Characterization of transposon insertion mutants in desulfovibrio vulgaris hilderborough by sequencing genomic DNA [abstract]

Abstract only availableTn5 transposon mutagenesis occurs by a mechanism in which a segment of DNA (transposon) encoded in a plasmid is inserted into genomic DNA (the target) by a conservative (cut-and-paste) mechanism. When the insertion position is in a coding sequence or regulatory region of DNA, the insertion results in a mutation. The plasmid pRL27 encodes a mini-Tn5 transposon, Tn5 transposase, and kanamycin resistance, (Metcalf, William W. et al, 2002 Arch Microbiol 178 :193-201) and was used to transform Desulfovibrio vulgaris Hildenborough by electroporation. Transposon insertion mutants were identified by their ability to grow in the presence of kanamycin. To identify the insertion sites of the transposons, in theory one should be able to sequence from the transposon into chromosomal DNA and identify the mutation site by comparison with the known genome. Unlike sequencing of plasmid DNA or PCR products, direct genomic sequencing has a limited success rate. Direct genomic sequencing is sensitive to DNA quality, interference of secondary DNA structures, salt concentration, and the availability of primer binding sites. Because of these difficulties, in our attempts to identify insertion sites of mini-Tn5, we examined template DNA quality as well as modifying sequencing reaction conditions. Our objective is to develop an effective, reliable method for sequencing genomic DNA to identify where transposon insertion sites have occurred in each mutant.Department of Energy Genomics: Genomes to Life Progra

Rapid automated characterization of transposon insertion mutants in Desulfovibrio vulgaris Hildenborough by srnPCR [abstract]

Abstract only availableTn5 transposon mutagenesis occurs by a mechanism in which a segment of DNA (transposon) encoded in a plasmid is inserted into genomic DNA (the target) by a conservative (cut-and-paste) mechanism (Fig. 2). When the insertion position is in a coding sequence or regulatory region of DNA, the insertion results in a mutation. The plasmid pRL27 (a generous gift from Bill Metcalf) encodes a mini-Tn5 transposon, Tn5 transposase, and kanamycin resistance (neo), and was used to transform Desulfovibrio vulgaris Hildenborough by electroporation. Transposon insertion mutants were identified by their ability to grow in the presence of kanamycin. To locate the insertion site of the transposon, in theory, one should be able to directly sequence from the transposon into chromosomal DNA (Fig. 3.1) and identify the mutation site by comparison with the known genome BLAST. Unlike sequencing of plasmid DNA or PCR products, direct genomic sequencing has a limited success rate. Therefore, a method of enriching the transposon-flanking sequence is needed. Nested semi-random PCR (Fig. 3.2) is an efficient and cost effective enrichment method. Sequencing these enriched products allows us to identify the transposon insertion site. The factors that influence characterization success rate are: frequency and location of priming sites, reaction volume, and reaction conditions (annealing temperature, extension time, etc.). By varying these factors, we have developed an efficient and reliable method for characterizing transposon insertion mutants. Utilizing high-throughput robotics and nested semi-random PCR, we have generated single gene mutants that may provide valuable biological data.U.S. Department of Energy Genomes to Life gran

BRCA2 polymorphic stop codon K3326X and the risk of breast, prostate, and ovarian cancers

Background: The K3326X variant in BRCA2 (BRCA2*c.9976A>T; p.Lys3326*; rs11571833) has been found to be associated with small increased risks of breast cancer. However, it is not clear to what extent linkage disequilibrium with fully pathogenic mutations might account for this association. There is scant information about the effect of K3326X in other hormone-related cancers. Methods: Using weighted logistic regression, we analyzed data from the large iCOGS study including 76 637 cancer case patients and 83 796 control patients to estimate odds ratios (ORw) and 95% confidence intervals (CIs) for K3326X variant carriers in relation to breast, ovarian, and prostate cancer risks, with weights defined as probability of not having a pathogenic BRCA2 variant. Using Cox proportional hazards modeling, we also examined the associations of K3326X with breast and ovarian cancer risks among 7183 BRCA1 variant carriers. All statistical tests were two-sided. Results: The K3326X variant was associated with breast (ORw = 1.28, 95% CI = 1.17 to 1.40, P = 5.9x10- 6) and invasive ovarian cancer (ORw = 1.26, 95% CI = 1.10 to 1.43, P = 3.8x10-3). These associations were stronger for serous ovarian cancer and for estrogen receptor–negative breast cancer (ORw = 1.46, 95% CI = 1.2 to 1.70, P = 3.4x10-5 and ORw = 1.50, 95% CI = 1.28 to 1.76, P = 4.1x10-5, respectively). For BRCA1 mutation carriers, there was a statistically significant inverse association of the K3326X variant with risk of ovarian cancer (HR = 0.43, 95% CI = 0.22 to 0.84, P = .013) but no association with breast cancer. No association with prostate cancer was observed. Conclusions: Our study provides evidence that the K3326X variant is associated with risk of developing breast and ovarian cancers independent of other pathogenic variants in BRCA2. Further studies are needed to determine the biological mechanism of action responsible for these associations

Analysis of cancer risk and BRCA1 and BRCA2 mutation prevalence in the kConFab familial breast cancer resource

INTRODUCTION: The Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) is a multidisciplinary, collaborative framework for the investigation of familial breast cancer. Based in Australia, the primary aim of kConFab is to facilitate high-quality research by amassing a large and comprehensive resource of epidemiological and clinical data with biospecimens from individuals at high risk of breast and/or ovarian cancer, and from their close relatives. METHODS: Epidemiological, family history and lifestyle data, as well as biospecimens, are collected from multiple-case breast cancer families ascertained through family cancer clinics in Australia and New Zealand. We used the Tyrer-Cuzick algorithms to assess the prospective risk of breast cancer in women in the kConFab cohort who were unaffected with breast cancer at the time of enrolment in the study. RESULTS: Of kConFab's first 822 families, 518 families had multiple cases of female breast cancer alone, 239 had cases of female breast and ovarian cancer, 37 had cases of female and male breast cancer, and 14 had both ovarian cancer as well as male and female breast cancer. Data are currently held for 11,422 people and germline DNAs for 7,389. Among the 812 families with at least one germline sample collected, the mean number of germline DNA samples collected per family is nine. Of the 747 families that have undergone some form of mutation screening, 229 (31%) carry a pathogenic or splice-site mutation in BRCA1 or BRCA2. Germline DNAs and data are stored from 773 proven carriers of BRCA1 or BRCA1 mutations. kConFab's fresh tissue bank includes 253 specimens of breast or ovarian tissue – both normal and malignant – including 126 from carriers of BRCA1 or BRCA2 mutations. CONCLUSION: These kConFab resources are available to researchers anywhere in the world, who may apply to kConFab for biospecimens and data for use in ethically approved, peer-reviewed projects. A high calculated risk from the Tyrer-Cuzick algorithms correlated closely with the subsequent occurrence of breast cancer in BRCA1 and BRCA2 mutation positive families, but this was less evident in families in which no pathogenic BRCA1 or BRCA2 mutation has been detected

Protocol for a randomised controlled trial of treatment of asymptomatic candidiasis for the prevention of preterm birth [ACTRN12610000607077]

<p>Abstract</p> <p>Background</p> <p>Prevention of preterm birth remains one of the most important challenges in maternity care. We propose a randomised trial with: a simple <it>Candida </it>testing protocol that can be easily incorporated into usual antenatal care; a simple, well accepted, treatment intervention; and assessment of outcomes from validated, routinely-collected, computerised databases.</p> <p>Methods/Design</p> <p>Using a prospective, randomised, open-label, blinded-endpoint (PROBE) study design, we aim to evaluate whether treating women with asymptomatic vaginal candidiasis early in pregnancy is effective in preventing spontaneous preterm birth. Pregnant women presenting for antenatal care <20 weeks gestation with singleton pregnancies are eligible for inclusion. The intervention is a 6-day course of clotrimazole vaginal pessaries (100 mg) and the primary outcome is spontaneous preterm birth <37 weeks gestation.</p> <p>The study protocol draws on the usual antenatal care schedule, has been pilot-tested and the intervention involves only a minor modification of current practice. Women who agree to participate will self-collect a vaginal swab and those who are culture positive for Candida will be randomised (central, telephone) to open-label treatment or usual care (screening result is not revealed, no treatment, routine antenatal care). Outcomes will be obtained from population databases.</p> <p>A sample size of 3,208 women with <it>Candida </it>colonisation (1,604 per arm) is required to detect a 40% reduction in the spontaneous preterm birth rate among women with asymptomatic candidiasis from 5.0% in the control group to 3.0% in women treated with clotrimazole (significance 0.05, power 0.8). Analyses will be by intention to treat.</p> <p>Discussion</p> <p>For our hypothesis, a placebo-controlled trial had major disadvantages: a placebo arm would not represent current clinical practice; knowledge of vaginal colonisation with <it>Candida </it>may change participants' behaviour; and a placebo with an alcohol preservative may have an independent affect on vaginal flora. These disadvantages can be overcome by the PROBE study design.</p> <p>This trial will provide definitive evidence on whether screening for and treating asymptomatic candidiasis in pregnancy significantly reduces the rate of spontaneous preterm birth. If it can be demonstrated that treating asymptomatic candidiasis reduces preterm births this will change current practice and would directly impact the management of every pregnant woman.</p> <p>Trial registration</p> <p>Australian New Zealand Clinical Trials Registry <a href="http://www.anzctr.org.au/ACTRN12610000607077.aspx">ACTRN12610000607077</a></p

Narrative Personae and Visual Signs: Reading Leonard’s intimate photo-memoir. a/b: Auto/Biography Studies.

In this paper, I look at Joanne Leonard’s Being in Pictures and engage in a critical dialogue with an assemblage of visual and textual narratives that comprise her intimate photo memoir. In doing this I draw on Hannah Arendt’s take on narratives as tangible traces of uniqueness and plurality, political traits par excellence in the cultural histories of the human condition. Being aware of my role as a reader/viewer/interpreter of a woman artist’s auto/biographical narratives, I move beyond dilemmas of representation or questions of unveiling “the real Leonard”. The artist is instead configured as a narrative persona, whose narratives respond to three interrelated themes of inquiry, namely the visualization of spatial technologies, vulnerability and the gendering of memory. Key words: gendered memories, narrative persona, spatial technologies, photo memoir, vulnerabilit

Homologous recombination DNA repair defects in PALB2- associated breast cancers

Abstract: Mono-allelic germline pathogenic variants in the Partner And Localizer of BRCA2 (PALB2) gene predispose to a high-risk of breast cancer development, consistent with the role of PALB2 in homologous recombination (HR) DNA repair. Here, we sought to define the repertoire of somatic genetic alterations in PALB2-associated breast cancers (BCs), and whether PALB2-associated BCs display bi-allelic inactivation of PALB2 and/or genomic features of HR-deficiency (HRD). Twenty-four breast cancer patients with pathogenic PALB2 germline mutations were analyzed by whole-exome sequencing (WES, n = 16) or targeted capture massively parallel sequencing (410 cancer genes, n = 8). Somatic genetic alterations, loss of heterozygosity (LOH) of the PALB2 wild-type allele, large-scale state transitions (LSTs) and mutational signatures were defined. PALB2-associated BCs were found to be heterogeneous at the genetic level, with PIK3CA (29%), PALB2 (21%), TP53 (21%), and NOTCH3 (17%) being the genes most frequently affected by somatic mutations. Bi-allelic PALB2 inactivation was found in 16 of the 24 cases (67%), either through LOH (n = 11) or second somatic mutations (n = 5) of the wild-type allele. High LST scores were found in all 12 PALB2-associated BCs with bi-allelic PALB2 inactivation sequenced by WES, of which eight displayed the HRD-related mutational signature 3. In addition, bi-allelic inactivation of PALB2 was significantly associated with high LST scores. Our findings suggest that the identification of bi-allelic PALB2 inactivation in PALB2-associated BCs is required for the personalization of HR-directed therapies, such as platinum salts and/or PARP inhibitors, as the vast majority of PALB2-associated BCs without PALB2 bi-allelic inactivation lack genomic features of HRD

Development and validation of a targeted gene sequencing panel for application to disparate cancers

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour’s molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy

Rare germline copy number variants (CNVs) and breast cancer risk.

Funder: CIHRGermline copy number variants (CNVs) are pervasive in the human genome but potential disease associations with rare CNVs have not been comprehensively assessed in large datasets. We analysed rare CNVs in genes and non-coding regions for 86,788 breast cancer cases and 76,122 controls of European ancestry with genome-wide array data. Gene burden tests detected the strongest association for deletions in BRCA1 (P = 3.7E-18). Nine other genes were associated with a p-value < 0.01 including known susceptibility genes CHEK2 (P = 0.0008), ATM (P = 0.002) and BRCA2 (P = 0.008). Outside the known genes we detected associations with p-values < 0.001 for either overall or subtype-specific breast cancer at nine deletion regions and four duplication regions. Three of the deletion regions were in established common susceptibility loci. To the best of our knowledge, this is the first genome-wide analysis of rare CNVs in a large breast cancer case-control dataset. We detected associations with exonic deletions in established breast cancer susceptibility genes. We also detected suggestive associations with non-coding CNVs in known and novel loci with large effects sizes. Larger sample sizes will be required to reach robust levels of statistical significance