Clinical and Genetic Features of Congenital Myasthenic Syndromes due to CHAT Mutations: Case Report and Literature Review.

Congenital myasthenic syndromes (CMS) are neuromuscular transmission disorders caused by mutations in genes encoding neuromuscular junction proteins. CMS due to choline acetyltransferase (CHAT) gene is characterized by episodic apnea. We report a case of a 12-month-old female patient presented with recurrent episodic apnea carrying a mutation in CHAT gene, p.I336T. Furthermore, we describe the genetic and clinical findings in 44 CMS patients due to CHAT mutations in the literature up to date. Episodes of apnea and respiratory insufficiency are the hallmarks of CHAT mutations. Clinical manifestations usually provoked by infections and fever. CMS due to CHAT mutations are rare, but it is important to diagnosis. Early diagnosis and appropriate treatment can improve morbidity and mortality.

Characterization of a novel BCHE "silent" allele: point mutation (p.Val204Asp) causes loss of activity and prolonged apnea with suxamethonium.

Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of suxamethonium leading to the discovery of a novel BCHE variant (c.695T>A, p.Val204Asp). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation disrupts the catalytic triad and determines a "silent" phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with heterozygous atypical silent genotype. Electrophoretic analysis of plasma BChE of the proband and his mother showed that patient has a reduced amount of tetrameric enzyme in plasma and that minor fast-moving BChE components: monomer, dimer, and monomer-albumin conjugate are missing. Kinetic analysis showed that the p.Val204Asp/p.Asp70Gly-p.Ala539Thr BChE displays a pure Michaelian behavior with BTC as the substrate. Both catalytic parameters Km = 265 µM for BTC, two times higher than that of the atypical enzyme, and a low Vmax are consistent with the absence of activity against suxamethonium. Molecular dynamic (MD) simulations showed that the overall effect of the mutation p.Val204Asp is disruption of hydrogen bonding between Gln223 and Glu441, leading Ser198 and His438 to move away from each other with subsequent disruption of the catalytic triad functionality regardless of the type of substrate. MD also showed that the enzyme volume is increased, suggesting a pre-denaturation state. This fits with the reduced concentration of p.Ala204Asp/p.Asp70Gly-p.Ala539Thr tetrameric enzyme in the plasma and non-detectable fast moving-bands on electrophoresis gels.

mPGES-1 and prostaglandin E2: vital role in inflammation, hypoxic response, and survival.

BACKGROUND: Apnea associated with infection and inflammation is a major medical concern in preterm infants. Prostaglandin E(2) (PGE(2)) serves as a critical mediator between infection and apnea. We hypothesize that alteration of the microsomal PGE synthase-1 (mPGES-1) PGE(2) pathway influences respiratory control and response to hypoxia. METHODS: Nine-d-old wild-type (WT) mice, mPGES-1 heterozygote (mPGES-1(+/-)), and mPGES-1 knockout (mPGES-1(-/-)) mice were used. Respiration was investigated in mice using flow plethysmography after the mice received either interleukin-1ß (IL-1ß) (10 µg/kg) or saline. Mice were subjected to a period of normoxia, subsequent exposure to hyperoxia, and finally either moderate (5 min) or severe hypoxia (until 1 min after last gasp). RESULTS: IL-1ß worsened survival in WT mice but not in mice with reduced or no mPGES-1. Reduced expression of mPGES-1 prolonged gasping duration and increased the number of gasps during hypoxia. Response to intracerebroventricular PGE(2) was not dependent on mPGES-1 expression. CONCLUSION: Activation of mPGES-1 is involved in the rapid and vital response to severe hypoxia as well as inflammation. Attenuation of mPGES-1 appears to have no detrimental effects, yet prolongs autoresuscitation efforts and improves survival. Consequently, inhibition of the mPGES-1 pathway may serve as a potential therapeutic target for the treatment of apnea and respiratory disorders.

Structure, activities and biomedical applications of human butyrylcholinesterase.

Human butyrylcholinesterase (BuChE) is a serine enzyme present in most organs and plasma. No clear physiological function has yet been assigned to BuChE, but it is a pharmacologically and toxicologically important enzyme that plays a role in degradation of numerous ester-containing drugs and poisonous esters. Thus, BuChE-based bioscavengers are an alternative for prophylaxis and treatments of intoxications by these compounds. Also, BuChE has been integrated in biosensors for detection of organophosphorus compounds and other cholinesterase inhibitors.

Intermittent hypoxia degrades HIF-2alpha via calpains resulting in oxidative stress: implications for recurrent apnea-induced morbidities.

Intermittent hypoxia (IH) occurs in many pathological conditions including recurrent apneas. Hypoxia-inducible factors (HIFs) 1 and 2 mediate transcriptional responses to low O(2). A previous study showed that HIF-1 mediates some of the IH-evoked physiological responses. Because HIF-2alpha is an orthologue of HIF-1alpha, we examined the effects of IH on HIF-2alpha, the O(2)-regulated subunit expression, in pheochromocytoma 12 cell cultures. In contrast to the up-regulation of HIF-1alpha, HIF-2alpha was down-regulated by IH. Similar down-regulation of HIF-2alpha was also seen in carotid bodies and adrenal medullae from IH-exposed rats. Inhibitors of calpain proteases (ALLM, ALLN) prevented IH-evoked degradation of HIF-2alpha whereas inhibitors of prolyl hydroxylases or proteosome were ineffective. IH activated calpain proteases and down-regulated the endogenous calpain inhibitor calpastatin. IH-evoked HIF-2alpha degradation led to inhibition of SOD2 transcription, resulting in oxidative stress. Over-expression of transcriptionally active HIF-2alpha prevented IH-evoked oxidative stress and restored SOD2 activity. Systemic treatment of IH-exposed rats with ALLM rescued HIF-2alpha degradation and restored SOD2 activity, thereby preventing oxidative stress and hypertension. These observations demonstrate that, unlike continuous hypoxia, IH leads to down-regulation of HIF-2alpha via a calpain-dependent signaling pathway and results in oxidative stress as well as autonomic morbidities.

Assay using succinyldithiocholine as substrate: the method of choice for the measurement of cholinesterase catalytic activity in serum to diagnose succinyldicholine sensitivity.

No comparative information is available concerning the ability of various cholinesterase (ChE) methods to identify succinyldicholine-sensitive patients, purely on the basis of the enzyme activity recorded in serum. Here, we evaluated six different methods for the measurement of ChE activity; 131 subjects were subdivided according to ChE phenotype and, therefore, to succinyldicholine sensitivity. ChE phenotype was determined by measuring dibucaine and fluoride numbers. DNA analysis was also performed to confirm correlation between the phenotype classification used in the study and the ChE genotype. The tested methods were significantly different in their ability to discriminate between the subjects with and without succinyldicholine-sensitive phenotypes. The succinyldithiocholine/5,5'-dithio-bis(2-nitrobenzoate) (DTNB) method showed the highest accuracy (area under the receiver operating characteristic (ROC) curve 0.97) followed by the propionylthiocholine/DTNB method (area under the ROC curve 0.94). On the other hand, the two methods using butyrylthiocholine as substrate and that employing benzoylcholine showed limited clinical utility in discriminating subjects at risk of prolonged apnea (area under the ROC curve < or = 0.9). Using the succinyldithiocholine method, a value < or = 23 U/l was approximately five times as likely to occur in a sensitive individual as in a normal one.

[Emergency from anesthesia in small children. From laryngospasm to prolonged apnea]. / Narkoseausleitung beim Kleinkind. Vom Laryngospasmus zur prolongierten Apnoe.

Postoperative laryngospasm during emergence from anaesthesia represents a potentially life-threatening complication. Even if this is successfully overcome using drug therapy, new, serious problems may develop. We report the case of a 3 1/2 -year-old boy of African descent weighing 15 kg who developed a laryngospasm during emergence from anaesthesia. Because the airway obstruction could not be controlled by deepening the anaesthesia again and administering anti-obstructive drugs, the boy was given 15 mg succinylcholine. Thereafter prolonged apnea developed such that the patient had to be admitted to the pediatric intensive care unit. The child was extubated 6 h later and the further course was normal so that he could be released from the hospital the following day. Further diagnostic study revealed a dibucaine-sensitive, fluoride-resistant pseudocholinesterase in the plasma, which is a rare form of atypical pseudocholinesterase, explaining the prolonged arousal phase after the administration of succinylcholine. Three significant aspects of this case are discussed: 1. risk factors and treatment of perioperative airway obstruction 2. factors and treatment of prolonged apnea, and 3. delayed arousal reactions and their management in an outpatient setting.

Neonatal hepatic steatosis by disruption of the adenosine kinase gene.

Neonatal hepatic steatosis (OMIM 228100) is a fatal condition of unknown etiology characterized by a pale and yellow liver and early postnatal mortality. In the present study, a deficit in adenosine-dependent metabolism is proposed as a causative factor. Physiologically, adenosine is efficiently metabolized to AMP by adenosine kinase (ADK), an enzyme highly expressed in liver. ADK not only ensures normal adenine nucleotide levels but also is essential for maintaining S-adenosylmethionine-dependent transmethylation processes, where adenosine, an obligatory product, has to be constantly removed. Homozygous Adk(-/-) mutants developed normally during embryogenesis. However, within 4 days after birth they displayed microvesicular hepatic steatosis and died within 14 days with fatty liver. Adenine nucleotides were decreased and S-adenosylhomocysteine, a potent inhibitor of transmethylation reactions, was increased in the mutant liver. Thus, a deficiency in adenosine metabolism is identified as a powerful contributor to the development of neonatal hepatic steatosis, providing a model for the rapid development of postnatally lethal fatty liver.

An explanation for the different inhibitory characteristics of human serum butyrylcholinesterase phenotypes deriving from inhibition of atypical heterozygotes.

The time course of inhibition of butyrylcholinesterase (EC 3.1.1.8) by the dimethylcarbamate Ro 02-0683 in sera taken from patients heterozygous for the usual (U), atypical (A), K or J variants was followed using propionylthiocholine as substrate. Data obtained were used to determine rate constants of inhibition together with the contribution made by each variant to total enzyme activity. The findings substantiate earlier reports that J and K mutations lead to quantitative changes in the concentration of usual enzyme in contrast to the qualitative changes of the atypical variant. The contribution of the atypical enzyme to the total activity in serum from UA, AK and AJ heterozygotes was respectively 17-20, 24-31 and 34-53%. The altered ratios of atypical to usual, K or J enzyme in UA, AK and AJ together with the constants on the usual enzyme alone, explain the differences in observed inhibitor numbers which enable these heterozygotes to be identified.

[3-hydroxy-3-methylglutaric aciduria and recurrent Reye-like syndrome]. / Aciduria 3-hidroxi-3-metilglutárica y síndrome Reye-like recurrente.

INTRODUCTION: 3-hydroxy-3-methylglutaryl-coenzyme A lyase deficiency (HMG-CoA lyase) is an inborn error of ketogenesis and Leucine catabolism. HMG-CoA lyase catalyses the final step in leucine degradation, converting HMG-CoA to acetyl-CoA and acetoacetic acid. Clinical manifestations include hepatomegaly, lethargy or coma and apnoea. Biochemically there is a characteristic absence of ketosis with hypoglycemia, acidosis, hipertransaminasemia and variable hyperammoniemia. The urinary organic acid profile includes elevated concentrations of 3-hydroxy-3-isovaleric, 3-hydroxy-3-methylglutaric, 3-methylglutaconic and 3-methylglutaric acids. CLINICAL CASE: Here, we report the case of a 17-year-old girl who presented in both ten months and five years of age a clinical picture characterized by lethargy leading to apnea and coma, hepatomegaly, hypoglycemia, metabolic acidosis, hyperammoniemia, elevated serum transaminases and absence of ketonuria. Diagnostic of Reye syndrome was suggested by hystopathologic finding of hepatic steatosis and clinical and biochemical data. As of 11 years old, laboratory investigations revealed carnitine deficiency and characteristic aciduria. Confirmatory enzyme diagnosis revealing deficiency of HMG-CoA lyase was made in cultured fibroblasts. CONCLUSION: Our report constitutes an example of the presentation of HMG-CoA lyase deficiency as recurrent Reye-like syndrome.

[Prolonged neuromuscular block caused by succinylcholine in a patient with normal cholinesterase activity]. / Blocco neuromuscolare prolungato da succinilcolina in un paziente con normale attività colinesterasica.

The authors describe a case of prolonged neuromuscular blockade following suxamethonlum in a patient with a normal cholinesterase activity and dibucaine number > or = 75%. In this case a peripheral nerve stimulator and capnography allowed neuromuscular blockade evaluation and fresh frozen plasma infusion led to a normal recovery from suxamethonium neuromuscular blockade. This report suggests a case of abnormal cholinesterase activity described as "silent variant".

Reversal of prolonged suxamethonium apnoea with fresh frozen plasma in a 6-week-old infant.

A period of apnoea lasting 480 minutes following suxamethonium in a 6-week-old male infant is described. Neuromuscular function recovered following the administration of fresh frozen plasma. The infant was found to be homozygous for atypical cholinesterase (E1a E1a). This is believed to be the youngest reported case of suxamethonium apnoea.

[Procedure for identifying atypical variants of pseudocholinesterase for the prevention of prolonged apnea induced by succinylcholine]. / Procedimento per l'individuazione delle varianti atipiche della pseudocolinesterasi ai fini della prevenzione dell'apnea protratta indotta dalla succinildicolina.

The simple Sclavo test based on succinyldicholine hydrolysis and on the following reaction with 5,5'-dithio-bis-2-nitrobenzoic acid for detection of the atypical patterns of serum cholinesterase has been investigated. A yellow staining appears but in presence of the atypical forms of the enzyme. Analysis of 4055 serum samples by the 430 Selective Analyzer Sclavo allowed the identification of one atypical homozygous patient. Prolonged apnoea induced by use of succinyldicholine as a neuromuscular blocking agent during anaesthesia may be avoided by use of this test.

[Phenotype of plasma cholinesterase in prolonged apnea and sensitivity to succinylcholine]. / Le phénotypage de la cholinestérase plasmatique dans les apnées prolongées à la succinylcholine.

90% of the injected dose of succinylcholine is hydrolysed by serum cholinesterase (E.C.3.I.I.8) Abnormal variants of serum cholinesterase lead to prolonged apnea. This report presents the results of 62 serum cholinesterase phenotyping including 12 cases of prolonged apneas. One clinical case of prolonged apnea in a patient homozygous for the atypical cholinesterase gene is presented with a study of his genealogy. The phenotypes were established on the basis of dibucaïne, fluorure, chloride and propranolol differential inhibition. The frequency and significance of the various phenotypes is discussed.

Is succinyldicholine the substrate of choice for the measurement of cholinesterase activity?

We have developed a succinyldicholine-based assay for serum cholinesterase (EC 3.1.1.8) to help establish whether patients with suspected sensitivity to drugs of this type have enzyme abnormalities that cannot be detected by conventional laboratory techniques. Although the method discriminates between cholinesterase activities of drug-sensitive and nonsensitive people as well as an assay involving propionylthiocholine does, it has not revealed cholinesterase abnormalities in patients who experienced prolonged apnea after succinyldicholine, in whom no enzyme defect could be demonstrated by other procedures. Of 50 individuals who were apneic for between 20 and 180 min, only one had a cholinesterase activity less than the mean for E1u homozygotes by more than 2.5 SD. We conclude that the number of patients who experience clinical problems due to enzyme abnormalities that at present go unrecognized is small. Consequently, although succinyldicholine might eventually become the substrate of choice for cholinesterase, its advantages over propionylthiocholine are not yet sufficient to encourage its use.