RESUMO
BACKGROUND: Gaps in our understanding of genetic susceptibility to malignant hyperthermia (MH) limit the application and interpretation of genetic diagnosis of the condition. Our aim was to define the prevalence and role of variants in the three genes implicated in MH susceptibility in the largest comprehensively phenotyped MH cohort worldwide. METHODS: We initially included one individual from each positive family tested in the UK MH Unit since 1971 to detect variants in RYR1, CACNA1S, or STAC3. Screening for genetic variants has been ongoing since 1991 and has involved a range of techniques, most recently next generation sequencing. We assessed the pathogenicity of variants using standard guidelines, including family segregation studies. The prevalence of recurrent variants of unknown significance was compared with the prevalence reported in a large database of sequence variants in low-risk populations. RESULTS: We have confirmed MH susceptibility in 795 independent families, for 722 of which we have a DNA sample. Potentially pathogenic variants were found in 555 families, with 25 RYR1 and one CACNA1S variants previously unclassified recurrent variants significantly over-represented (P<1×10-7) in our cohort compared with the Exome Aggregation Consortium database. There was genotype-phenotype discordance in 86 of 328 families suitable for segregation analysis. We estimate non-RYR1/CACNA1S/STAC3 susceptibility occurs in 14-23% of MH families. CONCLUSIONS: Our data provide current estimates of the role of variants in RYR1, CACNA1S, and STAC3 in susceptibility to MH in a predominantly white European population.
Assuntos
Hipertermia Maligna/epidemiologia , Hipertermia Maligna/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Canais de Cálcio/genética , Canais de Cálcio Tipo L , Estudos de Coortes , Simulação por Computador , Exoma , Família , Predisposição Genética para Doença , Testes Genéticos , Variação Genética , Humanos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Reino Unido/epidemiologiaRESUMO
BACKGROUND: . Missense variants in the ryanodine receptor 1 gene ( RYR1 ) are associated with malignant hyperthermia but only a minority of these have met the criteria for use in predictive DNA diagnosis. We examined the utility of a simplified method of segregation analysis and a functional assay for determining the pathogenicity of recurrent RYR1 variants associated with malignant hyperthermia. METHODS: . We identified previously uncharacterised RYR1 variants found in four or more malignant hyperthermia families and conducted simplified segregation analyses. An efficient cloning and mutagenesis strategy was used to express ryanodine receptor protein containing one of six RYR1 variants in HEK293 cells. Caffeine-induced calcium release, measured using a fluorescent calcium indicator, was compared in cells expressing each variant to that in cells expressing wild type ryanodine receptor protein. RESULTS.: We identified 43 malignant hyperthermia families carrying one of the six RYR1 variants. There was segregation of genotype with the malignant hyperthermia susceptibility phenotype in families carrying the p.E3104K and p.D3986E variants, but the number of informative meioses limited the statistical significance of the associations. HEK293 functional assays demonstrated an increased sensitivity of RyR1 channels containing the p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I compared with wild type, but cells expressing p.D3986E had a similar caffeine sensitivity to cells expressing wild type RyR1. CONCLUSIONS: . Segregation analysis is of limited value in assessing pathogenicity of RYR1 variants in malignant hyperthermia. Functional analyses in HEK293 cells provided evidence to support the use of p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I for diagnostic purposes but not p.D3986E.
Assuntos
Hipertermia Maligna/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Cafeína/farmacologia , Cálcio/metabolismo , Clonagem Molecular , Família , Predisposição Genética para Doença , Variação Genética , Genótipo , Células HEK293 , Humanos , Hipertermia Maligna/epidemiologia , Imagem Molecular , Mutagênese , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Curcumin (diferuoylmethane or 1,7-bis (4-hydroxy-3-methoxyphenol)-1,6-hepatadiene-3,5-dione) is the active ingredient of the spice turmeric. Curcumin has been shown to have a number of pharmacological and therapeutic uses. This study shows that curcumin is a potent inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca2+ channel (InsP3 receptor). In porcine cerebellar microsomes, the extent of InsP3-induced Ca2+ release (IICR) is almost completely inhibited by 50 microM curcumin (IC50 = 10 microM). As the extent of IICR cannot be restored back to control levels by the addition of excess InsP3 and since it has little effect on [3H]InsP3 binding to cerebellar microsomes, this inhibition is likely to be non-competitive in nature. IICR in cerebellar microsomes is biphasic consisting of a fast and slow component. The rate constants for the two components are both reduced by curcumin to similar extents (by about 70% of control values at 40 microM curcumin). In addition, curcumin also reduces agonist (ATP)-stimulated Ca2+ mobilization from intact HL-60 cells, indicating that curcumin is cell permeant. However, since it also affects intracellular Ca2+ pumps and possibly ryanodine receptors, it may lead to complex Ca2+ transient responses within cells, which may well explain some of its putative therapeutic properties.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Cálcio/metabolismo , Curcumina/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Canais de Cálcio/metabolismo , Cerebelo/metabolismo , Curcumina/metabolismo , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Cinética , Microssomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Suínos , TrítioRESUMO
Malignant hyperthermia (MH) is a life-threatening condition caused by exposure of susceptible individuals to volatile anaesthetics or suxamethonium. MH-susceptible individuals must avoid exposure to these drugs, so accurate and reproducible processes to remove residual anaesthetic agents from anaesthetic workstations are required. Activated charcoal filters (ACFs) have been used for this purpose. ACFs can reduce the time for preparing an anaesthetic workstation for MH patients. Currently, the only commercially available ACFs are the Vapor-Clean$trade; (Dynasthetics, Salt Lake City, UT, USA) filters which retail at approximately AUD$130 per set of two, both of which are to be used in a single anaesthetic. Anaesthetic workstations were saturated with anaesthetic vapours and connected to a Miran ambient air analyser (SapphRe XL, ThermoScientific, Waltham, MA, USA) to measure vapour concentration. Various scenarios were tested in order to determine the most economical configurations of machine flushing, component change and activated charcoal filter use. We found that placement of filters in an unprepared, saturated circuit was insufficient to safely prepare an anaesthetic workstation. Following flushing of the anaesthetic workstation with high-flow oxygen for 90 seconds, a circuit and soda lime canister change and the placement of an ACF on the inspiratory limb, we were able to safely prepare a workstation in less than three minutes. A single filter on the inspiratory limb was able to maintain a clean circuit for 12 hours, with gas flows dropped from 10 lpm to 3 lpm after 90 minutes or removal of the filter after 90 minutes if high gas flows were maintained.
Assuntos
Anestésicos Inalatórios/isolamento & purificação , Carvão Vegetal/farmacologia , Hipertermia Maligna/prevenção & controle , Filtração , HumanosRESUMO
Curcumin is a compound derived from the spice, tumeric. It is a potent inhibitor of the SERCA Ca2+ pumps (all isoforms), inhibiting Ca2+-dependent ATPase activity with IC50 values of between 7 and 15 microm. It also inhibits ATP-dependent Ca2+-uptake in a variety of microsomal membranes, although for cerebellar and platelet microsomes, a stimulation in Ca2+ uptake is observed at low curcumin concentrations (<10 microm). For the skeletal muscle isoform of the Ca2+ pump (SERCA1), the inhibition of curcumin is noncompetitive with respect to Ca2+, and competitive with respect to ATP at high curcumin concentrations ( approximately 10-25 microm). This was confirmed by ATP binding studies that showed inhibition in the presence of curcumin: ATP-dependent phosphorylation was also reduced. Experiments with fluorescein 5'-isothiocyanate (FITC)-labelled ATPase also suggest that curcumin stabilizes the E1 conformational state. The fact that FITC labels the nucleotide binding site of the ATPase (precluding ATP from binding), and the fact that curcumin affects FITC fluorescence indicate that curcumin must be binding to another site within the ATPase that induces a conformational change to prevent ATP from binding. This observation is interpreted, with the aid of recent structural information, as curcumin stabilizing the interaction between the nucleotide-binding and phosphorylation domains, precluding ATP binding.
Assuntos
ATPases Transportadoras de Cálcio/antagonistas & inibidores , Curcumina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/metabolismo , Estabilidade Enzimática/efeitos dos fármacos , Técnicas In Vitro , Cinética , Lipossomos , Permeabilidade , Fosforilação , Conformação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Coelhos , ATPases Transportadoras de Cálcio do Retículo SarcoplasmáticoRESUMO
One of the major mechanisms by which hormones elevate intracellular Ca(2+)levels is by generating the second messenger inositol 1,4, 5-trisphosphate (InsP(3)), which activates a Ca(2+)channel (InsP(3)receptor) located in the endoplasmic reticulum (ER). This study undertakes to identify the InsP(3)receptor subtypes (isoforms) in heart and aorta and to characterize their functional properties. The InsP(3)receptor isoforms were identified from rat heart and aorta tissues using both reverse-transcriptase polymerase chain reaction (RT-PCR) to assess the presence of mRNA for the different isoforms and immunochemistry using InsP(3)receptor isoform-specific antibodies. Functional studies included ligand binding experiments using [(3)H]InsP(3)and InsP(3)-induced Ca(2+)release studies using Fluo-3 as the Ca(2+)sensing dye. All three isoforms of the InsP(3)receptor were identified using RT-PCR and immunochemical analyses. [(3)H]InsP(3)binding studies using microsomes derived from these tissues showed that heart had a 3-fold lower abundance of InsP(3)receptors than aorta, while both have considerably lower abundance than the well characterized cerebellar microsomes. The affinity of the InsP(3)binding to the receptor was also different in the three tissues. In cerebellum the K(d)was 60 nM, while aorta had a much higher K(d)of 220 nM. Heart microsomes, appeared to show two classes of binding affinity with K(d)s of 150 nM and 60 nM. Furthermore, the effects of free [Ca(2+)] on [(3)H]InsP(3)binding levels were also different for the three tissues. InsP(3)binding to both cerebellar and aorta microsomes decreased by 90% and 60%, respectively, above 30 nM free [Ca(2+)], while InsP(3)binding to heart was relatively insensitive to changes in [Ca(2+)]. At maximal InsP(3)concentrations, aorta microsomes were able to release about 5% of the accumulated Ca(2+), compared to 25% by cerebellar microsomes. Heart microsomes, however, showed only very little InsP(3)-induced Ca(2+)release ( <0.5%). The EC(50)concentration for InsP(3)-induced Ca(2+)release was 1.2 micro M for aorta while that for cerebellum was 0.3 micro M. Known agonists of the cerebellar InsP(3)receptor such as 3-deoxy InsP(3)and adenophostin A were also able to mobilize Ca(2+)from aorta microsomes. In addition, the competitive antagonist heparin and the non-competitive antagonists of the cerebellar InsP(3)receptor, tetracaine and tetrahexylammonium chloride, were also able to block InsP(3)-induced Ca(2+)release from aorta microsomes.
Assuntos
Aorta/química , Canais de Cálcio/classificação , Miocárdio/química , Receptores Citoplasmáticos e Nucleares/classificação , Animais , Cálcio/metabolismo , Canais de Cálcio/fisiologia , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Isoformas de Proteínas/análise , Ratos , Receptores Citoplasmáticos e Nucleares/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SuínosRESUMO
Industrial alkylphenols in the environment may act as "xenoestrogens" to disrupt testicular development and decrease male fertility. Amongst possible targets for these compounds are testicular Sertoli cells, which nurture the developing sperm cells. We demonstrate that SERCA 2 and 3 Ca(2+) pumps are relatively abundant in rat testis microsomal membranes, and also in Sertoli, myoid, and TM4 cells (a Sertoli cell line). A number of estrogenic alkylphenols such as nonylphenol, octylphenol, bisphenol A, and butylated hydroxytoluene all inhibit testicular Ca(2+) ATPase in the low micromolar concentration range. These agents also mobilize intracellular Ca(2+) in intact TM4 cells in a manner consistent with the inhibition of ER Ca(2+) pumps. Alkylphenols dramatically decrease the viability of TM4 cells, an effect that is reversed by either a caspase inhibitor or by BAPTA, and is therefore consistent with Ca(2+)-dependent cell death via apoptosis. We postulate that alkylphenols disrupt testicular development by inhibiting ER Ca(2+) pumps, thus disturbing testicular Ca(2+) homeostasis.