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Innocenti F.,University of Chicago | Cooper G.M.,University of Washington | Cooper G.M.,Hudson Alpha Institute | Stanaway I.B.,University of Washington | And 18 more authors.
PLoS Genetics | Year: 2011

The discovery of expression quantitative trait loci ("eQTLs") can help to unravel genetic contributions to complex traits. We identified genetic determinants of human liver gene expression variation using two independent collections of primary tissue profiled with Agilent (n = 206) and Illumina (n = 60) expression arrays and Illumina SNP genotyping (550K), and we also incorporated data from a published study (n = 266). We found that ~30% of SNP-expression correlations in one study failed to replicate in either of the others, even at thresholds yielding high reproducibility in simulations, and we quantified numerous factors affecting reproducibility. Our data suggest that drug exposure, clinical descriptors, and unknown factors associated with tissue ascertainment and analysis have substantial effects on gene expression and that controlling for hidden confounding variables significantly increases replication rate. Furthermore, we found that reproducible eQTL SNPs were heavily enriched near gene starts and ends, and subsequently resequenced the promoters and 3′UTRs for 14 genes and tested the identified haplotypes using luciferase assays. For three genes, significant haplotype-specific in vitro functional differences correlated directly with expression levels, suggesting that many bona fide eQTLs result from functional variants that can be mechanistically isolated in a high-throughput fashion. Finally, given our study design, we were able to discover and validate hundreds of liver eQTLs. Many of these relate directly to complex traits for which liver-specific analyses are likely to be relevant, and we identified dozens of potential connections with disease-associated loci. These included previously characterized eQTL contributors to diabetes, drug response, and lipid levels, and they suggest novel candidates such as a role for NOD2 expression in leprosy risk and C2orf43 in prostate cancer. In general, the work presented here will be valuable for future efforts to precisely identify and functionally characterize genetic contributions to a variety of complex traits. © 2011 Innocenti et al. Source

Du J.,Purdue University | Tian Z.,Purdue University | Bowen N.J.,Georgia Institute of Technology | Schmutz J.,Hudson Alpha Institute | And 2 more authors.
Plant Cell | Year: 2010

Long terminal repeat (LTR) retrotransposons, the most abundant genomic components in flowering plants, are classifiable into autonomous and nonautonomous elements based on their structural completeness and transposition capacity. It has been proposed that selection is the major force for maintaining sequence (e.g., LTR) conservation between nonautonomous elements and their autonomous counterparts. Here, we report the structural, evolutionary, and expression characterization of a giant retrovirus-like soybean (Glycine max) LTR retrotransposon family, SNARE. This family contains two autonomous subfamilies, SAREA and SAREB, that appear to have evolved independently since the soybean genome tetraploidization event ~13 million years ago, and a nonautonomous subfamily, SNRE, that originated from SAREA. Unexpectedly, a subset of the SNRE elements, which amplified from a single founding SNRE element within the last ~3 million years, have been dramatically homogenized with either SAREA or SAREB primarily in the LTR regions and bifurcated into distinct subgroups corresponding to the two autonomous subfamilies. We uncovered evidence of region-specific swapping of nonautonomous elements with autonomous elements that primarily generated various nonautonomous recombinants with LTR sequences from autonomous elements of different evolutionary lineages, thus revealing a molecular mechanism for the enhancement of preexisting partnership and the establishment of new partnership between autonomous and nonautonomous elements. © 2010 American Society of Plant Biologists. Source

Guertin A.D.,Merck And Co. | Martin M.M.,Amgen | Roberts B.,Hudson Alpha Institute | Hurd M.,Merck And Co. | And 9 more authors.
Cancer Cell International | Year: 2012

Background: Inhibition of kinases involved in the DNA damage response sensitizes cells to genotoxic agents by abrogating checkpoint-induced cell cycle arrest. CHK1 and WEE1 act in a pathway upstream of CDK1 to inhibit cell cycle progression in response to damaged DNA. Therapeutic targeting of either CHK1 or WEE1, in combination with chemotherapy, is under clinical evaluation. These studies examine the overlap and potential for synergy when CHK1 and WEE1 are inhibited in cancer cell models.Methods: Small molecules MK-8776 and MK-1775 were used to selectively and potently inhibit CHK1 and WEE1, respectively.Results: In vitro, the combination of MK-8776 and MK-1775 induces up to 50-fold more DNA damage than either MK-8776 or MK-1775 alone at a fixed concentration. This requires aberrant cyclin-dependent kinase activity but does not appear to be dependent on p53 status alone. Furthermore, DNA damage takes place primarily in S-phase cells, implying disrupted DNA replication. When dosed together, the combination of MK-8776 and MK-1775 induced more intense and more durable DNA damage as well as anti-tumor efficacy than either MK-8776 or MK-1775 dosed alone. DNA damage induced by the combination was detected in up to 40% of cells in a treated xenograft tumor model.Conclusions: These results highlight the roles of WEE1 and CHK1 in maintaining genomic integrity. Importantly, the strong synergy observed upon inhibition of both kinases suggests unique yet complimentary anti-tumor effects of WEE1 and CHK1 inhibition. This demonstration of DNA double strand breaks in the absence of a DNA damaging chemotherapeutic provides preclinical rationale for combining WEE1 and CHK1 inhibitors as a cancer treatment regimen. © 2012 Guertin et al.; licensee BioMed Central Ltd. Source

Boddu R.,University of Alabama at Birmingham | Yang C.,University of Alabama at Birmingham | Yang C.,Center for Translational Science | O'Connor A.K.,Center for Translational Science | And 11 more authors.
Journal of Molecular Medicine | Year: 2014

Autosomal recessive polycystic kidney disease (ARPKD) results from mutations in the human PKHD1 gene. Both this gene, and its mouse ortholog, Pkhd1, are primarily expressed in renal and biliary ductal structures. The mouse protein product, fibrocystin/polyductin complex (FPC), is a 445-kDa protein encoded by a 67-exon transcript that spans >500 kb of genomic DNA. In the current study, we observed multiple alternatively spliced Pkhd1 transcripts that varied in size and exon composition in embryonic mouse kidney, liver, and placenta samples, as well as among adult mouse pancreas, brain, heart, lung, testes, liver, and kidney. Using reverse transcription PCR and RNASeq, we identified 22 novel Pkhd1 kidney transcripts with unique exon junctions. Various mechanisms of alternative splicing were observed, including exon skipping, use of alternate acceptor/donor splice sites, and inclusion of novel exons. Bioinformatic analyses identified, and exon-trapping minigene experiments validated, consensus binding sites for serine/arginine-rich proteins that modulate alternative splicing. Using site-directed mutagenesis, we examined the functional importance of selected splice enhancers. In addition, we demonstrated that many of the novel transcripts were polysome bound, thus likely translated. Finally, we determined that the human PKHD1 R760H missense variant alters a splice enhancer motif that disrupts exon splicing in vitro and is predicted to truncate the protein. Taken together, these data provide evidence of the complex transcriptional regulation of Pkhd1/PKHD1 and identified motifs that regulate its splicing. Our studies indicate that Pkhd1/PKHD1 transcription is modulated, in part by intragenic factors, suggesting that aberrant PKHD1 splicing represents an unappreciated pathogenic mechanism in ARPKD. © 2014, Springer-Verlag Berlin Heidelberg. Source

Preuss M.,Institute For Medizinische Biometrie Und Statistik | Preuss M.,University of Lubeck | Konig I.R.,Institute For Medizinische Biometrie Und Statistik | Thompson J.R.,University of Leicester | And 42 more authors.
Circulation: Cardiovascular Genetics | Year: 2010

Background-Recent genome-wide association studies (GWAS) of myocardial infarction (MI) and other forms of coronary artery disease (CAD) have led to the discovery of at least 13 genetic loci. In addition to the effect size, power to detect associations is largely driven by sample size. Therefore, to maximize the chance of finding novel susceptibility loci for CAD and MI, the Coronary ARtery DIsease Genome-wide Replication And Meta-analysis (CARDIoGRAM) consortium was formed. Methods and Results-CARDIoGRAM combines data from all published and several unpublished GWAS in individuals with European ancestry; includes >22 000 cases with CAD, MI, or both and >60 000 controls; and unifies samples from the Atherosclerotic Disease VAscular functioN and genetiC Epidemiology study, CADomics, Cohorts for Heart and Aging Research in Genomic Epidemiology, deCODE, the German Myocardial Infarction Family Studies I, II, and III, Ludwigshafen Risk and Cardiovascular Heath Study/AtheroRemo, MedStar, Myocardial Infarction Genetics Consortium, Ottawa Heart Genomics Study, PennCath, and the Wellcome Trust Case Control Consortium. Genotyping was carried out on Affymetrix or Illumina platforms followed by imputation of genotypes in most studies. On average, 2.2 million single nucleotide polymorphisms were generated per study. The results from each study are combined using meta-analysis. As proof of principle, we meta-analyzed risk variants at 9p21 and found that rs1333049 confers a 29% increase in risk for MI per copy (P=2×10-20). Conclusion-CARDIoGRAM is poised to contribute to our understanding of the role of common genetic variation on risk for CAD and MI. © 2010 American Heart Association, Inc. Source

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