Soledad Levano

Increasing the number of diagnostic mutations in malignant hyperthermia

Malignant hyperthermia (MH) is an autosomal dominant disorder characterized by abnormal calcium homeostasis in skeletal muscle in response to triggering agents. Today, genetic investigations on ryanodine receptor type 1 (RYR1) gene and α1 subunit of the dihydropyridine receptor (DHPR) (CACNA1S) gene have improved the procedures associated with MH diagnosis. In approximately 50% of MH cases a causative RYR1 mutation was found. Molecular genetic testing based on RYR1 mutations for MH diagnosis is challenging, because the causative mutations, most of which are private, are distributed throughout the RYR1 gene. A more comprehensive genetic testing procedure is needed. Therefore, we aim to expand the genetic information related to MH and to evaluate the effect of mutations on the MH phenotype. Performing an in‐depth mutation screening of the RYR1 transcript sequence in 36 unrelated MH susceptible (MHS) patients, we identified 17 novel, five rare, and eight non‐disease‐causing variants in 23 patients. The 13 remaining MHS patients presented no known variants, neither in RYR1 nor in the CACNA1S binding regions to RYR1. The 17 novel variants were found to affect highly conserved amino acids and were absent in 100 controls. Excellent genotype‐phenotype correlations were found by investigating 21 MHS families—a total of 186 individuals. Epstein‐Barr virus (EBV) lymphoblastoid cells carrying four of these novel mutations showed abnormal calcium homeostasis. The results of this study contribute to the establishment of a robust genetic testing procedure for MH diagnosis. Hum Mutat 0, 1–9, 2009.

Genotyping the butyrylcholinesterase in patients with prolonged neuromuscular block after succinylcholine.

Background:Succinylcholine remains the standard neuromuscular blocking drug for tracheal intubation in emergency situations. The short duration of action is due to its rapid hydrolytic degradation by butyrylcholinesterase (plasmacholinesterase). Multiple variants of this enzyme are known (A, F, S, H, J, K variants) with different effects on enzyme activity. This study was undertaken to evaluate the use of molecular genetic methods in patients with clinically prolonged neuromuscular block. Methods:Nine patients with a neuromuscular block of 14 min to 5 h were selected. All four exons of the butyrylcholinesterase were amplified by polymerase chain reaction and analyzed by automated sequencing. Molecular genetic results were compared with clinical relaxation time and with biochemical test results (total butyrylcholinesterase activity, dibucaine and fluoride inhibition). Results:Seven of nine patients were mutation carriers. Five of these had more than one mutation. The A and K variants were the most frequent variations. Three of four patients who were homozygous for the A variant were also carriers of the K allele. The authors identified one novel mutation (G1294T) introducing a stop codon at amino acid position 432. The duration of neuromuscular block was substantially different between patients with identical butyrylcholinesterase genotypes. Conclusions:Variations in the genetic sequence of butyrylcholinesterase are frequent in patients with prolonged duration of action of succinylcholine. Direct sequencing of the whole butyrylcholinesterase gene is an appropriate method for genotyping and, accordingly, should be used in future clinical studies with drugs metabolized by this enzyme (e.g., succinylcholine, mivacurium).

Enhanced excitation-coupled Ca2+ entry induces nuclear translocation of NFAT and contributes to IL-6 release from myotubes from patients with central core disease

Prolonged depolarization of skeletal muscle cells induces entry of extracellular calcium into muscle cells, an event referred to as excitation-coupled calcium entry. Skeletal muscle excitation-coupled calcium entry relies on the interaction between the 1,4-dihydropyridine receptor on the sarcolemma and the ryanodine receptor on the sarcoplasmic reticulum membrane. In this study, we directly measured excitation-coupled calcium entry by total internal reflection fluorescence microscopy in human skeletal muscle myotubes harbouring mutations in the RYR1 gene linked to malignant hyperthermia (MH) and central core disease (CCD). We found that excitation-coupled calcium entry is strongly enhanced in cells from patients with CCD compared with individuals with MH and controls. Furthermore, excitation-coupled calcium entry induces generation of reactive nitrogen species and enhances nuclear localization of NFATc1, which in turn may be responsible for the increased IL-6 released by myotubes from patients with CCD.

Simvastatin protects auditory hair cells from gentamicin-induced toxicity and activates Akt signaling in vitro

BackgroundInhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, known as statins, are commonly used as cholesterol-lowering drugs. During the past decade, evidence has emerged that statins also have neuroprotective effects. Research in the retina has shown that simvastatin, a commonly used statin, increases Akt phosphorylation in vivo, indicating that the PI3K/Akt pathway contributes to the protective effects achieved. While research about neuroprotective effects have been conducted in several systems, the effects of statins on the inner ear are largely unknown.ResultsWe evaluated whether the 3-hydroxy-3-methylglutaryl-coenzyme A reductase is present within the rat cochlea and whether simvastatin is able to protect auditory hair cells from gentamicin-induced apoptotic cell death in a in vitro mouse model. Furthermore, we evaluated whether simvastatin increases Akt phosphorylation in the organ of Corti. We detected 3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA in organ of Corti, spiral ganglion, and stria vascularis by reverse transcriptase-polymerase chain reaction (RT-PCR). Moreover, we observed a dose-dependent and significant reduction of hair cell loss in organs of Corti treated with simvastatin in addition to gentamicin, as compared to samples treated with gentamicin alone. The protective effect of simvastatin was reversed by addition of mevalonate, a downstream metabolite blocked by simvastatin, demonstrating the specificity of protection. Finally, Western blotting showed an increase in organ of Corti Akt phosphorylation after simvastatin treatment in vitro.ConclusionThese results suggest a neuroprotective effect of statins in the inner ear, mediated by reduced 3-hydroxy-3-methylglutaryl-coenzyme A reductase metabolism and Akt activation.

Annexin A1 is a biomarker of T-tubular repair in skeletal muscle of nonmyopathic patients undergoing statin therapy

Skeletal muscle complaints are a common consequence of cholesterol‐lowering therapy. Transverse tubular (T‐tubular) vacuolations occur in patients having statin‐associated myopathy and, to a lesser extent, in statin‐treated patients without myopathy. We have investigated quantitative changes in T‐tubular morphology and looked for early indicators of T‐tubular membrane repair in skeletal muscle biopsy samples from patients receiving cholesterol‐lowering therapy who do not have myopathic side effects. Gene expression and protein levels of incipient membrane repair proteins were monitored in patients who tolerated statin treatment without myopathy and in statin‐naive subjects. In addition, morphometry of the T‐tubular system was performed. Only the gene expression for annexin A1 was up‐regulated, whereas the expression of other repair genes remained unchanged. However, annexin A1 and dysferlin protein levels were significantly increased. In statin‐treated patients, the volume fraction of the T‐tubular system was significantly increased, but the volume fraction of the sarcoplasmic reticulum remained unchanged. A complex surface structure in combination with high mechanical loads makes skeletal muscle plasma membranes susceptible to injury. Ca2+‐dependent membrane repair proteins such as dysferlin and annexin A1 are deployed at T‐tubular sites. The up‐regulation of annexin A1 gene expression and protein points to this protein as a biomarker for T‐tubular repair.—Voigt, T., Sebald, H.‐J., Schoenauer, R., Levano, L., Girard, T., Hoppeler, H.H., Babiychuk, E.B., Draeger, A. Annexin A1 is a biomarker of T‐tubular repair in skeletal muscle of nonmyopathic patients undergoing statin therapy. FASEB J. 27, 2156–2164 (2013). www.fasebj.org

All Akt Isoforms (Akt1, Akt2, Akt3) Are Involved in Normal Hearing, but Only Akt2 and Akt3 Are Involved in Auditory Hair Cell Survival in the Mammalian Inner Ear

The kinase Akt is a key downstream mediator of the phosphoinositide-3-kinase signaling pathway and participates in a variety of cellular processes. Akt comprises three isoforms each encoded by a separate gene. There is evidence to indicate that Akt is involved in the survival and protection of auditory hair cells in vitro. However, little is known about the physiological role of Akt in the inner ear—especially in the intact animal. To elucidate this issue, we first analyzed the mRNA expression of the three Akt isoforms in the inner ear of C57/BL6 mice by real-time PCR. Next, we tested the susceptibility to gentamicin-induced auditory hair cell loss in isoform-specific Akt knockout mice compared to wild-types (C57/BL6) in vitro. To analyze the effect of gene deletion in vivo, hearing and cochlear microanatomy were evaluated in Akt isoform knockout animals. In this study, we found that all three Akt isoforms are expressed in the cochlea. Our results further indicate that Akt2 and Akt3 enhance hair cell resistance to ototoxicity, while Akt1 does not. Finally, we determined that untreated Akt1 and Akt2/Akt3 double knockout mice display significant hearing loss, indicating a role for these isoforms in normal hearing. Taken together, our results indicate that each of the Akt isoforms plays a distinct role in the mammalian inner ear.

A fulminant malignant hyperthermia episode in a patient with ryanodine receptor gene mutation p.Tyr522Ser.

A 37-yr-old patient scheduled for elective bursectomy developed fulminant malignant hyperthermia (MH) under sevoflurane anesthesia. The first sign was a dramatic increase in end-tidal CO(2). Symptomatic and specific therapy was rapidly instituted. Postoperative rhabdomyolysis was treated with veno-venous hemofiltration. The patient rejected open muscle biopsy for in vitro contracture testing. Therefore, molecular testing was performed. An infrequent MH causative mutation was identified on the ryanodine receptor gene. This case report confirms the causative nature of this mutation. It also shows that molecular genetic investigation may be as appropriate as in vitro contracture testing to confirm the diagnosis after a clinical episode of MH.

Rapid and accurate detection of atypical and Kalow variants in the butyrylcholinesterase gene using denaturing high performance liquid chromatography.

BACKGROUND:Variations in the butyrylcholinesterase (BCHE) gene can prolong neuromuscular block after the administration of the neuromuscular blocking drugs succinylcholine and mivacurium. The most frequent variations in the BCHE gene are the atypical (A) and Kalow (K) variants. We, therefore, developed a detection approach for these most common variants based on the method of denaturing high-performance liquid chromatography (dHPLC). METHODS:Forty-six subjects were included. Sixteen were known to carry either the normal sequence or different combinations of A- and K-variants and 30 test subjects were randomly selected from the general population. Three samples of the 16 were used to establish the dHPLC protocols. Results from dHPLC were blindly compared with the automated sequencing data. RESULTS:All A- and K-variants in their heterozygous and homozygous forms were unequivocally identified by dHPLC. For all 86 tested alleles, dHPLC results were 100% concordant with DNA sequencing results. Additional genetic variations were also detected, which included G344A encoding for the silent variant (S) and a known polymorphism, A1914G. CONCLUSIONS:The described dHPLC protocols offer a rapid and accurate screening method for the most common variants of BCHE. This screening tool might be useful in pharmacokinetic studies investigating drugs metabolized by BCHE, such as mivacurium and succinylcholine.

Predictors of the variability in neuromuscular block duration following succinylcholine: A prospective, observational study.

BACKGROUND The duration of neuromuscular block (NMB) following succinylcholine administration is characterised by a high interindividual variability. However, this has not yet been quantified in a large sample of surgical patients. The significance of underlying clinical factors is unknown. OBJECTIVE The objective of this study was to profile the variability in NMB duration following a standard dose of succinylcholine and to investigate contributing clinical and genetic factors. DESIGN A prospective, observational study. SETTING Tertiary referral centre. PATIENTS In a total of 1630 surgical patients undergoing a rapid sequence induction and intubation, clinical risk factors for a prolongation in NMB duration following succinylcholine were assessed. In a subset of 202 patients, additional biochemical and molecular genetic investigations of butyrylcholinesterase were performed. INTERVENTION A standard 1 mg kg−1 dose of succinylcholine after administration of an induction drug and an opioid. MAIN OUTCOME NMB duration measured as the time between administration of succinylcholine until reappearance of palpable muscular response to supramaximal transcutaneous ulnar nerve stimulation. RESULTS NMB varied from 80 s to 44 min with a median duration of 7.3 min. Sixteen percent of patients had NMB duration in excess of 10 min. A multivariable survival model identified physical status, sex, age, hepatic disease, pregnancy, history of cancer and use of etomidate or metoclopramide as independent risk factors for a prolonged NMB. Three novel butyrylcholinesterase variants were identified: p.Ile5Thr; p.Val178Ile; and p.Try231Ser. CONCLUSION Neuromuscular blockade duration in excess of 10 min occurred in 16% of a general surgical population following a single dose of succinylcholine. The multivariable model of clinical risk factors for prolonged NMB revealed a negative predictive value of 87%, thereby indicating that absence of such risk factors may reliably predict a shorter duration of NMB. In patients with clinical risk factors for a prolonged NMB or with butyrylcholinesterase mutations, an alternative to succinylcholine should be considered.

Dantrolene for severe rhabdomyolysis in Staphylococcus aureus toxic shock syndrome

rs12985668 c.594A>G p.Leu198 rs10406027 c.1077T>C p.Ala359 rs2288888 c.1668G>A p.Ser556 rs3745847 c.2286C>T p.Pro762 rs2228070 c.2979C>T p.Asn993 rs11083462 c.3456C>T p.Ile1152 rs2228071 c.7089C>T p.Cys2363 rs2229142 c.7863C>T p.His2621 Novel c.7864C>T p.Leu2622 rs2229144 c.7977G>A p.Thr2659 rs2960340 c.8118T>C p.Ile2706 rs2915951 c.8190T>C p.Asp2730 rs2915952 c.8337G>A p.Glu2779 rs2229146 c.8589T>C p.Ser2863 rs2071089 c.9186A>G p.Pro3062 rs2229145 c.10188C>T p.Asp3396 rs35959206 c.13671C>G p.Ser4557