Combined transcriptomic and proteomic studies reveal non-canonical signaling functions in the jasmonate pathway

Abstract Lipid derived signals mediate many stress and defense responses in multicellular eukaryotes. Among these are the jasmonates, potently active signaling compounds in plants. Jasmonic acid (JA) and 12-oxo-phytodienoic acid (OPDA) are the two best known members of the large jasmonate family. This thesis further investigates their roles as signals using genomic and proteomic approaches. The study is based on a simple genetic model involving two key genes. The first is ALLENE OXIDE SYNTHASE (AOS), encoding the most important enzyme in generating jasmonates. The second is CORONATINE INSENSITIVE 1 (COI1), a gene involved in all currently documented canonical signaling responses. We asked the simple question: do null mutations in AOS and COI1 have analogous effects on the transcriptome ? We found that they do not. If most COI1-dependent genes were also AOS-dependent, the expression of a zinc-finger protein was AOS-dependent but was unaffected by the coi1-1 mutation. We thus supposed that a jasmonate member, most probably OPDA, can alter gene expression partially independently of COI1. Conversely, the expression of at least three genes, one of these is a protein kinase, was shown to be COI1-dependent but did not require a functional AOS protein. We conclude that a non-jasmonate signal might alter gene expression through COIL Proteomic comparison of coi1-1 and aos plants confirmed these observations and highlighted probable protein degradation processes controlled by jasmonates and COI1 in the wounded leaf. This thesis revealed new functions for COI1 and for AOS-generated oxylipins in the jasmonate signaling pathway. Résumé Les signaux dérivés d'acides gras sont des médiateurs de réponses aux stress et de la défense des eucaryotes multicellulaires. Parmi eux, les jasmonates sont de puissants composés de sig¬nalisation chez les plantes. L'acide jasmonique (JA) et l'acide 12-oxo-phytodienoïc (OPDA) sont les deux membres les mieux caractérisés de la grande famille des jasmonates. Cette thèse étudie plus profondément leurs rôles de signalisation en utilisant des approches génomique et protéomique. Cette étude est basée sur un modèle génétique simple n'impliquant que deux gènes. Le premier est PALLENE OXYDE SYNTHASE (AOS) qui encode l'enzyme la plus importante pour la fabrication des jasmonates. Le deuxième est CORONATINE INSENSITIVE 1 (COI1) qui est impliqué dans la totalité des réponses aux jasmonates connues à ce jour. Nous avons posé la question suivante : est-ce que les mutations nulles dans les gènes AOS et COI1 ont des effets analogues sur le transcriptome ? Nous avons trouvé que ce n'était pas le cas. Si la majorité des gènes dépendants de COI1 sont également dépendants d'AOS, l'expression d'un gène codant pour une protéine formée de doigts de zinc n'est pas affectée par la mutation de COI1 tout en étant dépendante d'AOS. Nous avons donc supposé qu'un membre de la famille des jasmonates, probablement OPDA, pouvait modifier l'expression de certains gènes indépendamment de COI1. Inversement, nous avons montré que, tout en étant dépendante de COI1, l'expression d'au moins trois gènes, dont un codant pour une protéine kinase, n'était pas affectée par l'absence d'une protéine AOS fonctionnelle. Nous en avons conclu qu'un signal autre qu'un jasmonate devait modifier l'expression de certains gènes à travers COI1. La comparaison par protéomique de plantes aos et coi1-1 a confirmé ces observations et a mis en évidence un probable processus de dégradation de protéines contrôlé par les jasmonates et COU_ Cette thèse a mis en avant de nouvelles fonctions pour COI1 et pour des oxylipines générées par AOS dans le cadre de la signalisation par les jasmonates.

A genetic risk score combining 32 SNPs is associated with body mass index and improves obesity prediction in people with major depressive disorder.

BACKGROUND: Obesity is strongly associated with major depressive disorder (MDD) and various other diseases. Genome-wide association studies have identified multiple risk loci robustly associated with body mass index (BMI). In this study, we aimed to investigate whether a genetic risk score (GRS) combining multiple BMI risk loci might have utility in prediction of obesity in patients with MDD. METHODS: Linear and logistic regression models were conducted to predict BMI and obesity, respectively, in three independent large case-control studies of major depression (Radiant, GSK-Munich, PsyCoLaus). The analyses were first performed in the whole sample and then separately in depressed cases and controls. An unweighted GRS was calculated by summation of the number of risk alleles. A weighted GRS was calculated as the sum of risk alleles at each locus multiplied by their effect sizes. Receiver operating characteristic (ROC) analysis was used to compare the discriminatory ability of predictors of obesity. RESULTS: In the discovery phase, a total of 2,521 participants (1,895 depressed patients and 626 controls) were included from the Radiant study. Both unweighted and weighted GRS were highly associated with BMI (P <0.001) but explained only a modest amount of variance. Adding 'traditional' risk factors to GRS significantly improved the predictive ability with the area under the curve (AUC) in the ROC analysis, increasing from 0.58 to 0.66 (95% CI, 0.62-0.68; χ(2) = 27.68; P <0.0001). Although there was no formal evidence of interaction between depression status and GRS, there was further improvement in AUC in the ROC analysis when depression status was added to the model (AUC = 0.71; 95% CI, 0.68-0.73; χ(2) = 28.64; P <0.0001). We further found that the GRS accounted for more variance of BMI in depressed patients than in healthy controls. Again, GRS discriminated obesity better in depressed patients compared to healthy controls. We later replicated these analyses in two independent samples (GSK-Munich and PsyCoLaus) and found similar results. CONCLUSIONS: A GRS proved to be a highly significant predictor of obesity in people with MDD but accounted for only modest amount of variance. Nevertheless, as more risk loci are identified, combining a GRS approach with information on non-genetic risk factors could become a useful strategy in identifying MDD patients at higher risk of developing obesity.

Modulation of genetic associations with serum urate levels by body-mass-index in humans.

We tested for interactions between body mass index (BMI) and common genetic variants affecting serum urate levels, genome-wide, in up to 42569 participants. Both stratified genome-wide association (GWAS) analyses, in lean, overweight and obese individuals, and regression-type analyses in a non BMI-stratified overall sample were performed. The former did not uncover any novel locus with a major main effect, but supported modulation of effects for some known and potentially new urate loci. The latter highlighted a SNP at RBFOX3 reaching genome-wide significant level (effect size 0.014, 95% CI 0.008-0.02, Pinter= 2.6 x 10-8). Two top loci in interaction term analyses, RBFOX3 and ERO1LB-EDARADD, also displayed suggestive differences in main effect size between the lean and obese strata. All top ranking loci for urate effect differences between BMI categories were novel and most had small magnitude but opposite direction effects between strata. They include the locus RBMS1-TANK (men, Pdifflean-overweight= 4.7 x 10-8), a region that has been associated with several obesity related traits, and TSPYL5 (men, Pdifflean-overweight= 9.1 x 10-8), regulating adipocytes-produced estradiol. The top-ranking known urate loci was ABCG2, the strongest known gout risk locus, with an effect halved in obese compared to lean men (Pdifflean-obese= 2 x 10-4). Finally, pathway analysis suggested a role for N-glycan biosynthesis as a prominent urate-associated pathway in the lean stratum. These results illustrate a potentially powerful way to monitor changes occurring in obesogenic environment.

Chromosome 7 gain and DNA hypermethylation at the HOXA10 locus are associated with expression of a stem cell related HOX-signature in glioblastoma.

BACKGROUND: HOX genes are a family of developmental genes that are expressed neither in the developing forebrain nor in the normal brain. Aberrant expression of a HOX-gene dominated stem-cell signature in glioblastoma has been linked with increased resistance to chemo-radiotherapy and sustained proliferation of glioma initiating cells. Here we describe the epigenetic and genetic alterations and their interactions associated with the expression of this signature in glioblastoma. RESULTS: We observe prominent hypermethylation of the HOXA locus 7p15.2 in glioblastoma in contrast to non-tumoral brain. Hypermethylation is associated with a gain of chromosome 7, a hallmark of glioblastoma, and may compensate for tumor-driven enhanced gene dosage as a rescue mechanism by preventing undue gene expression. We identify the CpG island of the HOXA10 alternative promoter that appears to escape hypermethylation in the HOX-high glioblastoma. An additive effect of gene copy gain at 7p15.2 and DNA methylation at key regulatory CpGs in HOXA10 is significantly associated with HOX-signature expression. Additionally, we show concordance between methylation status and presence of active or inactive chromatin marks in glioblastoma-derived spheres that are HOX-high or HOX-low, respectively. CONCLUSIONS: Based on these findings, we propose co-evolution and interaction between gene copy gain, associated with a gain of chromosome 7, and additional epigenetic alterations as key mechanisms triggering a coordinated, but inappropriate, HOX transcriptional program in glioblastoma.

Single breath-hold 3D measurement of left atrial volume using compressed sensing cardiovascular magnetic resonance and a non-model-based reconstruction approach.

BACKGROUND: Left atrial (LA) dilatation is associated with a large variety of cardiac diseases. Current cardiovascular magnetic resonance (CMR) strategies to measure LA volumes are based on multi-breath-hold multi-slice acquisitions, which are time-consuming and susceptible to misregistration. AIM: To develop a time-efficient single breath-hold 3D CMR acquisition and reconstruction method to precisely measure LA volumes and function. METHODS: A highly accelerated compressed-sensing multi-slice cine sequence (CS-cineCMR) was combined with a non-model-based 3D reconstruction method to measure LA volumes with high temporal and spatial resolution during a single breath-hold. This approach was validated in LA phantoms of different shapes and applied in 3 patients. In addition, the influence of slice orientations on accuracy was evaluated in the LA phantoms for the new approach in comparison with a conventional model-based biplane area-length reconstruction. As a reference in patients, a self-navigated high-resolution whole-heart 3D dataset (3D-HR-CMR) was acquired during mid-diastole to yield accurate LA volumes. RESULTS: Phantom studies. LA volumes were accurately measured by CS-cineCMR with a mean difference of -4.73 ± 1.75 ml (-8.67 ± 3.54%, r2 = 0.94). For the new method the calculated volumes were not significantly different when different orientations of the CS-cineCMR slices were applied to cover the LA phantoms. Long-axis "aligned" vs "not aligned" with the phantom long-axis yielded similar differences vs the reference volume (-4.87 ± 1.73 ml vs. -4.45 ± 1.97 ml, p = 0.67) and short-axis "perpendicular" vs. "not-perpendicular" with the LA long-axis (-4.72 ± 1.66 ml vs. -4.75 ± 2.13 ml; p = 0.98). The conventional bi-plane area-length method was susceptible for slice orientations (p = 0.0085 for the interaction of "slice orientation" and "reconstruction technique", 2-way ANOVA for repeated measures). To use the 3D-HR-CMR as the reference for LA volumes in patients, it was validated in the LA phantoms (mean difference: -1.37 ± 1.35 ml, -2.38 ± 2.44%, r2 = 0.97). Patient study: The CS-cineCMR LA volumes of the mid-diastolic frame matched closely with the reference LA volume (measured by 3D-HR-CMR) with a difference of -2.66 ± 6.5 ml (3.0% underestimation; true LA volumes: 63 ml, 62 ml, and 395 ml). Finally, a high intra- and inter-observer agreement for maximal and minimal LA volume measurement is also shown. CONCLUSIONS: The proposed method combines a highly accelerated single-breathhold compressed-sensing multi-slice CMR technique with a non-model-based 3D reconstruction to accurately and reproducibly measure LA volumes and function.

Analysis of the genetic basis of periodic fever with aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome.

PFAPA syndrome is the most common autoinflammatory syndrome in children from Western countries. In spite of its strong familial clustering, its genetic basis and inheritance pattern are still unknown. We performed a comprehensive genetic study on 68 individuals from 14 families. Linkage analysis suggested a susceptibility locus on chromosome 8, but direct molecular sequencing did not support this initial statistical finding. Exome sequencing revealed the absence of any gene that was mutated in all patients. Exhaustive screening of genes involved in other autoinflammatory syndromes or encoding components of the human inflammasome showed no DNA variants that could be linked to PFAPA molecular pathology. Among these, the previously-reported missense mutation V198M in the NLRP3 gene was clearly shown not to co-segregate with PFAPA. Our results on this relatively large cohort indicate that PFAPA syndrome is unlikely to be a monogenic condition. Moreover, none of the several genes known to be involved in inflammation or in autoinflammatory disorders seem to be relevant, alone, to its etiology, suggesting that PFAPA results from oligogenic or complex inheritance of variants in multiple disease genes and/or non-genetic factors.

ASTRAL-R score predicts non-recanalisation after intravenous thrombolysis in acute ischaemic stroke.

Intravenous thrombolysis (IVT) as treatment in acute ischaemic strokes may be insufficient to achieve recanalisation in certain patients. Predicting probability of non-recanalisation after IVT may have the potential to influence patient selection to more aggressive management strategies. We aimed at deriving and internally validating a predictive score for post-thrombolytic non-recanalisation, using clinical and radiological variables. In thrombolysis registries from four Swiss academic stroke centres (Lausanne, Bern, Basel and Geneva), patients were selected with large arterial occlusion on acute imaging and with repeated arterial assessment at 24 hours. Based on a logistic regression analysis, an integer-based score for each covariate of the fitted multivariate model was generated. Performance of integer-based predictive model was assessed by bootstrapping available data and cross validation (delete-d method). In 599 thrombolysed strokes, five variables were identified as independent predictors of absence of recanalisation: Acute glucose > 7 mmol/l (A), significant extracranial vessel STenosis (ST), decreased Range of visual fields (R), large Arterial occlusion (A) and decreased Level of consciousness (L). All variables were weighted 1, except for (L) which obtained 2 points based on β-coefficients on the logistic scale. ASTRAL-R scores 0, 3 and 6 corresponded to non-recanalisation probabilities of 18, 44 and 74 % respectively. Predictive ability showed AUC of 0.66 (95 %CI, 0.61-0.70) when using bootstrap and 0.66 (0.63-0.68) when using delete-d cross validation. In conclusion, the 5-item ASTRAL-R score moderately predicts non-recanalisation at 24 hours in thrombolysed ischaemic strokes. If its performance can be confirmed by external validation and its clinical usefulness can be proven, the score may influence patient selection for more aggressive revascularisation strategies in routine clinical practice.

Present standards and future perspectives in the treatment of metastatic non-small cell lung cancer.

The development of novel effective immunotherapeutic agents and early clinical data hinting at significant activity in non-small cell lung cancer (NSCLC) has introduced yet another player in the field of management of advanced disease. At present, first-line cytotoxic chemotherapy is generally withheld pending results of molecular testing for any actionable genetic alteration that could lead to targeted treatment, and in their absence chemotherapy is prescribed as a default therapy. Phase III trials comparing head-to-head immune checkpoint inhibitors with standard platinum-based doublet chemotherapy are underway. Second-line chemotherapy is likewise being challenged in phase III trials, one of which having recently reported positive results in advanced squamous cell carcinoma. In tumors harboring actionable transforming genetic alterations such as EGFR mutations and ALK rearrangements, second- and third-generation inhibitors allow for multiple lines of targeted treatment beyond initial resistance, postponing the use of cytotoxic chemotherapy to very late lines of therapy. Chemotherapy as a longstanding but still present standard of care capable of prolonging survival, improving quality of life, and relieving symptoms sees its role increasingly restricted to clinical, immunological, and molecular subsets of patients where its activity and efficacy have never been tested prospectively.

Influence of MCHR2 and MCHR2-AS1 Genetic Polymorphisms on Body Mass Index in Psychiatric Patients and In Population-Based Subjects with Present or Past Atypical Depression.

Obesity development during psychotropic treatments represents a major health issue in psychiatry. Melanin-concentrating hormone receptor 2 (MCHR2) is a central receptor involved in energy homeostasis. MCHR2 shares its promoter region with MCHR2-AS1, a long antisense non-coding RNA. The aim of this study was to determine whether tagging single nucleotide polymorphisms (tSNPs) of MCHR2 and MCHR2-AS1 are associated with the body mass index (BMI) in the psychiatric and in the general population. The influence of MCHR2 and MCHR2-AS1 tSNPs on BMI was firstly investigated in a discovery psychiatric sample (n1 = 474). Positive results were tested for replication in two other psychiatric samples (n2 = 164, n3 = 178) and in two population-based samples (CoLaus, n4 = 5409; GIANT, n5 = 113809). In the discovery sample, TT carriers of rs7754794C>T had 1.08 kg/m2 (p = 0.04) lower BMI as compared to C-allele carriers. This observation was replicated in an independent psychiatric sample (-2.18 kg/m2; p = 0.009). The association of rs7754794C>T and BMI seemed stronger in subjects younger than 45 years (median of age). In the population-based sample, a moderate association was observed (-0.17 kg/m2; p = 0.02) among younger individuals (<45y). Interestingly, this association was totally driven by patients meeting lifetime criteria for atypical depression, i.e. major depressive episodes characterized by symptoms such as an increased appetite. Indeed, patients with atypical depression carrying rs7754794-TT had 1.17 kg/m2 (p = 0.04) lower BMI values as compared to C-allele carriers, the effect being stronger in younger individuals (-2.50 kg/m2; p = 0.03; interaction between rs7754794 and age: p-value = 0.08). This study provides new insights on the possible influence of MCHR2 and/or MCHR2-AS1 on obesity in psychiatric patients and on the pathophysiology of atypical depression.

Decades of population genetic research reveal the need for harmonization of molecular markers: the grey wolf Canis lupus as a case study

Following protection measures implemented since the 1970s, large carnivores are currently increasing in number and returning to areas from which they were absent for decades or even centuries. Monitoring programmes for these species rely extensively on non-invasive sampling and genotyping. However, attempts to connect results of such studies at larger spatial or temporal scales often suffer from the incompatibility of genetic markers implemented by researchers in different laboratories. This is particularly critical for long-distance dispersers, revealing the need for harmonized monitoring schemes that would enable the understanding of gene flow and dispersal dynamics. Based on a review of genetic studies on grey wolves Canis lupus from Europe, we provide an overview of the genetic markers currently in use, and identify opportunities and hurdles for studies based on continent-scale datasets. Our results highlight an urgent need for harmonization of methods to enable transnational research based on data that have already been collected, and to allow these data to be linked to material collected in the future. We suggest timely standardization of newly developed genotyping approaches, and propose that action is directed towards the establishment of shared single nucleotide polymorphism panels, next-generation sequencing of microsatellites, a common reference sample collection and an online database for data exchange. Enhanced cooperation among genetic researchers dealing with large carnivores in consortia would facilitate streamlining of methods, their faster and wider adoption, and production of results at the large spatial scales that ultimately matter for the conservation of these charismatic species.

Functional impact of genetic variation on gene expression

Numerous links between genetic variants and phenotypes are known and genome-wide association studies dramatically increased the number of genetic variants associated with traits during the last decade. However, how changes in the DNA perturb the molecular mechanisms and impact on the phenotype of an organism remains elusive. Studies suggest that many traitassociated variants are in the non-coding region of the genome and probably act through regulation of gene expression. During my thesis I investigated how genetic variants affect gene expression through gene regulatory mechanisms. The first chapter was a collaborative project with a pharmaceutical company, where we investigated genome-wide copy number variation (CNVs) among Cynomolgus monkeys (Macaca fascicularis) used in pharmaceutical studies, and associated them to changes in gene expression. We found substantial copy number variation and identified CNVs linked to tissue-specific expression changes of proximal genes. The second and third chapters focus on genetic variation in humans and its effects on gene regulatory mechanisms and gene expression. The second chapter studies two human trios, where the allelic effects of genetic variation on genome-wide gene expression, protein-DNA binding and chromatin modifications were investigated. We found abundant allele specific activity across all measured molecular phenotypes and show extended coordinated behavior among them. In the third chapter, we investigated the impact of genetic variation on these phenotypes in 47 unrelated individuals. We found that chromatin phenotypes are organized into local variable modules, often linked to genetic variation and gene expression. Our results suggest that chromatin variation emerges as a result of perturbations of cis-regulatory elements by genetic variants, leading to gene expression changes. The work of this thesis provides novel insights into how genetic variation impacts gene expression by perturbing regulatory mechanisms. -- De nombreux liens entre variations génétiques et phénotypes sont connus. Les études d'association pangénomique ont considérablement permis d'augmenter le nombre de variations génétiques associées à des phénotypes au cours de la dernière décennie. Cependant, comprendre comment ces changements perturbent les mécanismes moléculaires et affectent le phénotype d'un organisme nous échappe encore. Des études suggèrent que de nombreuses variations, associées à des phénotypes, sont situées dans les régions non codantes du génome et sont susceptibles d'agir en modifiant la régulation d'expression des gènes. Au cours de ma thèse, j'ai étudié comment les variations génétiques affectent les niveaux d'expression des gènes en perturbant les mécanismes de régulation de leur expression. Le travail présenté dans le premier chapitre est un projet en collaboration avec une société pharmaceutique. Nous avons étudié les variations en nombre de copies (CNV) présentes chez le macaque crabier (Macaca fascicularis) qui est utilisé dans les études pharmaceutiques, et nous les avons associées avec des changements d'expression des gènes. Nous avons découvert qu'il existe une variabilité substantielle du nombre de copies et nous avons identifié des CNVs liées aux changements d'expression des gènes situés dans leur voisinage. Ces associations sont présentes ou absentes de manière spécifique dans certains tissus. Les deuxième et troisième chapitres se concentrent sur les variations génétiques dans les populations humaines et leurs effets sur les mécanismes de régulation des gènes et leur expression. Le premier se penche sur deux trios humains, père, mère, enfant, au sein duquel nous avons étudié les effets alléliques des variations génétiques sur l'expression des gènes, les liaisons protéine-ADN et les modifications de la chromatine. Nous avons découvert que l'activité spécifique des allèles est abondante abonde dans tous ces phénotypes moléculaires et nous avons démontré que ces derniers ont un comportement coordonné entre eux. Dans le second, nous avons examiné l'impact des variations génétiques de ces phénotypes moléculaires chez 47 individus, sans lien de parenté. Nous avons observé que les phénotypes de la chromatine sont organisés en modules locaux, qui sont liés aux variations génétiques et à l'expression des gènes. Nos résultats suggèrent que la variabilité de la chromatine est due à des variations génétiques qui perturbent des éléments cis-régulateurs, et peut conduire à des changements dans l'expression des gènes. Le travail présenté dans cette thèse fournit de nouvelles pistes pour comprendre l'impact des différentes variations génétiques sur l'expression des gènes à travers les mécanismes de régulation.

Sociodemographic, behavioral and genetic determinants of allostatic load in a Swiss population-based study.

Allostatic load (AL) is a marker of physiological dysregulation which reflects exposure to chronic stress. High AL has been related to poorer health outcomes including mortality. We examine here the association of socioeconomic and lifestyle factors with AL. Additionally, we investigate the extent to which AL is genetically determined. We included 803 participants (52% women, mean age 48±16years) from a population and family-based Swiss study. We computed an AL index aggregating 14 markers from cardiovascular, metabolic, lipidic, oxidative, hypothalamus-pituitary-adrenal and inflammatory homeostatic axes. Education and occupational position were used as indicators of socioeconomic status. Marital status, stress, alcohol intake, smoking, dietary patterns and physical activity were considered as lifestyle factors. Heritability of AL was estimated by maximum likelihood. Women with a low occupational position had higher AL (low vs. high OR=3.99, 95%CI [1.22;13.05]), while the opposite was observed for men (middle vs. high OR=0.48, 95%CI [0.23;0.99]). Education tended to be inversely associated with AL in both sexes(low vs. high OR=3.54, 95%CI [1.69;7.4]/OR=1.59, 95%CI [0.88;2.90] in women/men). Heavy drinking men as well as women abstaining from alcohol had higher AL than moderate drinkers. Physical activity was protective against AL while high salt intake was related to increased AL risk. The heritability of AL was estimated to be 29.5% ±7.9%. Our results suggest that generalized physiological dysregulation, as measured by AL, is determined by both environmental and genetic factors. The genetic contribution to AL remains modest when compared to the environmental component, which explains approximately 70% of the phenotypic variance.