Alexandra Rousseau

ALLELE-SPECIFIC METHYLATED MULTIPLEX REAL TIME QUANTITATIVE PCR (ASMM RTQ-PCR), A POWERFUL METHOD FOR DIAGNOSING LOSS OF IMPRINTING OF THE 11p15 REGION IN RUSSELL SILVER AND BECKWITH WIEDEMANN SYNDROMES

Many human syndromes involve a loss of imprinting (LOI) due to a loss (LOM) or a gain of DNA methylation (GOM). Most LOI occur as mosaics and can therefore be difficult to detect with conventional methods. The human imprinted 11p15 region is crucial for the control of fetal growth, and LOI at this locus is associated with two clinical disorders with opposite phenotypes: Beckwith‐Wiedemann syndrome (BWS), characterized by fetal overgrowth and a high risk of tumors, and Russell‐Silver syndrome (RSS), characterized by intrauterine and postnatal growth restriction. Until recently, we have been using Southern blotting for the diagnosis of RSS and BWS. We describe here a powerful quantitative technique, allele‐specific methylated multiplex real‐time quantitative PCR (ASMM RTQ‐PCR), for the diagnosis of these two complex disorders. We first checked the specificity of the probes and primers used for ASMM RTQ‐PCR. We then carried out statistical validation for this method, on both retrospective and prospective populations of patients. This analysis demonstrated that ASMM RTQ‐PCR is more sensitive than Southern blotting for detecting low degree of LOI. Moreover, ASMM RTQ‐PCR is a very rapid, reliable, simple, safe, and cost effective method. Hum Mutat 32:249–258, 2011.

Cardiovascular findings and management in Turner syndrome: insights from a French cohort

OBJECTIVEnCongenital cardiovascular malformations and aortic dilatation are frequent in patients with Turner syndrome (TS). The objective of this study was to investigate the cardiovascular findings and management in a large cohort of patients, including children and adults.nnnDESIGN/METHODSnWe recruited 336 patients with TS from a network of tertiary centers. We reviewed their files, checking for cardiovascular events, cardiac valve abnormalities, and aortic diameters indexed to body surface area (BSA) from magnetic resonance imaging (n=110) or echocardiography (n=300).nnnRESULTSnInformative cardiovascular data were available for only 233 patients. Vascular surgery was reported in 7.4% of the cohort. The first cause of surgery was aortic coarctation, detected in 6.9% at a median age of 9.5 (range: 0-60) years. Bicuspid aortic valve (BAV) was detected in 21% at a median age of 20 years (25th-75th percentiles: 15-30). At least one aortic diameter exceeded 32 mm in 12% of the cohort. This was detected at a median age of 19 (7-30) years. When indexed to BSA, at least one aortic diameter exceeded 20 mm/m(2) in 39% of the cohort.nnnCONCLUSIONnOur study shows that cardiovascular monitoring for TS patients is currently insufficient in France. BAV is present at birth, but often remains undiagnosed until later in life. Therefore, improved management in cardiovascular monitoring is required and a more systematic approach should be taken.

Impact of age and gender interaction on circulating endothelial progenitor cells in healthy subjects

OBJECTIVEnTo assess the level of circulating endothelial progenitor cells (CEPC) in cycling women compared with men and menopausal women.nnnDESIGNnControlled clinical study.nnnSETTINGnHealthy, nonsmoking volunteers.nnnPATIENT(S)nTwelve women, aged 18-40 years, with regular menstrual cycles, 12 menopausal women, and two groups of 12 age-matched men were recruited. Women did not receive any hormone therapy.nnnINTERVENTION(S)nCollection of 20 mL of peripheral blood.nnnMAIN OUTCOME MEASURE(S)nThe number of CEPC, defined as (Lin-/7AAD-/CD34+/CD133+/KDR+) cells per 10(6) mononuclear cells (MNC), was measured by flow cytometry.nnnRESULT(S)nThe number of CEPC was significantly higher in cycling women than in age-matched men and menopausal women (26.5 per 10(6) MNC vs. 10.5 per 10(6) MNC vs. 10 per 10(6) MNC, respectively). The number of CEPC was similar in menopausal women, age-matched, and young men.nnnCONCLUSION(S)nThe number of CEPC is influenced by an age-gender interaction. This phenomenon may explain in part the better vascular repair and relative cardiovascular protection in younger women as compared with age-matched men.

A multicentric randomized controlled trial on the impact of lengthening the interval between neoadjuvant radiochemotherapy and surgery on complete pathological response in rectal cancer (GRECCAR-6 trial): rationale and design

BackgroundNeoadjuvant radiochemotherapy (RCT) is now part of the armamentarium of cancer of the lower and middle rectum. It is recommended in current clinical practice prior to surgical excision if the lesion is classified T3/T4 or N+. Histological complete response, defined by the absence of persistent tumor cell invasion and lymph node (ypT0N0) after pathological examination of surgical specimen has been shown to be an independent prognostic factor of overall survival and disease-free survival. Surgical excision is usually performed between 6 and 8xa0weeks after completion of CRT and pathological complete response rate ranges around 12%. In retrospective studies, a lengthening of the interval after RCT beyond 10xa0weeks was found as an independent factor increasing the rate of pathological complete response (between 26% and 31%), with a longer disease-free survival and without increasing the operative morbidity. The aim of the present study is to evaluate in 264 patients the rate of pathological complete response rate of rectal cancer after RCT by lengthening the time between RCT and surgery.Methods/designThe current study is a multicenter randomized trial in two parallel groups comparing 7 and 11xa0weeks of delay between the end of RCT and cancer surgery of rectal tumors.At the end of the RCT, surgery is planified and randomization is performed after patient’s written consent for participation. The histological complete response (ypT0N0) will be determined with analysis of the complete residual tumor and double reading by two pathologists blinded of the group of inclusion. Patients will be followed in clinics for 5xa0years after surgery. Participation in this trial does not change patient’s management in terms of treatment, investigations or visits. Secondary endpoints will include overall and disease free survival, rate of sphincter conservation and quality of mesorectal excision. The number of patients needed is 264.Trial registrationClinicalTrial.gov: NCT01648894

A Pharmacokinetic-Pharmacodynamic Model for Predicting the Impact of CYP2C9 and VKORC1 Polymorphisms on Fluindione and Acenocoumarol During Induction Therapy

Background and ObjectiveVitamin K epoxide reductase complex, subunit 1 (VKORC1) and cytochrome P450 2C9 (CYP2C9) polymorphisms are taken into account when predicting a safe oral dose of coumarin anticoagulant therapy, but little is known about the effects of genetic predictors on the response to fluindione and acenocoumarol. The aims of this study were to characterize the relationship between fluindione and acenocoumarol concentrations and the international normalized ratio (INR) response, and to identify genetic predictors that are important for dose individualization.MethodsFluindione concentrations, S- and R-acenocoumarol concentrations, the INR and genotype data from healthy subjects were used to develop a population pharmacokinetic-pharmacodynamic model in Monolix software. Twenty-four White healthy subjects were enrolled in the pharmacogenetic study. The study was an open-label, randomized, two-period cross-over study. The subjects received two doses of an oral anticoagulant: 20 mg of fluindione (period A) or 4 mg of acenocoumarol (period B). The pharmacokinetics and pharmacodynamics were studied from day 2 to day 3.ResultsA two-compartment model with a first-order input model was selected as the base model for the two drugs. The pharmacodynamic response was best described by an indirect action model with S-acenocoumarol concentrations and fluindione concentrations as the only exposure predictors of the INR response. Three covariates (CYP2C9 genotype, VKORC1 genotype and body weight) were identified as important predictors for the pharmacokinetic-pharmacodynamic model of S-acenocoumarol, and four covariates (CYP2C9 genotype, VKORC1 genotype, CYP1A2 phenotype and body weight) were identified as predictors for the pharmacokinetic-pharmacodynamic model of fluindione. Because some previous studies have shown a dose-response relationship between smoking exposure and the CYP1A2 phenotype, it was also noted that smokers have greater CYP1A2 activity.ConclusionDuring initiation of therapy, CYP2C9 and VKORC1 genetic polymorphisms are important predictors of fluindione and acenocoumarol pharmacokinetic-pharmacodynamic responses. Our result suggests that it is important to take the CYP1A2 phenotype into account to improve individualization of fluindione therapy, in addition to genetic factors.

Optimization and validation of the methods for the total mercury and methylmercury determination in breast milk

The objective of the work was to develop and validate methods for the total Hg and methylmercury (MeHg) in breast milk that could be further used to obtain first data on chemical contamination of French breast milk. For total Hg determination, the potential of two techniques, namely Advanced Mercury Analyzer (AMA) and ICP MS, was compared. For MeHg determination, ICP MS detection associated to a quantification by isotopic dilution was used and the potential of a preliminary separation by gas or liquid chromatography was evaluated and discussed. The optimization studies have shown that AMA for total Hg determination and HPLC - ID - ICP MS, after a preconcentration step by freeze-drying, for MeHg quantification were the most relevant methods to use for epidemiologic studies. The figures of merit for both methods were evaluated by means of accuracy profiles in terms of limits of quantification (1.82 and 1.35µg Hg/kg dry weight, corresponding to 0.22 and 0.16µg Hg/kg wet basis for total Hg and MeHg, respectively), repeatability (2-11% and 3-8% for total Hg and MeHg respectively), intermediate precision reproducibility (4-12% and 4-8% for total Hg and MeHg respectively) and trueness bias (-0.1-9% and -4-0% for total Hg and MeHg respectively). The methods were then applied to 180 breast milk samples. Total Hg concentrations ranged from <LD to 16.9µg Hg/kg wet basis (<LD to 142µg Hg/kg dry weight) and the MeHg contents from <LD to 0.43µg Hg/kg wet basis (<LD to 3.67µg Hg/kg dry weight).

PERC rule to exclude the diagnosis of pulmonary embolism in emergency low-risk patients: study protocol for the PROPER randomized controlled study.

BackgroundThe diagnosis of Pulmonary Embolism (PE) in the emergency department (ED) is crucial. As emergency physicians fear missing this potential life-threatening condition, PE tends to be over-investigated, exposing patients to unnecessary risks and uncertain benefit in terms of outcome. The Pulmonary Embolism Rule-out Criteria (PERC) is an eight-item block of clinical criteria that can identify patients who can safely be discharged from the ED without further investigation for PE. The endorsement of this rule could markedly reduce the number of irradiative imaging studies, ED length of stay, and rate of adverse events resulting from both diagnostic and therapeutic interventions. Several retrospective and prospective studies have shown the safety and benefits of the PERC rule for PE diagnosis in low-risk patients, but the validity of this rule is still controversial. We hypothesize that in European patients with a low gestalt clinical probability and who are PERC-negative, PE can be safely ruled out and the patient discharged without further testing.Methods/DesignThis is a controlled, cluster randomized trial, in 15 centers in France. Each center will be randomized for the sequence of intervention periods: a 6-month intervention period (PERC-based strategy) followed by a 6-month control period (usual care), or in reverse order, with 2xa0months of “wash-out” between the 2 periods. Adult patients presenting to the ED with a suspicion of PE and a low pre test probability estimated by clinical gestalt will be eligible. The primary outcome is the percentage of failure resulting from the diagnostic strategy, defined as diagnosed venous thromboembolic events at 3-month follow-up, among patients for whom PE has been initially ruled out.DiscussionThe PERC rule has the potential to decrease the number of irradiative imaging studies in the ED, and is reported to be safe. However, no randomized study has ever validated the safety of PERC. Furthermore, some studies have challenged the safety of a PERC-based strategy to rule-out PE, especially in Europe where the prevalence of PE diagnosed in the ED is high. The PROPER study should provide high-quality evidence to settle this issue. If it confirms the safety of the PERC rule, physicians will be able to reduce the number of investigations, associated subsequent adverse events, costs, and ED length of stay for patients with a low clinical probability of PE.Trial registrationNCT02375919.

Effect of two oral doses of 17β-estradiol associated with dydrogesterone on thrombin generation in healthy menopausal women: a randomized double-blind placebo-controlled study

Oral hormone therapy is associated with an increased risk of venous thrombosis. Drug agencies recommend the use of the lowest efficient dose to treat menopausal symptoms for a better risk/ratio profile, although this profile has not been totally investigated yet. The aim of the study was to compare the effect of the standard dose of 17β‐estradiol to a lower one on thrombin generation (TG).

The impact of nurse-driven targeted HIV screening in 8 emergency departments: study protocol for the DICI-VIH cluster-randomized two-period crossover trial

BackgroundIn 2010, to reduce late HIV diagnosis, the French national health agency endorsed non-targeted HIV screening in health care settings. Despite these recommendations, non-targeted screening has not been implemented and only physician-directed diagnostic testing is currently performed. A survey conducted in 2010 in 29 French Emergency Departments (EDs) showed that non-targeted nurse-driven screening was feasible though only a few new HIV diagnoses were identified, predominantly among high-risk groups. A strategy targeting high-risk groups combined with current practice could be shown to be feasible, more efficient and cost-effective than current practice alone.Methods/DesignDICI-VIH (acronym for nurse-driven targeted HIV screening) is a multicentre, cluster-randomized, two-period crossover trial. The primary objective is to compare the effectiveness of 2 strategies for diagnosing HIV among adult patients visiting EDs: nurse-driven targeted HIV screening combined with current practice (physician-directed diagnostic testing) versus current practice alone. Main secondary objectives are to compare access to specialist consultation and how early HIV diagnosis occurs in the course of the disease between the 2 groups, and to evaluate the implementation, acceptability and cost-effectiveness of nurse-driven targeted screening. The 2 strategies take place during 2 randomly assigned periods in 8 EDs of metropolitan Paris, where 42xa0% of France’s new HIV patients are diagnosed every year. All patients aged 18 to 64, not presenting secondary to HIV exposure are included. During the intervention period, patients are invited to fill a 7-item questionnaire (country of birth, sexual partners and injection drug use) in order to select individuals who are offered a rapid test. If the rapid test is reactive, a follow-up visit with an infectious disease specialist is scheduled within 72xa0h. Assuming an 80xa0% statistical power and a 5xa0% type 1 error, with 1.04 and 3.38 new diagnoses per 10,000 patients in the control and targeted groups respectively, a sample size of 140,000 patients was estimated corresponding to 8,750 patients per ED and per period. Inclusions started in June 2014. Results are expected by mid-2016.DiscussionThe DICI-VIH study is the first large randomized controlled trial designed to assess nurse-driven targeted HIV screening. This study can provide valuable information on HIV screening in health care settings.Trial registrationClinicalTrials.gov: NCT02127424xa0(29 April 2014).

Cross-checking to reduce adverse events resulting from medical errors in the emergency department: study protocol of the CHARMED cluster randomized study.

BackgroundMedical errors and preventable adverse events are a major cause of concern, especially in the emergency department (ED) where its prevalence has been reported to be roughly of 5–10xa0% of visits. Due to a short length of stay, emergency patients are often managed by a sole physician – in contrast with other specialties where they can benefit from multiples handover, ward rounds and staff meetings. As some studies report that the rate and severity of errors may decrease when there is more than one physician involved in the management in different settings, we sought to assess the impact of regular systematic cross-checkings between physicians in the ED.DesignThe CHARMED (Cross-checking to reduce adverse events resulting from medical errors in the emergency department) study is a multicenter cluster randomized study that aim to evaluate the reduction of the rate of severe medical errors with implementation of systematic cross checkings between emergency physician, compared to a control period with usual care. This study will evaluate the effect of this intervention on the rate of severe medical errors (i.e. preventable adverse events or near miss) using a previously described two-level chart abstraction. We made the hypothesis that implementing frequent and systematic cross checking will reduce the rate of severe medical errors from 10 to 6xa0% - 1584 patients will be included, 140 for each period in each center.DiscussionThe CHARMED study will be the largest study that analyse unselected ED charts for medical errors. This could provide evidence that frequent systematic cross-checking will reduce the incidence of severe medical errors.Trial registrationClinical Trials, NCT02356926