Platform Session 18: Plenary Session 1 †1 †3 Dissection of a Modifier Network in Marfan Syndrome Reveals New A Global Mechanism for Non-coding RNA Dependent Chromatin For- Therapeutic Targets. J. Doyle1, J. Pardo Habashi1, T. Holm1, D. Bedja1,H. mation in Mammals. A. Khalil1, 2, M. Guttman1, M. Huarte1, L. Goff1,M. Dietz1,2. 1) Institute of Genetic Medicine, Johns Hopkins School of Medicine, Kellis1, E. Lander1, 2, J. Rinn1, 2. 1) The Broad Institute of Harvard and MIT, Baltimore, MD; 2) Howard Hughes Medical Institute. Cambridge, MA; 2) Harvard Medical School, Boston, MA. Excessive TGFβ activity causes many of the manifestations of Marfan One of the most fundamental and unsolved problems in biology is: how syndrome (MFS), including aortic aneurysm, that can be attenuated by TGFβ does the same genome present in every cell of an organism encode a antagonism with neutralizing antibody or the angiotensin II (Ang II) type 1 multitude of different cellular states? While epigenetic regulation by chroma- receptor (AT1) blocker (ARB) losartan in MFS mice (Fbn1 genotype C1039G/ tin modifying complexes plays a key role in this process, it is not currently +). TGFβ signals through both canonical (pSmad2/3) and noncanonical known how these complexes are targeted to specific regions of the genome. (MAPK: Jnk, Erk, p38) cascades. To determine their relative contribution, Recently, we and others have found two large intergenic non-coding RNAs we introduced haploinsufficiency for Smad4 (a mediator of Smad-dependent (lincRNAs) known as HOTAIR and XIST to physically associate with the signaling) on the C1039G/+ background (S4+/-:C1039G/+). Unexpectedly, chromatin modifying complex PRC2 and guide it to specific genomic loci. S4+/-:C1039G/+ mice showed synthetic lethality due to aortic rupture com- Thus, we hypothesized that association of lincRNAs with chromatin modi- pared to S4+/+:C1039G/+ littermates. Both S4+/+:C1039G/+ and S4+/- fying complexes may be a general theme in the establishment and mainte- :C1039G/+ mice show marked and equivalent activation of the Smad2/3 nance of epigenetic states. To assay lincRNA interactions with chromatin and Erk1/2 cascades, which could not account for synthetic lethality. S4+/- modifying complexes on a genome-wide scale we developed RIP-Chip tech- :C1039G/+ mice uniquely show activation of Jnk, and a Jnk antagonist fully nology: RNA co-immunoprecipitation followed by hybridization to tilling rescues synthetic lethality. Both an Erk1/2 and Jnk1 antagonist protected arrays. Remarkably, we found that 20% of expressed lincRNAs are bound against aneurysm progression in S4+/+:C1039G/+ mice despite the fact by PRC2 in several human cell types. By examining several other chromatin that only Erk shows activation in this strain. Activation of MAPKs is dependent modifying complexes, we found that 56% of lincRNAs are associated with on both AT1 and TGFβ. These data show that Erk and Jnk signaling is these complexes. We then confirmed the localization of these lincRNAs to additive and interchangeable in the pathogenesis of ascending aortic aneu- chromatin by RNA in situ hybridization. Through loss-of-function experi- rysm. AngII signals through both the AT1 and AT2 receptors. Use of ACE ments, we found PRC2-associated lincRNAs to be required for proper inhibitors (ACEi, e.g. enalapril) inhibits both receptors, whereas selective expression of specific regions of the genome which are known to be regu- AT1 blockade is achieved with ARBs. AT2 signaling can induce apoptosis, lated by PRC2. We have also profiled lincRNAs across multiple cancer prompting the proposal that ACEi are preferable. To address this controversy types and discovered numerous lincRNAs that are both misregulated in we knocked out the AT2 gene in C1039G/+ mice (AT2KO:C1039G/+) and cancer and bound to chromatin modifying complexes. Moreover, p53 directly showed larger aortas and worsened vessel architecture compared to and temporally induces several chromatin-associated lincRNAs in response AT2+:C1039G/+ animals. A trial of enalapril in the MFS mice did not attenu- to DNA damage. Remarkably, these lincRNAs serve to regulate many key ate aortic growth, while losartan led to regression of aortic size. Much of genes in the p53 pathway. Together, these results point to a general mecha- the protection by losartan was lost in AT2KO:C1039G/+ mice, suggesting nism of lincRNA mediated regulation in key cancer processes via the guid- that shunting of signaling through AT2 upon AT1 blockade contributes to ance of chromatin modifying complexes. Furthermore, RIP-seq technology aortic rescue. Finally, we show that AT2 signaling mediates protection has revealed a plethora of small RNAs that map to peaks of various chroma- through inhibition of activation of Erk, which is increased in AT2KO:C1039G/ tin modifications. Our results are significant since: 1) This is the first genome- + mice. In contrast to losartan, enalapril fails to reduce Erk1/2 activation in wide characterization of ncRNAs interaction with chromatin modifying com- C1039G/+ mice. Taken together, these data define MAPK and AT2 signaling plexes, 2) They suggest that both small and large ncRNAs can establish as prognostic and therapeutic modifiers of vascular disease in MFS and epigenetic landscapes by guiding chromatin modifying complexes to specific provide rationale and incentive for a clinical trial of Erk and Jnk antagonists regions of the genome, and disruption of these interactions may represent and AT2 agonists in MFS. a novel paradigm in cancer etiology. 2 Next-Generation Mendelian Genetics by Exome Sequencing. J. Shend- ure1, S.B. Ng1, E.H. Turner1, P.D. Robertson1, A.W. Bigham2,C.Lee1, E.E. Eichler1, M. Bamshad2, D.A. Nickerson1. 1) Department of Genome Sciences, University of Washington, Seattle, WA; 2) Department of Pediat- rics, University of Washington, Seattle, WA. Rare monogenic diseases have been of incredible value to biomedical research, as the identification of the genes underlying phenotypes of interest has yielded fundamental, medically relevant insights into human biology. However, many rare disorders have yet to be solved. In many cases, this is because only a small number of cases/families are available, limiting the power of traditional gene mapping strategies. As most disease-causing variants affect coding sequences, comprehensive resequencing of all human genes has the potential to serve as a genome-wide scan for the underlying cause of a rare monogenic disease. While the routine sequencing of full human genomes continues to be cost prohibitive, the cost of sequencing all protein-coding regions (i.e., the “exome”, ~1% of a human genome) may soon be on par with that of dense genotyping arrays. We evaluated whether second-generation methods for targeted sequencing of the human exome could serve as a genome-wide scan for monogenic diseases that is indepen- dent of linkage data or other a priori assumptions. To that end, we carried out targeted capture and massively parallel sequencing of the exomes of twelve humans. These include eight HapMap individuals representing three populations, and four unrelated individuals with a rare dominantly inherited disorder, Freeman-Sheldon syndrome (FSS). We successfully interrogated ~96% of each human exome and demonstrate the sensitive and specific identification of rare and common variants in over 300 megabases (Mb) of coding sequence, capturing both single nucleotide substitutions and small insertion-deletions. Importantly, using FSS as a proof-of-concept, we show that candidate genes for monogenic disorders can be identified by exome sequencing of a small number of unrelated, affected individuals. Notably, however, this approach is critically dependent on the availability of adequate filters for common variants. In our analysis of FSS, the availability of the 8 HapMap exomes provided an essential supplement to dbSNP. Low-cost, high throughput technologies for exome resequencing have the potential to rapidly accelerate the discovery of candidate gene(s) and mutations that underlie rare monogenic diseases that have been resistant to conventional approaches. This strategy may also be applicable to diseases with more complex genetics through larger sample sizes and appropriate weighting of nonsynonymous variants by predicted functional impact. † Trainee Award Finalist Copyright © 2009 The American Society of Human Genetics. All rights reserved. 2 Platform Session 18: Plenary Session 4 5 Meta-analysis of >100,000 individuals identifies 63 new loci associated Long Range Effects of CNVs Mediated by cis-Acting Promoter Competi- with serum lipid concentrations. T.M. Teslovich1, K. Musunuru2,3, A.V. tion. K. Lower1, J. Hughes1, M. De Gobbi1, S. Henderson2, V. Viprakasit3, Smith4, S. Ripatti5,6, C. van Duijn7, J. Rotter8, D. Chasman9, E. Boerwinkle10, C. Fisher1, A. Goriely1, H. Ayyub1, J. Sloane-Stanley1, D. Vernimmen1,C. L.A. Cupples11, R. Krauss12, V. Gudnason4, D. Rader13, M. Sandhu14,15,J. Langford4, D. Garrick1, R. Gibbons1, D. Higgs1. 1) MRC Molecular Haematol- Kooner16, I. Borecki17, P. Munroe18, L. Pentonen2,5,19, M. Boehnke1,G. ogy Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Abecasis1, S. Kathiresan2,3, Global Lipids Genetics Consortium. 1) Center Oxford, UK; 2) National Haemoglobinopathy Reference Laboratory, Oxford for Statistical Genetics, Department of Biostatistics, University of Michigan, Radcliffe Hospitals NHS Trust, Oxford, UK; 3) Department of Paediatrics, Ann Arbor, MI; 2) Program in Medical and