium release compared to wild-type. The p.Val4849Ile variant associated with malignant hyperthermia was more sensitive to agonist than wild-type but did not reach statistical significance and two variants (p.Phe4857Ser and p.Asp4918Asn) associated with central core disease were completely inactive. CONCLUSIONS: The p.Val4849Ile variant should be considered a risk factor for malignant hyperthermia, while the p.Phe4857Ser and p.Asp4918Asn variants should be classified as pathogenic for central core disease.

Modulation of DNA topoisomerase II alpha promoter activity by members of the Sp (specificity protein) and NF-Y (nuclear factor Y) families of transcription factors.

Topo IIalpha (topoisomerase IIalpha) is a major target of several commonly used anticancer drugs and is subject to down-regulation at the transcriptional level in some drug-resistant cell lines and tumours in response to chemotherapy. Clinical resistance to such drugs has been correlated with down-regulation of topo IIalpha at transcription in some drug-resistant cell lines and tumours. Putative binding sites for a variety of transcription factors, including Sp1 (specificity protein 1) and NF-Y (nuclear factor Y) have previously been identified in the topo IIalpha promoter, but their functional significance and interactions have not been described following exposure to anti-cancer drugs. The binding of these factors to specific putative regulatory elements in the topo IIalpha promoter was studied using electrophoretic-mobility-shift assays. Sp1 was found to bind strongly to both distal and proximal GC-rich elements and NF-Y to ICB1 (the first inverted CCAAT box). The functional significance of transcription-factor binding was studied using transient transfection of HeLa cells using a luciferase reporter driven by a 617-bp minimal promoter containing point mutations in putative regulatory elements. Sp1 and NF-Y were both found to be transcriptional modulators with activator or repressor functions depending on protein/DNA context. Moreover, a functional interaction between Sp1 and NF-Y bound at proximal elements was observed.

Quantification of DNA by agarose gel electrophoresis and analysis of the topoisomers of plasmid and M13 DNA following treatment with a restriction endonuclease or DNA topoisomerase I.

A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of DNA with a restriction endonuclease and with a DNA topoisomerase. This session introduces students to the concept of DNA topoisomers, to the properties of different forms of DNA, and to the activity of restriction endonucleases and topoisomerases toward these forms. The exercise also involves measuring the size of linear duplex fragments of DNA by comparison of mobility with a ladder of double stranded DNA of known sizes.

Functional characterisation of the R2452W ryanodine receptor variant associated with malignant hyperthermia susceptibility.

Malignant hyperthermia (MH) is a pharmacogenetic disorder that manifests in susceptible individuals exposed to volatile anaesthetics. Over 400 variants in the ryanodine receptor 1 (RYR1) have been reported but relatively few have been definitively associated with susceptibility to MH. This is largely due to the technical challenges of demonstrating abnormal Ca(2+) release from the sarcoplasmic reticulum. This study focuses on the R2452W variant and its functional characterisation with the aim of classifying this variant as MH causative. HEK293 cells were transiently transfected with full-length human wildtype or R2452W mutant RYR1 cDNA. In addition, B-lymphoblastoid cells from blood and myoblasts propagated from in vitro contracture tests were extracted from patients positive for the R2452W variant. All cell lines generated were loaded with the ratiometric dye Fura-2 AM, stimulated with the RYR1-specific agonist 4-chloro-m-cresol and Ca(2+) release from the sarcoplasmic/endoplasmic reticulum was monitored by fluorescence emission. All cells expressing the RYR1 R2452W variant show a significantly higher Ca(2+) release in response to the agonist, 4-chloro-m-cresol, compared to cells expressing RYR1 WT. These results indicate that the R2452W variant results in a hypersensitive ryanodine receptor 1 and suggest that the R2452W variant in the ryanodine receptor 1 is likely to be causative of MH.

Allele-specific differences in ryanodine receptor 1 mRNA expression levels may contribute to phenotypic variability in malignant hyperthermia.

BACKGROUND: Malignant hyperthermia (MH) is a dominantly inherited skeletal muscle disorder that can cause a fatal hypermetabolic reaction to general anaesthetics. The primary locus of MH (MHS1 locus) in humans is linked to chromosome 19q13.1, the position of the gene encoding the ryanodine receptor skeletal muscle calcium release channel (RyR1). METHODS: In this study, an inexpensive allele-specific PCR (AS-PCR) assay was designed that allowed the relative quantification of the two RyR1 transcripts in heterozygous samples found to be susceptible to MH (MHS). Allele-specific differences in RyR1 expression levels can provide insight into the observed variable penetrance and variations in MH phenotypes between individuals. The presence/absence of the H4833Y mutation in RYR1 transcripts was employed as a marker that allowed discrimination between the two alleles. RESULTS: In four skeletal muscle samples and two lymphoblastoid cell lines (LCLs) from different MHS patients, the wild type allele was found to be expressed at higher levels than the mutant RyR1 allele. For both LCLs, the ratios between the wild type and mutant RYR1 alleles did not change after different incubation times with actinomycin D. This suggests that there are no allele-specific differences in RyR1 mRNA stability, at least in these cells. CONCLUSION: The data presented here revealed for the first time allele-specific differences in RYR1 mRNA expression levels in heterozygous MHS samples, and can at least in part contribute to the observed variable penetrance and variations in MH clinical phenotypes.