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Ann Arbor, MI, United States

Gillies C.E.,University of Michigan | Otto E.A.,University of Michigan | Vega-Warner V.,University of Michigan | Robertson C.C.,University of Michigan | And 8 more authors.
BMC Bioinformatics | Year: 2016

Background: Targeted sequencing of discrete gene sets is a cost effective strategy to screen subjects for monogenic forms of disease. One method to achieve this pairs microfluidic PCR with next generation sequencing. The PCR step of this pipeline creates challenges in accurate variant calling. This includes that most reads targeting a specific exon are duplicates that have been amplified from the PCR step. To reduce false positive variant calls from these experiments, previous studies have used threshold-based filtering of alternative allele depth ratio and manual inspection of the alignments. However even after manual inspection and filtering, many variants fail to be validated via Sanger sequencing. To improve the accuracy of variant calling from these experiments, we are challenged to design a variant filtering strategy that sufficiently models microfluidic PCR-specific issues. Results: We developed an open source variant filtering pipeline, targeted sequencing support vector machine ("tarSVM"), that uses a Support Vector Machine (SVM) and a new score the normalized allele dosage test to identify high quality variants from microfluidic PCR data. tarSVM maximizes training knowledge by selecting variants that are likely true and likely false variants by incorporating knowledge from the 1000 Genomes and the Exome Aggregation Consortium projects. tarSVM improves on previous approaches by synthesizing variant features from the Genome Analysis Toolkit and allele dosage information. We compared the accuracy of tarSVM versus existing variant quality filtering strategies on two cohorts (n=474 and n=1152), and validated our method on a third cohort (n=75). In the first cohort, our method achieved 84.5 % accuracy of predicting whether or not a variant would be validated with Sanger sequencing versus 78.8 % for the second most accurate method. In the second cohort, our method had an accuracy of 73.3 %, versus 61.5 % for the second best method. Finally, our method had a false discovery rate of 5 % for the validation cohort. Conclusions: tarSVM increases the accuracy of variant calling when using microfluidic PCR based targeted sequencing approaches. This results in higher confidence downstream analyses, and ultimately reduces the costs Sanger validation. Our approach is less labor intensive than existing approaches, and is available as an open source pipeline for read trimming, aligning, variant calling, and variant quality filtering on GitHub at https://github.com/christopher-gillies/TargetSpecificGATKSequencingPipeline. © 2016 Gillies et al. Source

Wettlaufer S.H.,150 West Medical Center Drive | Scott J.P.,150 West Medical Center Drive | McEachin R.C.,University of Michigan | Peters-Golden M.,150 West Medical Center Drive | Huang S.K.,150 West Medical Center Drive
American Journal of Respiratory Cell and Molecular Biology | Year: 2016

Myofibroblasts, the major effector cells in pathologic fibrosis, derive from the differentiation of fibroblasts driven by mediators such as transforming growth factor-β1 (TGF-β1) and biomechanical signals. Although the myofibroblast has traditionally been considered a terminally differentiated cell, the lipid mediator prostaglandin E2 (PGE2) has been shown to not only prevent but also reverse myofibroblast differentiation, as characterized by the ability of PGE2 to diminish expression of collagen I and α-smooth muscle actin in established myofibroblasts. Here, we use microarrays to examine the extent of transcriptomic changes that occur during TGF-β1-induced differentiation and PGE2-induced dedifferentiation of myofibroblasts. Normal primary human adult lung fibroblasts were cultured for 24 hours with or without TGF-β1 and treated for 48 hours with PGE2. Gene expression levels were assessed from total RNA on the Affymetrix U219 microarray. TGF-β1 up-regulated 588 genes and downregulated 689 genes compared with control cells. PGE2 reversed the expression of 363 (62%) of the TGF-β1-up-regulated genes and 345 (50%) of the TGF-β1-down-regulated genes. Genes up-regulated by TGF-β1 and reversed by PGE2 were enriched in annotations for Cell Adhesion, Contractile Fiber, and Actin Binding, whereas genes down-regulated by TGF-β1 but subsequently reversed by PGE2 were enriched in annotations for Glycoprotein, Polysaccharide Binding, and Regulation of Cell Migration. Surprisingly, the genes whose expression was affected by PGE2 differed between TGF-β1-induced myofibroblasts and undifferentiated fibroblasts. These data demonstrate the capacity of PGE2 to effect marked global alterations in the transcriptomic program of differentiated myofibroblasts and emphasize the considerable plasticity of these cells. Copyright © 2016 by the American Thoracic Society. Source

Yang Z.,University of Michigan | Yang Z.,830 North University Avenue | Yang Z.,150 West Medical Center Drive | Klionsky D.J.,University of Michigan | And 2 more authors.
Nature Cell Biology | Year: 2010

Macroautophagy (hereafter autophagy), or 'self-eating', is a conserved cellular pathway that controls protein and organelle degradation, and has essential roles in survival, development and homeostasis. Autophagy is also integral to human health and is involved in physiology, development, lifespan and a wide range of diseases, including cancer, neurodegeneration and microbial infection. Although research on this topic began in the late 1950s, substantial progress in the molecular study of autophagy has taken place during only the past 15 years. This review traces the key findings that led to our current molecular understanding of this complex process. Source

Han J.,Sanford Burnham Institute for Medical Research | Han J.,150 West Medical Center Drive | Back S.H.,150 West Medical Center Drive | Back S.H.,University of Ulsan | And 13 more authors.
Nature Cell Biology | Year: 2013

Protein misfolding in the endoplasmic reticulum (ER) leads to cell death through PERK-mediated phosphorylation of eIF2α, although the mechanism is not understood. ChIP-seq and mRNA-seq of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), key transcription factors downstream of p-eIF2α, demonstrated that they interact to directly induce genes encoding protein synthesis and the unfolded protein response, but not apoptosis. Forced expression of ATF4 and CHOP increased protein synthesis and caused ATP depletion, oxidative stress and cell death. The increased protein synthesis and oxidative stress were necessary signals for cell death. We show that eIF2α-phosphorylation-attenuated protein synthesis, and not Atf4 mRNA translation, promotes cell survival. These results show that transcriptional induction through ATF4 and CHOP increases protein synthesis leading to oxidative stress and cell death. The findings suggest that limiting protein synthesis will be therapeutic for diseases caused by protein misfolding in the ER. © 2013 Macmillan Publishers Limited. All rights reserved. Source

Shu L.,150 West Medical Center Drive | Vivekanandan-Giri A.,150 West Medical Center Drive | Pennathur S.,150 West Medical Center Drive | Shayman J.A.,150 West Medical Center Drive
Kidney International | Year: 2014

The endothelial dysfunction of Fabry disease results from α-galactosidase A deficiency leading to the accumulation of globotriaosylceramide. Vasculopathy in the α-galactosidase A null mouse is manifested as oxidant-induced thrombosis, accelerated atherogenesis, and impaired arterial reactivity. To better understand the pathogenesis of Fabry disease in humans, we generated a human cell model by using RNA interference. Hybrid endothelial cells were transiently transfected with small interfering RNA (siRNA) specifically directed against α-galactosidase A. Knockdown of α-galactosidase A was confirmed using immunoblotting and globotriaosylceramide accumulation. Endothelial nitric oxide synthase (eNOS) activity was correspondingly decreased by >60%. Levels of 3-nitrotyrosine (3NT), a specific marker for reactive nitrogen species and quantified using mass spectrometry, increased by 40- to 120-fold without corresponding changes in other oxidized amino acids, consistent with eNOS-derived reactive nitrogen species as the source of the reactive oxygen species. eNOS uncoupling was confirmed by the observed increase in free plasma and protein-bound aortic 3NT levels in the α-galactosidase A knockout mice. Finally, 3NT levels, assayed in biobanked plasma samples from patients with classical Fabry disease, were over sixfold elevated compared with age- and gender-matched controls. Thus, 3NT may serve as a biomarker for the vascular involvement in Fabry disease. © 2014 International Society of Nephrology. Source

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