Use of tree-based models to identify subgroups and increase power to detect linkage to cardiovascular disease traits

BACKGROUND: Our goal was to identify subgroups of sib pairs from the Framingham Heart Study data set that provided higher evidence of linkage to particular candidate regions for cardiovascular disease traits. The focus of this method is not to claim identification of significant linkage to a particular locus but to show that tree models can be used to identify subgroups for use in selected sib-pair sampling schemes. RESULTS: We report results using a novel recursive partitioning procedure to identify subgroups of sib pairs with increased evidence of linkage to systolic blood pressure and other cardiovascular disease-related quantitative traits, using the Framingham Heart Study data set provided by the Genetic Analysis Workshop 13. This procedure uses a splitting rule based on Haseman-Elston regression that recursively partitions sib-pair data into homogeneous subgroups. CONCLUSIONS: Using this procedure, we identified a subgroup definition for use as a selected sib-pair sampling scheme. Using the characteristics that define the subgroup with higher evidence for linkage, we have identified an area of focus for further study

U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP

U1 Adaptors are a recently reported novel approach for targeted reduction of mRNA transcripts. A U1 adaptor oligonucleotide comprising of a target-complimentary hybridization domain and a U1 recruitment domain, directs the U1 snRNP complex to the terminal exon of a targeted gene, subsequently inhibiting poly(A) tail addition and leading to degradation of that RNA species within the nucleus. Here, we present data demonstrating U1 adapter-mediated gene silencing can result in significant ‘off-target’ silencing effects as demonstrated by the reduction of multiple mRNA species that were not intended to be targeted. Our data suggest that a substantial portion of this U1 adaptor-mediated off-target mRNA reduction is the result of sequestration U1 snRNP at levels sufficient to affect splicing and processing of non-target transcripts

Genomic Analysis of wig-1 Pathways

Background: Wig-1 is a transcription factor regulated by p53 that can interact with hnRNP A2/B1, RNA Helicase A, and dsRNAs, which plays an important role in RNA and protein stabilization. in vitro studies have shown that wig-1 binds p53 mRNA and stabilizes it by protecting it from deadenylation. Furthermore, p53 has been implicated as a causal factor in neurodegenerative diseases based in part on its selective regulatory function on gene expression, including genes which, in turn, also possess regulatory functions on gene expression. In this study we focused on the wig-1 transcription factor as a downstream p53 regulated gene and characterized the effects of wig-1 down regulation on gene expression in mouse liver and brain. Methods and Results: Antisense oligonucleotides (ASOs) were identified that specifically target mouse wig-1 mRNA and produce a dose-dependent reduction in wig-1 mRNA levels in cell culture. These wig-1 ASOs produced marked reductions in wig-1 levels in liver following intraperitoneal administration and in brain tissue following ASO administration through a single striatal bolus injection in FVB and BACHD mice. Wig-1 suppression was well tolerated and resulted in the reduction of mutant Htt protein levels in BACHD mouse brain but had no effect on normal Htt protein levels nor p53 mRNA or protein levels. Expression microarray analysis was employed to determine the effects of wig-1 suppression on genome-wide expression in mouse liver and brain. Reduction of wig-1 caused both down regulation and up regulation of several genes

Application of stellar photometry to the analysis of microarray images

Improvements in the analysis of microarray images are critical for accurately quantifying gene expression levels. The acquisition of accurate spot intensities directly influences the results and interpretation of statistical analyses. This dissertation discusses the implementation of a novel approach to the analysis of cDNA microarray images. We use a stellar photometric model, the Moffat function, to quantify microarray spots from nylon microarray images. The inherent flexibility of the Moffat shape model makes it ideal for quantifying microarray spots. We apply our novel approach to a Wilms\u27 tumor microarray study and compare our results with a fixed-circle segmentation approach for spot quantification. Our results suggest that different spot feature extraction methods can have an impact on the ability of statistical methods to identify differentially expressed genes. We also used the Moffat function to simulate a series of microarray images under various experimental conditions. These simulations were used to validate the performance of various statistical methods for identifying differentially expressed genes. Our simulation results indicate that tests taking into account the dependency between mean spot intensity and variance estimation, such as the smoothened t-test, can better identify differentially expressed genes, especially when the number of replicates and mean fold change are low. The analysis of the simulations also showed that overall, a rank sum test (Mann-Whitney) performed well at identifying differentially expressed genes. Previous work has suggested the strengths of nonparametric approaches for identifying differentially expressed genes. We also show that multivariate approaches, such as hierarchical and k-means cluster analysis along with principal components analysis, are only effective at classifying samples when replicate numbers and mean fold change are high. Finally, we show how our stellar shape model approach can be extended to the analysis of 2D-gel images by adapting the Moffat function to take into account the elliptical nature of spots in such images. Our results indicate that stellar shape models offer a previously unexplored approach for the quantification of 2D-gel spots

wig-1 knockdown does not affect p53 mRNA or protein levels following wig-1 ASO treatment in mice.

<p><i>A) mRNA and protein were extracted from BACHD</i> striatum of mice treated with 75 µg of wig-1 ASO, PBS, or control ASO. B) p53 mRNA and protein levels in mouse FVB striatum following ASO injection with increasing doses of wig-1 ASO, and C) Effects of wig-1 ASO treatment on p53 mRNA and protein levels in mouse liver. Data are expressed as means ± SEM (<i>n</i> = 4).</p

Genes up-regulated in BACHD mouse brain following wig-1 ASO treatment.

<p>Ingenuity™ Pathway Analysis was used to ascribe up-regulated genes with potential functions (additional genes can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029429#pone.0029429.s003" target="_blank">Figure S3</a>).</p

Mouse Wig-1 ASOs specifically reduce wig-1 mRNA levels in vitro and in vivo.

<p>A) oligonucleotides were 20 nucleotides in length and chemically modified with phosphorothioate in the backbone and 2′-<i>O</i>-methoxyethyl (MOE) on the wings with a central deoxy gap (“5-10-5” design). B) b.END cells were transfected with indicated concentration of wig-1 ASOs. RNA was extracted 24 hours after transfection and analyzed by RT-PCR to determine wig-1 mRNA levels. C) Male <i>BALB/c</i> mice were injected intraperitoneally with 4 different wig-1 ASOs at 100 mg/kg body weight per week or with saline for 4 weeks. Total RNA was prepared from liver, and used for real-time quantitative RT-PCR analysis to evaluate wig-1 mRNA levels. Data are expressed as means ± SEM (<i>n</i> = 4, ***p≤<i>0.01</i>).</p

Heatmap of the <i>log2</i> normalized intensities of the significantly differentially expressed 260 gene probes on the array.

<p>The columns represent the PBS, ASO treated, and negative control samples and the rows represent the 260 differentially expressed gene probes on the array. The heatmap shows that the majority of the differentially expressed genes show mostly upregulation after ASO treatment. This is shown by the transition of probe intensities to higher values relative to the PBS and negative control samples for the 260 gene probes. The dendrogram on the left of the heatmap shows the clustering of genes having similar profiles across samples. The dendrogram on top of the heatmap shows the clustering of the samples. The PBS and negative control samples clustered together whereas the ASO treated samples clustered separately.</p

Up-regulation of Gpnmb mRNA levels following wig-1 ASO treatment.

<p>A) Gpnmb mRNA levels in BACHD striatum; B) FVB striatum, and C) BALB/c liver. Data are expressed as means ± SEM (<i>n</i> = 4; *p≤0.1; **<i>p≤0.05</i>).</p

Genes down-regulated in BACHD mouse brain following wig-1 ASO treatment.

<p>Ingenuity™ Pathway Analysis was used to ascribe down-regulated genes with potential functions (additional genes can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029429#pone.0029429.s002" target="_blank">Figure S2</a>).</p