Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel heart-specific long non-coding RNAs.

AIM: Heart disease is recognized as a consequence of dysregulation of cardiac gene regulatory networks. Previously, unappreciated components of such networks are the long non-coding RNAs (lncRNAs). Their roles in the heart remain to be elucidated. Thus, this study aimed to systematically characterize the cardiac long non-coding transcriptome post-myocardial infarction and to elucidate their potential roles in cardiac homoeostasis. METHODS AND RESULTS: We annotated the mouse transcriptome after myocardial infarction via RNA sequencing and ab initio transcript reconstruction, and integrated genome-wide approaches to associate specific lncRNAs with developmental processes and physiological parameters. Expression of specific lncRNAs strongly correlated with defined parameters of cardiac dimensions and function. Using chromatin maps to infer lncRNA function, we identified many with potential roles in cardiogenesis and pathological remodelling. The vast majority was associated with active cardiac-specific enhancers. Importantly, oligonucleotide-mediated knockdown implicated novel lncRNAs in controlling expression of key regulatory proteins involved in cardiogenesis. Finally, we identified hundreds of human orthologues and demonstrate that particular candidates were differentially modulated in human heart disease. CONCLUSION: These findings reveal hundreds of novel heart-specific lncRNAs with unique regulatory and functional characteristics relevant to maladaptive remodelling, cardiac function and possibly cardiac regeneration. This new class of molecules represents potential therapeutic targets for cardiac disease. Furthermore, their exquisite correlation with cardiac physiology renders them attractive candidate biomarkers to be used in the clinic.

Genome-wide association study of metabolic traits reveals novel gene-metabolite-disease links.

Metabolic traits are molecular phenotypes that can drive clinical phenotypes and may predict disease progression. Here, we report results from a metabolome- and genome-wide association study on (1)H-NMR urine metabolic profiles. The study was conducted within an untargeted approach, employing a novel method for compound identification. From our discovery cohort of 835 Caucasian individuals who participated in the CoLaus study, we identified 139 suggestively significant (P<5×10(-8)) and independent associations between single nucleotide polymorphisms (SNP) and metabolome features. Fifty-six of these associations replicated in the TasteSensomics cohort, comprising 601 individuals from São Paulo of vastly diverse ethnic background. They correspond to eleven gene-metabolite associations, six of which had been previously identified in the urine metabolome and three in the serum metabolome. Our key novel findings are the associations of two SNPs with NMR spectral signatures pointing to fucose (rs492602, P = 6.9×10(-44)) and lysine (rs8101881, P = 1.2×10(-33)), respectively. Fine-mapping of the first locus pinpointed the FUT2 gene, which encodes a fucosyltransferase enzyme and has previously been associated with Crohn's disease. This implicates fucose as a potential prognostic disease marker, for which there is already published evidence from a mouse model. The second SNP lies within the SLC7A9 gene, rare mutations of which have been linked to severe kidney damage. The replication of previous associations and our new discoveries demonstrate the potential of untargeted metabolomics GWAS to robustly identify molecular disease markers.

Behind the Scenes of an Ant Genome Project

Dramatic improvements in DNA sequencing technologies have led to amore than 1,000-fold reduction in sequencing costs over the past five years.Genome-wide research approaches can thus now be applied beyond medicallyrelevant questions to examine the molecular-genetic basis of behavior,development and unique life histories in almost any organism. A first step foran emerging model organism is usually establishing a reference genomesequence. I offer insight gained from the fire ant genome project. First, I detailhow the project came to be and how sequencing, assembly and annotationstrategies were chosen. Subsequently, I describe some of the issues linked toworking with data from recently sequenced genomes. Finally, I discuss anapproach undertaken in a follow-up project based on the fire ant genomesequence.

Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study.

BACKGROUND & AIMS: Hepatitis C virus (HCV) induces chronic infection in 50% to 80% of infected persons; approximately 50% of these do not respond to therapy. We performed a genome-wide association study to screen for host genetic determinants of HCV persistence and response to therapy. METHODS: The analysis included 1362 individuals: 1015 with chronic hepatitis C and 347 who spontaneously cleared the virus (448 were coinfected with human immunodeficiency virus [HIV]). Responses to pegylated interferon alfa and ribavirin were assessed in 465 individuals. Associations between more than 500,000 single nucleotide polymorphisms (SNPs) and outcomes were assessed by multivariate logistic regression. RESULTS: Chronic hepatitis C was associated with SNPs in the IL28B locus, which encodes the antiviral cytokine interferon lambda. The rs8099917 minor allele was associated with progression to chronic HCV infection (odds ratio [OR], 2.31; 95% confidence interval [CI], 1.74-3.06; P = 6.07 x 10(-9)). The association was observed in HCV mono-infected (OR, 2.49; 95% CI, 1.64-3.79; P = 1.96 x 10(-5)) and HCV/HIV coinfected individuals (OR, 2.16; 95% CI, 1.47-3.18; P = 8.24 x 10(-5)). rs8099917 was also associated with failure to respond to therapy (OR, 5.19; 95% CI, 2.90-9.30; P = 3.11 x 10(-8)), with the strongest effects in patients with HCV genotype 1 or 4. This risk allele was identified in 24% of individuals with spontaneous HCV clearance, 32% of chronically infected patients who responded to therapy, and 58% who did not respond (P = 3.2 x 10(-10)). Resequencing of IL28B identified distinct haplotypes that were associated with the clinical phenotype. CONCLUSIONS: The association of the IL28B locus with natural and treatment-associated control of HCV indicates the importance of innate immunity and interferon lambda in the pathogenesis of HCV infection.

Quality control and conduct of genome-wide association meta-analyses.

Rigorous organization and quality control (QC) are necessary to facilitate successful genome-wide association meta-analyses (GWAMAs) of statistics aggregated across multiple genome-wide association studies. This protocol provides guidelines for (i) organizational aspects of GWAMAs, and for (ii) QC at the study file level, the meta-level across studies and the meta-analysis output level. Real-world examples highlight issues experienced and solutions developed by the GIANT Consortium that has conducted meta-analyses including data from 125 studies comprising more than 330,000 individuals. We provide a general protocol for conducting GWAMAs and carrying out QC to minimize errors and to guarantee maximum use of the data. We also include details for the use of a powerful and flexible software package called EasyQC. Precise timings will be greatly influenced by consortium size. For consortia of comparable size to the GIANT Consortium, this protocol takes a minimum of about 10 months to complete.

A mega-analysis of genome-wide association studies for major depressive disorder.

Prior genome-wide association studies (GWAS) of major depressive disorder (MDD) have met with limited success. We sought to increase statistical power to detect disease loci by conducting a GWAS mega-analysis for MDD. In the MDD discovery phase, we analyzed more than 1.2 million autosomal and X chromosome single-nucleotide polymorphisms (SNPs) in 18 759 independent and unrelated subjects of recent European ancestry (9240 MDD cases and 9519 controls). In the MDD replication phase, we evaluated 554 SNPs in independent samples (6783 MDD cases and 50 695 controls). We also conducted a cross-disorder meta-analysis using 819 autosomal SNPs with P<0.0001 for either MDD or the Psychiatric GWAS Consortium bipolar disorder (BIP) mega-analysis (9238 MDD cases/8039 controls and 6998 BIP cases/7775 controls). No SNPs achieved genome-wide significance in the MDD discovery phase, the MDD replication phase or in pre-planned secondary analyses (by sex, recurrent MDD, recurrent early-onset MDD, age of onset, pre-pubertal onset MDD or typical-like MDD from a latent class analyses of the MDD criteria). In the MDD-bipolar cross-disorder analysis, 15 SNPs exceeded genome-wide significance (P<5 × 10(-8)), and all were in a 248 kb interval of high LD on 3p21.1 (chr3:52 425 083-53 822 102, minimum P=5.9 × 10(-9) at rs2535629). Although this is the largest genome-wide analysis of MDD yet conducted, its high prevalence means that the sample is still underpowered to detect genetic effects typical for complex traits. Therefore, we were unable to identify robust and replicable findings. We discuss what this means for genetic research for MDD. The 3p21.1 MDD-BIP finding should be interpreted with caution as the most significant SNP did not replicate in MDD samples, and genotyping in independent samples will be needed to resolve its status.

Thirty new loci for age at menarche identified by a meta-analysis of genome-wide association studies.

To identify loci for age at menarche, we performed a meta-analysis of 32 genome-wide association studies in 87,802 women of European descent, with replication in up to 14,731 women. In addition to the known loci at LIN28B (P = 5.4 × 10⁻⁶⁰) and 9q31.2 (P = 2.2 × 10⁻³³), we identified 30 new menarche loci (all P < 5 × 10⁻⁸) and found suggestive evidence for a further 10 loci (P < 1.9 × 10⁻⁶). The new loci included four previously associated with body mass index (in or near FTO, SEC16B, TRA2B and TMEM18), three in or near other genes implicated in energy homeostasis (BSX, CRTC1 and MCHR2) and three in or near genes implicated in hormonal regulation (INHBA, PCSK2 and RXRG). Ingenuity and gene-set enrichment pathway analyses identified coenzyme A and fatty acid biosynthesis as biological processes related to menarche timing.

Genome-wide association and genetic functional studies identify autism susceptibility candidate 2 gene (AUTS2) in the regulation of alcohol consumption.

Alcohol consumption is a moderately heritable trait, but the genetic basis in humans is largely unknown, despite its clinical and societal importance. We report a genome-wide association study meta-analysis of ∼2.5 million directly genotyped or imputed SNPs with alcohol consumption (gram per day per kilogram body weight) among 12 population-based samples of European ancestry, comprising 26,316 individuals, with replication genotyping in an additional 21,185 individuals. SNP rs6943555 in autism susceptibility candidate 2 gene (AUTS2) was associated with alcohol consumption at genome-wide significance (P = 4 × 10(-8) to P = 4 × 10(-9)). We found a genotype-specific expression of AUTS2 in 96 human prefrontal cortex samples (P = 0.026) and significant (P < 0.017) differences in expression of AUTS2 in whole-brain extracts of mice selected for differences in voluntary alcohol consumption. Down-regulation of an AUTS2 homolog caused reduced alcohol sensitivity in Drosophila (P < 0.001). Our finding of a regulator of alcohol consumption adds knowledge to our understanding of genetic mechanisms influencing alcohol drinking behavior.

A genome-wide association study of early menopause and the combined impact of identified variants.

Early menopause (EM) affects up to 10% of the female population, reducing reproductive lifespan considerably. Currently, it constitutes the leading cause of infertility in the western world, affecting mainly those women who postpone their first pregnancy beyond the age of 30 years. The genetic aetiology of EM is largely unknown in the majority of cases. We have undertaken a meta-analysis of genome-wide association studies (GWASs) in 3493 EM cases and 13 598 controls from 10 independent studies. No novel genetic variants were discovered, but the 17 variants previously associated with normal age at natural menopause as a quantitative trait (QT) were also associated with EM and primary ovarian insufficiency (POI). Thus, EM has a genetic aetiology which overlaps variation in normal age at menopause and is at least partly explained by the additive effects of the same polygenic variants. The combined effect of the common variants captured by the single nucleotide polymorphism arrays was estimated to account for ∼30% of the variance in EM. The association between the combined 17 variants and the risk of EM was greater than the best validated non-genetic risk factor, smoking.

Systems biology of plants : from intraspecific genome variation to computational morphodynamics

Recently, the introduction of second generation sequencing and further advance-ments in confocal microscopy have enabled system-level studies for the functional characterization of genes. The degree of complexity intrinsic to these approaches needs the development of bioinformatics methodologies and computational models for extracting meaningful biological knowledge from the enormous amount of experi¬mental data which is continuously generated. This PhD thesis presents several novel bioinformatics methods and computational models to address specific biological questions in Plant Biology by using the plant Arabidopsis thaliana as a model system. First, a spatio-temporal qualitative analysis of quantitative transcript and protein profiles is applied to show the role of the BREVIS RADIX (BRX) protein in the auxin- cytokinin crosstalk for root meristem growth. Core of this PhD work is the functional characterization of the interplay between the BRX protein and the plant hormone auxin in the root meristem by using a computational model based on experimental evidence. Hyphotesis generated by the modelled to the discovery of a differential endocytosis pattern in the root meristem that splits the auxin transcriptional response via the plasma membrane to nucleus partitioning of BRX. This positional information system creates an auxin transcriptional pattern that deviates from the canonical auxin response and is necessary to sustain the expression of a subset of BRX-dependent auxin-responsive genes to drive root meristem growth. In the second part of this PhD thesis, we characterized the genome-wide impact of large scale deletions on four divergent Arabidopsis natural strains, through the integration of Ultra-High Throughput Sequencing data with data from genomic hybridizations on tiling arrays. Analysis of the identified deletions revealed a considerable portion of protein coding genes affected and supported a history of genomic rearrangements shaped by evolution. In the last part of the thesis, we showed that VIP3 gene in Arabidopsis has an evo-lutionary conserved role in the 3' to 5' mRNA degradation machinery, by applying a novel approach for the analysis of mRNA-Seq data from random-primed mRNA. Altogether, this PhD research contains major advancements in the study of natural genomic variation in plants and in the application of computational morphodynamics models for the functional characterization of biological pathways essential for the plant. - Récemment, l'introduction du séquençage de seconde génération et les avancées dans la microscopie confocale ont permis des études à l'échelle des différents systèmes cellulaires pour la caractérisation fonctionnelle de gènes. Le degrés de complexité intrinsèque à ces approches ont requis le développement de méthodologies bioinformatiques et de modèles mathématiques afin d'extraire de la masse de données expérimentale générée, des information biologiques significatives. Ce doctorat présente à la fois des méthodes bioinformatiques originales et des modèles mathématiques pour répondre à certaines questions spécifiques de Biologie Végétale en utilisant la plante Arabidopsis thaliana comme modèle. Premièrement, une analyse qualitative spatio-temporelle de profiles quantitatifs de transcripts et de protéines est utilisée pour montrer le rôle de la protéine BREVIS RADIX (BRX) dans le dialogue entre l'auxine et les cytokinines, des phytohormones, dans la croissance du méristème racinaire. Le noyau de ce travail de thèse est la caractérisation fonctionnelle de l'interaction entre la protéine BRX et la phytohormone auxine dans le méristème de la racine en utilisant des modèles informatiques basés sur des preuves expérimentales. Les hypothèses produites par le modèle ont mené à la découverte d'un schéma différentiel d'endocytose dans le méristème racinaire qui divise la réponse transcriptionnelle à l'auxine par le partitionnement de BRX de la membrane plasmique au noyau de la cellule. Cette information positionnelle crée une réponse transcriptionnelle à l'auxine qui dévie de la réponse canonique à l'auxine et est nécessaire pour soutenir l'expression d'un sous ensemble de gènes répondant à l'auxine et dépendant de BRX pour conduire la croissance du méristème. Dans la seconde partie de cette thèse de doctorat, nous avons caractérisé l'impact sur l'ensemble du génome des délétions à grande échelle sur quatre souches divergentes naturelles d'Arabidopsis, à travers l'intégration du séquençage à ultra-haut-débit avec l'hybridation génomique sur puces ADN. L'analyse des délétions identifiées a révélé qu'une proportion considérable de gènes codant était affectée, supportant l'idée d'un historique de réarrangement génomique modelé durant l'évolution. Dans la dernière partie de cette thèse, nous avons montré que le gène VÏP3 dans Arabidopsis a conservé un rôle évolutif dans la machinerie de dégradation des ARNm dans le sens 3' à 5', en appliquant une nouvelle approche pour l'analyse des données de séquençage d'ARNm issue de transcripts amplifiés aléatoirement. Dans son ensemble, cette recherche de doctorat contient des avancées majeures dans l'étude des variations génomiques naturelles des plantes et dans l'application de modèles morphodynamiques informatiques pour la caractérisation de réseaux biologiques essentiels à la plante. - Le développement des plantes est écrit dans leurs codes génétiques. Pour comprendre comment les plantes sont capables de s'adapter aux changements environnementaux, il est essentiel d'étudier comment leurs gènes gouvernent leur formation. Plus nous essayons de comprendre le fonctionnement d'une plante, plus nous réalisons la complexité des mécanismes biologiques, à tel point que l'utilisation d'outils et de modèles mathématiques devient indispensable. Dans ce travail, avec l'utilisation de la plante modèle Arabidopsis thalicinci nous avons résolu des problèmes biologiques spécifiques à travers le développement et l'application de méthodes informatiques concrètes. Dans un premier temps, nous avons investigué comment le gène BREVIS RADIX (BRX) régule le développement de la racine en contrôlant la réponse à deux hormones : l'auxine et la cytokinine. Nous avons employé une analyse statistique sur des mesures quantitatives de transcripts et de produits de gènes afin de démontrer que BRX joue un rôle antagonisant dans le dialogue entre ces deux hormones. Lorsque ce-dialogue moléculaire est perturbé, la racine primaire voit sa longueur dramatiquement réduite. Pour comprendre comment BRX répond à l'auxine, nous avons développé un modèle informatique basé sur des résultats expérimentaux. Les simulations successives ont mené à la découverte d'un signal positionnel qui contrôle la réponse de la racine à l'auxine par la régulation du mouvement intracellulaire de BRX. Dans la seconde partie de cette thèse, nous avons analysé le génome entier de quatre souches naturelles d'Arabidopsis et nous avons trouvé qu'une grande partie de leurs gènes étaient manquant par rapport à la souche de référence. Ce résultat indique que l'historique des modifications génomiques conduites par l'évolution détermine une disponibilité différentielle des gènes fonctionnels dans ces plantes. Dans la dernière partie de ce travail, nous avons analysé les données du transcriptome de la plante où le gène VIP3 était non fonctionnel. Ceci nous a permis de découvrir le rôle double de VIP3 dans la régulation de l'initiation de la transcription et dans la dégradation des transcripts. Ce rôle double n'avait jusqu'alors été démontrée que chez l'homme. Ce travail de doctorat supporte le développement et l'application de méthodologies informatiques comme outils inestimables pour résoudre la complexité des problèmes biologiques dans la recherche végétale. L'intégration de la biologie végétale et l'informatique est devenue de plus en plus importante pour l'avancée de nos connaissances sur le fonctionnement et le développement des plantes.

Genome-wide association study of recurrent major depressive disorder in two European case-control cohorts.

Major depressive disorder (MDD) is a highly prevalent disorder with substantial heritability. Heritability has been shown to be substantial and higher in the variant of MDD characterized by recurrent episodes of depression. Genetic studies have thus far failed to identify clear and consistent evidence of genetic risk factors for MDD. We conducted a genome-wide association study (GWAS) in two independent datasets. The first GWAS was performed on 1022 recurrent MDD patients and 1000 controls genotyped on the Illumina 550 platform. The second was conducted on 492 recurrent MDD patients and 1052 controls selected from a population-based collection, genotyped on the Affymetrix 5.0 platform. Neither GWAS identified any SNP that achieved GWAS significance. We obtained imputed genotypes at the Illumina loci for the individuals genotyped on the Affymetrix platform, and performed a meta-analysis of the two GWASs for this common set of approximately half a million SNPs. The meta-analysis did not yield genome-wide significant results either. The results from our study suggest that SNPs with substantial odds ratio are unlikely to exist for MDD, at least in our datasets and among the relatively common SNPs genotyped or tagged by the half-million-loci arrays. Meta-analysis of larger datasets is warranted to identify SNPs with smaller effects or with rarer allele frequencies that contribute to the risk of MDD.

A genome-wide association study of resistance to HIV infection in highly exposed uninfected individuals with hemophilia A.

Human genetic variation contributes to differences in susceptibility to HIV-1 infection. To search for novel host resistance factors, we performed a genome-wide association study (GWAS) in hemophilia patients highly exposed to potentially contaminated factor VIII infusions. Individuals with hemophilia A and a documented history of factor VIII infusions before the introduction of viral inactivation procedures (1979-1984) were recruited from 36 hemophilia treatment centers (HTCs), and their genome-wide genetic variants were compared with those from matched HIV-infected individuals. Homozygous carriers of known CCR5 resistance mutations were excluded. Single nucleotide polymorphisms (SNPs) and inferred copy number variants (CNVs) were tested using logistic regression. In addition, we performed a pathway enrichment analysis, a heritability analysis, and a search for epistatic interactions with CCR5 Δ32 heterozygosity. A total of 560 HIV-uninfected cases were recruited: 36 (6.4%) were homozygous for CCR5 Δ32 or m303. After quality control and SNP imputation, we tested 1 081 435 SNPs and 3686 CNVs for association with HIV-1 serostatus in 431 cases and 765 HIV-infected controls. No SNP or CNV reached genome-wide significance. The additional analyses did not reveal any strong genetic effect. Highly exposed, yet uninfected hemophiliacs form an ideal study group to investigate host resistance factors. Using a genome-wide approach, we did not detect any significant associations between SNPs and HIV-1 susceptibility, indicating that common genetic variants of major effect are unlikely to explain the observed resistance phenotype in this population.

Meta-analysis of genome-wide association data identifies a risk locus for major mood disorders on 3p21.1.

The major mood disorders, which include bipolar disorder and major depressive disorder (MDD), are considered heritable traits, although previous genetic association studies have had limited success in robustly identifying risk loci. We performed a meta-analysis of five case-control cohorts for major mood disorder, including over 13,600 individuals genotyped on high-density SNP arrays. We identified SNPs at 3p21.1 associated with major mood disorders (rs2251219, P = 3.63 x 10(-8); odds ratio = 0.87; 95% confidence interval, 0.83-0.92), with supportive evidence for association observed in two out of three independent replication cohorts. These results provide an example of a shared genetic susceptibility locus for bipolar disorder and MDD.