Advances in the metabolic profiling of acidic compounds in children's urines achieved by comprehensive two-dimensional gas chromatography.

The main objective of this work was to evaluate a comprehensive two-dimensional gas chromatographic (GCxGC) coupled to quadrupole mass spectrometry (qMS) method in the field of biomarker candidates' discovery. To this purpose we developed a GCxGC-qMS method suitable for the separation of organic acids and other classes of compounds with silylable polar hydrogen such as sugars, amino-acids, and vitamins. As compared to those obtained by a widely used 1D-GC method, the urinary chromatographic profiles performed by the proposed 2D-GC method exhibit higher resolution and sensitivity, leading to the detection of up to 92 additional compounds in some urine samples including some well-known biomarkers. In order to validate the proposed method we focused on three metabolites of interest with various functional groups and polarities including CH3-malonic acid (MMA: biomarker of methylmalonic acidemia), 3-hydroxy-3-methyl-glutaric acid (3-OHMGA: biomarker of 3-hydroxy-3-methylglutaric acidemia), and phenylpiruvic acid (PhPA: marker of phenylketonuria). While these three metabolites can be considered as representative of organic acids classically determined by 1D-GC, they cannot be representative of new detected metabolites. Thus, we also focused on quinolic acid (QUIN), taken as an example of biomarker not detected at basal levels with the classical 1D GC-qMS method. In order to obtain sufficient recoveries for all tested compounds, we developed a sample preparation protocol including a step of urea removal followed by two extraction steps using two solvents of different polarity and selectivity. Recoveries with the proposed method reached more than 80% for all targeted compounds and the linearity was satisfactory up to 50µmol/L. The CVs of the within-run and within-laboratory precisions were less than 8% for all tested compounds. The limits of quantification (LOQs) were 0.6µmol/L for MMA, 0.4µmol/L for 3-OHMGA, 0.7µmol/L for PhPA, and 1µmol/L for QUIN. The LOQs of these metabolites obtained by a classical GC-MS method under the same chromatographic conditions were 5µmol/L for MMA, 4µmol/L for 3-OHMGA, 6µmol/L for PhPA while QUIN was below the limit of detection. As compared to 1D-GC, these results highlight the enhanced detectability of urine metabolites by the 2D-GC technique. Our results also show that for each new detected compound it is necessary to develop and validate an appropriate sample preparation procedure.

[Lung diseases in children associated with inherited disorders of surfactant metabolism]. / Pathologies respiratoires de l'enfant associées à des anomalies héréditaires du métabolisme du surfactant.

Pulmonary surfactant is a unique mixture of lipids and specific proteins that reduces surface tension at the air-liquid interface, preventing collapse of the lung at the end of expiration. Recessive loss-of-function mutations of pulmonary surfactant protein B (SP-B) was initially described in infants who develop respiratory failure at birth. More recently, mutations in other constitutive surfactant proteins like surfactant protein C or implied in its metabolism like ATP-binding cassette, sub-family A, member 3 (ABCA3) or NK2 homeobox (NKX2-1) were identified in newborn with respiratory distress but also in children with diffuse infiltrative pneumonia. Intra-alveolar accumulation of protein related to surfactant dysfunction leads to cough, hypoxemia and radiological abnormalities including ground-glass opacities and lung cysts. The clinical and radiological features associated with these genetic disorders, along with their treatment and outcome, are reviewed.

Biallelic nonsense mutations in the otogelin-like gene (OTOGL) in a child affected by mild to moderate hearing impairment.

Hearing impairment is characterized by great genetic heterogeneity. We report the identification, by whole exome sequencing, of two different nonsense mutations (c.1558C>T; p.Gln520 and c.2773C>T; p.Arg925) in the otogelin-like gene (OTOGL), in a child affected by mild to moderate isolated deafness. Parental genotypes allowed us to conclude that these mutations are present in the compound heterozygous state in the patient. In addition, our clinical data establish that the tectorial membrane and/or the outer hair cells are defective in this form of deafness.

Apolipoprotein E polymorphism interacts with cigarette smoking in progression of multiple sclerosis.

BACKGROUND AND PURPOSE: The influence of apolipoprotein E (ApoE) polymorphism on clinical severity of multiple sclerosis (MS) is still controversial. Cigarette smoking has been suggested to influence the progression of disability in these patients. In this study, we aimed to investigate whether an interaction of smoking with the ApoE polymorphism influences the progression of disability in MS patients. METHODS: Smoking history from 205 female patients with MS was obtained. Clinical data collected include age at onset, disease duration, annual relapse rate, the Expanded Disability Status Scale (EDSS) and the Multiple Sclerosis Severity Score (MSSS). ApoE polymorphism was examined in all patients and stratified according to smoking status and associations with the clinical data investigated. RESULTS: There were no significant associations between cigarette smoking and any of the clinical characteristics in the whole group of patients. In women carrying the ApoE E4 isoform, smokers had a lower EDSS (P = 0.033) and MSSS (P = 0.023) in comparison with non-smokers. CONCLUSION: Our data suggest that in women with MS carrying the ApoE E4 isoform, cigarette smoking may have a protective influence on disease progression and accumulation of disability. These findings need to be confirmed by future large longitudinal studies.

New surfactant protein C gene mutations associated with diffuse lung disease.

BACKGROUND: Mutations in the surfactant protein C gene (SFTPC) have been recently associated with the development of diffuse lung disease, particularly sporadic and familial interstitial lung disease (ILD). OBJECTIVE: We have investigated the prevalence and the spectrum of SFTPC mutations in a large cohort of infants and children with diffuse lung disease and suspected with surfactant dysfunction. METHOD AND RESULTS: 121 children were first screened for the common SFTPC mutation, p.Ile73Thr (I73T). Ten unrelated patients were shown to carry this mutation. The I73T mutation was inherited in six cases, and appeared de novo in four. The 111 patients without the I73T mutation were screened for the entire coding sequence of SFTPC. Of these, eight (seven unrelated) subjects were shown to carry a novel mutant allele of SFTPC. All these seven new mutations are located in the BRICHOS domain except the p.Val39Ala (V39A) mutation, which is in the surfactant protein C (SP-C) mature peptide. CONCLUSIONS: Our results confirm that SFTPC mutations are a frequent cause of diffuse lung disease, and that I73T is the most frequent SFTPC mutation associated with diffuse lung disease.

Screening of SLC26A4 (PDS) gene in Pendred's syndrome: a large spectrum of mutations in France and phenotypic heterogeneity.

Sensorineural hearing defect and goiter are common features of Pendred's syndrome. The clinical diagnosis of Pendred's syndrome remains difficult because of the lack of sensitivity and specificity of the thyroid signs. The identification of PDS as the causative gene allowed molecular screening and enabled a re-evaluation of the syndrome to identify potential diagnostic characteristics. This report presents the clinical and genotypic findings of 30 French families, for whom a diagnosis of Pendred's syndrome had been made. Twenty-seven families had at least one mutated allele. Twenty-eight different mutations were identified, 11 of which had never been previously reported. The main clinical characteristics were: early hearing loss, fluctuation in terms of during deafness evolution, and the presence of an enlarged vestibular aqueduct.

[Comparison of plasma total homocysteine determinations in 9 French hospital laboratories by using 6 different methods]. / Comparaison du dosage de l'homocystéine plasmatique totale dans neuf laboratoires par six techniques différentes.

A lot of methods are now available for total plasma homocysteine (tHcy) determination. Commercial kits using immunoassay, easier to use, begin to supplant in-house laboratory methods. Our aim is to evaluate the interchangeability of tHcy measurements in 9 French hospital laboratories. Six different method types were used: 2 gas chromatography-mass spectrometry (GC-MS), 2 HPLC with fluorescence detection subdivided in one in-house method and one commercial kit (Bio-Rad ), 3 fluorescence polarization immunoassays (FPIA), 1 enzyme immunoassay, 1 amino acid analyser, 1 capillary electrophoresis coupled with laser-induced fluorescence detection (EC-LIF). Each laboratory analysed 41 patient's plasma samples in which 8 samples contained added homocystine. Results were analysed for imprecision, recovery, and methodological differences. The mean among-laboratory imprecision (CV) ranged from 12.5 to 18% in function of plasma sample type and was identical to the mean among-method variation. In terms of recovery, we obtained underestimated results with immunoassays. The bias relative to the GC-MS method was less than 12.5% except for two laboratories, one using FPIA assay and the other EC-LIF. In conclusion, the interchangeability of tHcy results between laboratories is not satisfactory and does not allow us to evaluate cardiovascular risk linked to moderate increases of tHcy.

[Genetic testing for cystic fibrosis: evaluation of the Elucigene CF20 kit in blood and buccal cells]. / Diagnostic moléculaire de la mucoviscidose: evaluation de la trousse Elucigene CF20 dans le sang et les cellules buccales.

Routine determination of mutations in cystic fibrosis requires accurate, rapid, reliable and low-cost methods, permitting the simultaneous detection of multiple mutations. The Elucigene CF20 kit developped by Cellmark Diagnostics, uses multiplex ARMS, which allows the screening for 20 CFTR gene mutations (deltaF508, G542X, N1303K, 1717-1G>A, G551D, W1282X, R553X, deltaI507, 1078delT, 2183AA>G, 3849+10kbC>T, R1162X, 621+1G>T, R334W, R347P, 3659delC, R117H, S1251N, E60X, A455E ) in a work day without specific instrumentation. The kit distinguishes between homozygotes and heterozygotes for deltaF508, but not for rare mutations. The kit detects from 68 to 92% of defective alleles in Caucasians. We evaluate the kit in a blind study in two independent laboratories. Thirty blood samples and thirty mouthwash samples from CF patients, carriers and unaffected individuals were analysed by the Elucigene CF20 kit. All the samples were previously analysed by denaturing gradient gel electrophoresis and sequencing. The Elucigene CF20 kit consists of three multiplexes. Each mutiplex contains ARMS specific primers for six to eight mutations and two control reactions. The absence of the upper control fragment indicates that a repeat test is required. We demonstrated a first time amplification rate of 98.3%: of the 60 samples tested, one required a reamplification. Results compared with the reference method demonstrated that in all cases where one or more of the 20 mutations detected by the kit were present in the test set, the kit accurately identified them. Reproducibility was assessed by repeating the analysis of a blood and mouthwash sample five times. Cross reactivity between R117C and R117H, R117P and R117H, R347P and R347H, deltaI507 and deltaF508, G551D and R553X were evaluated. Only a cross reactivity between R347P and R347H was observed. The kit is specially useful for first line study of patients and carrier identification.

[Constitutional deficiency of pulmonary surfactant protein B: clinical presentation, histologic and molecular diagnosis]. / Déficit constitutionnel en protéine B du surfactant pulmonaire: présentation clinique, diagnostic histologique et moléculaire.

We report a female full-term infant with fatal respiratory failure of early onset due to inherited SP-B deficiency. Lung biopsy was performed at 18 days after birth, with histopathological characterization indicating congenital alveolar proteinosis. Immunohistochemical studies of lung tissue revealed the absence of SP-B and the presence of intra-alveolar SP-A normal quantities. Analysis of genomic DNA showed homozygosity for the 121ins2 mutation of the SFTPB gene. The infant died 21 days after birth. Both parents were heterozygotes for the mutation. Chorionic villus sampling was performed at the first trimester of the following pregnancy. Restriction analysis of amplified fetal DNA, studies of microsatellite segregation and direct sequencing led to the diagnosis of homozygosity for the parental wild-type allele. The diagnosis of congenital SP-B deficiency should be suspected whenever an early and acute respiratory failure in a term or near-term infant does not resolve after five days of age: diagnostic confirmation can be easily and rapidly obtained with the analysis of genomic DNA and immunohistochemical characterization of lung tissue.

[Caveolae membrane domains, specialized transmembrane exchange zones implicated in cell signalling]. / Des groupements lipidiques membranaires (caveolae), zones d'échanges transmembranaires privilégiées à l'origine de signaux intracellulaires.

Caveolae are small pockets or invaginations localized at the plasma membrane. They are enriched in glycosphingolipids, cholesterol, sphingomyelin and lipid-enchored membrane proteins, and they are characterized by a light buoyant density and resistance to solubilization by Triton X-100 at 4 C. Caveolins are the principal protein components of caveolae and play an important structural role in the formation of caveolae membranes. Numerous molecules involved in cell signalling have been identified in caveolae, suggesting that these structures may serve to compartimentalize, modulate and integrate signalling events at the cell surface. Depletion of membrane cholesterol disrupts the formation and function of caveolae, suggesting that these membrane microdomains are involved in a range of biological processes. Moreover, exposure of endothelial cells to high levels of cholesterol upregulates the caveolin abundance in caveolae, and decreases nitric oxide synthesis, suggesting that this may be an early event in atherogenesis. Alteration in the expression of caveolin genes has also been implicated in human diseases such as cancers, diabetes, Alzheimer's disease and muscular distrophy.

[Lipoprotein(a): risk factor for atherosclerotic vascular disease important to take into account in practice]. / La lipoprotéine(a), intérêt de son dosage pour évaluer le risque cardiovasculaire.

Coronary artery disease is a leading cause of death in France. Some of its risk factors are well identified such as age, smoking, high blood pressure and dyslipidemia, but some others such as lipoprotein (a) (Lp(a)) are still under investigation. Lp(a) is an LDL-like particle to which is linked an apolipoprotein (a). The latter shows a high sequence homology with plasminogen that gives Lp(a) thrombogenic properties in addition to its atherogenic capacity. Many epidemiological studies have shown that a high plasma level of Lp(a) is a risk factor for coronary, cerebral and peripheral atherosclerosis. Out of thirteen prospective studies, ten have confirmed this result. The negative results from the three remaining studies were probably due to either the inadequate storage of the samples or the preventive drug treatment given to the patients during the studies and to the lack of standardization of Lp(a) assays. More over it has been shown that beside high plasma Lp(a) level, the presence of a low molecular weight Apo(a) isoform is also related to a higher incidence of coronary artery disease. This review of the literature clearly demonstrates the relationship between Lp(a) and atherosclerosis, and the need to measure Lp(a) in order to better evaluate the risk of atherosclerotic vascular disease especially in patients with a hyper LDLemia an early cardio- or cerebrovascular disease or a family history of atherosclerosis. Management of patients with high Lp(a) concentrations should be directed at minimizing all other risk factors for atherosclerotic disease.

Acquired circulating anticoagulant with anti-factor V activity in AIDS: first case report.

An acquired circulating anticoagulant with anti-factor V activity appeared in a 29 year old AIDS patient with widespread Kaposi's sarcoma following 21 days of fresh frozen plasma therapy for haemolytic and uraemic syndrome. Residual factor V activity was very low (< 5% of normal). However, the inhibitor was of low titre (0.5 Bethesda Units/ml), while antigenic factor V levels remained at 100%. Dot blotting with human factor V and polyvalent and specific immunoglobulin antisera showed the antibody to belong to the IgG class. Haemostatic tests in vitro were only partly corrected by addition of washed human platelets and despite transfusion of large amounts of platelets the patient died from massive pulmonary haemorrhage. This would appear to be the first documented case of an anti-factor V inhibitor occurring in an AIDS patient.