Characterization of four BCHE mutations associated with prolonged effect of suxamethonium.

Butyrylcholinesterase (BChE) 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 the characterization of four BCHE mutations associated with prolonged effect of suxamethonium (amino acid numbering based on the matured enzyme): p.20delValPheGlyGlyThrValThr, p.Leu88His, p.Ile140del and p.Arg386Cys. Expression of recombinant BCHE mutants, kinetic analysis and molecular dynamics were undertaken to understand how these mutations induce BChE deficiency. Three of the mutations studied (p.20delValPheGlyGlyThrValThr, p.Ile140del and p.Arg386Cys) lead to a "silent" BChE phenotype. Recombinant BCHE expression studies for these mutants revealed BChE activity levels comparable to untransfected cells. Only the last one (hBChE-L88H) presented BChE activity in the transfected cell culture medium. This BChE mutant (p.Leu88His) is associated with a lower kcat value compare to the wild-type enzyme. Molecular dynamics simulations analyses suggest that a destabilization of a structure implicated in enzyme activity (Ω-loop) can explain the modification of the kinetic parameter of the mutated protein.

[The patient billing cost in medical biology: advantages and disadvantages]. / La facturation coût-patient en biologie médicale: avantages et inconvénients.

Control of health expenditures in relation with staff reduction have led hospital or private labs to seek out growing productivity associated with limited costs. Consolidation and harmonization of equipment have to be implemented, as well as work positions, with the aid of computers. Analytical and preanalytical choices often converge towards the same manufacturer, and it is tempting to simplify also order, receipt and invoicing procedures. This cost optimization requires invoicing taking into account real activity of the lab, establishing "pay per patient" rather than "pay per kit". These changes may also apply to smaller analyzers such as those employed for haemostasis that are deployed in several sites, in aim to reduce costs. We describe the principles of this new mode of invoicing proposed by Roche Diagnostics, for which one may bring out main advantages and inconveniences.

Evaluation of Systematic Blood Testing at the Time of Recruitment to the Armed Forces: Retrospective Monocentric Study Among 726 French Army Soldiers.

INTRODUCTION: In the French armed forces, the biological checkup required during the recruitment process comprises a urinalysis (urinary dipstick), a complete blood count (CBC), and measurement of serum levels of aspartate aminotransferase, alanine aminotransferase, fasting blood glucose, and creatinine. This study aimed to evaluate the benefits of this biological checkup and to determine the most relevant parameters. MATERIALS AND METHODS: We conducted a monocentric retrospective study of all standardized and systematically conducted blood tests (CBC and measurement of aspartate aminotransferase, alanine aminotransferase, fasting blood glucose, and creatinine) over a 15-month period among 726 French Army recruits. RESULTS: The population included mainly young males (85.4%, mean age 21.6 years). More than half (54.1%) of the blood tests had at least one abnormal parameter, most often concerning the CBC. Anemia occurred in 5.3% of the population and was mostly normocytic. Microcytosis was mostly not associated with anemia (72.3% of cases). Lymphopenia occurred in 20.1% of the population and was mostly mild. Eosinophilia was present in 5.1% of the population and was never severe. Thrombocytopenia occurred in 0.7% of the population and was never severe. Serum levels of aminotransferases were elevated in 8.1% of the population. Fasting plasma glucose averaged 84 mg/dL (SD: 0.07) ranging from 64 to 123 mg/dL, was abnormal in 0.4% of the population, and one case of diabetes was diagnosed. Serum creatinine concentration was elevated in 0.7% of the population. CONCLUSION: CBCs gave useful information but iron deficiency was common and insufficiently detected by this single analysis. Assessing aminotransferase levels without screening for viral hepatitis and systematic measurement of fasting plasma glucose levels did not appear to be efficient. In addition, the only interest in systematic measurement of creatinine serum levels was to obtain a reference level for long-term follow-up. In addition to the urinary dipstick, the systematic biological checkup at recruitment could be limited to a CBC with measurement of plasma ferritin levels and Hepatitis B virus serology, providing that any CBC abnormalities, in particular cytopenia, eosinophilia, and microcytosis, are systematically investigated. For a public health approach, systematic screening for other sexually transmitted infections could be proposed.

Military community: a privileged site for clinical research: Epidemiological Study of Metabolic Syndrome Risk Factors in the Military Environment.

The metabolic syndrome is considered to be an important public health problem. The Epidemiological Study of Metabolic Syndrome Risk Factors in the Military Environment is a prospective epidemiological study that is designed to identify clinical and laboratory parameters of metabolic syndrome and cardiovascular risk factors with an initial 1-year cross-sectional study followed by a 10-year follow-up and patient care. One hundred eight-five (9%) of 2,045 military personnel subjects presented at least three of the five National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) criteria. They were significantly older (42.2 +/- 8.5 years) than the other subjects (37.3 +/- 8.7 years, p < 0.001), had a higher body mass index (BMI) (29.5 +/- 3.4 vs. 24.8 +/- 2.9, p < 0.001), and a greater body weight at age 20 (75.4 +/- 11 vs. 70.4 +/- 8.5 kg, p < 0.001). Smoking, little physical activity, and family histories of diabetes and arterial hypertension were more frequent in these subjects. Total plasma cholesterol and C-reactive protein were higher. Plasma insulin and BMI (r = 0.456, p < 0.0001) and plasma insulin and waist circumference (r = 0.446, p < 0.0001) were well correlated. Plasma insulin and homeostasis model assessment increased with the number of metabolic syndrome criteria. These results demonstrate a strong association with insulin resistance. Men with several risk factors require specific care especially for hypertension and dyslipidemia that will be evaluated during the follow-up period. Genotyping of subjects having metabolic syndrome vs. controls for genes, presumably involved should enlarge the area of exploration of this syndrome.

[Myoglobin: still a useful biomarker in 2017?] / Y a-t-il un intérêt au dosage de la myoglobine en 2017 ?

The clinical biologist plays a role as a consultant for the relevant use of biological examination. Advisory activities of the medical laboratory may help physician in diagnosis or therapeutic algorithm, avoiding redundant ordering or useless tests. In this context, we performed a review of literature about the clinically interest of myoglobin assays. The indications of myoglobin's assays appear fairly limited. It is no longer mentioned in the European guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation. In patients with rhabdomyolysis myoglobin is neither a diagnostic nor a prognostic criterion. Its interest in predicting the occurrence of acute renal failure is also discussed. The most recent clinico-biological score (such as the McMahon score) do not integrate it. In this context, we decided to stop performing myoglobin assay.

MDR3 mutations associated with intrahepatic and gallbladder cholesterol cholelithiasis: an update.

BACKGROUND: The recurrent microlithiasis represents one of the most frequent clinical forms of lithiasis of the bile ducts. This affection is characterized by the presence of cholesterolic microgallstones on hepatic canaliculars, and belongs to a heterogeneous group of autosomal recessive liver disorders. Radiological diagnosis can be confirmed by analysis of MDR3 gene, coding a protein involved in physiologic translocation of phospholipids in bile. Discovery of MDR3 mutations is of particular interest, since normally associated with good effectiveness of medication by ursodesoxycholic acid. AIM: To review MDR3 mutations in humans associated with recurrent cholesterol microlithiasis and to suggest a practical approach for MDR3 gene analysis. RESULTS: 48 mutations of MDR3 gene have been reported in humans to date, from which 43 (89.5%) in the coding region, and 5 splice site mutations have been associated to cholesterol cholelithiasis. 21 (43.8%) of the 43 precited mutations are located in only 8 exons on 28, near transmembrane or nucleotide binding domains of the protein. From the 22 remaining described mutations, 9 (18.8%) are restricted to exon 14. We suggest therefore to start analysis of MDR3 gene by screening exons 6, 7, 9, 10, 12, 14, 17, 23 and 24 with an appropriate protocol in this diagnosis associated with effective treatment. In conclusion such therapeutic orientation is valuable, since recurrent cholesterolic microlithiasis occurs relatively early in life, and by the fact that recurrence of symptoms may occur despite cholecystectomy, or shock-wave therapy.

[Acute pancreatitis after treatment by celecoxib]. / Pancréatite aiguë survenant après la prise de celecoxib.

Drug-induced pancreatitis injury due to celecoxib, a first generation Cox-2 inhibitor, has been rarely reported. We describe one case of severe pancreatitis after treatment with celecoxib for 3 months in a woman. No aetiology has been found for pancreatitis. The role of celecoxib in the etiology of colitis was considered probable. This report and a few other cases in the literature suggest to seek a pancreatitis in the event of pains abdominal when there is a catch of the cyclooxygenase-2 selective non-steroidal anti-inflammatory drug inhibitor.

Diagnosis of genetic predisposition for lactose intolerance by high resolution melting analysis.

Lactose, the principle sugar in milk, is a disaccharide hydrolyzed by intestinal lactase into glucose and galactose, which are absorbed directly by diffusion in the intestine. The decline of lactase expression (or hypolactasia) in intestinal microvilli after weaning is a normal phenomenon in mammals known as lactase deficiency. It is observed in nearly 75% of the world population and is an inherited autosomal recessive trait with incomplete penetrance. It is caused by SNPs in a regulatory element for lactase gene. In Indo-European, lactase deficiency is associated with rs4982235 SNP (or -13910C>T). The aim of this study is to describe a method based on high resolution melting for rapidly detecting genetic predisposition to lactose intolerance. Analytical performance of the assay was assessed by evaluating within and betwwen-run precision and by comparing the results (n = 50 patients) obtained with the HRM assay to those obtained with the gold standard (Sanger sequencing of the region of interest). In silico prediction of HRM curves was performed to evaluate the potential impact of the other SNPs described within the PCR product on the HRM analytical performances. The assay has good performance (CV <0.2% during the between-run study). A perfect agreement with the gold standard method was observed. The presence of other polymorphisms within the amplified sequence is detected, the misclassification risk is low. This assay can be used for rapidly diagnosing genetic predisposition to lactose intolerance.

[Urinary dipsticks must not be used to detect diabetes-induced incipient nephropathy]. / Inefficacité des bandelettes urinaires dans la recherche de néphropathie incipiens chez les diabétiques.

INTRODUCTION: Microalbuminuria is an early indication of diabetic nephropathy in patients with Type 1 diabetes and a marker of cardiovascular in patients with type 2 diabetes. It must therefore be assessed annually in these patients. We sought to determine whether semiquantitative determination of proteinuria with urinary dipsticks was useful for this purpose. METHOD: This analysis of consecutive urinary samples among diabetic patients excluded those with dipstick results positive either for leukocyturia or nitrituria, to avoid false positives due to urinary infection. We assessed the reliability of the dipsticks in comparison with conventional microalbuminuria and proteinuria assays. RESULTS: The study included 230 patients. Positive dipstick results had good positive (95.7%) and negative (93.9%) predictive values. Low levels of microalbuminuria, however - those that lead to early adjustment of treatment, were much more difficult to identify: the negative predictive value was only 73.7% and proteinuria was no longer correlated with microalbuminuria. DISCUSSION: Urinary dipsticks cannot replace conventional assays for microalbuminuria or proteinuria.

Butyrylcholinesterase deficiency.

Butyrylcholinesterase (EC 3.1.1.8; BChE) is a sister enzyme of acetylcholinesterase. Though BChE lacks obvious physiological functions, it is of toxicological and pharmacological importance in detoxifying or catabolising ester-containing drugs. Furthermore, individuals deficient in BChE appear asymptomatic, apart from a heightened sensitivity to the muscle relaxants suxamethonium and mivacurium, two BChE substrates used as myorelaxant. Although many acquired conditions may affect BChE activity, BChE deficiency is mainly due to mutations in the BCHE gene (OMIM 177400). Currently, more than 70 natural mutations have been documented in human BCHE. They have an adverse effect on BChE activity by affecting the catalytic functioning or the protein expression. However, the atypical variant (rs1799807) is the most frequently involved in prolonged apnea.

[Sexual abuse and chemical submission, a present-day problem]. / Agressions sexuelles et soumissions chimiques, un problème d'actualité.

CHEMICAL SUBMISSION: Certain forms of sexual abuse have recourse to molecules in order to obtain the victim's submission. These fast-acting, muscle-relaxing and sedative molecules, provoke anterograde amnesia that makes subsequent legal enquiries difficult. These products can be combined with alcohol or other addictive substances, therefore facilitating their administration, without the victim knowing, and enhancing their effects. THE MOLECULES USED: Among these molecules, hydroxybutyrate and its derivatives as well as benzodiazepines, with flunitrazepam, are frequently mentioned and detected. However, many other sedative molecules can be used to obtain chemical submission. MANAGEMENT OF THE VICTIMS: Knowledge of the facts and concomitant symptoms facilitates the medical and medico-legal management of the victims, generally encountered in hospital emergency wards. The quality and quantity of toxicological samples permit the use of efficient and validated techniques in organizing medico-legal enquiries.

[Rapid detection of BCHE atypical variant (p.Asp70Gly) by high resolution melting curve analysis]. / Génotypage rapide du variant atypique du gène BCHE (p.Asp70Gly) par une technique HRM.

Butyrylcholinesterase (BChE) deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium). Although many acquired conditions may affect BChE activity, BChE deficiency is mainly due to mutations in the BCHE gene (MIM 177400). Though close to 70 natural mutations have been documented in human BCHE, the atypical variant (rs1799807) is the most frequently involved in prolonged apnea. We describe an HRM method for the detection of this variant. Thirty-four patients with known genotype [5 wild-type (U/U), 12 heterozygous (U/A), 17 homozygous (A/A) - A: atypical allele of BCHE, U: usual allele of BCHE -] were screened with the HRM analysis. Within and between-run precision were also evaluated. In silico prediction of HRM curves was performed in order to evaluate the potential impact of the other SNPs described within the PCR product on the HRM diagnostic accuracy. HRM analysis for the BCHE atypical variant genotyping is a simple, rapid, sensitive and low cost method.

Characterization of a novel butyrylcholinesterase point mutation (p.Ala34Val), "silent" with mivacurium.

Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivarcurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of mivacurium leading to the discovery of a novel BCHE variant (c.185C>T, p.Ala34Val). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation remote from the active center determines the "silent" phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with a heterozygous enzyme of type atypical/silent. Kinetic analysis with succinyldithiocholine (SCdTC) as the substrate showed that Ala34Val BChE was inactive against this substrate. However, with BTC, the mutant enzyme was active, displaying an unexpected activation by excess substrate. Competitive inhibition of BTC by mivacurium gave a Ki=1.35 mM consistent with the lack of activity with the related substrate SCdTC, and with the clinical data. Molecular dynamic simulations revealed the mechanism by which mutation Ala34Val determines the silent phenotype: a chain of intramolecular events leads to disruption of the catalytic triad, so that His438 no longer interacts with Ser198, but instead forms hydrogen bonds either with residues Glu197 and Trp82, or peripheral site residue Tyr332. However, at high BTC concentration, initial binding of substrate to the peripheral site triggers restoration of a functional catalytic triad, and activity with BTC.

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.

[Cooperations between biology laboratories of the establishments of healthcare]. / Coopérations entre laboratoires de biologie médicale des établissements de santé

In France, the cooperations between biological laboratories of the healthcare establishments increased after those realized in the private laboratories. The biologists are confronted with various hypotheses of organization. They are often complex because they may preserve the quality of the care and their continuity while realizing financial economies. These economies are mostly based on the global reduction in the staff and in the equipments by mutualising the biological tests with varying degrees. We describe the various elements to be taken into account (staff, activities, budget, quality, transport, materials) and propose many scenarios of cooperations, from a unique central shape to the transfer of very specialized tests, with their advantages and their inconveniences. The management of human aspects in these cooperations is determining to facilitate their success as well as a reliable preliminary inventory of fixtures.