A Genome Wide Meta-Analysis Study for Identification of Common Variation Associated with Breast Cancer Prognosis

Objective

Genome wide association studies (GWAs) of breast cancer mortality have identified few potential associations. The concordance between these studies is unclear. In this study, we used a meta-analysis of two prognostic GWAs and a replication cohort to identify the strongest associations and to evaluate the loci suggested in previous studies. We attempt to identify those SNPs which could impact overall survival irrespective of the age of onset.

Methods

To facilitate the meta-analysis and to refine the association signals, SNPs were imputed using data from the 1000 genomes project. Cox-proportional hazard models were used to estimate hazard ratios (HR) in 536 patients from the POSH cohort (Prospective study of Outcomes in Sporadic versus Hereditary breast cancer) and 805 patients from the HEBCS cohort (Helsinki Breast Cancer Study). These hazard ratios were combined using a Mantel-Haenszel fixed effects meta-analysis and a p-value threshold of 5×10⁻⁸ was used to determine significance. Replication was performed in 1523 additional patients from the POSH study.

Results

Although no SNPs achieved genome wide significance, three SNPs have significant association in the replication cohort and combined p-values less than 5.6×10⁻⁶. These SNPs are; rs421379 which is 556 kb upstream of ARRDC3 (HR = 1.49, 95% confidence interval (CI) = 1.27–1.75, P = 1.1×10⁻⁶), rs12358475 which is between ECHDC3 and PROSER2 (HR = 0.75, CI = 0.67–0.85, P = 1.8×10⁻⁶), and rs1728400 which is between LINC00917 and FOXF1.

Conclusions

In a genome wide meta-analysis of two independent cohorts from UK and Finland, we identified potential associations at three distinct loci. Phenotypic heterogeneity and relatively small sample sizes may explain the lack of genome wide significant findings. However, the replication at three SNPs in the validation cohort shows promise for future studies in larger cohorts. We did not find strong evidence for concordance between the few associations highlighted by previous GWAs of breast cancer survival and this study.     

Internal Sense of Direction: Sensing and Signaling from Cytoplasmic Chemoreceptors

SUMMARY

Chemoreceptors sense environmental signals and drive chemotactic responses in Bacteria and Archaea. There are two main classes of chemoreceptors: integral inner membrane and soluble cytoplasmic proteins. The latter were identified more recently than integral membrane chemoreceptors and have been studied much less thoroughly. These cytoplasmic chemoreceptors are the subject of this review. Our analysis determined that 14% of bacterial and 43% of archaeal chemoreceptors are cytoplasmic, based on currently sequenced genomes. Cytoplasmic chemoreceptors appear to share the same key structural features as integral membrane chemoreceptors, including the formations of homodimers, trimers of dimers, and 12-nm hexagonal arrays within the cell. Cytoplasmic chemoreceptors exhibit varied subcellular locations, with some localizing to the poles and others appearing both cytoplasmic and polar. Some cytoplasmic chemoreceptors adopt more exotic locations, including the formations of exclusively internal clusters or moving dynamic clusters that coalesce at points of contact with other cells. Cytoplasmic chemoreceptors presumably sense signals within the cytoplasm and bear diverse signal input domains that are mostly N terminal to the domain that defines chemoreceptors, the so-called MA domain. Similar to the case for transmembrane receptors, our analysis suggests that the most common signal input domain is the PAS (Per-Arnt-Sim) domain, but a variety of other N-terminal domains exist. It is also common, however, for cytoplasmic chemoreceptors to have C-terminal domains that may function for signal input. The most common of these is the recently identified chemoreceptor zinc binding (CZB) domain, found in 8% of all cytoplasmic chemoreceptors. The widespread nature and diverse signal input domains suggest that these chemoreceptors can monitor a variety of cytoplasmically based signals, most of which remain to be determined.     

Differential Regulation of Cellular Senescence and Differentiation by Prolyl Isomerase Pin1 in Cardiac Progenitor Cells

Autologous c-kit⁺ cardiac progenitor cells (CPCs) are currently used in the clinic to treat heart disease. CPC-based regeneration may be further augmented by better understanding molecular mechanisms of endogenous cardiac repair and enhancement of pro-survival signaling pathways that antagonize senescence while also increasing differentiation. The prolyl isomerase Pin1 regulates multiple signaling cascades by modulating protein folding and thereby activity and stability of phosphoproteins. In this study, we examine the heretofore unexplored role of Pin1 in CPCs. Pin1 is expressed in CPCs in vitro and in vivo and is associated with increased proliferation. Pin1 is required for cell cycle progression and loss of Pin1 causes cell cycle arrest in the G₁ phase in CPCs, concomitantly associated with decreased expression of Cyclins D and B and increased expression of cell cycle inhibitors p53 and retinoblastoma (Rb). Pin1 deletion increases cellular senescence but not differentiation or cell death of CPCs. Pin1 is required for endogenous CPC response as Pin1 knock-out mice have a reduced number of proliferating CPCs after ischemic challenge. Pin1 overexpression also impairs proliferation and causes G₂/M phase cell cycle arrest with concurrent down-regulation of Cyclin B, p53, and Rb. Additionally, Pin1 overexpression inhibits replicative senescence, increases differentiation, and inhibits cell death of CPCs, indicating that cell cycle arrest caused by Pin1 overexpression is a consequence of differentiation and not senescence or cell death. In conclusion, Pin1 has pleiotropic roles in CPCs and may be a molecular target to promote survival, enhance repair, improve differentiation, and antagonize senescence.     

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the ''larva chip''. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3^rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.     

Agrobacterium tumefaciens– mediated transformation of soybean [Glycine max (L.) Merrill.] using immature zygotic cotyledon explants

Agrobacterium tumefaciens -mediated transformation of soybean [Glycine max (L.) Merrill. cv. Jack] using immature zygotic cotyledons was investigated to identify important factors that affected transformation efficiency and resulted in the production of transgenic soybean somatic embryos. The factors evaluated were initial immature zygotic cotyledon size, Agrobacterium concentration during inoculation and co-culture and the selection regime. Our results showed that 8- to 10-mm zygotic cotyledons exhibited a higher transformation rate, as indicated by transient GUS gene expression, whereas the smaller zygotic cotyledons, at less than 5 mm, died shortly after co-cultivation. However, the smaller zygotic cotyledon explants were found to have a higher embryogenic potential. Analysis of Agrobacterium and immature cotyledon explant interactions involved two Agrobacterium concentrations for the inoculation phase and three co-culture regimes. No differences in explant survival or somatic embyogenic potential were observed between the two Agrobacterium concentrations tested. Analysis of co-culture regimes revealed that the shorter co-culture times resulted in higher explant survival and higher somatic embryo production on the explants, whereas the co-culture time of 4 days severely reduced survival of the cotyledon explants and lowered their embryogenic potential. Analysis of selection regimes revealed that direct placement of cotyledon explants on hygromycin 25 mg/l was detrimental to explant survival, whereas 10 mg/l gave continued growth and subsequent somatic embryo development and plant regeneration. The overall transformation frequency in these experiments, from initial explant to whole plant, was 0.03 %. Three fertile soybean plants were obtained during the course of these experiments. Enzymatic GUS assays and Southern blot hybridizations confirmed the integration of T-DNA and expression of the GUS-intron gene in the three primary transformants. Analysis of 48 progeny revealed that three copies of the transgene were inherited as a single Mendelian locus. Received: 6 December 1999 / Revised: 11 February 2000 / Accepted: 14 March 2000     

Comparison of different methods of measuring 8-oxoguanine as a marker of oxidative DNA damage. ESCODD (European Standards Committee on Oxidative DNA Damage)

We are attempting to resolve some of the problems encountered in measuring 8-hydroxy-2'-deoxyguanosine (8-oxodG) in human cellular DNA as a marker of oxidative stress. Samples of authentic 8-oxodG were distributed, and participating laboratories undertook to analyse this material within a specified period. Most HPLC procedures gave values for 8-oxodG within ±40% of the target, as did two of four GC-MS procedures, and both LC-MS-MS methods. Calf thymus DNA samples containing increasing amounts of 8-oxodG were also distributed for analysis. Fewer than half the procedures tested were able to detect the dose response; those that were successful tended to be procedures with low coefficients of variation. For the analysis of 8-oxodG in human cells, where it is likely to be present at much lower concentrations than in the calf thymus DNA, it is crucial to reduce analytical variation to a minimum; a coefficient of variation of less than 10% should be the aim, to give reasonable precision. HPLC with amperometric electrochemical detection is not recommended, as it is less sensitive than coulometric detection. Immunological detection, ³²P-postlabelling and LC-MS-MS are alternative approaches to measurement of 8-oxodG in DNA that, on the grounds of precision and detection of dose response, cannot at present be recommended.