The NEI/NCBI dbGAP database: Genotypes and haplotypes that may specifically predispose to risk of neovascular age-related macular degeneration

<p>Abstract</p> <p>Background</p> <p>To examine if the significantly associated SNPs derived from the genome wide allelic association study on the AREDS cohort at the NEI (dbGAP) specifically confer risk for neovascular age-related macular degeneration (AMD). We ascertained 134 unrelated patients with AMD who had one sibling with an AREDS classification 1 or less and was past the age at which the affected sibling was diagnosed (268 subjects). Genotyping was performed by both direct sequencing and Sequenom iPLEX system technology. Single SNP analyses were conducted with McNemar's Test (both 2 × 2 and 3 × 3 tests) and likelihood ratio tests (LRT). Conditional logistic regression was used to determine significant gene-gene interactions. LRT was used to determine the best fit for each genotypic model tested (additive, dominant or recessive).</p> <p>Results</p> <p>Before release of individual data, <it>p</it>-value information was obtained directly from the AREDS dbGAP website. Of the 35 variants with <it>P </it>< 10^-6examined, 23 significantly modified risk of neovascular AMD. Many variants located in tandem on 1q32-q22 including those in <it>CFH</it>, <it>CFHR4</it>, <it>CFHR2</it>, <it>CFHR5</it>, <it>F13B</it>, <it>ASPM </it>and <it>ZBTB </it>were significantly associated with AMD risk. Of these variants, single SNP analysis revealed that <it>CFH </it>rs572515 was the most significantly associated with AMD risk (P < 10^-6). Haplotype analysis supported our findings of single SNP association, demonstrating that the most significant haplotype, GATAGTTCTC, spanning <it>CFH</it>, <it>CFHR4</it>, and <it>CFHR2 </it>was associated with the greatest risk of developing neovascular AMD (<it>P </it>< 10^-6). Other than variants on 1q32-q22, only two SNPs, rs9288410 (<it>MAP2</it>) on 2q34-q35 and rs2014307 (<it>PLEKHA1</it>/<it>HTRA1</it>) on 10q26 were significantly associated with AMD status (<it>P </it>= .03 and <it>P </it>< 10^-6respectively). After controlling for smoking history, gender and age, the most significant gene-gene interaction appears to be between rs10801575 (<it>CFH</it>) and rs2014307 (<it>PLEKHA1</it>/<it>HTRA1</it>) (<it>P </it>< 10^-11). The best genotypic fit for rs10801575 and rs2014307 was an additive model based on LRT. After applying a Bonferonni correction, no other significant interactions were identified between any other SNPs.</p> <p>Conclusion</p> <p>This is the first replication study on the NEI dbGAP SNPs, demonstrating that alleles on 1q, 2q and 10q may predispose an individual to AMD.</p

Identifying subtypes of patients with neovascular age-related macular degeneration by genotypic and cardiovascular risk characteristics

<p>Abstract</p> <p>Background</p> <p>One of the challenges in the interpretation of studies showing associations between environmental and genotypic data with disease outcomes such as neovascular age-related macular degeneration (AMD) is understanding the phenotypic heterogeneity within a patient population with regard to any risk factor associated with the condition. This is critical when considering the potential therapeutic response of patients to any drug developed to treat the condition. In the present study, we identify patient subtypes or clusters which could represent several different targets for treatment development, based on genetic pathways in AMD and cardiovascular pathology.</p> <p>Methods</p> <p>We identified a sample of patients with neovascular AMD, that in previous studies had been shown to be at elevated risk for the disease through environmental factors such as cigarette smoking and genetic variants including the complement factor H gene (<it>CFH</it>) on chromosome 1q25 and variants in the <it>ARMS2</it>/HtrA serine peptidase 1 (<it>HTRA1</it>) gene(s) on chromosome 10q26. We conducted a multivariate segmentation analysis of 253 of these patients utilizing available epidemiologic and genetic data.</p> <p>Results</p> <p>In a multivariate model, cigarette smoking failed to differentiate subtypes of patients. However, four meaningfully distinct clusters of patients were identified that were most strongly differentiated by their cardiovascular health status (histories of hypercholesterolemia and hypertension), and the alleles of <it>ARMS2</it>/<it>HTRA1 </it>rs1049331.</p> <p>Conclusions</p> <p>These results have significant personalized medicine implications for drug developers attempting to determine the effective size of the treatable neovascular AMD population. Patient subtypes or clusters may represent different targets for therapeutic development based on genetic pathways in AMD and cardiovascular pathology, and treatments developed that may elevate CV risk, may be ill advised for certain of the clusters identified.</p

Increased Throughput by Parallelization of Library Preparation for Massive Sequencing

Background: Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing. Methodology/Principal Findings: In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling. Conclusions/Significance: We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsiz

Prognostic factors for patients with hepatic metastases from breast cancer

Median survival from liver metastases secondary to breast cancer is only a few months, with very rare 5-year survival. This study reviewed 145 patients with liver metastases from breast cancer to determine factors that may influence survival. Data were analysed using Kaplan–Meier survival curves, univariate and multivariate analysis. Median survival was 4.23 months (range 0.16–51), with a 27.6% 1-year survival. Factors that significantly predicted a poor prognosis on univariate analysis included symptomatic liver disease, deranged liver function tests, the presence of ascites, histological grade 3 disease at primary presentation, advanced age, oestrogen receptor (ER) negative tumours, carcinoembryonic antigen of over 1000 ng ml−1 and multiple vs single liver metastases. Response to treatment was also a significant predictor of survival with patients responding to chemo- or endocrine therapy surviving for a median of 13 and 13.9 months, respectively. Multivariate analysis of pretreatment variables identified a low albumin, advanced age and ER negativity as independent predictors of poor survival. The time interval between primary and metastatic disease, metastases at extrahepatic sites, histological subtype and nodal stage at primary presentation did not predict prognosis. Awareness of the prognostic implications of the above factors may assist in selecting the most appropriate treatment for these patients

Addressing challenges in the production and analysis of illumina sequencing data

Advances in DNA sequencing technologies have made it possible to generate large amounts of sequence data very rapidly and at substantially lower cost than capillary sequencing. These new technologies have specific characteristics and limitations that require either consideration during project design, or which must be addressed during data analysis. Specialist skills, both at the laboratory and the computational stages of project design and analysis, are crucial to the generation of high quality data from these new platforms. The Illumina sequencers (including the Genome Analyzers I/II/IIe/IIx and the new HiScan and HiSeq) represent a widely used platform providing parallel readout of several hundred million immobilized sequences using fluorescent-dye reversible-terminator chemistry. Sequencing library quality, sample handling, instrument settings and sequencing chemistry have a strong impact on sequencing run quality. The presence of adapter chimeras and adapter sequences at the end of short-insert molecules, as well as increased error rates and short read lengths complicate many computational analyses. We discuss here some of the factors that influence the frequency and severity of these problems and provide solutions for circumventing these. Further, we present a set of general principles for good analysis practice that enable problems with sequencing runs to be identified and dealt with

Automated Workflow for Preparation of cDNA for Cap Analysis of Gene Expression on a Single Molecule Sequencer

Background: Cap analysis of gene expression (CAGE) is a 59 sequence tag technology to globally determine transcriptional starting sites in the genome and their expression levels and has most recently been adapted to the HeliScope single molecule sequencer. Despite significant simplifications in the CAGE protocol, it has until now been a labour intensive protocol. Methodology: In this study we set out to adapt the protocol to a robotic workflow, which would increase throughput and reduce handling. The automated CAGE cDNA preparation system we present here can prepare 96 ‘HeliScope ready ’ CAGE cDNA libraries in 8 days, as opposed to 6 weeks by a manual operator.We compare the results obtained using the same RNA in manual libraries and across multiple automation batches to assess reproducibility. Conclusions: We show that the sequencing was highly reproducible and comparable to manual libraries with an 8 fold increase in productivity. The automated CAGE cDNA preparation system can prepare 96 CAGE sequencing samples simultaneously. Finally we discuss how the system could be used for CAGE on Illumina/SOLiD platforms, RNA-seq and fulllengt

Complement Inhibition Promotes Endogenous Neurogenesis and Sustained Anti-Inflammatory Neuroprotection following Reperfused Stroke

The restoration of blood-flow following cerebral ischemia incites a series of deleterious cascades that exacerbate neuronal injury. Pharmacologic inhibition of the C3a-receptor ameliorates cerebral injury by attenuating post-ischemic inflammation. Recent reports also implicate C3a in the modulation of tissue repair, suggesting that complement may influence both injury and recovery at later post-ischemic time-points.To evaluate the effect of C3a-receptor antagonism on post-ischemic neurogenesis and neurological outcome in the subacute period of stroke, transient focal cerebral ischemia was induced in adult male C57BL/6 mice treated with multiple regimens of a C3a receptor antagonist (C3aRA).Low-dose C3aRA administration during the acute phase of stroke promotes neuroblast proliferation in the subventricular zone at 7 days. Additionally, the C3a receptor is expressed on T-lymphocytes within the ischemic territory at 7 days, and this cellular infiltrate is abrogated by C3aRA administration. Finally, C3aRA treatment confers robust histologic and functional neuroprotection at this delayed time-point.Targeted complement inhibition through low-dose antagonism of the C3a receptor promotes post-ischemic neuroblast proliferation in the SVZ. Furthermore, C3aRA administration suppresses T-lymphocyte infiltration and improves delayed functional and histologic outcome following reperfused stroke. Post-ischemic complement activation may be pharmacologically manipulated to yield an effective therapy for stroke

The Time Course of Segmentation and Cue-Selectivity in the Human Visual Cortex

Texture discontinuities are a fundamental cue by which the visual system segments objects from their background. The neural mechanisms supporting texture-based segmentation are therefore critical to visual perception and cognition. In the present experiment we employ an EEG source-imaging approach in order to study the time course of texture-based segmentation in the human brain. Visual Evoked Potentials were recorded to four types of stimuli in which periodic temporal modulation of a central 3° figure region could either support figure-ground segmentation, or have identical local texture modulations but not produce changes in global image segmentation. The image discontinuities were defined either by orientation or phase differences across image regions. Evoked responses to these four stimuli were analyzed both at the scalp and on the cortical surface in retinotopic and functional regions-of-interest (ROIs) defined separately using fMRI on a subject-by-subject basis. Texture segmentation (tsVEP: segmenting versus non-segmenting) and cue-specific (csVEP: orientation versus phase) responses exhibited distinctive patterns of activity. Alternations between uniform and segmented images produced highly asymmetric responses that were larger after transitions from the uniform to the segmented state. Texture modulations that signaled the appearance of a figure evoked a pattern of increased activity starting at ∼143 ms that was larger in V1 and LOC ROIs, relative to identical modulations that didn't signal figure-ground segmentation. This segmentation-related activity occurred after an initial response phase that did not depend on the global segmentation structure of the image. The two cue types evoked similar tsVEPs up to 230 ms when they differed in the V4 and LOC ROIs. The evolution of the response proceeded largely in the feed-forward direction, with only weak evidence for feedback-related activity

Overexpression of HTRA1 Leads to Ultrastructural Changes in the Elastic Layer of Bruch's Membrane via Cleavage of Extracellular Matrix Components

Variants in the chromosomal region 10q26 are strongly associated with an increased risk for age-related macular degeneration (AMD). Two potential AMD genes are located in this region: ARMS2 and HTRA1 (high-temperature requirement A1). Previous studies have suggested that polymorphisms in the promotor region of HTRA1 result in overexpression of HTRA1 protein. This study investigated the role of HTRA1 overexpression in the pathogenesis of AMD. Transgenic Htra1 mice overexpressing the murine protein in the retinal pigment epithelium (RPE) layer of the retina were generated and characterized by transmission electron microscopy, immunofluorescence staining and Western Blot analysis. The elastic layer of Bruch's membrane (BM) in the Htra1 transgenic mice was fragmented and less continuous than in wild type (WT) controls. Recombinant HTRA1 lacking the N-terminal domain cleaved various extracellular matrix (ECM) proteins. Subsequent Western Blot analysis revealed an overexpression of fibronectin fragments and a reduction of fibulin 5 and tropoelastin in the RPE/choroid layer in transgenic mice compared to WT. Fibulin 5 is essential for elastogenesis by promoting elastic fiber assembly and maturation. Taken together, our data implicate that HTRA1 overexpression leads to an altered elastogenesis in BM through fibulin 5 cleavage. It highlights the importance of ECM related proteins in the development of AMD and links HTRA1 to other AMD risk genes such as fibulin 5, fibulin 6, ARMS2 and TIMP3