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Prostate cancer (PC) is the most frequently diagnosed non-skin cancer in the world. Previous studies have shown that genomic alterations represent the most common mechanism for molecular alterations responsible for the development and progression of PC. This highlights the importance of identifying functional genomic variants for early detection in high-risk PC individuals. Great efforts have been made to identify common protein-coding genetic variations; however, the impact of non-coding variations, including regulatory genetic variants, is not well understood. Identification of these variants and the underlying target genes will be a key step in improving the detection and treatment of PC. To gain an understanding of the functional impact of genetic variants, and in particular, regulatory variants in PC, we developed an integrative pipeline (AGV) that uses whole genome/exome sequences, GWAS SNPs, chromosome conformation capture data, and ChIP-Seq signals to investigate the potential impact of genomic variants on the underlying target genes in PC. We identified 646 putative regulatory variants, of which 30 significantly altered the expression of at least one protein-coding gene. Our analysis of chromatin interactions data (Hi-C) revealed that the 30 putative regulatory variants could affect 131 coding and non-coding genes. Interestingly, our study identified the 131 protein-coding genes that are involved in disease-related pathways, including Reactome and MSigDB, for most of which targeted treatment options are currently available. Notably, our analysis revealed several non-coding RNAs, including RP11-136K7.2 and RAMP2-AS1, as potential enhancer elements of the protein-coding genes CDH12 and EZH1, respectively. Our results provide a comprehensive map of genomic variants in PC and reveal their potential contribution to prostate cancer progression and development.

High blood pressure (BP) is the major risk factor for cardiovascular disease. Genome-wide association studies have identified genetic variants for BP, but functional insights into causality and related molecular mechanisms lag behind. We functionally characterize 4,608 genetic variants in linkage with 135 BP loci in vascular smooth muscle cells and cardiomyocytes by massively parallel reporter assays. High densities of regulatory variants at BP loci (i.e., ULK4, MAP4, CFDP1, PDE5A) indicate that multiple variants drive genetic association. Regulatory variants are enriched in repeats, alter cardiovascular-related transcription factor motifs, and spatially converge with genes controlling specific cardiovascular pathways. Using heuristic scoring, we define likely causal variants, and CRISPR prime editing finally determines causal variants for KCNK9, SFXN2, and PCGF6, which are candidates for developing high BP. Our systems-level approach provides a catalog of functionally relevant variants and their genomic architecture in two trait-relevant cell lines for a better understanding of BP gene regulation. We thank the Melé and Maass labs for intellectual input, Dr. Steven Erwood from Dr. Ronald Cohn’s lab for guidance in applying CRISPR prime editing, The Centre for Applied Genomics, The Structural & Biophysical Core Facility, and The Imaging Facility, The Hospital for Sick Children, Toronto, Canada, for assistance with high-throughput sequencing, luminescence detection, and imaging. We thank Dovetail Genomics, LLC, 100 Enterprise Way, Suite A101, Scotts Valley, CA 95066, USA, for generating Omni-C libraries and for the collaborative support throughout the project. W.O. was supported by a Fundació la Marató grant (ref. 321/C/2019), K.K. was supported by an OGS fellowship, J.W.L.B. was supported by a CGS-D fellowship, and D.F.L. was supported by an Ontario Genomics-CANSSI Ontario Postdoctoral Fellowship in Genome Data Science. This project was supported by Canada’s New Frontiers in Research Fund (NFRFE-2018-01305), the Canadian Institutes of Health Research (CIHR PJT 173542 [P.G.M.] and PJT 175034 [S.M., J.E.]), CIHR ENP 161429 under the frame of ERA PerMed (S.M.), the Ted Rogers Centre for Heart Research (S.M., J.E.), and the Heart and Stroke Foundation of Canada. J.E. holds a Canada Research Chair Tier 1 in Stem Cell Models of Childhood Disease, S.M. holds the Heart and Stroke Foundation of Canada & Robert M. Freedom Chair in Cardiovascular Science, M. Melé was supported by a Ramon y Cajal fellowship (RYC-2017-22249), and P.G.M. holds a Canada Research Chair Tier 2 in Non-coding Disease Mechanisms. Peer Reviewed "Article signat per 14 autors/es: Winona Oliveros, Kate Delfosse, Daniella F. Lato , Katerina Kiriakopulos, Milad Mokhtaridoost, Abdelrahman Said, Brandon J. McMurray, Jared W.L. Browning, Kaia Mattioli, Guoliang Meng, James Ellis, Seema Mital, Marta Melé, Philipp G. Maass"

A diverse range of loss-of-function variants in the SPINK1 gene (encoding pancreatic secretory trypsin inhibitor) has been identified in patients with chronic pancreatitis (CP). The haplotype harboring the SPINK1 c.101A>G (p.Asn34Ser or N34S) variant (rs17107315:T>C) is one of the most important heritable risk factors for CP as a consequence of its relatively high prevalence worldwide (population allele frequency ≈ 1%) and its considerable effect size (odds ratio ≈ 11). The causal variant responsible for this haplotype has been intensively investigated over the past two decades. The different hypotheses tested addressed whether the N34S missense variant has a direct impact on enzyme structure and function, whether c.101A>G could affect pre-mRNA splicing or mRNA stability, and whether another variant in linkage disequilibrium with c.101A>G might be responsible for the observed association with CP. Having reviewed the currently available genetic and experimental data, we conclude that c.-4141G>T (rs142703147:C>A), which disrupts a PTF1L-binding site within an evolutionarily conserved HNF1A-PTF1L cis-regulatory module located ∼4 kb upstream of the SPINK1 promoter, can be designated as the causal variant beyond reasonable doubt. This case illustrates the difficulties inherent in determining the identity of the causal variant underlying an initially identified disease association.

Objective To identify and characterize genetic loci associated with the risk of developing ANCA-associated vasculitides (AAV). Methods Genetic association analyses were performed after Illumina sequencing of 1853 genes and subsequent replication with genotyping of selected single nucleotide polymorphisms in a total cohort of 1110 Scandinavian cases with granulomatosis with polyangiitis or microscopic polyangiitis, and 1589 controls. A novel AAV-associated single nucleotide polymorphism was analysed for allele-specific effects on gene expression using luciferase reporter assay. Results PR3-ANCA+ AAV was significantly associated with two independent loci in the HLA-DPB1/HLA-DPA1 region [rs1042335, P = 6.3 × 10−61, odds ratio (OR) 0.10; rs9277341, P = 1.5 × 10−44, OR 0.22] and with rs28929474 in the SERPINA1 gene (P = 2.7 × 10−10, OR 2.9). MPO-ANCA+ AAV was significantly associated with the HLA-DQB1/HLA-DQA2 locus (rs9274619, P = 5.4 × 10−25, OR 3.7) and with a rare variant in the BACH2 gene (rs78275221, P = 7.9 × 10−7, OR 3.0), the latter a novel susceptibility locus for MPO-ANCA+ granulomatosis with polyangiitis/microscopic polyangiitis. The rs78275221-A risk allele reduced luciferase gene expression in endothelial cells, specifically, as compared with the non-risk allele. Conclusion We identified a novel susceptibility locus for MPO-ANCA+ AAV and propose that the associated variant is of mechanistic importance, exerting a regulatory function on gene expression in specific cell types.

A diverse range of loss-of-function variants in the SPINK1 gene (encoding pancreatic secretory trypsin inhibitor) has been identified in patients with chronic pancreatitis (CP). The haplotype harboring the SPINK1 c.101A>G (p.Asn34Ser or N34S) variant (rs17107315:T>C) is one of the most important heritable risk factors for CP as a consequence of its relatively high prevalence worldwide (population allele frequency ≈ 1%) and its considerable effect size (odds ratio ≈ 11). The causal variant responsible for this haplotype has been intensively investigated over the past two decades. The different hypotheses tested addressed whether the N34S missense variant has a direct impact on enzyme structure and function, whether c.101A>G could affect pre-mRNA splicing or mRNA stability, and whether another variant in linkage disequilibrium with c.101A>G might be responsible for the observed association with CP. Having reviewed the currently available genetic and experimental data, we conclude that c.-4141G>T (rs142703147:C>A), which disrupts a PTF1L-binding site within an evolutionarily conserved HNF1A-PTF1L cis-regulatory module located ∼4 kb upstream of the SPINK1 promoter, can be designated as the causal variant beyond reasonable doubt. This case illustrates the difficulties inherent in determining the identity of the causal variant underlying an initially identified disease association.

Single nucleotide polymorphisms (SNPs) within the regulatory elements of a gene can alter gene expression, making these SNPs of prime importance for candidate gene association studies. We aimed to determine whether such regulatory variants are associated with clinical outcomes in three cohorts of patients with prostate cancer. We used RegulomeDB to identify potential regulatory variants based on in silico predictions and reviewed genome‐wide experimental findings. Overall, 131 putative regulatory SNPs with the highest confidence score on predicted functionality were investigated in two independent localized prostate cancer cohorts totalling 458 patients who underwent radical prostatectomy. The statistically significant SNPs identified in these two cohorts were then tested in an additional cohort of 504 patients with advanced prostate cancer. We identified one regulatory SNPs, rs1646724, that are consistently associated with increased risk of recurrence in localized disease (P = .003) and mortality in patients with advanced prostate cancer (P = .032) after adjusting for known clinicopathological factors. Further investigation revealed that rs1646724 may affect expression of SLC35B4, which encodes a glycosyltransferase, and that down‐regulation of SLC35B4 by transfecting short hairpin RNA in DU145 human prostate cancer cell suppressed proliferation, migration and invasion. Furthermore, we found increased SLC35B4 expression correlated with more aggressive forms of prostate cancer and poor patient prognosis. Our study provides robust evidence that regulatory genetic variants can affect clinical outcomes.

The first part of my thesis deals with the experimental characterization of Plasmodium falciparum submicroscopic infections in asymptomatic Congolese women during childbirth. Malaria remains a major public health problem in the world with about 219 million cases and 435,000 deaths a year, mostly in sub-Saharan Africa (90%). In the area of high transmission, malaria infection is mainly characterized by the onset of maternal anemia and the presence of parasites in the placenta. A high proportion of individuals living in malaria-endemic areas harbor parasites undetectable by microscopy, but which can nevertheless be detected using molecular tools such as PCR. Morbidity and mortality related to gestational malaria has decreased since the introduction of intermittent preventive treatment with sulfadoxine-pyrimethamine (IPT-SP). In the endemic area, there is a decrease in the prevalence of placental malaria and an increase in the average weight of the newborn. Nevertheless, the plasmodial infection in the pregnant woman persists and is not controlled. In order to understand the causes of this persistence, I characterized the parasitic populationsof P. falciparum in Congolese pregnant women from southern Brazzaville on IPT-SPand I analyzed their genetic profile in the peripheral blood, placental blood and umbilical cord. The evaluation of the frequency of the plasmodial infection has shown that the treatment correlates with a decrease in the frequency of microscopic infections and an increase in the frequency of submicroscopic infections with a moderate genetic diversity of P. falciparum. Age, pregnancy, and doses of SP do not interfere with the multiplicity of infections that are similar in all three types of blood. These results contribute to the understanding of parasite dynamics in peripheral, placental and umbilical cord blood and circulating parasite populationin Congo.The second part of my thesis involved the use of bioinformatic approaches to detect regulatory variants associated with susceptibility to severe malaria. Recent advances in sequencing technologies enable to identify an increasingly broad spectrum of variants in the human genome. However, the identification of regulatory variants associated with complex diseases remains a challenge, particularly for identifying functionally relevant variants in non-coding regions. We have developed a bioinformatic method to predict regulatory variants of noncoding regions associated with a disease and acting on transcriptional regulation. The approach is based on the integration of various elements of information col-lected automatically from Ensembl database, dbSNP and GWAS catalog, and on the selection of variations that may affect regulation, by combining specialized bioinformatics tools: analysis Regulatory Sequence Analysis Tools and ChIP-seq (ReMap) data. To do this, we developed an analysis workflow in the R statistics language, which invokes remote resources (Web services). The tool is designed generically and can be adapted for the study of regulatory variants of any disease documented in the GWAS catalog. In order to facilitate its use by a biologist, the tool automatically generates (in R markdown) an analysis report illustrated by figures and tables. I tested the tool with an example case of severe malaria that showed that on a set of 375 candidate variants, the tool predicts eleven potential regulatory variants that alter the binding sites of transcription factors. Three of these candidate variants (rs1541253, rs1541254, rs1541255) associated with the ATP2B4 gene (plasma membrane calcium-transporting ATPase 4, encodes the main calcium pump of erythrocytes) are being experimentally validated for their impact on promoter activity., La première partie de ma thèse porte sur la caractérisation expérimentale des infections submicroscopiques à Plasmodium falciparum chez les femmes Congolaises asymptomatiques lors de l’accouchement. Le paludisme demeure un problème majeur de santé publique dans le monde avecenviron 219 millions de cas et 435 000 décès par an, majoritairement en Afrique sub-saharienne (90%). Dans la zone de forte transmission, l’infection palustre se caractérise principalement par le déclenchement d’une anémie maternelle et par la présence de parasites dans le placenta. Une forte proportion d’individus vivant dans des zones d’endémie palustre héberge des parasites indétectables par microscopie, mais qui peuvent néanmoins être détectées à l’aide d’outils moléculaires tels que la PCR. La morbi-mortalité liée au paludisme gestationnel a diminué depuis la mise en place du traitement préventif intermittent à base de la sulfadoxine-pyriméthamine (TPI-SP). Dans la zone endémique, on observe une baisse de la prévalence du paludisme placentaire et l’augmentation du poids moyen du nouveau-né. Malgré tout, l’infection plasmodiale chez la femme enceinte persiste et n’est pas maîtrisée.Dans le but de comprendre les causes de cette persistance, j’ai caractérisé les populations parasitaires de P. falciparum chez les femmes enceintes Congolaises du sud de Brazzaville sous TPI-SP et j’ai analysé leur profil génétique dans le sang périphérique, placentaire et du cordon ombilical. L’évaluation de la fréquence de l’infection plasmodiale a montré qu’il y’a une baisse de la fréquence des infections microscopiques chez les femmes sous traitement préventif intermittent et une augmentation de la fréquence des infections submicroscopiques avec une diversité génétique modérée de P. falciparum. L’âge, la gravidité et les doses de SP n’interfèrent pas avec la multiplicité des infections qui est similaire dans lestrois types de sang. Ces résultats contribuent à la compréhension de la dynamique des parasites dans le sang périphérique, placentaire et du cordon ombilical et de la population parasitaire en circulation au Congo.La seconde partie de ma thèse a consisté à utiliser des approches bioinformatiques pour détecter les variants régulateurs associés à la susceptibilité au paludisme sévère. Les progrès récents des technologies de séquençage ont permis d’identifier un spectre de plus en plus large de variants dans le génome humain. Cependant, l’identification des variants régulateurs associés à des maladies complexes reste un défi, en particulier pour identifier des variants pertinents du point de vue fonctionnel dans des régions non-codantes. Nous avons développé une méthodebioinformatique de prédiction des variants régulateurs des régions non-codantes associés à une maladie et agissant sur la régulation transcriptionnelle. L’approche repose sur l’intégration d’éléments d’informations collectées automatiquement à partir des bases de données Ensembl, dbSNP et GWAS catalog, et sur la sélection des variations susceptibles d’affecter la régulation, en combinant des outils bioinformatiques spécialisés : analyse de motifs (Regulatory Sequence Analysis Tools) et de données ChIP-seq (ReMap). Pour ce faire, nous avons développé unworkflow d’analyse dans le langage de statistiques R pour invoquer des ressources à distance (Web services). L’outil est conçu de façon générique, et peut s’adapter pour l’étude des variants régulateurs de n’importe quelle maladie documentée dans le GWAS catalog. Afin de faciliter son utilisation par un biologiste, l’outil génère automatiquement (en R markdown) un rapport d’analyse illustré par des figures et tableaux. J’ai testé l’outil avec un cas d’exemple du paludisme sévère qui a montré que sur un ensemble de 375 variants candidats, l’outil prédit 11 potentiels variantsrégulateurs qui altèrent les sites de liaisons des facteurs de transcription. Trois de ces variants candidats (rs1541253, rs1541254, rs1541255) associés au gène ATP2B4 (ATPase 4 transportant le calcium sur la membrane plasmique, code pour la pompe de calcium principale des érythrocytes) sont en cours de validation expérimentale pour leur impact sur l’activité du promoteur.

Introduction We hypothesized that common Alzheimer's disease (AD)-associated variants within the triggering receptor expressed on myeloid ( TREM ) gene cluster influence disease through gene expression. Methods Expression microarrays on temporal cortex and cerebellum from ∼400 neuropathologically diagnosed subjects and two independent RNAseq replication cohorts were used for expression quantitative trait locus analysis. Results A variant within a DNase hypersensitive site 5′ of TREM2 , rs9357347-C, associates with reduced AD risk and increased TREML1 and TREM2 levels (uncorrected P = 6.3 × 10 −3 and 4.6 × 10 −2 , respectively). Meta-analysis on expression quantitative trait locus results from three independent data sets ( n = 1006) confirmed these associations (uncorrected P = 3.4 × 10 −2 and 3.5 × 10 −3 , Bonferroni-corrected P = 6.7 × 10 −2 and 7.1 × 10 −3 , respectively). Discussion Our findings point to rs9357347 as a functional regulatory variant that contributes to a protective effect observed at the TREM locus in the International Genomics of Alzheimer's Project genome-wide association study meta-analysis and suggest concomitant increase in TREML1 and TREM2 brain levels as a potential mechanism for protection from AD.

Genome-wide association studies (GWASes) revealed several single-nucleotide polymorphisms (SNPs) in the human 17q12-21 locus associated with autoimmune diseases. However, follow-up studies are still needed to identify causative SNPs directly mediating autoimmune risk in the locus. We have chosen six SNPs in high linkage disequilibrium with the GWAS hits that showed the strongest evidence of causality according to association pattern and epigenetic data and assessed their functionality in a local genomic context using luciferase reporter system. We found that rs12946510, rs4795397, rs12709365, and rs8067378 influenced the reporter expression level in leukocytic cell lines. The strongest effect visible in three distinct cell types was observed for rs12946510 that is predicted to alter MEF2A/C and FOXO1 binding sites.

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect affecting 135,000 newborns worldwide each year. While a multifactorial etiology has been suggested as the cause, despite decades of research, the genetic underpinnings of NSCLP remain largely unexplained. In our previous genome-wide linkage study of a large NSCLP African-American family, we identified a candidate locus at 8q21.3-24.12 (LOD = 2.98). This region contained four genes, Frizzled-6 (FZD6), Matrilin-2 (MATN2), Odd-skipped related 2 (OSR2) and Solute Carrier Family 25, Member 32 (SLC25A32). FZD6 was located under the maximum linkage peak. In this study, we sequenced the coding and noncoding regions of these genes in two affected family members, and identified a rare variant in intron 1 of FZD6 (rs138557689; c.-153 + 432A>C). The variant C allele segregated with NSCLP in this family, through affected and unaffected individuals, and was found in one other NSCLP African-American family. Functional assays showed that this allele creates an allele-specific protein-binding site and decreases promoter activity. We also observed that loss and gain of fzd6 in zebrafish contributes to craniofacial anomalies. FZD6 regulates the WNT signaling pathway, which is involved in craniofacial development, including midfacial formation and upper labial fusion. We hypothesize, therefore, that alteration in FZD6 expression contributes to NSCLP in this family by perturbing the WNT signaling pathway.

It remains challenging to predict regulatory variants in particular tissues or cell types due to highly context-specific gene regulation. By connecting large-scale epigenomic profiles to expression quantitative trait loci (eQTLs) in a wide range of human tissues/cell types, we identify critical chromatin features that predict variant regulatory potential. We present cepip, a joint likelihood framework, for estimating a variant's regulatory probability in a context-dependent manner. Our method exhibits significant GWAS signal enrichment and is superior to existing cell type-specific methods. Furthermore, using phenotypically relevant epigenomes to weight the GWAS single-nucleotide polymorphisms, we improve the statistical power of the gene-based association test., published_or_final_version

Genetic variation in C-type lectins influences infectious disease susceptibility but remains poorly understood. We used allelic mRNA expression imbalance (AEI) technology for surfactant protein (SP)-A1, SP-A2, SP-D, dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), macrophage mannose receptor (MRC1) and Dectin-1, expressed in human macrophages and/or lung tissues. Frequent AEI, an indicator of regulatory polymorphisms, was observed in SP-A2, SP-D and DC-SIGN. AEI was measured for SP-A2 in 38 lung tissues using four marker single-nucleotide polymorphisms (SNPs) and was confirmed by next-generation sequencing of one lung RNA sample. Genomic DNA at the SP-A2 DNA locus was sequenced by Ion Torrent technology in 16 samples. Correlation analysis of genotypes with AEI identified a haplotype block, and, specifically, the intronic SNP rs1650232 (30% minor allele frequency); the only variant consistently associated with an approximately twofold change in mRNA allelic expression. Previously shown to alter a NAGNAG splice acceptor site with likely effects on SP-A2 expression, rs1650232 generates an alternative splice variant with three additional bases at the start of exon 3. Validated as a regulatory variant, rs1650232 is in partial linkage disequilibrium with known SP-A2 marker SNPs previously associated with risk for respiratory diseases including tuberculosis. Applying functional DNA variants in clinical association studies, rather than marker SNPs, will advance our understanding of genetic susceptibility to infectious diseases.

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits. This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants). Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells. DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers. The primer extension assay is carried out using the MassARRAY (Sequenom) platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results. © 2010 Journal of Visualized Experiments.