Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations

A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP. It will continue to be important to use complementary methods for mutational analysis.     

Prevalence and genetic diversity of Staphylococcus aureus small-colony variants in cystic fibrosis patients

Staphylococcus aureus small-colony variants (SCVs) are being isolated more frequently in cystic fibrosis (CF) patients. We aimed to determine the prevalence of S. aureus SCVs and their phenotypic and genotypic properties in CF patients admitted to a university hospital. Specimens of 248 patients were examined during a period of 11 months. Colonies supposed to be SCVs were evaluated on Columbia blood agar, mannitol salt agar, and brain–heart infusion agar with 5% NaCl (BHIA 5% NaCl). Strains were confirmed by S. aureus nucA PCR. Antibiotic susceptibilities of SCVs and simultaneously isolated S. aureus strains were determined for oxacillin, gentamicin, trimethoprim–sulphamethoxazole, vancomycin, ciprofloxacin, linezolid, and tigecycline. Genetic relatedness between SCVs and normal S. aureus strains was determined with a pulsed-field gel electrophoresis (PFGE) method. S. aureus SCVs were detected in 20 of 248 patients (8.1%). The highest SCV isolation rate was obtained with MSA, followed by BHIA 5% NaCl. Auxotrophism for thymidine was demonstrated in six SCVs. The tigecycline susceptibilities of 48 SCV strains isolated in this study showed higher MIC values than those of 33 simultaneously isolated normal S. aureus strains. Whereas SCVs and normal S. aureus strains showed identical genotypes in 14 of the patients, five patients showed different genotypes. This first study from Turkey evaluating S. aureus SCVs in CF patients has indicated the importance of considering and reporting SCVs in chronic infections such as CF. The presence of SCVs will probably indicate persistent infection, and this might impact on antibiotic treatment decisions, as they are more resistant to antibiotics.     

Acute changes of coagulation and fibrinolysis parameters after experimental thromboembolic stroke and thrombolytic therapy

Thrombolysis is the only effective pharmaceutical therapy in acute ischemic stroke in humans but has a high risk of intracerebral hemorrhage. We aimed to establish an animal model to study changes of coagulation and fibrinolytic parameters during thromboembolic ischemic stroke and thrombolysis with recombinant tissue plasminogen activator (rt-PA). We used a thromboembolic stroke model in the rat. Animals were treated with rt-PA thrombolysis (n=10) and compared with untreated (n=10), sham operated (n=10) and control animals (n=20). Coagulation parameters (APTT, PT, TT, fibrinogen, AT III, TAT) and fibrinolytic parameters (t-PA antigen concentration, t-PA activity, PAI-1 concentration, PAI activity, plasminogen, antiplasmin) were measured at two time points (2.5 and 5h after stroke induction) with a battery of commercially available test kits. We observed an (1) initiation of coagulation and inhibition of fibrinolysis by the operation procedure itself, (2) simultaneous activation of fibrinolysis and its inhibitors after stroke induction and (3) potent initiation of fibrinolysis and consumption of fibrinolysis inhibitors after rt-PA therapy of stroke. We established a model system to monitor coagulation and fibrinolysis during thrombolytic therapy of stroke in the rat. This model may be used to study the influence of these parameters on hemorrhagic stroke transformation and outcome in experimental stroke in future.     

Phosphorylation states of cell cycle and DNA repair proteins can be altered by the nsSNPs

Background

Sentinel lymph node (SLN) biopsy is an effective tool for axillary staging in patients with invasive breast cancer. This procedure has been recently proposed as part of the treatment for patients with ductal carcinoma in situ (DCIS), because cases of undetected invasive foci and nodal metastases occasionally occur. However, the indications for SLN biopsy in DCIS patients are controversial. The aim of the present study was therefore to assess the incidence of SLN metastases in a series of patients with a diagnosis of pure DCIS.

Methods

A retrospective evaluation was made of a series of 102 patients who underwent SLN biopsy, and had a final histologic diagnosis of pure DCIS. Patients with microinvasion were excluded from the analysis. The patients were operated on in five Institutions between 1999 and 2004. Subdermal or subareolar injection of 30–50 MBq of 99 m-Tc colloidal albumin was used for SLN identification. All sentinel nodes were evaluated with serial sectioning, haematoxylin and eosin staining, and immunohistochemical analysis for cytocheratin.

Results

Only one patient (0.98%) was SLN positive. The primary tumour was a small micropapillary intermediate-grade DCIS and the SLN harboured a micrometastasis. At pathologic revision of the specimen, no detectable focus of microinvasion was found.

Conclusion

Our findings indicate that SLN metastases in pure DCIS are a very rare occurrence. SLN biopsy should not therefore be routinely performed in patients who undergo resection for DCIS. SLN mapping can be performed, as a second operation, in cases in which an invasive component is identified in the specimen. Only DCIS patients who require a mastectomy should have SLN biopsy performed at the time of breast operation, since in these cases subsequent node mapping is not feasible.     

Angiolipoma of the neck: a case report

Angiolipomas of the neck are extremely rare; to our knowledge, only 2 cases have been previously reported. We report a new case, which occurred in a 28-year-old woman. The mass was totally resected, and the patient showed no recurrence at the 18-month follow-up.     

Maternal erythrocyte malondialdehyde level in preeclampsia prediction: a longitudinal study

We aimed to determine the value of maternal erythrocyte malondialdehyde levels in the prediction of preeclampsia. 110 healthy women were included in this prospective study. Maternal erythrocyte malondialdehyde levels were measured at each trimester of pregnancy (10-14, 20-25 and 30-35 gestational weeks). On follow-up, patients were assigned to two groups as normotensive women and preeclamptic patients. Preeclampsia had developed in eight (8.9%) of the 90 pregnant women who completed the study. Preeclamptic patients were diagnosed between 36 and 39 gestational weeks (36.8 +/- 1.0 weeks). Malondialdehyde levels of preeclamptic patients increased significantly in the third trimester (p < 0.05), while there was no difference between values of malondialdehyde in the first and second trimester. Malondialdehyde levels were significantly higher in the patients who developed preeclampsia than in those who did not in the third trimester (p < 0.05). With the use of the receiver operating characteristics (ROC) 35.98 nmol malondialdehyde/gm hemoglobin was found to be a cut-off value predictive for the development of preeclampsia in the third trimester. However, cut-off values in the first and second trimesters could not be found. The sensitivity, specificity, positive and negative predictive values were 89, 75, 29 and 98%, respectively. Preeclampsia risk was found to increase nearly 24 times in values above 35.98 nmol malondialdehyde/ gm hemoglobin. Our results showed that maternal erythrocyte malondialdehyde could predict patients within a few weeks prior to onset of clinical symptoms of preeclampsia in the third trimester. There is no evidence of enhanced early lipid peroxidation in pregnancies with late onset preeclampsia.     

Homocysteine-induced enhancement of spontaneous contractions of myometrium isolated from pregnant women

BACKGROUND: Although many associations have been demonstrated between hyperhomocysteinemia and pregnancy complications, such as spontaneous abortion, preterm labor, preeclampsia and low birthweight, it is still not clear whether hyperhomocysteinemia is the cause or the consequence of these pregnancy complications. The aim of this study was to investigate the effects of homocysteine on the spontaneous contractility of isolated pregnant human myometrium. METHODS: Myometrium samples obtained from women undergoing elective caesarean section were suspended in a jacketed organ bath containing Krebs' solution at 37 degrees C (pH 7.4), continuously gassed with 95% O2 and 5% CO2. After manifestation of spontaneous contractions under 2 g of resting tension, homocysteine was applied to the organ bath, and amplitude and frequency of contractions were evaluated at 20-min intervals. Statistical analysis of amplitude and frequency of the contractions was performed using the Kruskal-Wallis analysis of variance test. RESULTS: Application of 0.1 mm homocysteine had no significant effect on either frequency (4.63 +/- 0.42 vs. 4.01 +/- 0.53 for the control period; p > 0.05) or amplitude (3.20 +/- 0.07 g vs. 3.15 +/- 0.07 g; p > 0.05) of spontaneous contractions, while 1 and 2 mm homocysteine significantly increased the frequency (8.54 +/- 0.62 vs. 3.9 +/- 0.44, p < 0.02, and 12.32 +/- 0.72 vs. 3.96 +/- 0.51, p < 0.001, respectively) of spontaneous contractions but caused only a modest change in amplitude (2.92 +/- 0.04 g vs. 2.87 +/- 0.05 g, p > 0.05, and 3.02 +/- 0.06 g vs. 2.93 +/- 0.05 g, p > 0.05, respectively). CONCLUSION: Results from this study indicate for the first time that homocysteine causes enhancement of spontaneous contractions of myometrium derived from pregnant women.     

Asthma in children: Management practices among paediatricians and family physicians

OBJECTIVE:

To ascertain the variation in asthma management practices among paediatricians and family physicians to determine how to improve care.

DESIGN:

Questionnaire study of paediatricians and family physicians that focused on the use of beta₂-agonists, inhaled corticosteroids, patient asthma education, quantitative measurements of airflow and diagnostic investigations for asthma. Case scenarios were used in the questionnaire.

RESULTS:

The response rate was 66% (415 of 632) among paediatricians and 42% (1156 of 2750) among family physicians. In general, both groups followed consensus guidelines. There were some differences in management practices among paediatricians and family physicians. Paediatricians were more likely to develop an action plan and less likely to use xanthines or inhaled anticholinergic agents. However, family physicians were more likely to use spirometry or home peak expiratory flow rates to make a diagnosis of asthma.

CONCLUSION:

Family physicians and paediatricians require a different focus on educational interventions to improve the care of children with asthma.     

Pulmonary cystic hydatid disease in Ireland

Background Hydatid disease is rare in Ireland and its incidence and prevalence are unknown. Most cases are diagnosed by a combination of clinical findings, morphological features on imaging and by serological testing. Aims We describe an Irish case of pulmonary hydatid disease detected at bronchoscopy by bronchoalveolar lavage, and discuss the diagnosis and treatment of the disorder.