Evaluation of Phi29-based whole-genome amplification for microarray-based comparative genomic hybridisation

For the optimal performance of high throughput genomic technologies sufficient yields of high-quality DNA are crucial. Following microdissection, most samples fail to produce sufficient quantities of DNA for genome-wide experiments. Various PCR-based amplification methods have been used, but these usually produce nonuniform representations of the genome. Bacteriophage Phi29 DNA polymerase random-primed DNA amplification is based on isothermal multiple displacement amplification. We sought to define the genome representation of this method in a bacterial artificial chromosome microarray comparative genomic hybridisation (aCGH) platform. Test genomic female DNA was amplified using Phi29 amplification at four different starting concentrations (0.5, 5, 10 and 50 ng). These products were combined with unamplified and amplified genomic female DNA as reference. In addition, 50 ng of DNA from five microdissected breast cancer frozen samples, were amplified using the same method. Three combinations were performed: unamplified test with unamplified reference, amplified test with unamplified reference and both amplified tumour and reference DNA. aCGH was performed with an in-house 16 K BAC platform (a resolution of approximately 100 Kb). Pearson's correlation tests and hierarchical clustering were performed to compare the profiles obtained. aCGH profiles obtained with amplified test and unamplified reference female genomic DNA showed copy number biases throughout the genome. These biases were more conspicuous with smaller amounts of starting material and mapped to regions of known copy number polymorphisms. When similar concentrations of test and reference DNA were amplified, the biases were significantly reduced, rendering accurate profiles. For the tumours, representative profiles were obtained when both test and reference DNA were amplified. Phi29 amplification induces copy number biases and unamplified material remains the gold standard for copy number analysis. For accurate results using Phi29 amplification, samples subjected to aCGH analysis should be combined with reference DNA amplified with the same method, using similar amounts of starting template.     

Gender differences in negative mood states in secondary school students: health survey in Catalonia (Spain)

Objective

To determine the prevalence of negative mood states in adolescents according to gender, to analyze variability among schools, and to evaluate the associated factors.

Methods

A cross-sectional study with a cluster design was carried out. We administered the High-school students health survey to a sample of 9,340 students (aged 14-16 years) in the third and fourth year of Compulsory Secondary Education in Catalonia, Spain, during the 2005-6 academic year. The main outcome measure was evidence of a negative mood state. A multilevel logistic regression model stratified by gender was used to identify the factors associated with negative mood states and to determine variability among distinct schools.

Results

Approximately 19% of adolescents reported evidence of a negative mood state, with a higher prevalence in girls (25%). The most significant factors associated with negative mood states were “use of tranquilizers” and “having eating disorders” in girls and “not exercising” and “poor self-perception of health status” in boys. In both genders, variability was found among schools in the prevalence of negative mood states (girls: variance = 0.078; p <0.001; boys: variance = 0.079; p = 0.012).

Conclusions

The prevalence of negative mood states in adolescent boys and girls was high. Differences were observed between genders in the factors related to these health states. The variability observed in the prevalence of negative mood states among distinct schools could not be explained by the study variables. Our results emphasize the association between the use of tranquilizers and negative mood states.     

Effectiveness and Safety of Air-Filled Balloon Heliosphere BAG® in 82 Consecutive Obese Patients

Background

Intragastric balloon is a widely used technique to treat obesity that is considered to be more efficient than conservative treatment before bariatric surgery. To describe air-filled balloon (Heliosphere BAG®) effectiveness [absolute weight loss, body mass index (BMI) loss, percentage of body weight loss (BWL), percentage of excess weight loss (EWL)] and complications 6 months after its insertion.

Methods

Eighty-four consecutive intragastric balloons were placed endoscopically. Individualized nutritional counseling was given. The follow-up was carried out in an endocrinology outpatient clinic. Due to the weight or height data missed in two cases, only 82 patients were included in this report, 63 women with a mean age 39 years (SD, 11.1); mean BMI, 39.1 kg/m² (SD, 5.8). The median follow-up was 182 days.

Results

The mean weight loss and BMI loss were 14.5 kg (SD, 8.2); and 5.3 kg/m² (SD, 2.8), respectively (for difference, p?10%. The percentage of EWL reached 33.2% (SD, 19.2). After adjusting by sex and initial BMI, absolute weight loss (p?=?0.033), BMI loss (p?=?0.034), percentage of BWL (p?=?0.034), and percentage of EWL (p?=?0.034) were inversely related to age. Absolute weight loss and BMI loss were greater in higher initial BMI, but the percentage of EWL was lower. Two spontaneous deflations occurred (3%), but only one surgical early removal (1.2%) was required. Nausea and vomiting developed in 7.4% of the patients during the first week.

Conclusions

Air-filled Heliosphere BAG® has been effective in achieving a relevant loss of body weight.

     

Consultation on interactions between National Regulatory Authorities and National Immunization Technical Advisory Groups

A consultation during the Developing Country Vaccine Regulators' Network meeting of May 2010 considered the interactions between the National Immunization Technical Advisory Group (NITAG) and the National Regulatory Authority (NRA) in various countries. This meeting was co-hosted by the WHO and the Supporting Independent Immunization and Vaccine Advisory Committees Initiative implemented by the Agence de Médecine Préventive in partnership with the International Vaccine Institute. Representatives from Developing Country Vaccine Regulators' Network and representatives from several additional countries' regulatory authorities met representatives from NITAGs and/or the National Immunization Program from these countries (Brazil, Canada, China, Cuba, France, Indonesia, Iran, South Africa, Thailand, Vietnam and the USA). The objectives of the workshop included a discussion on the issues of NRA-NITAG interaction, the assessment of the advantages of different models of interaction and proposals for an optimal coordination process for market authorization and recommendations for use of vaccines. It was concluded that there is need for increased and more formal interactions between NRAs and NITAGs, a clear framework establishing a formal interaction and early interactions before market authorization. NRA experts being at the same time NITAG ex officio members and vice versa are solutions which can be adopted by countries. The NRA issues the license based on the evidence submitted by the manufacturer. The NITAG makes recommendations based on scientific evidence, public health needs and policy, and consideration of the license conditions. If there is a need to make recommendations that are not covered by the license evidence then there should be interactions between NITAG, NRA and the license holder to encourage the license-holder to submit appropriate evidence, or to ensure that the justification for the off-label recommendation is communicated to the users of the medicine.     

Genomic analysis of the HER2/TOP2A amplicon in breast cancer and breast cancer cell lines

HER2 and TOP2A are targets for the therapeutic agents trastuzumab and anthracyclines and are frequently amplified in breast cancers. The aims of this study were to provide a detailed molecular genetic analysis of the 17q12-q21 amplicon in breast cancers harbouring HER2/TOP2A co-amplification and to investigate additional recurrent co-amplifications in HER2/TOP2A-co-amplified cancers. In total, 15 breast cancers with HER2 amplification, 10 of which also harboured TOP2A amplification, as defined by chromogenic in situ hybridisation, and 6 breast cancer cell lines known to be amplified for HER2 were subjected to high-resolution microarray-based comparative genomic hybridisation analysis. This revealed that the genomes of 12 cases were characterised by at least one localised region of clustered, relatively narrow peaks of amplification, with each cluster confined to a single chromosome arm (ie 'firestorm' pattern) and 3 cases displayed many narrow segments of duplication and deletion affecting the vast majority of chromosomes (ie 'sawtooth' pattern). The smallest region of amplification (SRA) on 17q12 in the whole series extended from 34.73 to 35.48 Mb, and encompassed HER2 but not TOP2A. In HER2/TOP2A-co-amplified samples, the SRA extended from 34.73 to 36.54 Mb, spanning a region of approximately 1.8 Mb. Apart from HER2 and TOP2A, this region encompassed four additional genes whose expression levels as defined by quantitative real-time PCR are significantly higher in HER2/TOP2A-co-amplified vs HER2-amplified breast cancers: CASC3, CDC6, RARA and SMARCE1. Of the cell lines studied, SKBR3 and UACC812 showed HER2/TOP2A co-amplification. In conclusion, this is the first detailed genome-wide characterisation of HER2/TOP2A-amplified breast cancers; cell lines were identified that can be used to model these cancers in vitro. The 17q12 amplicon is complex and harbours multiple genes that may be associated with breast cancer development and progression, and potentially exploitable as therapeutic targets.