Publications by authors named "Jun Z Li"

82 Publications

The phenotypic spectrum associated with OTX2 mutations in humans.

Eur J Endocrinol 2021 05 25;185(1):121-135. Epub 2021 May 25.

Section of Molecular Basis of Rare Disease, Genetics and Genomic Medicine Research & Teaching Department, UCL Great Ormond Street Institute of Child Health, London, UK.

Objective: The transcription factor OTX2 is implicated in ocular, craniofacial, and pituitary development.

Design: We aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 expression in the human brain, with a view to investigate the mechanism of action.

Methods: We screened patients from the UK (n = 103), international centres (n = 24), and Brazil (n = 282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants.

Results: Two chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities; however, studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary.

Conclusions: OTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin.
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http://dx.doi.org/10.1530/EJE-20-1453DOI Listing
May 2021

Impact of between-tissue differences on pan-cancer predictions of drug sensitivity.

PLoS Comput Biol 2021 02 25;17(2):e1008720. Epub 2021 Feb 25.

Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, United States of America.

Increased availability of drug response and genomics data for many tumor cell lines has accelerated the development of pan-cancer prediction models of drug response. However, it is unclear how much between-tissue differences in drug response and molecular characteristics may contribute to pan-cancer predictions. Also unknown is whether the performance of pan-cancer models could vary by cancer type. Here, we built a series of pan-cancer models using two datasets containing 346 and 504 cell lines, each with MEK inhibitor (MEKi) response and mRNA expression, point mutation, and copy number variation data, and found that, while the tissue-level drug responses are accurately predicted (between-tissue ρ = 0.88-0.98), only 5 of 10 cancer types showed successful within-tissue prediction performance (within-tissue ρ = 0.11-0.64). Between-tissue differences make substantial contributions to the performance of pan-cancer MEKi response predictions, as exclusion of between-tissue signals leads to a decrease in Spearman's ρ from a range of 0.43-0.62 to 0.30-0.51. In practice, joint analysis of multiple cancer types usually has a larger sample size, hence greater power, than for one cancer type; and we observe that higher accuracy of pan-cancer prediction of MEKi response is almost entirely due to the sample size advantage. Success of pan-cancer prediction reveals how drug response in different cancers may invoke shared regulatory mechanisms despite tissue-specific routes of oncogenesis, yet predictions in different cancer types require flexible incorporation of between-cancer and within-cancer signals. As most datasets in genome sciences contain multiple levels of heterogeneity, careful parsing of group characteristics and within-group, individual variation is essential when making robust inference.
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http://dx.doi.org/10.1371/journal.pcbi.1008720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906305PMC
February 2021

Investigating rare pathogenic/likely pathogenic exonic variation in bipolar disorder.

Mol Psychiatry 2021 Jan 22. Epub 2021 Jan 22.

HudsonAlpha Institute for Biotechnology, Huntsville, AL, 35806, USA.

Bipolar disorder (BD) is a serious mental illness with substantial common variant heritability. However, the role of rare coding variation in BD is not well established. We examined the protein-coding (exonic) sequences of 3,987 unrelated individuals with BD and 5,322 controls of predominantly European ancestry across four cohorts from the Bipolar Sequencing Consortium (BSC). We assessed the burden of rare, protein-altering, single nucleotide variants classified as pathogenic or likely pathogenic (P-LP) both exome-wide and within several groups of genes with phenotypic or biologic plausibility in BD. While we observed an increased burden of rare coding P-LP variants within 165 genes identified as BD GWAS regions in 3,987 BD cases (meta-analysis OR = 1.9, 95% CI = 1.3-2.8, one-sided p = 6.0 × 10), this enrichment did not replicate in an additional 9,929 BD cases and 14,018 controls (OR = 0.9, one-side p = 0.70). Although BD shares common variant heritability with schizophrenia, in the BSC sample we did not observe a significant enrichment of P-LP variants in SCZ GWAS genes, in two classes of neuronal synaptic genes (RBFOX2 and FMRP) associated with SCZ or in loss-of-function intolerant genes. In this study, the largest analysis of exonic variation in BD, individuals with BD do not carry a replicable enrichment of rare P-LP variants across the exome or in any of several groups of genes with biologic plausibility. Moreover, despite a strong shared susceptibility between BD and SCZ through common genetic variation, we do not observe an association between BD risk and rare P-LP coding variants in genes known to modulate risk for SCZ.
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http://dx.doi.org/10.1038/s41380-020-01006-9DOI Listing
January 2021

Regulation of meiotic progression by Sertoli-cell androgen signaling.

Mol Biol Cell 2020 12 7;31(25):2841-2862. Epub 2020 Oct 7.

The Jackson Laboratory, Bar Harbor, ME 04609.

Androgen receptor (AR) signaling in Sertoli cells is known to be important for germ-cell progression through meiosis, but the extent to which androgens indirectly regulate specific meiotic stages is not known. Here, we combine synchronization of spermatogenesis, cytological analyses and single-cell RNAseq (scRNAseq) in the ertoli-ell ndrogen eceptor nockut (SCARKO) mutant and control mice, and demonstrate that SCARKO mutant spermatocytes exhibited normal expression and localization of key protein markers of meiotic prophase events, indicating that initiation of meiotic prophase is not androgen dependent. However, spermatocytes from SCARKO testes failed to acquire competence for the meiotic division phase. ScRNAseq analysis of wild-type and SCARKO mutant testes revealed a molecular transcriptomic block in an early meiotic prophase state (leptotene/zygotene) in mutant germ cells, and identified several misregulated genes in SCARKO Sertoli cells, many of which have been previously implicated in male infertility. Together, our coordinated cytological and scRNAseq analyses identified germ-cell intrinsic and extrinsic genes responsive to Sertoli-cell androgen signaling that promotes cellular states permissive for the meiotic division phase.
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http://dx.doi.org/10.1091/mbc.E20-05-0334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851862PMC
December 2020

A Novel Recurrent Genetic Variant Is Associated With a Dysplasia-Associated Arterial Disease Exhibiting Dissections and Fibromuscular Dysplasia.

Arterioscler Thromb Vasc Biol 2020 11 17;40(11):2686-2699. Epub 2020 Sep 17.

Division of Cardiovascular Medicine, Department of Internal Medicine (H.L.H., Y.W., M.-L.Y., K.L.H., J.L., A.E.K., S.K.G.), University of Michigan Medical School, Ann Arbor.

Objective: While rare variants in the gene have been associated with classical Ehlers-Danlos syndrome and rarely with arterial dissections, recurrent variants in underlying a systemic arteriopathy have not been described. Monogenic forms of multifocal fibromuscular dysplasia (mFMD) have not been previously defined. Approach and Results: We studied 4 independent probands with the pathogenic variant c.1540G>A, p.(Gly514Ser) who presented with arterial aneurysms, dissections, tortuosity, and mFMD affecting multiple arteries. Arterial medial fibroplasia and smooth muscle cell disorganization were confirmed histologically. The c.1540G>A variant is predicted to be pathogenic in silico and absent in gnomAD. The c.1540G>A variant is on a shared 160.1 kb haplotype with 0.4% frequency in Europeans. Furthermore, exome sequencing data from a cohort of 264 individuals with mFMD were examined for variants. In this mFMD cohort, c.1540G>A and 6 additional relatively rare variants predicted to be deleterious in silico were identified and were associated with arterial dissections (=0.005).

Conclusions: c.1540G>A is the first recurring variant recognized to be associated with arterial dissections and mFMD. This variant presents with a phenotype reminiscent of vascular Ehlers-Danlos syndrome. A shared haplotype among probands supports the existence of a common founder. Relatively rare genetic variants predicted to be deleterious by in silico analysis were identified in ≈2.7% of mFMD cases, and as they were enriched in patients with arterial dissections, may act as disease modifiers. Molecular testing for should be considered in patients with a phenotype overlapping with vascular Ehlers-Danlos syndrome and mFMD.
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http://dx.doi.org/10.1161/ATVBAHA.119.313885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953329PMC
November 2020

Chromosome 1q21.2 and additional loci influence risk of spontaneous coronary artery dissection and myocardial infarction.

Nat Commun 2020 09 4;11(1):4432. Epub 2020 Sep 4.

Division of Cardiovascular Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.

Spontaneous coronary artery dissection (SCAD) is a non-atherosclerotic cause of myocardial infarction (MI), typically in young women. We undertook a genome-wide association study of SCAD (N = 270/N = 5,263) and identified and replicated an association of rs12740679 at chromosome 1q21.2 (P = 2.19 × 10, OR = 1.8) influencing ADAMTSL4 expression. Meta-analysis of discovery and replication samples identified associations with P < 5 × 10 at chromosome 6p24.1 in PHACTR1, chromosome 12q13.3 in LRP1, and in females-only, at chromosome 21q22.11 near LINC00310. A polygenic risk score for SCAD was associated with (1) higher risk of SCAD in individuals with fibromuscular dysplasia (P = 0.021, OR = 1.82 [95% CI: 1.09-3.02]) and (2) lower risk of atherosclerotic coronary artery disease and MI in the UK Biobank (P = 1.28 × 10, HR = 0.91 [95% CI :0.89-0.93], for MI) and Million Veteran Program (P = 9.33 × 10, OR = 0.95 [95% CI: 0.94-0.96], for CAD; P = 3.35 × 10, OR = 0.96 [95% CI: 0.95-0.98] for MI). Here we report that SCAD-related MI and atherosclerotic MI exist at opposite ends of a genetic risk spectrum, inciting MI with disparate underlying vascular biology.
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http://dx.doi.org/10.1038/s41467-020-17558-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474092PMC
September 2020

Murine models of IDH-wild-type glioblastoma exhibit spatial segregation of tumor initiation and manifestation during evolution.

Nat Commun 2020 07 22;11(1):3669. Epub 2020 Jul 22.

Gilbert Family Neurofibromatosis Institute, Children's National Hospital, Washington, DC, 20010, USA.

Recent characterization of spatiotemporal genomic architecture of IDH-wild-type multifocal glioblastomas (M-GBMs) suggests a clinically unobserved common-ancestor (CA) with a less aggressive phenotype, generating highly genetically divergent malignant gliomas/GBMs in distant brain regions. Using serial MRI/3D-reconstruction, whole-genome sequencing and spectral karyotyping-based single-cell phylogenetic tree building, we show two distinct types of tumor evolution in p53-mutant driven mouse models. Malignant gliomas/GBMs grow as a single mass (Type 1) and multifocal masses (Type 2), respectively, despite both exhibiting loss of Pten/chromosome 19 (chr19) and PI3K/Akt activation with sub-tetraploid/4N genomes. Analysis of early biopsied and multi-segment tumor tissues reveals no evidence of less proliferative diploid/2N lesions in Type 1 tumors. Strikingly, CA-derived relatively quiescent tumor precursors with ancestral diploid/2N genomes and normal Pten/chr19 are observed in the subventricular zone (SVZ), but are distantly segregated from multi focal Type 2 tumors. Importantly, PI3K/Akt inhibition by Rictor/mTORC2 deletion blocks distant dispersal, restricting glioma growth in the SVZ.
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http://dx.doi.org/10.1038/s41467-020-17382-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376246PMC
July 2020

Single-Cell RNA Sequencing of Human, Macaque, and Mouse Testes Uncovers Conserved and Divergent Features of Mammalian Spermatogenesis.

Dev Cell 2020 08 5;54(4):529-547.e12. Epub 2020 Jun 5.

Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA; Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI, USA; Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA; Department of Urology, University of Michigan, Ann Arbor, MI, USA. Electronic address:

Spermatogenesis is a highly regulated process that produces sperm to transmit genetic information to the next generation. Although extensively studied in mice, our current understanding of primate spermatogenesis is limited to populations defined by state-specific markers from rodent data. As between-species differences have been reported in the duration and differentiation hierarchy of this process, it remains unclear how molecular markers and cell states are conserved or have diverged from mice to man. To address this challenge, we employ single-cell RNA sequencing to identify transcriptional signatures of major germ and somatic cell types of the testes in human, macaque, and mice. This approach reveals similarities and differences in expression throughout spermatogenesis, including the stem/progenitor pool of spermatogonia, markers of differentiation, potential regulators of meiosis, RNA turnover during spermatid differentiation, and germ cell-soma communication. These datasets provide a rich foundation for future targeted mechanistic studies of primate germ cell development and in vitro gametogenesis.
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http://dx.doi.org/10.1016/j.devcel.2020.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7879256PMC
August 2020

Whole-exome sequencing identifies rare variants in STAB2 associated with venous thromboembolic disease.

Blood 2020 07;136(5):533-541

Department of Pediatrics and.

Deep vein thrombosis and pulmonary embolism, collectively defined as venous thromboembolism (VTE), are the third leading cause of cardiovascular death in the United States. Common genetic variants conferring increased varying degrees of VTE risk have been identified by genome-wide association studies (GWAS). Rare mutations in the anticoagulant genes PROC, PROS1 and SERPINC1 result in perinatal lethal thrombosis in homozygotes and markedly increased VTE risk in heterozygotes. However, currently described VTE variants account for an insufficient portion of risk to be routinely used for clinical decision making. To identify new rare VTE risk variants, we performed a whole-exome study of 393 individuals with unprovoked VTE and 6114 controls. This study identified 4 genes harboring an excess number of rare damaging variants in patients with VTE: PROS1, STAB2, PROC, and SERPINC1. At STAB2, 7.8% of VTE cases and 2.4% of controls had a qualifying rare variant. In cell culture, VTE-associated variants of STAB2 had a reduced surface expression compared with reference STAB2. Common variants in STAB2 have been previously associated with plasma von Willebrand factor and coagulation factor VIII levels in GWAS, suggesting that haploinsufficiency of stabilin-2 may increase VTE risk through elevated levels of these procoagulants. In an independent cohort, we found higher von Willebrand factor levels and equivalent propeptide levels in individuals with rare STAB2 variants compared with controls. Taken together, this study demonstrates the utility of gene-based collapsing analyses to identify loci harboring an excess of rare variants with functional connections to a complex thrombotic disease.
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http://dx.doi.org/10.1182/blood.2019004161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393257PMC
July 2020

Landscape of Intercellular Crosstalk in Healthy and NASH Liver Revealed by Single-Cell Secretome Gene Analysis.

Mol Cell 2019 08;75(3):644-660.e5

Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA; Department of Cell & Developmental Biology, University of Michigan Medical Center, Ann Arbor, MI 48109, USA. Electronic address:

Cell-cell communication via ligand-receptor signaling is a fundamental feature of complex organs. Despite this, the global landscape of intercellular signaling in mammalian liver has not been elucidated. Here we perform single-cell RNA sequencing on non-parenchymal cells isolated from healthy and NASH mouse livers. Secretome gene analysis revealed a highly connected network of intrahepatic signaling and disruption of vascular signaling in NASH. We uncovered the emergence of NASH-associated macrophages (NAMs), which are marked by high expression of triggering receptors expressed on myeloid cells 2 (Trem2), as a feature of mouse and human NASH that is linked to disease severity and highly responsive to pharmacological and dietary interventions. Finally, hepatic stellate cells (HSCs) serve as a hub of intrahepatic signaling via HSC-derived stellakines and their responsiveness to vasoactive hormones. These results provide unprecedented insights into the landscape of intercellular crosstalk and reprogramming of liver cells in health and disease.
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http://dx.doi.org/10.1016/j.molcel.2019.07.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262680PMC
August 2019

Genome-wide linkage analysis and whole-exome sequencing identifies an ITGA2B mutation in a family with thrombocytopenia.

Br J Haematol 2019 08 23;186(4):574-579. Epub 2019 May 23.

Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.

Hereditary thrombocytopenias can be subclassified based on mode of inheritance and platelet size. Here we report a family with autosomal dominant (AD) thrombocytopenia with normal platelet size. Linkage analysis and whole exome sequencing identified the R1026W substitution in ITGA2B as the causative defect. The same mutation has been previously reported in 7 Japanese families/patients with AD thrombocytopenia, but all of these patients had macrothrombocytopenia. This is the first report of a family with AD thrombocytopenia with normal platelet size resulting from mutation in ITGA2B. ITGA2B mutations should therefore be included in the differential diagnosis of this latter disorder.
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http://dx.doi.org/10.1111/bjh.15961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679728PMC
August 2019

Variants in myelin regulatory factor (MYRF) cause autosomal dominant and syndromic nanophthalmos in humans and retinal degeneration in mice.

PLoS Genet 2019 05 2;15(5):e1008130. Epub 2019 May 2.

Department of Ophthalmology and Visual Sciences, W.K. Kellogg Eye Center, University of Michigan, Ann Arbor, MI, United States of America.

Nanophthalmos is a rare, potentially devastating eye condition characterized by small eyes with relatively normal anatomy, a high hyperopic refractive error, and frequent association with angle closure glaucoma and vision loss. The condition constitutes the extreme of hyperopia or farsightedness, a common refractive error that is associated with strabismus and amblyopia in children. NNO1 was the first mapped nanophthalmos locus. We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor (MYRF c.3376-1G>A), a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization. This variant produced a stable RNA transcript, leading to a frameshift mutation p.Gly1126Valfs*31 in the C-terminus of the protein. In addition, we identified an early truncating MYRF frameshift mutation, c.769dupC (p.S264QfsX74), in a patient with extreme axial hyperopia and syndromic features. Myrf conditional knockout mice (CKO) developed depigmentation of the retinal pigment epithelium (RPE) and retinal degeneration supporting a role of this gene in retinal and RPE development. Furthermore, we demonstrated the reduced expression of Tmem98, another known nanophthalmos gene, in Myrf CKO mice, and the physical interaction of MYRF with TMEM98. Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development.
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http://dx.doi.org/10.1371/journal.pgen.1008130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527243PMC
May 2019

Genome-wide association study identifies 30 loci associated with bipolar disorder.

Nat Genet 2019 05 1;51(5):793-803. Epub 2019 May 1.

Department of Psychiatry, Weill Cornell Medical College, New York, NY, USA.

Bipolar disorder is a highly heritable psychiatric disorder. We performed a genome-wide association study (GWAS) including 20,352 cases and 31,358 controls of European descent, with follow-up analysis of 822 variants with P < 1 × 10 in an additional 9,412 cases and 137,760 controls. Eight of the 19 variants that were genome-wide significant (P < 5 × 10) in the discovery GWAS were not genome-wide significant in the combined analysis, consistent with small effect sizes and limited power but also with genetic heterogeneity. In the combined analysis, 30 loci were genome-wide significant, including 20 newly identified loci. The significant loci contain genes encoding ion channels, neurotransmitter transporters and synaptic components. Pathway analysis revealed nine significantly enriched gene sets, including regulation of insulin secretion and endocannabinoid signaling. Bipolar I disorder is strongly genetically correlated with schizophrenia, driven by psychosis, whereas bipolar II disorder is more strongly correlated with major depressive disorder. These findings address key clinical questions and provide potential biological mechanisms for bipolar disorder.
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http://dx.doi.org/10.1038/s41588-019-0397-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956732PMC
May 2019

Extended regions of suspected mis-assembly in the rat reference genome.

Sci Data 2019 04 23;6(1):39. Epub 2019 Apr 23.

Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, USA.

We performed whole-genome sequencing for eight inbred rat strains commonly used in genetic mapping studies. They are the founders of the NIH heterogeneous stock (HS) outbred colony. We provide their sequences and variant calls to the rat genomics community. When analyzing the variant calls we identified regions with unusually high levels of heterozygosity. These regions are consistent across the eight inbred strains, including Brown Norway, which is the basis of the rat reference genome. These regions show higher read depths than other regions in the genome and contain higher rates of apparent tri-allelic variant sites. The evidence suggests that these regions may correspond to duplicated segments that were incorrectly overlaid as a single segment in the reference genome. We provide masks for these regions of suspected mis-assembly as a resource for the community to flag potentially false interpretations of mapping or functional results.
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http://dx.doi.org/10.1038/s41597-019-0041-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478900PMC
April 2019

COQ4 Mutation Leads to Childhood-Onset Ataxia Improved by CoQ10 Administration.

Cerebellum 2019 Jun;18(3):665-669

Molecular & Behavioral Neuroscience Institute, University of Michigan, 5061 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI, 48109-2200, USA.

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http://dx.doi.org/10.1007/s12311-019-01011-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536000PMC
June 2019

Genome-Wide Association Transethnic Meta-Analyses Identifies Novel Associations Regulating Coagulation Factor VIII and von Willebrand Factor Plasma Levels.

Circulation 2019 01;139(5):620-635

Institute of Cardiovascular and Medical Sciences (P.W.), University of Glasgow, UK.

Background: Factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are associated with risk of arterial and venous thrombosis and with hemorrhagic disorders. We aimed to identify and functionally test novel genetic associations regulating plasma FVIII and VWF.

Methods: We meta-analyzed genome-wide association results from 46 354 individuals of European, African, East Asian, and Hispanic ancestry. All studies performed linear regression analysis using an additive genetic model and associated ≈35 million imputed variants with natural log-transformed phenotype levels. In vitro gene silencing in cultured endothelial cells was performed for candidate genes to provide additional evidence on association and function. Two-sample Mendelian randomization analyses were applied to test the causal role of FVIII and VWF plasma levels on the risk of arterial and venous thrombotic events.

Results: We identified 13 novel genome-wide significant ( P≤2.5×10) associations, 7 with FVIII levels ( FCHO2/TMEM171/TNPO1, HLA, SOX17/RP1, LINC00583/NFIB, RAB5C-KAT2A, RPL3/TAB1/SYNGR1, and ARSA) and 11 with VWF levels ( PDHB/PXK/KCTD6, SLC39A8, FCHO2/TMEM171/TNPO1, HLA, GIMAP7/GIMAP4, OR13C5/NIPSNAP, DAB2IP, C2CD4B, RAB5C-KAT2A, TAB1/SYNGR1, and ARSA), beyond 10 previously reported associations with these phenotypes. Functional validation provided further evidence of association for all loci on VWF except ARSA and DAB2IP. Mendelian randomization suggested causal effects of plasma FVIII activity levels on venous thrombosis and coronary artery disease risk and plasma VWF levels on ischemic stroke risk.

Conclusions: The meta-analysis identified 13 novel genetic loci regulating FVIII and VWF plasma levels, 10 of which we validated functionally. We provide some evidence for a causal role of these proteins in thrombotic events.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.034532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6438386PMC
January 2019

Helmsman: fast and efficient mutation signature analysis for massive sequencing datasets.

BMC Genomics 2018 Nov 28;19(1):845. Epub 2018 Nov 28.

Department of Biostatistics, University of Michigan, Ann Arbor, MI, USA.

Background: The spectrum of somatic single-nucleotide variants in cancer genomes often reflects the signatures of multiple distinct mutational processes, which can provide clinically actionable insights into cancer etiology. Existing software tools for identifying and evaluating these mutational signatures do not scale to analyze large datasets containing thousands of individuals or millions of variants.

Results: We introduce Helmsman, a program designed to perform mutation signature analysis on arbitrarily large sequencing datasets. Helmsman is up to 300 times faster than existing software. Helmsman's memory usage is independent of the number of variants, resulting in a small enough memory footprint to analyze datasets that would otherwise exceed the memory limitations of other programs.

Conclusions: Helmsman is a computationally efficient tool that enables users to evaluate mutational signatures in massive sequencing datasets that are otherwise intractable with existing software. Helmsman is freely available at https://github.com/carjed/helmsman .
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http://dx.doi.org/10.1186/s12864-018-5264-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6263557PMC
November 2018

Analyses of LMNA-negative juvenile progeroid cases confirms biallelic POLR3A mutations in Wiedemann-Rautenstrauch-like syndrome and expands the phenotypic spectrum of PYCR1 mutations.

Hum Genet 2018 Dec 19;137(11-12):921-939. Epub 2018 Nov 19.

Warren Alpert Medical School of Brown University, Providence, RI, USA.

Juvenile segmental progeroid syndromes are rare, heterogeneous disorders characterized by signs of premature aging affecting more than one tissue or organ starting in childhood. Hutchinson-Gilford progeria syndrome (HGPS), caused by a recurrent de novo synonymous LMNA mutation resulting in aberrant splicing and generation of a mutant product called progerin, is a prototypical example of such disorders. Here, we performed a joint collaborative study using massively parallel sequencing and targeted Sanger sequencing, aimed at delineating the underlying genetic cause of 14 previously undiagnosed, clinically heterogeneous, non-LMNA-associated juvenile progeroid patients. The molecular diagnosis was achieved in 11 of 14 cases (~ 79%). Furthermore, we firmly establish biallelic mutations in POLR3A as the genetic cause of a recognizable, neonatal, Wiedemann-Rautenstrauch-like progeroid syndrome. Thus, we suggest that POLR3A mutations are causal for a portion of under-diagnosed early-onset segmental progeroid syndromes. We additionally expand the clinical spectrum associated with PYCR1 mutations by showing that they can somewhat resemble HGPS in the first year of life. Moreover, our results lead to clinical reclassification in one single case. Our data emphasize the complex genetic and clinical heterogeneity underlying progeroid disorders.
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http://dx.doi.org/10.1007/s00439-018-1957-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6652186PMC
December 2018

Genome Analyses of >200,000 Individuals Identify 58 Loci for Chronic Inflammation and Highlight Pathways that Link Inflammation and Complex Disorders.

Am J Hum Genet 2018 11;103(5):691-706

Department of Epidemiology and Prevention, Public Health Sciences, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA.

C-reactive protein (CRP) is a sensitive biomarker of chronic low-grade inflammation and is associated with multiple complex diseases. The genetic determinants of chronic inflammation remain largely unknown, and the causal role of CRP in several clinical outcomes is debated. We performed two genome-wide association studies (GWASs), on HapMap and 1000 Genomes imputed data, of circulating amounts of CRP by using data from 88 studies comprising 204,402 European individuals. Additionally, we performed in silico functional analyses and Mendelian randomization analyses with several clinical outcomes. The GWAS meta-analyses of CRP revealed 58 distinct genetic loci (p < 5 × 10). After adjustment for body mass index in the regression analysis, the associations at all except three loci remained. The lead variants at the distinct loci explained up to 7.0% of the variance in circulating amounts of CRP. We identified 66 gene sets that were organized in two substantially correlated clusters, one mainly composed of immune pathways and the other characterized by metabolic pathways in the liver. Mendelian randomization analyses revealed a causal protective effect of CRP on schizophrenia and a risk-increasing effect on bipolar disorder. Our findings provide further insights into the biology of inflammation and could lead to interventions for treating inflammation and its clinical consequences.
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http://dx.doi.org/10.1016/j.ajhg.2018.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218410PMC
November 2018

Statistical Detection of Relatives Typed with Disjoint Forensic and Biomedical Loci.

Cell 2018 10 11;175(3):848-858.e6. Epub 2018 Oct 11.

Department of Biology, Stanford University, Stanford, CA 94305, USA. Electronic address:

In familial searching in forensic genetics, a query DNA profile is tested against a database to determine whether it represents a relative of a database entrant. We examine the potential for using linkage disequilibrium to identify pairs of profiles as belonging to relatives when the query and database rely on nonoverlapping genetic markers. Considering data on individuals genotyped with both microsatellites used in forensic applications and genome-wide SNPs, we find that ∼30%-32% of parent-offspring pairs and ∼35%-36% of sib pairs can be identified from the SNPs of one member of the pair and the microsatellites of the other. The method suggests the possibility of performing familial searches of microsatellite databases using query SNP profiles, or vice versa. It also reveals that privacy concerns arising from computations across multiple databases that share no genetic markers in common entail risks, not only for database entrants, but for their close relatives as well.
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http://dx.doi.org/10.1016/j.cell.2018.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240431PMC
October 2018

Extremely rare variants reveal patterns of germline mutation rate heterogeneity in humans.

Nat Commun 2018 09 14;9(1):3753. Epub 2018 Sep 14.

Department of Biostatistics, University of Michigan, Ann Arbor, MI, 48109, USA.

A detailed understanding of the genome-wide variability of single-nucleotide germline mutation rates is essential to studying human genome evolution. Here, we use ~36 million singleton variants from 3560 whole-genome sequences to infer fine-scale patterns of mutation rate heterogeneity. Mutability is jointly affected by adjacent nucleotide context and diverse genomic features of the surrounding region, including histone modifications, replication timing, and recombination rate, sometimes suggesting specific mutagenic mechanisms. Remarkably, GC content, DNase hypersensitivity, CpG islands, and H3K36 trimethylation are associated with both increased and decreased mutation rates depending on nucleotide context. We validate these estimated effects in an independent dataset of ~46,000 de novo mutations, and confirm our estimates are more accurate than previously published results based on ancestrally older variants without considering genomic features. Our results thus provide the most refined portrait to date of the factors contributing to genome-wide variability of the human germline mutation rate.
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http://dx.doi.org/10.1038/s41467-018-05936-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138700PMC
September 2018

A Comprehensive Roadmap of Murine Spermatogenesis Defined by Single-Cell RNA-Seq.

Dev Cell 2018 09 23;46(5):651-667.e10. Epub 2018 Aug 23.

Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA; Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI, USA; Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, USA; Department of Urology, University of Michigan, Ann Arbor, MI, USA. Electronic address:

Spermatogenesis requires intricate interactions between the germline and somatic cells. Within a given cross section of a seminiferous tubule, multiple germ and somatic cell types co-occur. This cellular heterogeneity has made it difficult to profile distinct cell types at different stages of development. To address this challenge, we collected single-cell RNA sequencing data from ∼35,000 cells from the adult mouse testis and identified all known germ and somatic cells, as well as two unexpected somatic cell types. Our analysis revealed a continuous developmental trajectory of germ cells from spermatogonia to spermatids and identified candidate transcriptional regulators at several transition points during differentiation. Focused analyses delineated four subtypes of spermatogonia and nine subtypes of Sertoli cells; the latter linked to histologically defined developmental stages over the seminiferous epithelial cycle. Overall, this high-resolution cellular atlas represents a community resource and foundation of knowledge to study germ cell development and in vivo gametogenesis.
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http://dx.doi.org/10.1016/j.devcel.2018.07.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713459PMC
September 2018

Inference of cell type content from human brain transcriptomic datasets illuminates the effects of age, manner of death, dissection, and psychiatric diagnosis.

PLoS One 2018 17;13(7):e0200003. Epub 2018 Jul 17.

Molecular and Behavioral Neuroscience Institute. University of Michigan, Ann Arbor, MI, United States of America.

Psychiatric illness is unlikely to arise from pathology occurring uniformly across all cell types in affected brain regions. Despite this, transcriptomic analyses of the human brain have typically been conducted using macro-dissected tissue due to the difficulty of performing single-cell type analyses with donated post-mortem brains. To address this issue statistically, we compiled a database of several thousand transcripts that were specifically-enriched in one of 10 primary cortical cell types in previous publications. Using this database, we predicted the relative cell type content for 833 human cortical samples using microarray or RNA-Seq data from the Pritzker Consortium (GSE92538) or publicly-available databases (GSE53987, GSE21935, GSE21138, CommonMind Consortium). These predictions were generated by averaging normalized expression levels across transcripts specific to each cell type using our R-package BrainInABlender (validated and publicly-released on github). Using this method, we found that the principal components of variation in the datasets strongly correlated with the predicted neuronal/glial content of the samples. This variability was not simply due to dissection-the relative balance of brain cell types appeared to be influenced by a variety of demographic, pre- and post-mortem variables. Prolonged hypoxia around the time of death predicted increased astrocytic and endothelial gene expression, illustrating vascular upregulation. Aging was associated with decreased neuronal gene expression. Red blood cell gene expression was reduced in individuals who died following systemic blood loss. Subjects with Major Depressive Disorder had decreased astrocytic gene expression, mirroring previous morphometric observations. Subjects with Schizophrenia had reduced red blood cell gene expression, resembling the hypofrontality detected in fMRI experiments. Finally, in datasets containing samples with especially variable cell content, we found that controlling for predicted sample cell content while evaluating differential expression improved the detection of previously-identified psychiatric effects. We conclude that accounting for cell type can greatly improve the interpretability of transcriptomic data.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200003PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049916PMC
January 2019

Mutations in VPS13D lead to a new recessive ataxia with spasticity and mitochondrial defects.

Ann Neurol 2018 06 30;83(6):1075-1088. Epub 2018 Jun 30.

Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI.

Objective: To identify novel causes of recessive ataxias, including spinocerebellar ataxia with saccadic intrusions, spastic ataxias, and spastic paraplegia.

Methods: In an international collaboration, we independently performed exome sequencing in 7 families with recessive ataxia and/or spastic paraplegia. To evaluate the role of VPS13D mutations, we evaluated a Drosophila knockout model and investigated mitochondrial function in patient-derived fibroblast cultures.

Results: Exome sequencing identified compound heterozygous mutations in VPS13D on chromosome 1p36 in all 7 families. This included a large family with 5 affected siblings with spinocerebellar ataxia with saccadic intrusions (SCASI), or spinocerebellar ataxia, recessive, type 4 (SCAR4). Linkage to chromosome 1p36 was found in this family with a logarithm of odds score of 3.1. The phenotypic spectrum in our 12 patients was broad. Although most presented with ataxia, additional or predominant spasticity was present in 5 patients. Disease onset ranged from infancy to 39 years, and symptoms were slowly progressive and included loss of independent ambulation in 5. All but 2 patients carried a loss-of-function (nonsense or splice site) mutation on one and a missense mutation on the other allele. Knockdown or removal of Vps13D in Drosophila neurons led to changes in mitochondrial morphology and impairment in mitochondrial distribution along axons. Patient fibroblasts showed altered morphology and functionality including reduced energy production.

Interpretation: Our study demonstrates that compound heterozygous mutations in VPS13D cause movement disorders along the ataxia-spasticity spectrum, making VPS13D the fourth VPS13 paralog involved in neurological disorders. Ann Neurol 2018.
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http://dx.doi.org/10.1002/ana.25220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105379PMC
June 2018

Growth hormone deficiency with advanced bone age: phenotypic interaction between GHRH receptor and CYP21A2 mutations diagnosed by sanger and whole exome sequencing.

Arch Endocrinol Metab 2017 Dec;61(6):633-636

Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular LIM42, Disciplina de Endocrinologia, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP), São Paulo, SP Brasil.

Isolated growth hormone deficiency (IGHD) is the most common pituitary hormone deficiency and, clinically, patients have delayed bone age. High sequence similarity between CYP21A2 gene and CYP21A1P pseudogene poses difficulties for exome sequencing interpretation. A 7.5 year-old boy born to second-degree cousins presented with severe short stature (height SDS -3.7) and bone age of 6 years. Clonidine and combined pituitary stimulation tests revealed GH deficiency. Pituitary MRI was normal. The patient was successfully treated with rGH. Surprisingly, at 10.8 years, his bone age had advanced to 13 years, but physical exam, LH and testosterone levels remained prepubertal. An ACTH stimulation test disclosed a non-classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency explaining the bone age advancement and, therefore, treatment with cortisone acetate was added. The genetic diagnosis of a homozygous mutation in GHRHR (p.Leu144His), a homozygous CYP21A2 mutation (p.Val282Leu) and CYP21A1P pseudogene duplication was established by Sanger sequencing, MLPA and whole-exome sequencing. We report the unusual clinical presentation of a patient born to consanguineous parents with two recessive endocrine diseases: non-classic congenital adrenal hyperplasia modifying the classical GH deficiency phenotype. We used a method of paired read mapping aided by neighbouring mis-matches to overcome the challenges of exome-sequencing in the presence of a pseudogene.
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http://dx.doi.org/10.1590/2359-3997000000311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5806044PMC
December 2017

Genetic variants in as well as smoking are major determinants of plasma ADAMTS13 levels.

Blood Adv 2017 Jun 19;1(15):1037-1046. Epub 2017 Jun 19.

Department of Pediatrics and Communicable Disease.

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) in circulating blood, limiting the size of VWF multimers and regulating VWF activity. Abnormal regulation of VWF contributes to bleeding and to thrombotic disorders. ADAMTS13 levels in plasma are highly variable among healthy individuals, although the heritability and the genetic determinants of this variation are unclear. We performed genome-wide association studies of plasma ADAMTS13 concentrations in 3244 individuals from 2 independent cohorts of healthy individuals. The heritability of ADAMTS13 levels was between 59.1% (all individuals) and 83.5% (siblings only), whereas tobacco smoking was associated with a decrease in plasma ADAMTS13 levels. Meta-analysis identified common variants near the locus on chromosome 9q34.2 that were significantly associated with ADAMTS13 levels and collectively explained 20.0% of the variance. The top single nucleotide polymorphism (SNP), rs28673647, resides in an intron of (β, 6.7%; = 1.3E-52). Conditional analysis revealed 3 additional independent signals represented by rs3739893 (β, -22.3%; = 1.2E-30) and rs3124762 (β, 3.5%; = 8.9E-9) close to and rs4075970 (β, 2.4%; = 6.8E-9) on 21q22.3. Linkage analysis also identified the region around (9q34.2) as the top signal (LOD 3.5), consistent with our SNP association analyses. Two nonsynonymous variants in the top 2 independent linkage disequilibrium blocks (Q448E and A732V) were identified and characterized in vitro. This study uncovered specific common genetic polymorphisms that are key genetic determinants of the variation in plasma ADAMTS13 levels in healthy individuals.
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http://dx.doi.org/10.1182/bloodadvances.2017005629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728318PMC
June 2017

Mutation landscape and intra-tumor heterogeneity of two MANECs of the esophagus revealed by multi-region sequencing.

Oncotarget 2017 Sep 27;8(41):69610-69621. Epub 2017 Jun 27.

Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Laboratory of Genetics, Peking University Cancer Hospital and Institute, Beijing 100142, PR China.

Mixed adenoneuroendocrine carcinoma (MANEC) in the esophagus is an infrequent but highly malignant cancer with few known genomic alterations. We conducted whole-exome sequencing and whole-genome SNP genotyping for 4-6 tumor subregions and 5-6 adjacent normal tissue sites and 1-3 lymph node metastases in two esophageal MANECs to detect somatic mutations and copy number alterations, and to explore their spatial heterogeneity and underlying clonal structure. mutation, deletion or LOH, and amplification appeared in all regions in both tumors. Mutations falling in known cancer genes tended to show higher variant allele frequencies than those not falling in these genes in at least one of the cases. Phylogenetic analyses of the samples and underlying subclones suggested extensive migration across different tumor regions and from some regions to the lymph nodes. Lymph node metastases appeared to have been seeded by both early founder cells as well as subsequent, locally emerging daughter clones. A phenotypically normal tissue site carried most of the mutations found in neighboring tumor samples, implying field cancerization. Understanding such complex genetic heterogeneity within each patient will be important for guiding clinical decisions.
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http://dx.doi.org/10.18632/oncotarget.18678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5642503PMC
September 2017

Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci.

Proc Natl Acad Sci U S A 2017 09 21;114(36):9659-9664. Epub 2017 Aug 21.

Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109;

Factor V Leiden ( ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized -ethyl--nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for ( ) and haploinsufficient for tissue factor pathway inhibitor ( ). deficiency enhanced the survival of mice, demonstrating that lethality is genetically suppressible. ENU-mutagenized males and females were crossed to generate 6,729 progeny, with 98 offspring surviving until weaning. Sixteen lines, referred to as "modifier of Factor 5 Leiden ()," exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in identified a chromosome 3 locus containing the tissue factor gene (). Although no ENU-induced mutation was identified, haploinsufficiency for ( ) suppressed lethality. Whole-exome sequencing in identified an gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of lethality ( = 1.7 × 10), suggesting that is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent knockin/knockout demonstrated that haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for Our findings identify and the axis as key regulators in determining thrombosis balance in the setting of and also suggest a role for in this process.
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http://dx.doi.org/10.1073/pnas.1705762114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5594664PMC
September 2017

Post-mortem molecular profiling of three psychiatric disorders.

Genome Med 2017 07 28;9(1):72. Epub 2017 Jul 28.

HudsonAlpha Institute for Biotechnology, 601 Genome Way, Huntsville, AL, 35806, USA.

Background: Psychiatric disorders are multigenic diseases with complex etiology that contribute significantly to human morbidity and mortality. Although clinically distinct, several disorders share many symptoms, suggesting common underlying molecular changes exist that may implicate important regulators of pathogenesis and provide new therapeutic targets.

Methods: We performed RNA sequencing on tissue from the anterior cingulate cortex, dorsolateral prefrontal cortex, and nucleus accumbens from three groups of 24 patients each diagnosed with schizophrenia, bipolar disorder, or major depressive disorder, and from 24 control subjects. We identified differentially expressed genes and validated the results in an independent cohort. Anterior cingulate cortex samples were also subjected to metabolomic analysis. ChIP-seq data were used to characterize binding of the transcription factor EGR1.

Results: We compared molecular signatures across the three brain regions and disorders in the transcriptomes of post-mortem human brain samples. The most significant disease-related differences were in the anterior cingulate cortex of schizophrenia samples compared to controls. Transcriptional changes were assessed in an independent cohort, revealing the transcription factor EGR1 as significantly down-regulated in both cohorts and as a potential regulator of broader transcription changes observed in schizophrenia patients. Additionally, broad down-regulation of genes specific to neurons and concordant up-regulation of genes specific to astrocytes was observed in schizophrenia and bipolar disorder patients relative to controls. Metabolomic profiling identified disruption of GABA levels in schizophrenia patients.

Conclusions: We provide a comprehensive post-mortem transcriptome profile of three psychiatric disorders across three brain regions. We highlight a high-confidence set of independently validated genes differentially expressed between schizophrenia and control patients in the anterior cingulate cortex and integrate transcriptional changes with untargeted metabolite profiling.
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http://dx.doi.org/10.1186/s13073-017-0458-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5534072PMC
July 2017