Detection and characterisation of large SERPINC1 deletions in type I inherited antithrombin deficiency.

Methods routinely used for investigating the molecular basis of antithrombin (AT) deficiency do not detect large SERPINC1 rearrangements. Between 2000 and 2008, 86 probands suspected of having AT-inherited type I deficiency were screened for SERPINC1 mutations in our laboratory. Mutations causally linked to the deficiency were identified by sequencing analysis in 63 probands. We present here results of multiplex ligation-dependent probe amplification (MLPA) analysis performed in 22 of the 23 remaining probands, in whom sequencing had revealed no mutation. Large deletions, present at the heterozygous state, were detected in 10 patients: whole gene deletions in 5 and partial deletions removing either exon 6 (n = 2), exons 1-2 (n = 1) or exons 5-7 (n = 2) in 5 others. Exon 6 partial deletions are a 2,769-bp deletion and a 1,892-bp deletion associated with a 10-bp insertion, both having 5' and/or 3' breakpoints located within Alu repeat elements. In addition, we identified the 5' breakpoint of a previously reported deletion of exons 1-2 within an extragenic Alu repeat. Distinct mutational mechanisms explaining these Alu sequence-related deletions are proposed. Overall, in this series, large deletions detected by MLPA explain almost half of otherwise unexplained type I AT-inherited deficiency cases.

Molecular bases of antithrombin deficiency: twenty-two novel mutations in the antithrombin gene.

Antithrombin (AT) is a major physiological inhibitor of hemostasis. We report 22 novel antithrombin gene (SERPINC1) mutations associated with antithrombin deficiency in 17 French and five German families. They were all present at the heterozygous state. Nine missense mutations accounted for type I deficiency, defined by equally low antithrombin activity and antigen level. Most of them (7/9) affected highly conserved serpin residues and were associated with venous thrombosis occurring at a young age (before age 32). One splice site, one nonsense mutation, three small deletions and one insertion were also identified as a cause for type I antithrombin deficiency. Seven other missense mutations were identified in type II or unclassified AT deficiency; g.5270C>T (p.T147I, T115I) and g.5281A>T (p.I151F, I119F) change residues in the heparin binding region, g.13267C>G (p.P439A, P407A) and g.13271T>C (p.F440S, F408S) affect amino acids in the pleiotropic region, g.2372G>A (p.G25D, G-8D) changes a signal peptide amino acid, g.2456G>C (p.C53S, C21S) affects one of the three disulfide bonds of the protein, and g.7585A>T (p.M347K, M315K) changes a nonconserved residue on strand 2C.

Use of N-terminal prohormone brain natriuretic peptide assay for etiologic diagnosis of acute dyspnea in elderly patients.

BACKGROUND: B-type peptide assay (brain natriuretic peptide [BNP] and N-terminal prohormone brain natriuretic peptide [NT-proBNP]) is useful for the diagnosis of heart failure (HF), but few data are available on the use of these markers in elderly subjects. The aim of this study was to evaluate NT-proBNP assay for the diagnosis of acute left HF in patients older than 70 years hospitalized for acute dyspnea. METHODS: We prospectively enrolled 256 elderly patients with acute dyspnea. They were categorized by 2 cardiologists unaware of NT-proBNP values into a cardiac dyspnea subgroup (left HF) and a noncardiac dyspnea subgroup (all other causes). RESULTS: Mean age was 81 +/- 7 years, and 52% of the patients were women. The diagnoses made in the emergency setting were incorrect or uncertain in 45% of cases. The median NT-proBNP value was higher (P < .0001) in patients with cardiac dyspnea (n = 142; 7906 pg/mL) than in patients with noncardiac dyspnea (n = 112; 1066 pg/mL). The area under the receiver operating characteristic curve was 0.86 (95% CI 0.81-0.91). At a cutoff of 2000 pg/mL, NT-proBNP had a sensitivity of 86%, a specificity of 71%, and an overall accuracy of 80% for cardiac dyspnea. The use of 2 cutoffs (< 1200 and > 4500 pg/mL) resulted in an 8% error rate and a gray area englobing 32% of values. CONCLUSION: NT-proBNP appears to be a sensitive and specific means of distinguishing pulmonary from cardiac causes of dyspnea in elderly patients. An optimal diagnostic strategy requires the use of 2 cutoffs and further investigations of patients with values in the gray area.

Protein C and protein S assessment in hospital laboratories: which strategy and what role for DNA sequencing?

This paper presents a critical assessment of protein C (PC) and protein S (PS) functional and immunological approaches with regard to DNA sequencing in a large hospital recruitment for thrombosis exploration in more than 1700 consecutive patients. After examination of clinical status and PC and PS phenotype, a genotypic study was implemented for 17 PC-deficient and 28 PS-deficient patients (activity < 70%). Sixty-five percent of the genotyped PC-deficient patients were found to have heterozygous mutations. Among the < 70% values, decreases in PC activity without gene mutation were always slight (mean value 64 +/- 7%) while patients presenting a PC gene mutation had a mean 50 +/- 17% activity (P < 0.05). Among the eight PC mutations found, only one has previously been described. A novel mutation in the promoter region (-1522), located in the HNF-1 site and associated with the Y226H heterozygous mutation, was found in a 9-month-old girl with 4% PC activity. Determination of PS functional activity was considerably improved by contemporaneous measurement of calibration and samples in a single step. Only 50% of the genotyped PS-deficient patients demonstrated heterozygous alterations of the gene. The benefit of sequencing to identify putative causal mutations was only 39% in PS-deficient women, while it was 90% in men. Among the nine PS mutations found, six have not yet been published. In the present paper, we explain our methodological choices and diagnostic strategy.

Admission NT-proBNP levels, renal insufficiency and age as predictors of mortality in elderly patients hospitalized for acute dyspnea.

BACKGROUND: Assay of baseline B-type peptide (BNP and NT-proBNP) is useful for heart failure (HF) prognostication. In contrast, the prognostic value of NT-proBNP assay performed on admission of elderly subjects for acute dyspnea is uncertain. The aim of this study was to determine the vital prognostic value of NT-proBNP assay and other relevant variables available on admission in elderly patients hospitalized for acute dyspnea. METHODS: 254 patients over 70 years of age who were initially hospitalized with acute dyspnea were prospectively studied. The log-rank test and Cox proportional-hazards regression models were used to determine the prognostic value of NT-proBNP and creatinine clearance, measured within 24 h of initial admission, as well as age, gender, vascular risk factors and other clinical variables. RESULTS: Mean age was 81+/-7 years, and 52% of the patients were women. During a median follow-up of 34 months, 134 patients (55%) died and 9 patients (4%) were lost to follow-up. The median survival time was 25 months, and almost half the deaths occurred during the first 6 months. In multivariate analysis the following three variables were independently associated with mortality (shown with their accompanying hazard ratios (HR)): NT-proBNP>2856 pg/mL (median), HR=1.6[95%CI:1.3-5.2]; creatinine clearance <30 mL/min, HR=1.7[95%CI:1.2-2.5]; and age>80 years, HR=1.7[95%CI:1.1-2.6]. The median survival time among patients with an admission NT-proBNP level of >2856 pg/mL (median) was 14 months, compared to >36 months in the rest of the population. CONCLUSION: The admission NT-proBNP level, age, and creatinine clearance are predictive of vital outcome in elderly patients hospitalized for acute dyspnea.

Prehospital administration of enoxaparin before primary angioplasty for ST-elevation acute myocardial infarction.

We hypothesized that primary percutaneous coronary intervention (PCI) could be performed with prehospital injections of enoxaparin for ST segment elevation myocardial infarction (STEMI). Enoxaparin has been studied in combination with fibrinolysis in STEMI, but has not been evaluated as anticoagulant regimen for primary PCI. In a prospective registry, 143 consecutive patients with STEMI received prehospital 0.5 mg/kg intravenous (i.v.) bolus followed by 1 mg/kg subcutaneous enoxaparin before immediate transport for PCI. We focused on anti-Xa activities before and after PCI, bleedings, infarct-related artery patency, and major adverse cardiac events at day 30. Anti-Xa activity was at the target level (>0.5 IU/ml) in 99% of patients during PCI, and in 100% 4 hr after injections; over-anticoagulation (>1.5 IU/ml) was noted in 9 and 2%, respectively at start and 4 hr after injections. Bleeding complications with enoxaparin were rare: major in 1.4% (no intracranial hemorrhages), minor in 2.1%. A patent infarct-related artery (TIMI 2 + 3) was observed in 40.6% of the patients before PCI. TIMI 3 flow was obtained in 88.1% of the cases after PCI. Major adverse cardiac events at 30 days occurred in 5.6% of cases: death 2.8%, reinfarction 3.5%, and target lesion revascularisation 3.5%. Prehospital i.v. and subcutaneous enoxaparin provides simple and rapid anticoagulation for PCI in STEMI patients. Enoxaparin dose needs to be reduced regarding the 9% of over-anticoagulation. This study suggests the potential of enoxaparin as an alternative anticoagulant regimen for primary PCI.

Intrathecal bupivacaine protects against extension of lesions in an acute photochemical spinal cord injury model.

PURPOSE: The photochemical spinal-cord injury model reproduces extensive secondary lesions that occur after spinal injury. We have evaluated in 27 rats the functional, electrophysiological and anatomical consequences of a photochemical spinal-cord lesion induced before or after intrathecal injection of bupivacaine. METHODS: After randomization, nine rats received 20 micro L of intrathecal bupivacaine 0.5% 15 min before a photochemical spinal-cord lesion (Group I) and eight rats received 20 micro L intrathecal bupivacaine 15 min after such a lesion (Group II). Ten rats received 20 micro L of saline 15 min before the photochemical injury (control group). Paraplegia was tested on days one, three, five, seven, nine, 12, 15 and 18 using an evaluation of hindlimb movements and an inclined plane stability test. Sensory block was evaluated by the animal's response when each hindlimb was brought into contact with a hot plate. Sympathetic injury was evaluated in terms of bladder voiding dysfunction. On day 18, residual somatosensory evoked potentials (SEP) were measured and the area of the intact spinal cord was determined using a digitalized system. RESULTS: Early paraplegia recovery was found in the two bupivacaine groups (P < 0.05). On day 12, motor recovery was complete in both bupivacaine groups whereas recovery was not complete on day 18 in the control group. Compared to the control group, inclined plane stability recovered earlier in Groups I and II, from day three to day 15. Sensory and sympathetic block scores were not different in the three groups. Nevertheless, SEP latencies were longer and amplitudes were lower in control group rats compared with the two bupivacaine groups on day 18. The intact spinal-cord cross-sectional area around the lesion was not different in the three groups. CONCLUSION: Twenty microlitres of intrathecal bupivacaine before or after acute photochemical spinal injury improves hindlimb motor recovery and SEP parameters in rats.

The effects of spread of block and adrenaline on cardiac output after epidural anesthesia in young children: a randomized, double-blind, prospective study.

UNLABELLED: Epidural anesthesia is considered to be without significant hemodynamic consequence in young children. However, conversely to adults, few studies have investigated cardiac output. Using transesophageal Doppler monitoring of cardiac output, we prospectively investigated hemodynamic alterations in 48 children (median age, 22.5 mo) receiving sevoflurane general anesthesia combined with caudal or thoracolumbar epidural anesthesia. They were randomly assigned to receive 0.8 mL/kg of plain local anesthetic mixture (lidocaine 1% + bupivacaine 0.25% (50/50) + 1 microg/mL of fentanyl) or 1 mL/kg of the same mixture with 5 microg/mL of adrenaline. No significant hemodynamic alteration was elicited in caudal and thoracolumbar groups receiving the plain mixture except a moderate decrease in heart rate. Conversely, a mixture with adrenaline added provoked a significant decrease in mean arterial blood pressure by 14% and 17%, in systemic vascular resistance by 24% and 40%, and an increase in cardiac output by 20% and 34% in caudal and thoracolumbar groups, respectively. The adrenaline effect was greater by the thoracolumbar than the caudal approach. In young children, epidural anesthesia induces an increase in cardiac output only when adrenaline is added to local anesthetics, probably through its systemic absorption from the epidural space. IMPLICATIONS: Epidural anesthesia may induce significant hemodynamic changes, well documented in adults. Using noninvasive hemodynamic monitoring in children, we reported an increase in cardiac output and a decrease in arterial blood pressure only when epinephrine was added to epidurally-injected local anesthetics.