Multi-omics approaches to sickle cell disease heterogeneity

La drépanocytose est une maladie causée par une seule mutation dans le gène de la bêta-globine. Les complications liées à la maladie se manifestent sur le plan génétique, épigénique, transcriptionnel, et métabolique. Les approches intégratives des technologies de séquençage à haut-débit permettent de comprendre le mécanisme pathologique et de découvrir des thérapies en lien avec la maladie. Dans cette thèse, j’intègre divers jeux de données omiques et j’applique des méthodes statistiques pour élaborer de nouvelles hypothèses et analyser les données. Dans les deux premières études, je combine les résultats des études d'association pangénomique d'hémoglobine fœtale (HbF) et des globules rouges denses déshydratés (DRBC) avec l'expression génique, l'interaction chromatinienne, les bases de données relatives aux maladies et les cibles médicamenteuses sélectionnées par des experts. Cette approche intégrative a révélé trois nouveaux loci sur le chromosome 10 (BICC1), le chromosome 19 (KLF1) et le chromosome 22 (CECR2) comme régulateurs de l'HbF. Pour l’étude sur la densité de globules rouges, quatre cibles médicamenteuses (BCL6, LRRC32, KNCJ14 et LETM1) ont été identifiées comme des modulateurs potentiels de la sévérité. Dans la troisième étude, j’intégre la métabolomique à la génomique pour établir une relation causale entre la L-glutamine et les crises douleurs en utilisant la randomisation mendélienne. En outre, nous avons identifié 66 biomarqueurs pour 6 complications liées à la drépanocytose et le débit de filtration glomérulaire estimé (DFGe). Enfin, dans la dernière étude j’ai appliqué une approche de clustering aux métabolites que j’ai ensuite combiné aux données de génotype. J’ai découvert des changements métabolomiques mettant en évidence des familles de métabolites impliqués dans les dysfonctionnements rénaux et hépatiques, en plus de confirmer le rôle d'une classe d'acides gras dans la formation en faucille des globules rouges. Ce travail met en évidence l'importance des approches multi-omiques pour découvrir de nouveaux mécanismes biologiques et étudier les maladies humaines.Sickle cell disease is a monogenic disorder caused by a point mutation in the beta-globin gene. The complications related to the disease are characterized by a broad spectrum of distinct genetic, epigenetic, transcriptional, and metabolomic states. Integrative high-throughput technologies approaches to sickle cell disease pathophysiology are crucial to understanding complications mechanisms and uncovering therapeutic interventions. In this thesis, I integrate various omics datasets and apply statistical methods to derive new hypotheses and analyze data. I combine genome-wide association studies results of fetal hemoglobin (HbF) and dehydrated dense red blood cells (DRBC) with gene expression, chromatin interaction, disease-relevant databases, and expert-curated drug targets. This integrative approach revealed three novel loci on chromosome 10 (BICC1), chromosome 19 (KLF1) and chromosome 22 (CECR2) as key modulators of HbF. For DRBC, four drug targets (BCL6, LRRC32, KNCJ14, and LETM1) were identified as potential severity modifiers. Using mendelian randomization, I integrated metabolomics with genomics in the third study to establish a potential causal relationship between L-glutamine and painful crisis. Additionally, we identified 66 biomarkers for 6 SCD-related complications and estimated glomerular filtration rate (eGFR). Finally, the last study applied a clustering framework to metabolites which I then combined with genotypes. I found specific metabolomics changes highlighting families of metabolites involved in renal and liver dysfunction and confirming the role of a class of fatty acids in red blood cell sickling. This work highlights the importance of multi-omics approaches to unearth new biology and study human diseases

The genetics of red blood cell density, a biomarker of clinical severity in sickle cell disease

L’anémie falciforme est l’une des maladies du sang les plus répandues chez l’homme. Les complications liées à la maladie sont systémiques. Influant virtuellement tous les organes du corps, cette affection provoque des crises de douleurs imprévisibles et aigües dont les complications mènent parfois à la mort. Le processus à travers lequel un globule rouge sain prend la forme d’une faucille est bien décrit dans la littérature; sous désoxygénation, l’eau et les solutés se retirent des globules rouges, la concentration d’hémoglobine S augmente et nous donne des globules rouges denses et déshydratés qui par la suite deviennent falciformes. Les traitements d’aujourd’hui sont pour la plupart expérimentaux et coûteux. De plus, leurs efficacités à long terme varient d’un patient à l’autre. Il est donc impératif de trouver un biomarqueur qui est à la fois abordable et qui améliore la santé des malades de façon systématique. La densité des globules rouges est un biomarqueur largement ignoré par la communauté médicale dans le contexte de la drépanocytose. Aborder l’étude de la sévérité de cette maladie en se concentrant sur la densité des globules rouges nous met en position d’identifier des traitements pour réhydrater les érythrocytes et leur rendre leur forme originale de disque biconcave. Plusieurs études cliniques et physiologiques se sont penchées sur ce biomarqueur sans explorer le volet génétique. Nous avons cherché à éclaircir cet aspect en menant une étude d’association pangénomique et en examinant les séquences exomiques d’individus avec des mesures de densité extrême. Notre étude d’association pangénomique n’a pas conduit à la découverte de nouveau loci, probablement parce que la taille de notre échantillon, et donc notre puissance statistique, était limitée. En revanche, à travers notre approche de priorisation, nous avons découvert un marqueur intronique qui contrôle l’expression d’ATP2B4, la protéine principale de transport de calcium dans les hématies. Notre séquençage exomique a identifié deux mutations rares faux-sens chez un même patient; l’une dans ATP1B2, un transporteur de Na+/K+, et l’autre dans SPTB, le gène du β-spectrin. Ces mutations expliqueraient pourquoi ce patient a le pourcentage de densité le plus élevé parmi tous nos patients séquencés, et pourquoi il vit avec plusieurs complications de la maladie. Finalement, nous avons localisé une mutation faux-sens rare chez deux patients avec un indice élevé de densité de globule rouge, dans PIEZO1, le canal ionique mécano-sensitif. La mutation est prédite délétère par deux algorithmes de prédiction de fonction protéique.Sickle cell disease is one of most common blood disorder amongst human. The complications associated with the disease are systemic. They damage virtually all the organs of the body, causing severe, unpredictable pain episodes, which repercussions can eventually lead to death. The process through which a biconcave, healthy red blood cell assumes a crescent-shape is well described in the literature; under deoxygenation, as water and solutes leave erythrocytes the concentration of hemoglobin S increases thus giving us dense dehydrated cells and subsequently sickled cells. Today’s current therapies are for the most part experimental, costly, and vary widely in their long-term effectiveness from patient to patient. There is, therefore, a pressing need, to identify a biomarker that is cost-effective and provides positive health outcomes to patients. The density of red blood cell is a biological indicator largely ignored by the medical community in sickle cell disease. Exploring erythrocytes density can facilitate the development of new therapies by targeting channels to rehydrate cells back to their normal shape. Clinical and physiological characterizations of this phenotype exist in many studies, but the genetic characterization is absent. We attempted to elucidate the genetic underpinning of this phenotype, by conducting a genome-wide scan, and examining the whole-exome sequences of individuals with extreme red blood cell density. Our genome-wide association study did not highlight any new loci due to our limited statistical power reflected by the cohort’s small sample size. However, our prioritization approach highlighted an intronic variant that controls the expression of ATP2B4, the main calcium pump in erythrocytes. Our whole-exome sequencing experiment pointed out two rare missense mutations in the same patient; one in ATP1B2, a Na+/K+ transporter, and the other in SPTB, the β-spectrin gene. These variants could explain why he has the highest measured density of red blood cells amongst all of our sequenced patients, and why this person experiences several of the disease-related complications. Another rare missense mutation in two patients with elevated levels of dense cells was discovered in PIEZO1, the mechanosensitive ion channel. The mutation is predicted to be deleterious by both protein function prediction algorithms

Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci

Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants from genome-wide association studies largely cluster in regulatory DNA. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating mutagenesis libraries with single or combinatorial nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, a locus associated with red blood cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false positive regions, which emphasizes the importance of off-target analysis in design of saturating mutagenesis experiments. Taken together, these data establish a widely applicable high-throughput and high-resolution methodology to reliably identify minimal functional sequences within large regions of disease- and trait-associated DNA

HLA allele-calling using multi-ancestry whole-exome sequencing from the UK Biobank identifies 129 novel associations in 11 autoimmune diseases

Abstract The human leukocyte antigen (HLA) region on chromosome 6 is strongly associated with many immune-mediated and infection-related diseases. Due to its highly polymorphic nature and complex linkage disequilibrium patterns, traditional genetic association studies of single nucleotide polymorphisms do not perform well in this region. Instead, the field has adopted the assessment of the association of HLA alleles (i.e., entire HLA gene haplotypes) with disease. Often based on genotyping arrays, these association studies impute HLA alleles, decreasing accuracy and thus statistical power for rare alleles and in non-European ancestries. Here, we use whole-exome sequencing (WES) from 454,824 UK Biobank (UKB) participants to directly call HLA alleles using the HLA-HD algorithm. We show this method is more accurate than imputing HLA alleles and harness the improved statistical power to identify 360 associations for 11 auto-immune phenotypes (at least 129 likely novel), leading to better insights into the specific coding polymorphisms that underlie these diseases. We show that HLA alleles with synonymous variants, often overlooked in HLA studies, can significantly influence these phenotypes. Lastly, we show that HLA sequencing may improve polygenic risk scores accuracy across ancestries. These findings allow better characterization of the role of the HLA region in human disease

Whole-genome sequencing in diverse subjects identifies genetic correlates of leukocyte traits: The NHLBI TOPMed program

Many common and rare variants associated with hematologic traits have been discovered through imputation on large-scale reference panels. However, the majority of genome-wide association studies (GWASs) have been conducted in Europeans, and determining causal variants has proved challenging. We performed a GWAS of total leukocyte, neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts generated from 109,563,748 variants in the autosomes and the X chromosome in the Trans-Omics for Precision Medicine (TOPMed) program, which included data from 61,802 individuals of diverse ancestry. We discovered and replicated 7 leukocyte trait associations, including (1) the association between a chromosome X, pseudo-autosomal region (PAR), noncoding variant located between cytokine receptor genes (CSF2RA and CLRF2) and lower eosinophil count; and (2) associations between single variants found predominantly among African Americans at the S1PR3 (9q22.1) and HBB (11p15.4) loci and monocyte and lymphocyte counts, respectively. We further provide evidence indicating that the newly discovered eosinophil-lowering chromosome X PAR variant might be associated with reduced susceptibility to common allergic diseases such as atopic dermatitis and asthma. Additionally, we found a burden of very rare FLT3 (13q12.2) variants associated with monocyte counts. Together, these results emphasize the utility of whole-genome sequencing in diverse samples in identifying associations missed by European-ancestry-driven GWASs